DOMAIN 2 CELL ARCHITECTURE AND GROWTH

Modulation of Chemical
Composition and Other
Parameters of the Cell at Different
Exponential Growth Rates
HANS BREMER1 AND PATRICK P. DENNIS2
1
Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson,
TX 75083-0688
2
Department of Biochemistry and Molecular Biology, University of British Columbia,
Vancouver, British Columbia V6T 1Z3, Canada

Received: 1 January 2008
Accepted: 30 March 2008
Posted: 7 October 2008
Supercedes previous posting at EcoSal.org.
Editor: James M. Slauch, University of Illinois at
Urbana-Champaign, Urbana, IL
Citation: Ecosal Plus 2013; doi: 10.1128/
ecosal.5.2.3
Correspondence: Hans Bremer bremer3@
attglobal.net and Patrick P. Dennis pdennis@nsf.
gov
Copyright: ASM Press. All rights
reserved.2008
doi: 10.1128/ecosal.5.2.3
ABSTRACT This review begins by briefly presenting the history of research on the
chemical composition and other parameters of cells of E. coli and S. enterica at different
exponential growth rates. Studies have allowed us to determine the in vivo strength of
promoters and have allowed us to distinguish between factor-dependent transcriptional
control of the promoter and changes in promoter activity due to changes in the concen-
tration of free functional RNA polymerase associated with different growth conditions. The
total, or bulk, amounts of RNA and protein are linked to the growth rate, because most
bacterial RNA is ribosomal RNA (rRNA). Since ribosomes are required for protein synthesis,
their number and their rate of function determine the rate of protein synthesis and
cytoplasmic mass accumulation. Many mRNAs made in the presence of amino acids have
strong ribosome binding sites whose presence reduces the expression of all other active
genes. This implies that there can be profound differences in the spectrum of gene
activities in cultures grown in different media that produce the same growth rate. Five
classes of growth-related parameters that are generally useful in describing or establishing
the macromolecular composition of bacterial cultures are described in detail in this review.
A number of equations have been reported that describe the macromolecular composition
of an average cell in an exponential culture as a function of the culture doubling time and
five additional parameters: the C- and D-periods, protein per origin (PO), ribosome activity,
and peptide chain elongation rate.
HISTORY
Schaechter et al. (1) first demonstrated that the macromolecular composi-
tion of the bacterial cell was related to its metabolic activity and that RNA-
containing particles were involved in the synthesis of protein. When they
examined the variations in growth and composition of exponential Salmo-
nella enterica serovar Typhimurium cultures in different media, they realized
that the cellular contents of DNA, RNA, and protein at a given temperature
depended only on the growth rate and not on the nutrient supplement in the
growth medium used to achieve that growth rate. They defined the expo-
nential growth rate, , in doublings per hour. The growth rate is pro-
portional to the reciprocal of the culture doubling time, , which is usually
expressed in minutes, so that = 60/. They also found (i) that fast-growing

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Bremer and Dennis
bacteria are larger and contain more DNA, RNA, and
protein than slow-growing bacteria, (ii) that the amounts
of these macromolecules per cell are exponential func-
tions of growth rate, and (iii) that the exponents of these
functions are different for different macromolecules. The
last finding implies that the relative proportions of the
different macromolecules change with growth rate; at a
given temperature, RNA and ribosome concentrations
increase with increasing growth rate, the DNA concen-
tration decreases, and the protein concentration remains
almost constant. When changing the temperature rather
than the nutrient content of the growth medium, the
DNA, RNA, and protein concentrations remained in-
variant at varying growth rates.
These early studies of bacterial physiology are docu-
mented by Maale and Kjeldgaard (2). The statement of
observations in terms of simple mathematical relation-
ships was characteristic for the Copenhagen approach,
in which calculated constants, proportionalities, and
quadratic or exponential functions suggested special
control mechanisms. Many of these relationships later
turned out to be more complex than originally imagined.
Nevertheless, these propositions stimulated thought and
led to more sophisticated observations. Many of the
fundamental problems posed by the Copenhagen group
are still far from being solved.
A theoretical basis for explaining the empirical re-
lationships of the early Copenhagen school was not
available until Cooper and Helmstetter (3) derived a
formula for determining the average amount of DNA
per cell in an exponential culture as a function of the
time required to replicate the chromosome (C), the time
period between termination of a round of replication
and the following cell division (D), and the culture
doubling time (). This theory also included the im-
portant concept of overlapping rounds of chromosome
replication, where a new round of replication is initiated
before the previous round is completed. This occurs
when C is greater than and explains how bacteria are
able to grow with a doubling time shorter than the time
required to replicate their chromosome.
Donachie (4) extended this theory by introducing the
concept of the initiation mass, which he defined as the
cell mass per replication origin at the time of initiation.
Based on this idea, he derived a formula for the average
mass per cell, or amount of protein per cell, as a function
of C,D, , and an additional parameter, mass or protein
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per replication origin (MO or PO, respectively). The
Cooper-Helmstetter equation predicts the amount of
DNA per cell as a function of C, D, and , and the
additional parameter, PO, links the amount of DNA to
the amount of protein in the cell.
At about the same time, Schleif (5) and Maale (6) began
to establish a theoretical relationship between the
amounts of protein and RNA in the cell and the two
parameters, cp and r, which define the function of ri-
bosomes: r is the fraction of total ribosomes actively
engaged in peptide chain elongation; cp is the rate of
peptide chain elongation. Based on these relationships,
Churchward et al. (7) were able to describe the global
cell composition in terms of DNA, RNA, and protein
content as a function of the doubling time, , and the
five additional parameters, C,D,Po, r, and cp. These
mathematical relationships define the connections be-
tween the various physiological parameters, but do not
explain the underlying biological process, since the re-
lationships are derived entirely from the definition of the
exponential growth function and of the parameters used,
without requiring any observed data. Therefore, the re-
lationships do not help to understand how the para-
meters are determined in the cell.
Despite almost 50 years of intensive study in many
different laboratories, the mechanisms by which the
growth medium affects the synthesis of ribosomes and
their rate of function (i.e., the two factors that determine
the growth rate; see Growth rate dependency of the
macromolecular composition, below), are not yet un-
derstood. This means that the important problem of the
control of bacterial growth is far from being solved.
However, considerable progress has been made during
the past 10 years in the determination of absolute pro-
moter activities (in transcripts initiated per minute per
promoter) for ribosomal RNA (rRNA) and ribosomal
protein (r-protein) genes at different growth rates. This
determination requires a combination of transcription
and replication information. Such studies have allowed
us to determine the in vivo strength of promoters and to
define the promoter control in terms of changing Mi-
chaelis-Menten constants, which, in turn, allows one to
distinguish between factor-dependent transcriptional
control of the promoter and changes in promoter ac-
tivity due to changes in the concentration of free func-
tional RNA polymerase associated with different growth
conditions. The latter affects all unsaturated promoters,
both controlled and constitutive, to the same extent.
 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates
These new developments have been recently reviewed
(8).
GROWTH RATE DEPENDENCY OF THE
MACROMOLECULAR COMPOSITION
Relationship between Macromolecular
Composition and Growth Rate
The total, or bulk, amounts of RNA and protein are
linked to the growth rate, because most bacterial RNA is
ribosomal RNA (rRNA). Since ribosomes are required
for protein synthesis, their number and their rate of
function determine the rate of protein synthesis and
cytoplasmic mass accumulation. Mathematically, the
exponential growth rate equals the product of the
number of ribosomes per amount of total protein pres-
ent (Nr/P, representing the ribosome concentration)
times the rate of protein synthesis per average ribosome,
known as ribosome efficiency (er; see equation 18 in
Table 6).
The idea that the exponential growth rate, , is limited
by only two factors, Nr/P and er, might seem overly
simplistic, as one can think of many other potential
bottle necks besides translation that could limit the
growth rate. However, the relationship between Nr/P, er,
and (see equation 18 in Table 6) is not based on any
model or assumption (other than that most Escher-
ichia coli proteins are stable). Instead it follows directly
from the definition of exponential functions. As long as
steady-state exponential cultures are considered, the re-
lationship is necessarily correct (for details in the deri-
vation of the formula, see reference 8). The values of Nr/
P and er, of course, depend on many physiological fac-
tors which, in turn, depend at least in part on the
composition of the growth medium.
In the original work by Schaechter et al. (1) the ribosome
efficiencies observed during growth in different media
were quite similar, especially during growth in amino
acid-supplemented media, when ribosomes are assumed
to function at or near their maximum rate (see Peptide
chain elongation rate, below). Therefore, Schaechter et
al. (1) assumed a general constancy of the rate of pro-
tein synthesis/particle, i.e., a constant value of er. Under
such conditions the growth rate is fully determined by
the value of Nr/P, which reflects the ratio of total RNA/
total protein. Therefore, when (at constant er) a partic-
ular growth medium produces a particular value of Nr/P,
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this value also sets the growth rate. This is the main
reason for Maale's conclusion that the macromolecular
composition depends only on the growth rate, and not
on the growth medium. In the following we suggest that
the situation is more complex and that this idea is not
universally applicable.
Growth Medium-Dependent Control
Since the macromolecular composition of bacteria ap-
peared to depend only on the growth rate (see above),
observed parameters pertaining to the physiology of
bacterial cultures were most often expressed as functions
of the growth rate. However, in some instances this can
be misleading. For example, the synthesis of many
bacterial mRNAs and enzymes can be induced by ex-
ogenous nutrients without producing a significant
change in the growth rate or in the general physiology
and macromolecular composition of the bacterial cul-
ture. Evidently, the growth rate does not determine the
physiological parameters of the bacteria; rather vice
versa, certain physiological parameters determine the
growth rate. Therefore, instead of using the term
growth rate-dependent control (e.g., of the synthesis of
ribosomes or of other components), we prefer to use the
term growth medium-dependent control.
An important observation in this context is the follow-
ing. When the rate of mRNA synthesis (per genome
equivalent of DNA) was compared in six cultures, three
in minimal media with different carbon sources (succi-
nate, glycerol, and glucose) and the other three cultures
in the same media supplemented with all 20 amino
acids, the mRNA synthesis rates could not be plotted as
a single function of growth rate, but only as two separate
curves, one for the three minimal media, and a second
curve for the three amino acid-supplemented media
(10). In another study cultures were grown in two sets of
media, one with both different carbon sources and
amino acid content as in the experiments on which and
below are based, and the other with different carbon
sources in the presence of all amino acids. Here the
strength and activity of a ribosomal RNA (rRNA) pro-
moter, plotted as function of growth rate, gave a dif-
ferent curve for each set (11). For cultures grown in the
presence of all amino acids, it was found that, inde-
pendent of the growth rate, (i) the rate of ribosome
function is near maximal (12), (ii) the accumulation of
the effector ppGpp (an inhibitor of rRNA synthesis; see
also section about RNA polymerase activity below) is
3
Bremer and Dennis
close to zero, and (iii) the rRNA promoters have their
maximum strength (8, 11). (Promoter strength is de-
fined as the promoter activity per free RNA polymerase
concentration under conditions when the promoter is
far from being saturated with polymerase.)
These observations show that two cultures grown in
different media may produce the same growth rate but
have different macromolecular compositions; this con-
flicts with Maale's conclusion above. One might argue
that mRNA represents only a few percent of total RNA,
so that different rates of mRNA synthesis do not sig-
nificantly affect the overall composition of the bacteria.
However, all mRNAs compete for ribosome binding.
Many mRNAs made in the presence of amino acids (i.e.,
when the mRNAs for amino acid biosynthesis are re-
pressed) have strong ribosome binding sites whose
presence reduces the expression of all other active genes
(see Translation frequency of mRNA, below, and the
discussion of Fig. 3 in reference 8). This implies that
there can be profound differences in the spectrum of
gene activities in cultures grown in different media that
produce the same growth rate.
Different Compositions in Cultures Growing at the
Same Rate in Different Media
Why did we find differences in the macromolecular
composition of cultures growing at equal rates but in
different growth media when the Copenhagen school
did not observe this result? There are several possible
reasons; the most apparent are as follows. (i) In part, the
difference reflects the choice of media. Schaechter et al.
(1) used mostly media that contained only amino acids
as nutrients. We have not used media that contain only
amino acids, because the amino acids preferentially used
as a carbon source (i.e., serine or aspartate) might be-
come exhausted, so that the bacteria have to switch to
another amino acid for a carbon source, which makes it
impossible to maintain prolonged balanced growth. (ii)
In the original work only four parameters (expressed
per cell) were measured: mass (optical density units),
RNA, DNA, and nuclei. We have also measured
mRNA, RNA polymerase, peptide chain elongation, as
well as DNA replication and other cell division para-
meters. This leads to a more complete description of the
composition, where differences will become apparent
that are not necessarily apparent when only the four
original parameters are observed. (iii) There also seems
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to be a difference in the growth rates observed by
Schaechter et al. (1) for Salmonella and our results ob-
tained with E. coli B/r. We found a growth rate of 1.0
doubling/h in a medium with all amino acids plus suc-
cinate as a carbon source (10), whereas Schaechter et al.
(1) found almost twice that growth rate in a medium
containing all amino acids but without an additional
carbon source (their Table 1: = 2.0 for Casamino acids
medium and = 1.83 for 20 amino acids medium). We
cannot explain this difference; perhaps some amino acid
used as a carbon source by the bacteria in the experi-
ments of Schaechter et al. was superior to succinate.
Growth Medium-Dependent and Growth
Rate-Dependent Control
Since our aim is to understand the control of the bac-
terial growth rate, it is apparent from the above dis-
cussion that we cannot logically make the growth rate an
independent, freely chosen parameter. Nevertheless, for
both historical and practical reasons, we have kept the
tradition in Table 2 and Table 3 below to describe
physiological parameters as functions of growth rate.
The observations mentioned above indicate that the
single functions of growth rate represented by the data in
these tables were only possible by the particular choice of
media used: minimal media with different carbon
sources (succinate, glycerol, glucose) with glucose pro-
ducing the fastest growth, followed by media containing
glucose and amino acids, and additional supplements
(glucose-amino acids, and LB-glucose) resulting in
higher growth rates than obtained with glucose alone.
The data in the tables were then obtained by drawing
smoothed best-fit curves through the points from par-
ticular sets of published experiments (indicated in the
footnotes to the tables), so that they appear to represent
continuous functions of growth rate. Actual data, even if
precisely determined, generally deviate somewhat from
those values and do not necessarily produce a smooth
curve.
Since the tables are based on a selective choice of media,
the interpretation of the values in these tables requires
some caveats. As an example, if we consider two cul-
tures, one in glycerol minimal medium, the other one in
succinate-amino acids medium, both grow at a rate of
approximately 1.0 doubling/h. In the first case, about
50% of the total rate of RNA synthesis is mRNA, and it
the second case it is only about 20% (from Fig. 9 in
 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates

Table 1 Parameters related to the growth and macromolecular composition of bacterial cells
Class No. Parameter Symbol Value Reference
I 1 Deoxyribonucleotide residues per genome kbp/genome 4,700 13
2 Ribonucleotide residues per rRNA precursor nucl/prib 6,000 14
3 Ribonucleotide residues per 70S ribosome nucl/rib 4,566 14
4 Amino acid residues per 70 ribosome aa/rib 7.336 15
5 Ribonucleotide residues per tRNA nucl/tRNA 80 16
6 Amino acid residue s per RNA polymerase core aa/pol 3,707 1719
II 7 Fraction of total RNA that is stable RNA fs 0.98 20, 21
8 Fraction of stable RNA that is tRNA ft 0.14 22, 23
9 Fraction of active ribosomes r 0.85 Table 3
III 10 Fraction of total protein that is rRNA protein r 0.080.23 Table 3
11 Fraction of total protein that is RNA polymerase p 0.0090.016 Table 3
12 Fraction of active RNAP synthesizing stable RNA s 0.240.86 Table 3
13 Fraction of active RNA polymerase p 0.140.33 Table 3
IV 14 Peptide chain elongation rate cp 1322 aa/s Table 3
15 Stable RNA chain elongation rate cs 85 nucl/s Table 3
16 mRNA chain elongation rate cm 3956 nucl/s Table 3
17 DNA chain elongation rate cd 5801,190 bp/s Table 3
V 18 Time to replicate the chromosome C 3367 min Table 3
19 Time between termination of replication and division D 2330 min Table 3
20 Protein per replication origin PO 3.51084.4108 aa Table 2

reference 10). The data for = 1.0 doubling/h in Table 2
and Table 3 below refer only to cultures grown in
glycerol minimal medium; i.e., they are not valid for
cultures in succinate-amino acids medium that grow at
the same rate.
To bridge the discrepancy between more complex
multifactorial control and growth rate-dependent con-
trol inherent in these tables, we suggest that the growth
rate values in the tables should be understood as re-
presenting particular media that produce growth rates
close to the values shown. Since the growth rate values
in the tables were rounded to constant intervals of 0.5
doubling/h which do not exactly match the observed
growth rates in the media used, most of the parameter
values shown had to be obtained by interpolation. For
this purpose, smooth functions of growth rate were as-
sumed for the growth media chosen. Partly because of
the history effect described below, and partly because
different growth media reflect qualitative, rather than
gradually changing quantitative, differences in the nu-
trients, this assumption may be only approximately
correct.
The Physiological History of a Culture Affects Its
Growth and Composition
Even a given growth medium does not fully determine
the growth rate and macromolecular composition of a
bacterial culture. In addition, effects of the history of the
particular starter culture that is used to inoculate an
experimental culture can be of importance. Certain
physiological controls are apparently set shortly after
an experimental culture is started by dilution from an-
other, generally stationary (overnight) culture; these
controls lead to a certain exponential growth rate asso-
ciated with a certain macromolecular composition after
at least 10 generations (see Exponential growth, be-
low). This particular balanced physiological state is
maintained as long as the culture is kept under non-
restricting conditions, meaning that all nutrient factors
in the medium, including oxygen, are present at non-
limiting concentrations, and it may be prolonged in-
definitely by dilutions with fresh (prewarmed) medium
(28). The effects of the history of the starter cultures
from which experimental cultures are prepared have
hardly been studied in the past and are at present
somewhat beyond the control of the experimenter.

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 6
Bremer and Dennis

Table 2 Macromolecular composition of exponentially growing E. coli B/r as a function of growth rate at 37Ca
At t (min) and (doublings/h)
t, 100 t, 60 t, 40 t, 30 t, 24 t, 20
Parameter Symbol Units , 0.6 , 1.0 , 1.5 , 2.0 , 2.5 , 3.0 Observed parameters Footnote
Protein/mass PM 1017 aa/OD460 5.8 5.5 5.1 4.8 4.5 4.0 P, M b
16
RNA/mass RM 10 nucl./OD460 3.3 3.8 4.4 5.3 6.3 6.7 R, M c
8
DNA/mass GM 10 genomes/OD460 12.0 9.1 7.8 6.8 6.7 6.8 G, M d
Cell no./mass CM 108 cells/OD460 7.7 4.6 3.1 2.2 1.9 1.7 GM, GC e
(P+R+D)/ mass PRDM g/OD460 128 124 119 118 118 111 f

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Protein/ genome PG 108 aa residues 4.8 6.0 6.6 7.1 6.7 5.9 PM,GM g
7
RNA/ genome RG 10 nucl. residues 2.8 4.1 5.6 7.8 9.4 9.9 RM, GM h

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Origins/ genome OG no./genome equ. 1.3 1.4 1.7 1.6 1.7 1.7 C i

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Protein/origin PO 108 aa residues 3.9 4.4 4.4 4.4 4.1 3.5 PM,OG j
8
Protein/cell PC 10 aa residues 7.6 11.9 16.4 21.5 24.0 23.7 PM,CM k
9
PC (g) g/10 cells 136 214 295 387 431 426 l
RNA/cell RC 107 nucl. residues 4.3 8.1 14.0 23.8 33.3 39.6 RM, CM m
9
RC (g) g/10 cells 23 44 76 128 180 214 n
DNA/cell GC Genome equ./cell 1.6 2.0 2.5 3.0 3.6 4.0 C, D o
GC (g) g/109 cells 7.6 9.5 12.0 14.7 17.2 19.4 p
9
Mass/cell MC OD460 units/10 cells 1.3 2.2 3.2 4.5 5.3 5.9 CM q
MC (g) g dry wt./109 cells 226 374 555 774 921 1,023 g/OD460 r
9
Sum (P+R+D) PRDC g/10 cells 167 267 383 530 628 659 PM, RM, GM (g) s
Origins/cell OC no./cell 2.0 2.7 3.8 4.9 5.9 6.7 C, D t
Termini/cell TC no./cell 1.2 1.4 1.5 1.7 1.9 2.1 D u
Repl. forks/cell FC no./cell 1.5 2.7 4.4 6.2 7.8 9.2 C, D v

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 a
With the exception of the values for the D-period, he data in this table are based on newer experiments (12) that deviate somewhat from the data based on earlier experiments resented in the previous
editions of this chapter. The table now includes data for the maximum growth rate at 3.0 doublings/h in LB medium. All values are from nonradioactive assays that have been calibrated as described
(reference 12; see also text for details and variability of values).
b
Protein was determined with a colorimetric assay (24), calibrated and corrected for nonlinearity as described in reference 12. The protein/mass values are taken from Table 2 of reference 25, based on
the smoothed curve drawn in Fig. 4a of reference 12.
c
The RNA/mass values are taken from Table 2 of reference 25, based on the smoothed curve drawn in Fig. 4b in reference 12.
d
The DNA/mass values are taken from the smoothed curve drawn in Fig. 4c of reference 12.
e
The cells/mass values were calculated: CM = GM/GC (see footnotes d and o of this table for GM and GC, respectively).
f
The sum of the weights (in g) of protein, RNA, and DNA per cell was calculated: PRDM = CM PRDC (see footnotes e and s of this table for CM and PRDC, respectively).
g
The protein/genome values were calculated: PG = PM/GM (see footnotes b and d of this table for PM and GM, respectively).
h
The RNA/genome values were calculated: RG = RM/GM (see footnotes c and d of this table for RM and GM, respectively).

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i
The origins/genome values were calculated from C (Table 3), using the relationship (29): OG = ln2(C/)/[1 2 (C/)].
j
The protein/origin values were calculated: PO = PG/OG (see footnotes g and i of this table for PG and OG, respectively).
k
The protein/cell values were calculated: PC = PM/CM (see footnotes b and e of this table for PM and CM, respectively).
l
The protein/cell values in g/109 cells were calculated from the PC values given in 108 aa residues (see footnote k above), the molecular weight of an average amino acid residue in E. coli protein (= 108
g/mol [26]) and Avogadro's number (NA = 6 1023 molecules/mol): PC (g/109 cells) = 109 106 PC 108/NA, where the factors 109 and 106 correspond to the number of cells considered and the number of
g/g, respectively.
m
The RNA/cell values were calculated: RC = RM/CM (see footnotes c and e of this table for RM and CM, respectively).
n
The RNA/cell values in g/109 cells were calculated from the RC where the ribosome efficiency, er = r cp values given in 107 nucleotide residues (see footnote m above), the molecular weight of an
average nucleotide residue in E. coli RNA (= 324 g/mol [26]) and Avogadro's number (NA = 6 1023 molecules/mol): RC (g/109 cells) = 109 106 RC 324/NA, where the factors 109 and 106 correspond to
the number of cells considered and the number of g/g, respectively.
o
The DNA/cell values were calculated from the number of replication forks per cell (FC; see footnote v below) and C (from Table 3), using the relationship (reference 27; equation 3 in Table 5 below): GC
= (/ ln2) FC /2C.
p
The DNA/cell values in g/109 cells were calculated from the GC values given in genome equivalents per cell (see footnote o above), the number of DNA base pairs per genome (Table 1 above: 4.7 106),
the molecular weight of an average base pair in E. coli DNA (= 618 g/mol [26]) and Avogadro's number (NA = 6 1023 molecules/mol): GC (g/109 cells) = 109 (4.7106) 106 GC 618/NA, where the factors
109 and 106 correspond to the number of cells considered and the number of g/g, respectively.
q
The mass/cell values were calculated from the reciprocal of the cells/mass values (in 108 cells per OD460 unit; see footnote e above): MC = 10/CM, where the factor 10 accounts for the fact that 109, rather
than 108 cells were considered.

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r
The mass/cell values in g dry weight per 109 cells were calculated from MC (mass in OD460 units per 109 cells; see footnote q above) and the dry weight of 1.0 OD460 units of bacteria (= 173 g;
reference 28): MC (g dry weight per 109 cells) = MC 173.

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s
The sum of the weights of protein, RNA and DNA (in g per 109 cells) was calculated by addition of the individual values for protein, RNA and DNA in g per 109 cells (see footnotes l, n, and p
above): PRDC = PC (g) + RC (g) + GC (g).

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t
The average number of replication origins per cell was calculated from C and D (see Table 3 below): OC = 2(C+D)/ (reference 29; equation 7 in Table 5 below).
u
The average number of replication termini per cell was calculated from D (Table 3 below): TC = 2D/ (reference 27; equation 8 in Table 5 below).
v
The average number of replication forks per cell was calculated as the difference of replication origins and termini (reference 27; see also equation 10 of Table 5 below), where the factor of 2 accounts
for the fact that every initiation of replication at the origin creates one fork pair during bidirectional replication: FC = 2(OC TC).

7
Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates
 8
a
Table 3 Parameters pertaining to the macromolecular synthesis rates in exponentially growing E. coli B/r as a function of growth rate at 37C
Bremer and Dennis

At t (min) and (doublings/h)

t, 100 t, 60 t, 40 t, 30 t, 24 t, 20 Observed
Parameter Symbol Units , 0.6 , 1.0 , 1.5 , 2.0 , 2.5 , 3.0 parameters Footnote
RNAP/total protein p % 0.90 1.10 1.30 1.45 1.55 1.60 p a
3
RNAP molec./cell Np 10 RNAP/cell 1.8 3.5 5.7 8.4 10.0 10.2 p, PC b
RNAP activity p % 15.5 16.8 17.6 21.9 28.2 36.2 rs, rm, cs, cm, Np c
Active RNAP/cell Nap RNAP/cell 285 592 1,010 1,840 2,820 3,700 c
Stable RNA synthesized per total RNA synth. rs/rt % 41 52 68 78 85 90 rs/rt d
Active RNAP synthesizing stable RNA s % 24 36 56 69 79 86 rs/rt, cs, cm e
rRNA chain elongation cs nucl./s 85 85 85 85 85 852 Indirect f
mRNA chain elongation cm nucl./s 39 45 50 53 55 56 Indirect g
5
Rate of stable RNA synthesis/cell rs 10 nucl/min/cell 3.5 11 29 65 113 161 RC h
5
Rate of mRNA synthesis/cell rm 10 nucl/min/cell 5.1 10.2 13.5 18.2 19.9 17.9 rs, rs/rt i
mRNA lifetime m min 1.9 2.0 2.1 2.2 2.3 2.4 Indirect j
5
mRNA/cell Rm 10 nucl/ cell 10 20 28 40 46 43 rm, m k

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ppGpp concn ppGpp/M pmol/OD460 55 38 22 15 10 6 ppGpp/M l
ppGpp/P pmol/1017 aa 9.5 6.9 4.3 3.1 2.2 1.5 ppGpp/M, PM l

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r-prot./total protein r % 7.7 9.2 11.6 15.0 18.8 22.7 r m

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Ribosome activity r % 85 85 85 85 85 85 Indirect n
Peptide chain elong. cp aa resid./s 13 18 21 22 22 22 Indirect o
3
Ribosomes/cell Nr 10 ribosomes/cell 8 15 26 44 61 73 RC, fs, ft p
tRNA/cell Nt 103 tRNA/cell 74 139 241 408 571 680 Nr, ft q
rrn genes/ genome Nrrn/G No./ genome 7.9 8.4 8.8 9.1 9.3 9.4 C r
rrn genes/cell Nrrn No./ cell 12.4 16.5 22.0 27.6 32.9 37.5 C, D s
Init. rate at rrn gene irrn init/min/gene 4 10 20 37 54 68 Nr, Nrrn t
Distance of ribos. on mRNA dr nucl/ribosome 142 160 128 107 88 69 Rm, r, Nr u
Translat./ mRNA Ntrans Ribosomes 16 20 30 37 45 57 rm, PC v
RNA pol./ ribosome Np/Nr % 23 24 22 19 16 14 Nr, Np w
DNA chain elong. cd bp/s 584 658 762 883 1,023 1,186 C, kbp/G x
C-period C min 67 60 51 44 38 33 Indirect y
D-period D min 30 27 25 24 23 22 Indirect z

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 a
The fraction of total protein that is core RNA polymerase was calculated from the -and -0subunit content determined by sodium dodecyl sulfategel electrophoresis (172). The value for = 3.0 was
obtained by extrapolation.
b
The number of core RNA polymerase per cell was calculated from p (this table), PC (Table 2), and the number of amino acid residues per core RNA polymerase (aa/pol; Table 1): Np = PC p /(aa/pol).
c
The fraction of total RNA polymerase that is actively transcribing was calculated from values in this table, using the relationship: p = (rs/cs + rm/cm)/Np. The number of actively transcribing RNA
polymerase molecules per cell (Nap) was then found: Nap = p Np (see footnote b for Np).
d
The fraction of the total RNA synthesis rate that is stable RNA was determined by hybridization of pulse-labeled RNA to an rDNA probe and correction for tRNA (30, 31).
e
The fraction of active RNA polymerase synthesizing stable RNA was calculated: s = 1/{1 + [1/(rs/rt) 1] (cs/cm)}, using the values for rs/rt, cs, and cm in this table.
f
The stable RNA (or rRNA) chain elongation rate was determined from the 5S rRNA or tRNA labeling after rifampin addition (3236).
g
The mRNA chain elongation rate was determined by analyzing the pulse labeling kinetics after size fractionation (37) and by the time lag between induction of transcription of specific mRNAs (lacZ,
infB) and the appearance of specific hybridization to DNA probes from the 3 ends of the respective genes (36).
h
The stable RNA synthesis rate per cell was calculated from the data in Tables 1 and 2: rs = (ln 2/) RC fs 1.2, where the factor 1.2 corrects for the 20% of the rRNA and tRNA primary transcripts that

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are unstable spacer or flanking sequences.
i
The mRNA synthesis rate per cell was calculated from the data in this table: rm = rs {[1/(rs/rt)] 1}.
j
The mRNA lifetimes represent the functional life of lacZ mRNA, given by the average time of the first endonucleolytic cleavage close to the 5 end after transcript initiation by induction with lac
inducer (IPTG). This time was determined by analyzing the induction kinetics of -galactosidase and independently by analyzing the kinetics of residual -galactosidase accumulation after stopping
transcript initiation with rifampin (38). Different mRNAs are assumed to have different functional lifetimes; the lacZ mrNA lifetimes were assumed to be representative for bulk mRNA. The data are taken
from Table 2 of reference 25, which were based on observations reported in reference 38.
k
The amount of mRNA per cell was calculated from the data in this table: Rm = rm m.
l
Measurement of ppGpp was by A260 after separation of nucleotides by high-pressure liquid chromatography (39); ppGpp/P = (ppGpp/M)/PM.
m
The differential rate of rprotein synthesis equals the fraction of total protein that is r-protein. This fraction was calculated from the number of ribosomes per cell (Nr, this table, footnote p), the number
of amino acid residues per ribosome (aa/rib, Table 1), and the amount of total protein per cell (PC, Table 2): r = Nr (aa/rib)/PC.
n
The fraction of active ribosomes was measured as fraction of ribosomes in polysomes, with a correction for active 70S ribosomes; this fraction was found to be approximately constant, at about 0.8
(40). Here we have assumed the slightly higher value of 0.85 from Table 2 in reference 25 to make the calculated values for the peptide chain elongation rate consistent with values obtained by other
methods (see footnote o below).
o
The peptide chain elongation rate was calculated from the amount of protein per cell (Pc, Table 2) and the number of active ribosomes (r Nr; from the values in this table, footnotes n and p): cp = (ln /
) PC / (r Nr). This relationship is equivalent to Equation 5 in Table 5 below). cp has also been measured more directly by analyzing the size distribution of pulse-labeled polypeptides (41).
p
The number of ribosomes per cell was determined from the values in Tables 1 and 2: Nr = RC fs (1 ft)/(nucl./rib), where fs, ft, and nucl./rib are defined in Table 1.
q
The number of tRNA molecules per cell was calculated from the amount of RNA per cell (RC, Table above) and the values for fs, ft, and nucl./tRNA in Table 1: Nt = RC fs ft /(nucl./tRNA).

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r
The number of Nrrn genes per genome, Nrrn/G, was calculated from the C-period (see footnote v below) and the map locations of the 7 rrn genes on the chromosome (at 87, 89.5, 85, 72, 90.5, 57, and 5
min, respectively), using Equations 11 and 12 from Table 5 below. (For details see also Table 1 in reference 8).

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s
The number of rrn genes per cell was calculated from the number of rrn genes per genome (footnote r above) and the number of genome equivalents per average cell (Table 2): Nrrn = Nrrn/G GC.
t
The rate of transcript initiation at the each rrn gene was calculated from the number of ribosomes per cell (footnote p above) and the number of rrn genes per cell (footnote s above): irrn = (ln 2/) Nr/

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Nrrn.
u
The average nucleotide distance between ribosomes on mRNA was calculated from the amount of mRNA (footnote k above) and the number of ribosomes per cell (footnote p above): dr = RM/(r Nr).
v
The average number of translations per mRNA was calculated from the amounts of protein (PC, Table 2) and of mRNA per cell (RM, footnote k above): Ntrans = 3 (ln 2/) PC/Rm, where the factor of 3
is the coding ratio, i.e., 3 mRNA nucleotides per amino acid residue. The calculation does not account for untranslated regions in the mRNA.
w
The number of RNA polymerase molecules per ribosome (given in percent) was calculated from the ratio Np/Nr (footnotes b and p of this Table).
x
The rate of DNA chain elongation was calculated from the number of DNA base pairs per chromosome (kbp/genome, Table 1) and the C-period (footnote x below): cd = (kbp/genome)/2C. The factor
of 2 in the denominator represents the fact that each of the two replisomes generated at the initiation of replication at oriC replicates a half-chromosome.
y
The C-period was first calculated from the number of replication origins per genome, measured as the factor increase in DNA after stopping initiation of replication with rifampin (reference 12; see
equation in footnote i of Table 2 above). For different growth rates, those calculated values can be closely approximated by the function C = 80 2(/2.35); the points scattered by less than 10% around this
function, which was identical for the E. coli strains B/r and K used. Therefore, this exponential relationship has been used to generate the values for C in this table. For consistency, these smoothed C
values were then used to calculate the origins/genome values in Table 2. The C-period has also been determined from age-fractionated cultures (42), synchronized cultures (43), and flow-cytometric data
(44, 45). Those methods are considered to be less accurate, because they are influenced by large cell-to-cell variations in the D-periods.
z
The average D-period was determined by treating cells with sodium azide, which stops replication but does not prevent the division of cells that are already in the D-period at the time of the replication
stop (46). The D-period has also been determined in age-fractionated and synchronized cultures, as well as from flow-cytometric data (4244, 47).

9
Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates
Bremer and Dennis
This history effect causes variations in the data, i.e., both
in the exponential growth rate and in the macromolec-
ular composition, from cultures prepared on different
days, i.e., with different overnight starter cultures. This is
especially true for cultures grown in nutritionally poor
media, as observed variations in the growth rate for a
given medium tend to increase with decreasing nutri-
tional value of the carbon source used in minimal media.
For example, doubling times were found to vary between
42 and 48 min in glucose minimal medium, or between
55 and 70 min in glycerol minimal medium, and be-
tween 67 and 113 min in succinate minimal medium
(28, 51). An average growth rate for a given minimal
medium cannot be clearly defined, because both the
observed average and extent of variation in the growth
rate may depend on particular ways different experi-
menters prepare their overnight cultures. In one study
with four succinate cultures (51), the ribosome concen-
tration (Nr/P) for the fastest growing culture (0.90
doubling/h) was lower than the values observed in the
other, more slowly growing cultures, but the lower ri-
bosome concentration was balanced by a higher rate of
ribosome function (er), which was close to the value
normally found only in cultures growing in amino acid-
supplemented media. The varying growth rates in min-
imal media imply varying rates (per total amount of
protein, or per cell volume) of amino acid biosynthesis,
of importing and processing the exogenous carbon
source, and of respiration. This raises the (unanswered)
question: what limits the growth rate of a particular
culture in a particular medium?
An implication of these observations is that bacteria in
nutritionally poor media generally do not grow at their
maximum possible rate, i.e., they appear to partly
downregulate their potential growth rate. Why this
could be advantageous for the bacteria is discussed in the
section Optimal cell composition for maximal growth,
below.
Cultures grown in minimal media may not only show
variable growth rates, but also somewhat different
macromolecular compositions at the same growth rate.
This latter variability relates to the fact that the growth
rate equals the product of the two factors, ribosome
concentration and function (Nr/P and er; see above and
equation 18 in Table 6 below). During fast growth in
rich media, ribosomes function at or near their maxi-
mum, so that the only variable factor that determines the
growth rate is the ribosome concentration, Nr/P. In
10
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contrast, during slow growth in poor media, the rate of
ribosome function is generally submaximal, so that dif-
ferent pairs of Nr/P and er may produce the same growth
rate, i.e., either fewer ribosomes that function somewhat
faster or more ribosomes that function more slowly.
Either combination requires the same rate of amino acid
biosynthesis and leads to the same rate of protein syn-
thesis and growth.
What causes the cell to choose a particular pair of values
for Nr/P and er in a given minimal medium is not known.
However, when several experimental cultures are started
at the same time from the same stationary overnight
culture, they generally behave identically; i.e., they assume
the same exponential growth rate and show the same
macromolecular composition. Apparently certain physi-
ological parameters are affected by events during the
preceding stationary phase (28). As a result, Nr/P and er
values observed in a particular experiment might differ
from the values observed in a similar study on another
day. The product of Nr/P and er may either remain similar
to produce a similar growth rate, or within certain limits,
differ to produce a different growth rate. In general, longer
durations in stationary phase are associated with a longer
initial lag before growth resumes after inoculation of an
experimental culture and with a somewhat lower final
growth rate. The reason for this is not known since these
effects have never been systematically studied. The most
reproducible results are obtained when the duration of the
stationary phase in the starter culture used to inoculate the
experimental culture is kept at a minimum. This can be
achieved in various ways, for example by minimizing the
inoculum used for the overnight starter culture, so that it
reaches stationary phase only a few hours before it is used
to inoculate the experimental cultures.
When stationary cultures in different media are incu-
bated for several days and monitored by flow cytometry,
the average size of their cells and the chromosome
number per cell is found to gradually decrease. For ex-
ample, a stationary culture in LB medium contains ini-
tially large cells with four or eight chromosomes, but
after further incubation, different subpopulations arise
with smaller cells containing only one or two chromo-
some(s) (52). It is not surprising, therefore, that the
condition of a particular stationary culture affects the
subsequent growth of an experimental culture derived
from it. A systematic study of these effects will be nec-
essary in the future to fully understand the control of
bacterial growth. A further discussion about the control
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 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates
of the bacterial growth rate is found in Mathematical
description of cell composition and growth, below.
The observations described above might suggest that the
growth rate is perhaps not the only factor that de-
termines the macromolecular composition, but at least it
is one among other factors. However, the causal arrow
can only go in one direction, from the cell environment
via genetic background (species, strain) and initial con-
ditions (history) to composition and growth rate, and
not in the opposite direction. The following analogy
might help to clarify this idea: a unique relationship
exists between the volume of the mercury within a
thermometer and the temperature surrounding it.
However, to say that the volume of mercury in the
thermometer determines the temperature is ambigu-
ous, because causally the weather produces the outside
temperature which, in turn, determines the volume of the
mercury, not the other way around.
OBSERVED CELL COMPOSITION OF E. COLI B/R
Cell Growth-Related Parameters
In Table 1, a number of growth-related parameters are
listed that are generally useful in describing or estab-
lishing the macromolecular composition of bacterial
cultures. These parameters can be divided into five
classes: (i) structural parameters that are inherently
constant and do not vary with the growth rate, like the
number of rRNA nucleotides in a 70S ribosome; (ii)
partition factors that are essentially invariant and growth
rate independent, like the fraction of total RNA that is
stable rRNA and tRNA; (iii) other partition parameters
that change as a function of the exponential growth rate
and have substantial effects on cell composition, like the
fraction of active RNA polymerase synthesizing rRNA
and tRNA; (iv) kinetic parameters describing functional
activities (the values of some of the parameters are es-
sentially invariant, whereas others approach a maximum
or biological limit value, like the peptide chain elonga-
tion rate); (v) chromosome replication and cell division
parameters that in general do not limit the exponential
growth rate, like the C-period.
The values of the fraction of total RNA that is stable RNA
(fs) and the fraction of total stable RNA that is tRNA (ft)
in class ii need some comment. The value of fs = 0.98
means that 98% of all bacterial RNA is rRNA and tRNA,
whereas 2% is mRNA. The value of 98%, which cannot
be measured with the precision necessary to distinguish
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it from 100% (total RNA), was obtained instead as the
difference between measurements of total RNA (100%)
and mRNA. Since there is no hybridization probe for
total mRNA, the 2% value for mRNA was estimated
indirectly from the rate of mRNA synthesis and the av-
erage mRNA lifetime. The rate of mRNA synthesis was
found from the (observable) rate of stable RNA accu-
mulation and from the fraction of the total RNA syn-
thesis rate that is stable RNA, rs/rt (see Table 3; obtained
by hybridization of pulse-labeled RNA to an rDNA hy-
bridization probe, taking into account the hybridization
efficiency and the fraction of total stable RNA that is
tRNA, ft). The proportion of the mRNA synthesis rate is
then equal to the difference (1 rs/rt). To obtain the
amount of mRNA, the average lifetime of an average
mRNA must also be determined. This was done by a
complex evaluation of the pulse-labeling kinetics of total
RNA and rRNA for bacteria grown in glucose minimal
medium at 1.3 doublings/h: under those conditions, 60%
of the total RNA synthesis was mRNA synthesis, the
average life of mRNA was 1.0 0.2 min, and the amount
of mRNA was calculated to be 2.3% of the total RNA
(20). Thus, fs is equal to about 98%, as given in Table 1. In
another approach based on measurements of the average
life of lacZ mRNA at different growth rates, the relative
abundance of mRNA has been estimated to decrease
from about 2.2% at = 0.6 to 1.1% at = 3.0 (25). This
suggests that fs may increase slightly with growth rate
from 0.98 to 0.99.
The value ft = 0.14 in Table 1 means that 14% of the total
stable RNA is tRNA and 86% is rRNA. Stable RNA
refers to the sum of 23S, 16S, and 5S rRNA and all tRNA
species, excluding unstable spacers in the primary rRNA
and tRNA transcripts. The value of ft = 0.14 corresponds
to about 9 tRNA molecules per ribosome. During slow
growth in nutritionally poor media, the number of tRNA
molecules per ribosome is somewhat higher, and this
number was found to decrease from about 12 at = 0.4
to a constant plateau of about 7 at growth rates faster
than 1.0 doubling/h (53). The higher tRNA values dur-
ing slow growth can be attributed to two growth rate-
related phenomenaan instability of some newly made
rRNA at slow growth rates and a slight increase in the
ratio of rRNA to tRNA genes at fast growth rates due to
increased chromosome branching associated with
shorter intervals between initiations of chromosome
replication (see Fig. 1). In the first instance, at the very
low growth rate of 1 doubling/10 h, 70% of newly made
rRNA was found to be degraded (54); this unstable
11
Bremer and Dennis
Figure 1 Relationships between growth rate, cell size, chromosome
replication, transcription, and macromolecular composition. (Left)
Average cell size (mass per cell, Table 2) for E. coli B/r growing with a
doubling time, , ranging from 100 to 20 min (growth rate, , ranging
from 0.6 to 3.0 doublings/h) is depicted by the shaded ovals. An
idealized cell cycle with the major cell cycle events, ranging from cell
age 0.0 (a newborn daughter cell) to 1.0 (a dividing mother cell), is
presented for each growth rate. The position of an average cell of age
0.41 (defined so that 50% of the cells in the population are younger
and 50% are older) is indicated by A. The cell ages at initiation (I)
and termination (T) of chromosome replication are also indicated.
The dashed portion of the age axis indicates a period during which
there is no DNA replication (no replication forks on the chromo-
some). The light line portions represent periods where there are two
forks per chromosome structure, and the heavy line portions indicate
the age periods during which there are six forks per chromosome
structure. After termination, there are two chromosome structures
per cell, which are segregated to the daughter cells at the subsequent
cell division (at age 1.0). (Center) Average structure of the replicating
chromosome or chromosomes in the culture. For 24- and 20-min cell
cycles ( =24 or 20 min; bottom portion), the chromosome pattern
indicates that replication has reinitiated and that each of these
chromosome structures has multiple (six) replication forks. The
amount of DNA in these structures in genome equivalents (G) is
indicated (values from Table 2). The numbers of origins (O), termini
(T), and forks (F) in this average genome are also indicated (from
Table 2). (Right) The synthesis rates of rRNA (rR), tRNA (tR), r-
protein mRNA (rpm), and other mRNA (om), expressed as a percent
of total transcription, and the macromolecular composition are il-
lustrated in bar graph form. The stable RNA fraction of the total
12
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fraction diminishes as cells grow faster and at growth
rates above 1 doubling/h, virtually all of the newly made
rRNA is believed to be stable. In the second instance, an
analysis of the location of seven rrn transcription units
and 81 different tRNA genes on the E. coli chromosome
indicates that the average rrn unit is located about 10
min (using the 100-minute genetic scale) from the origin
of replication, oriC, whereas the average tRNA gene is
located about 20 min from oriC. This means that with
increasing growth rate, the dosage of rrn genes increases
slightly faster than the dosage of tRNA genes due to the
increase in the degree of chromosome branching (be-
tween = 0.6 and 3.0 the ratio of gene dosages, rRNA/
tRNA, is expected to increases by about 15%). During
growth in succinate minimal medium at a rate of 0.67
doubling/h, the accumulation of tRNA relative to that of
rRNA was 10 to 15% higher than during twofold faster
growth in glucose minimal medium (22), so that perhaps
for the lowest growth rate of 0.6 doubling/h in Table 2
and Table 3 below, a 10 to 15% upward correction would
need to be made for the value of ft.
We assume that the bulk of tRNA is coregulated with
rRNA transcription. This notion, primarily based on the
observed constancy of ft at higher growth rates, is further
supported by observations that the rates of rRNA and
bulk tRNA transcription decrease coordinately in the
presence of extra rrn genes (55) and increase coordi-
nately during chloramphenicol treatment, which mimics
the relaxed response or an internal nutritional shift-up
(10, 56). Furthermore, most (although not all) tRNA
genes have P1 and P2 promoters similar to those of the
rrn genes and are subject to stringent control like rRNA
genes (57). Therefore, ft in Table 1 could also be defined
as the fraction of the stable RNA synthesis rate that is
tRNA synthesis, which would then be valid for all
growth rates.
In this context, it should be noted that Emilsson and
Kurland (58) have measured the abundance of five
leucine and three methionine isoacceptor tRNAs as a
function of growth rate. They found that the ratios of
Leu1 and Leu3, and fMet1, fMet2, and Met3 tRNAs to
transcription increases with increasing growth rate, the r-protein
mRNA increases as a fraction of the total mRNA synthesis rate.
Relative amounts of protein (P), DNA (D), RNA (R), and other
components (O) as percent of the total cell mass are from the data in
Table 2.
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 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates
ribosomes remained relatively constant between growth
rates of 0.5 and 2.1 doublings/h. The Leu1 tRNA reads
the major CUG codon and is encoded by four identical
genes whereas the Leu3 tRNA reads the minor codon
CUA as well as the major CUG codon (through wobble
base pairing) and is encoded by a single-copy gene. In
contrast, the ratios of Leu2, Leu4, and Leu5 tRNAs to
ribosomes decrease five- to eightfold as the growth rate
is increased from 0.5 to 2.1 doublings/h. These three
tRNAs read the minor CUU, CUC, UUG, and UUA
leucine codons that are infrequent in highly expressed
proteins. Each of the three tRNAs is encoded by single-
copy genes. These results demonstrate that the syn-
thesis of at least some of the tRNAs reading the most
abundant codons is coordinate with the synthesis of
ribosomes, whereas at least some of the tRNAs reading
minor codons are more highly expressed at slow
growth rates where they would be required for the
synthesis of proteins containing a higher frequency of
minor codons.
Reference Units
Physiological parameters describing the cell composi-
tion, like the amounts or synthesis rates of particular
components, require a reference unit such as per cell,
per cell mass, per amount of protein, per genome,
per microgram of dry weight, etc. This means that for
any quantitative measurement of a particular cell
component, at least two parameters have to be quan-
titatively measured, representing the desired component
of interest and the reference unit. Therefore, the accu-
racy of any physiological measurement is also affected
by the accuracy with which the reference unit can be
determined.
The primary reference unit for our measurements from
exponential cultures is the cell mass density, given in
units of turbidity of the culture, i.e., per OD460 (optical
density unit at 460 nm), because determination of the
optical density is simpler, faster, and more accurate than
that of other units. For E. coli B/r bacteria grown in
glycerol minimal medium at 1.0 doubling/h and in
glucose-amino acids medium at 2.1 doublings/h, we
found their dry weight (after washing them free of ad-
hering growth medium) to be 173 and 172 g, respec-
tively, per OD460 unit of culture (28). For the calculations
of cell mass in micrograms in Table 2 below, we have
therefore used a constant value of 173 g/OD460 for all
media (see Table 2, footnote r).
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The per mass values have also been used to express
approximate relative intracellular concentrations, be-
cause the average cell volume per mass unit changes
relatively little with growth conditions. For E. coli B/r
growing in glucose minimal medium (at 1.33 doublings/
h) and in glucose-amino acids medium (at 2.14 dou-
blings/h) average cell volumes of 0.35 and 0.29 l per
OD460 unit, respectively, were found (Table 1 in refer-
ence 59). This corresponds to a 17% decrease in volume
per mass for a 1.6-fold increase in the growth rate.
However, using data from cultures grown under similar
conditions, volume/mass values of 0.32 and 0.25 l per
OD460 unit, respectively, have been estimated for these
two growth rates (60), corresponding to a 22% differ-
ence. The reason for the different results is the variability
of the average cell size in cultures grown on different
days under seemingly identical conditions (see Cell
volumes and cytoplasmic concentrations, below).
When parameters are to be expressed with other refer-
ence units, they are found from additional measure-
ments of the wanted reference parameter per mass.
For example, the amount of protein per average cell is
obtained by dividing the amount of protein/mass by
the number of cells/mass.
There has been a tendency in the literature for authors to
state that rRNA or ribosomes accumulate in proportion
to the square of the growth rate. This, of course, is un-
suitable because the unit of reference is not specified. As
first pointed out by Maale (6), the rate of RNA accu-
mulation per genome equivalent of DNA does increase
with the square of the growth rate, (61). This reflects
the fact that the amount of RNA per genome is pro-
portional to the growth rate (at least above growth rates
of 0.6 doubling/h). Since in any exponential system the
synthesis rate is proportional to the amount, and the
amount is already proportional to , it follows that the
rate must be proportional to 2. However, since the ratio
RNA/DNA reflects both controls of chromosome repli-
cation and RNA synthesis, this square relationship has
no particular significance for ribosome control itself; the
relationship no longer holds in certain replication con-
trol mutants that alter DNA content but show no change
in the growth rate or rRNA control (7, 62).
In the following, the limitations in the accuracy of
measurements of the three most frequently used refer-
ence units, i.e., per mass, per cell, and per cell volume,
are discussed.
13
Bremer and Dennis
Optical density of bacterial cultures
In contrast to normal absorbance measurements of so-
lutions, measurements of the OD of a bacterial culture (i.
e., of the turbidity caused by scattering of light by bac-
terial cells), depend somewhat on the particular spec-
trophotometer used (i.e., features of the light beam and
the measuring cuvette), so that OD measurements from
different laboratories do not always reflect exactly the
same culture densities. The amounts of macromolecules
per OD460 unit in Table 2 below are based on newer data
(12) that deviate somewhat from the values given in
previous editions of this table. This is partly due to the
use of different spectrophotometers and partly due to a
refinement of the method to determine the OD.
The OD of a culture is not always an exact measure of
the cell mass density: the higher the cell density, the
greater the deviation of the observed OD from the
true OD because of cell shadowing and backscattering.
In earlier work, this effect was partly taken into account
by measuring the OD of diluted culture samples. This
causes some inaccuracies due to variation in pipetting
volumes associated with the dilution and because the
lower OD values measured (closer to the background
OD) have inherently a greater variability. For the new
data in Table 2 below, the relationship between observed
and true OD was first independently determined for the
bacterial strain used; it was found that the deviation
from the true OD increases in proportion to the square
of the true OD. Using this mathematical relationship,
the corrected OD (including subtraction of the back-
ground OD of the cell-free growth medium) was cal-
culated by a computer spreadsheet (see reference 12 for
details). From these corrected values, the spreadsheet
calculated the best-fit exponential growth function. The
corrected OD values from samples taken over two to
three doubling times deviated by less than 1% from this
exponential function. The best-fit function thus pro-
vided an accurate value for the growth rate (). For
determinations of particular cell components, like pro-
tein, RNA, or DNA, the exact OD at the sampling times
calculated from the best-fit exponential growth function
was used to obtain macromolecular amounts per OD-
unit of culture mass (12).
We have now extended the data range in Table 2 and
Table 3 from the maximum growth rate of 2.5 doublings/
h in the previous edition up to 3.0 doublings/h in this
edition. The previous data were based on experiments in
which glucose-amino acids was the most nutritious
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growth medium used. That medium typically gives a
growth rate of 2.14 doublings/h for E. coli B/r, so that the
values given for 2.5 doublings/h were actually obtained
by extrapolation. The inclusion of values for a growth
rate of 3.0 doublings/h in the new tables was possible by
using experimental cultures grown in Luria-Bertani (LB)
medium (supplemented with 0.2% glucose; reference 12).
In this medium the bacteria grow at what appears to be
their maximum rate of 3.0 doublings/h (at 37C). How-
ever, this cannot be done by following the OD at 460 nm,
because of the high background absorbance of the LB
medium alone, without bacteria, at this wavelength.
Therefore, OD600 units were used as a reference for cul-
tures grown in LB medium. By monitoring both the
OD460 and the OD600 of cultures growing in different
media, parallel growth curves were obtained, differing by
a factor of 1.60 0.03 (OD460/OD600). This factor was
then used to convert OD600 units into OD460 units for
cultures in LB medium. The use of LB medium excludes
the use of radioactive assays because the nutrients in the
medium dilute by an unknown factor the specific ra-
dioactivity of the base, nucleoside, or amino acid pre-
cursor used to label the macromolecules in the cell. For
that reason nonradioactive colorimetric assays were used
for the basic measurements of DNA, RNA, or protein.
Cell numbers
A seemingly natural reference is the average cell.
When it was first observed in Maale's laboratory that
fast-growing cells are much larger than slowly growing
cells (reference 1; see also the next section), Maale
proposed not to use per cell, but instead to use a
genome equivalent of DNA (per genome) as the
standard reference unit for physiological data from
bacteria. He thought that DNA was somehow limiting
reactions that were important for determining the
growth rate and macromolecular composition. The per
genome reference is no longer used, since later studies
have shown that DNA is generally not limiting the rate
of transcription (see below). For historical reasons per
genome values for the cell composition are still shown in
Table 2 below.
The per cell values in Table 2 and Table 3 below were
obtained from the primary values determined per OD by
division with the cell numbers per OD. An average cell
refers to a particular size between two cell divisions,
which is found by taking the so-called age distribution
into account (29) (see Cell composition at a defined cell
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 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates
age, below). Because of relatively large variations of the
times between consecutive cell divisions, the actual
predivision and postdivision cell volumes in a culture
vary by more than a factor of two in a non-Gaussian
fashion (29, 63).
The values for mass per cell (i.e., the reciprocal of cell
numbers per OD) in Table 2 were obtained in two ways.
First, the number of cells at a given OD was determined
with an electronic particle counter equipped with a 20-
m orifice, which allows the counting of small cells
present in nutritionally poor media. The size distribution
displayed by the particle counter helps to visualize and
to avoid counting the background from debris in the
growth or dilution medium (each has to be carefully
filtered). The size distribution also indicates when newly
divided cells are temporarily sticking together to form
snakes. This degree of stickiness often depends on
uncontrolled conditions within a particular culture, so
that measured cell numbers per OD unit may vary from
culture to culture. The E. coli strain B/r used for the data
in Table 2 and Table 3 below exhibits a relatively tight
size distribution and is less prone to stickiness than
many of the more widely used K-12 strains (see below).
A second, indirect method consists of calculating cells/
mass as the quotient DNA/mass and DNA/cell, where
DNA/mass is measured directly and DNA/cell is ob-
tained from the C- and D-periods (Table 3), using the
Cooper-Helmstetter formula (see above; equation 3 in
Table 5 below). This latter method has been used for the
data in Table 2 and Table 3 to make all values in the
tables consistent with the theoretical relationships con-
necting them in various ways.
Cell volumes and cytoplasmic concentrations
For biochemical studies the most relevant reference
seems to be the average cell volume, because this allows
one to estimate intracellular molar concentrations by
division of the average per cell values of a given com-
pound with the volume per average cell. The average
cell volume has been determined in two ways: first, from
the volume distribution obtained with a particle size
analyzer calibrated with latex spheres of known volume
(64), and second, by measuring the cell dimensions in
electron microscopic pictures (63).
The particle counter equipped with a size analyzer
(made by Coulter Electronics) that has been used for
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our studies with E. coli B/r measures the temporary
reduction in the electric conductivity between two vo-
lumes of salt solution separated by a small hole (or-
ifice) through which the particles are being sucked; i.e.,
while a particle passes through the hole, the electrical
resistance of the solution in the narrow channel formed
by the orifice temporarily increases. The electronic
pulse generated in this manner is assumed to be
proportional to the volume of the passing particle, i.e.,
to the volume of the solution displaced by the particle.
These counters were originally developed for much
larger eukaryotic cells; to sufficiently increase their
sensitivity for much smaller bacteria, the orifice had to
be reduced to the smallest available size of 20 m di-
ameter. This is still much wider than the bacteria with
dimensions of about 1 m, and it is not clear how ac-
curately the electronic pulses generated in this manner
reflect exact cell volumes, and to what extent the dif-
ference in the shapes of the elongated bacteria and the
round latex spheres used for calibration might affect
these distributions.
The volume distributions of bacteria are highly asym-
metric, with a peak close to the minimum volume and a
longer trail toward larger size bacteria. This reflects the
age distribution of the cells, i.e., the average young cell
just after division is half the size and twice as frequent as
the average older cell before division. The volume dis-
tributions for cultures grown at 1.30 doublings/h in
glucose minimal medium and at 2.14 doublings/h in
glucose amino acids-medium were found to have peaks
at 0.60 and 0.81 m3 (Fig. 4 in reference 64). Assuming
that these peaks represent the most frequent cells just
after division, their cell volumes were multiplied with the
factor 2 ln2 (= 1.39) to obtain the average cell volume,
i.e. by taking the age distribution into account (see Age
distribution and the concept of the average cell, below).
With this factor, average cell volumes for E. coli B/r in
these two media were estimated to be 0.83 and 1.12 m3,
respectively (Table 1 of reference 64).
By use of the method of electron microscopy for bacteria
grown in glucose minimal medium at 1.33 doublings/
h and fixed with formaldehyde, a similar volume dis-
tribution was obtained from measurements of the widths
and lengths (and assuming half-spherical ends) of 959
cells (Fig. 8 in reference 63); the average volume was 0.73
m3; i.e., somewhat smaller than the value of 0.83 m3
obtained with the particle counter. Because of distortions
of the shape of the bacteria during the preparation for
15
Bremer and Dennis
the electron microscopy, such volume determinations
might also be not entirely accurate.
By using the data above, approximate cell volumes for E.
coli B/r growing at 1.0 and 2.5 doublings/h have been
estimated by extrapolation (60). For this purpose, the
cell volumes of 0.83 m3 and 1.12 m3 for = 1.33 and
2.14 doublings/h (see above) were first divided by the
mass/cell values to calculate volume per mass ratios
(0.32 and 0.25 l, respectively), which were then linearly
extrapolated to 1.0 and 2.5 doublings/h to give 0.34 and
0.22 l, respectively, of combined cell volumes per OD460
unit of culture mass. Multiplication with the averaged
mass/cell values for these two growth rates (1.84 and
5.47 OD460 units per 109 cells, calculated indirectly from
protein/mass, protein/oriC, and oriC/cell, setting 1 m3
= 109 l) gave 0.63 and 1.20 m3 for the average cell
volume in exponential cultures growing at 1.0 and 2.5
doublings/h, respectively (60).
The average cell volumes estimated in this manner may
not be precise because average cell sizes vary consider-
ably for cultures grown under the same conditions on
different days, presumably reflecting a variability in the
average D-period (see DNA replication and cell divi-
sion, below). As described below (Exponential
growth), flow-cytometric data indicate that the average
cell sizes may begin to decrease in exponential cultures
at very low cell densities, long before the exponential
accumulation of mass and major macromolecules begins
to decrease during the approach to stationary phase
(52).
Since a substantial fraction of the cell volume is taken up
by the nucleoid, the actual space available for most
biochemical reactions within the cell is much smaller
than the total cell volume, and the macromolecular
concentrations calculated from the total cell volume are
correspondingly underestimated (see below).
The cell volume appears to be largely determined by its
protein content. At the two growth rates of 1.0 and 2.5
doublings/h, where the average volume increases from
0.63 to 1.20 m3 (see above), the average amounts of
protein per cell correspond to 1.2 109 and 2.4 109 amino
acid residues, respectively (see Table 2). This means that,
at least within this 2.5-fold range of growth rates, every
cubic micrometer of (total) cell volume is associated with
about 2 109 amino acid residues in protein.
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The average amount of protein per cell (PC) (and thus
also the average cell volume) increases with increasing
growth rate in proportion to the average number of
replication origins per cell. This is seen as follows: protein
per cell equals the product of protein per replication
origin (PO) times the number of origins per cell (OC).
Thus, the volume of an average cell in a culture of the E.
coli strain B/r (VolC) can be described by the formula:
VolC = (POOC/2109) m3, where the values for PO and OC
at different growth rates can be found in Table 2.
PO expresses the control of initiation of replication (see
above) and is approximately constant for E. coli B/r
during exponential growth at different rates (reference
12; Table 2), whereas OC depends on the C- and D-
periods and on the doubling time: OC = 2(C+D)/ (see
equation 7 in Table 5). Substituting the original data of
Helmstetter and Cooper (where the sum of C and D was
found to be equal to about 60 min at growth rates above
1.0 doubling/h; reference 42), we obtain OC = 260/ = 2.
This implies that the average cell volume increases
nearly exponentially with increasing growth rate, ap-
proximated by the function 2.
If approaches zero, the values for OC in Table 2 ex-
trapolate to 1.0 (i.e., 20 = 1). This would mean that most
cells at close to zero are expected to have one non-
replicating chromosome with one origin. However, de-
terminations of C and D for very slowly growing E. coli
B/r suggest that OC approaches a lowest value of about
1.25, which was observed at 0.08 doubling/h (rather than
1.06 = 20.08; reference 44). Assuming further that the
amount of protein per origin remains approximately
constant under these conditions, equal to about 4 108
amino acid residues (Table 2), the formula above gives
an approximate minimum volume of (B/r) bacteria
growing very slowly in poor media (but still exponen-
tially under balanced conditions), equal to 0.25 m3.
Although our data showed that C and D decrease
somewhat with increasing growth rate (see Table 3),
these considerations show that the increasing cell vo-
lumes with increasing growth rate are the result of the
controls of DNA replication and cell division and an
implication of the exponential growth function. There-
fore, any parameter (like protein, RNA, or DNA, or their
rates of synthesis) observed under different growth
conditions and expressed per cell reflects not only the
specific control of the parameter under study but also
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 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates
Figure 2 Amounts and synthesis rates of molecular components in
bacteria growing exponentially at rates between 0.6 and 2.5 dou-
blings/h. The values of the RNA-to-protein (R/P; panel a) and DNA-
to protein (G/P; panel b) ratios were calculated from lines 1, 2, and 3
in Table 2. The ribosome efficiency (i.e., the protein synthesis rate per
average ribosome; panel c, left ordinate) was calculated from the
number of ribosomes per cell (Table 3) and the rate of protein
synthesis per cell. The latter was obtained fromthe amount of protein
per cell (Table 2) using the first-order rate equation. The peptide
chain elongation rates (panel c, right ordinate) are 1.25-fold higher
than the ribosome efficiency values and account for the fact that only
about 80%of the ribosomes are active at any instant. The fraction of
the total RNA synthesis rate that is stable RNA ormRNA (rs or rm;
panel d) is from line 5, Table 3. The rates of stable RNA and mRNA
synthesis per amount of protein (rs/P or rm/P; panel e) were calcu-
lated from the data in Table 3, divided by the amount of protein per
cell (Table 2). The ppGpp per protein value (ppGpp/P; panel f) is
from Table 3. The cell age at which chromosome replication is ini-
tiated at oriC (ai in fractions of a generation; panel g) is calculated
from C and D (Table 3) and equation 14 in Table 5. The protein (or
mass) per cell at replication initiation (panel h) was calculated from
the initiation age (ai; panel g) and the cell mass immediately after cell
division (age zero; i.e., a = 0), using equation 17 in Table 5. The latter
was obtained from the average protein or mass content of cells (lines
10 or 13, respectively; Table 2), using equation 16 of Table 5. The
number of replication origins at the time of replication initiation (Oi;
panel i) was obtained from the values of C and D (Table 3), using
equation 15 of Table 5. The initiation mass (panel j), given as protein
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the control of DNA replication and cell division and the
exponential growth function in general. This justifies
Maale's concern about the use of the per cell refer-
ence and his search for a more suitable reference unit.
However, now we see that his proposal to use per ge-
nome as a standard reference is subject to similar defi-
ciencies (see above).
In the tables below, molar concentrations were not cal-
culated. Because of the high component densities within
the cytoplasm, and because a substantial fraction of the
total cell volume is occupied by the densely packed
chromosomal DNA in the nucleoid, most cell compo-
nents are not expected to be evenly distributed
throughout the cell volume. As was pointed out by
Maale and Kjeldgaard (2), large enzyme complexes,
including RNA polymerase and ribosomes, might not
move freely between the DNA strands within the nu-
cleoid. Therefore, local concentrations of many (espe-
cially larger) cellular compounds are expected to differ
from the cellular average. This makes estimates of molar
concentrations within the cell (and, in some instances,
the extrapolation from in vitro data to the in vivo situ-
ation) somewhat tenuous. These effects, known as
crowding, may be taken into account by estimating a
volume fraction that is available for most biochemical
reactions in the cell (65).
The following observed example illustrates the uneven
distribution of molecules within the cells. When radio-
active uracil is used to label RNA, the UMP residues in
the newly synthesized RNA have a much higher specific
radioactivity than the simultaneously determined (av-
erage) specific radioactivity of the UTP precursor pool
(66). Apparently most transcriptionally active sections
on the DNA are located close to the sites for UTP
synthesis, presumably near the cell periphery where
uracil transport from the growth medium involves a
conversion to UMP which is quickly phosphorylated to
UDP and UTP.
Use of the amount of protein as a reference unit
Since average cell volumes for given exponential cultures
are difficult to determine and since values per cell, per
genome, or per mass (i.e., per turbidity unit of culture)
(or mass) per replication origin at the time of replication initiation,
was obtained as the quotient of the values for Pi (or Mi) and Oi shown
in panels h and i.
17
Bremer and Dennis
have little physiological significance, one might ask: is
there any better choice? We believe that the use of total
protein as a standard reference (see also Fig. 2) has
several advantages: (i) as was seen above, the amount of
protein per cell volume is approximately constant, so
that the amounts of cellular components per amount of
protein closely reflect cellular concentrations; (ii) two
parameters referring to total protein, the ribosome
concentration (ribosomes per protein, Nr/P) and the
initiation mass (P/oriC, reciprocal of oriC concentra-
tion) have a special significance for bacterial physiology
(see History and Relationship between macromolec-
ular composition and growth rate, above); and (iii) the
protein reference is already used as the fraction of total
protein that is ribosomal protein (r) or RNA poly-
merase (p; see Table 3) and as specific enzyme activities
for gene products with enzymatic function.
Macromolecular Composition at Different
Growth Rates
Exponential growth
The parameter values in the tables below refer to the
condition of balanced, steady-state exponential
growth. Balanced means that every component in the
medium (including oxygen provided by aeration or
shaking) is present at saturating, nonlimiting con-
centrations; and steady state means that the bacteria
have grown for at least ten generations in a given me-
dium (i.e., at least a 1,000-fold dilution from an over-
night culture). In this condition, the rate of
accumulation of every component relative to its total
amount in the culture is constant in time and its value is
the same for every component. If the growth conditions
for bacteria are changed, e.g., by dilution of a stationary
culture into fresh medium, or after a nutritional shift-up,
then the new steady state is approached with the func-
tion (1 2t/), which means it takes ten doubling times
until all parameters have reached 99.9% of the change to
their final new steady-state value (28), although some
parameters may reach their final value immediately after
a medium shift (see Control of the growth rate, below).
The above definition of exponential growth refers to
cultures containing large numbers of asynchronously
growing cells. Components within single cells that in-
crease stepwise during the cell cycle, like the copy
number of a particular gene, can be assumed to increase
exponentially when a significant volume of culture is
considered. If a single cell were isolated from such an
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exponential culture and cultured separately under the
same conditions, the cellular mass would continue to
increase exponentially, but the cell number would at first
double in a stepwise manner. Moreover, rounds of
replication in the initial daughter cells would be initiated
synchronously. It is unclear how long this synchrony
would persist. As far as we are aware, parameters that
vary and contribute to the gradual loss of synchrony in a
culture started from a single exponentially growing cell,
and the length of time it would take until the culture
could be considered truly exponential and asynchronous
has never been studied in detail. One possible factor
contributing to asynchrony might be the positioning of
the division plane; if the division is somewhat imprecise,
the daughter cells would have slightly different initial
masses and the triggering of the subsequent initiation of
rounds of replication would be slightly out of phase.
In some published experimental work, authors have
diluted their overnight culture much less than 1,000-
fold, so that the culture begins to approach the new
stationary phase before reaching true steady-state ex-
ponential growth. The term midlog phase is frequently
used to indicate the time when samples were taken for
measurement (i.e., the time at which the mass density
plotted on a log scale changes from increasing to de-
creasing). This inflection point does not define steady-
state exponential growth, and the use of the term midlog
phase is misleading, because once the steady state is
reached, it should remain in that state for a finite period
of time as required for the analysis. The period of ex-
ponential growth can be extended either by a greater
initial dilution of the overnight culture, or later, before
oxygen becomes limiting, by successive dilutions with
prewarmed, conditioned medium (28).
kerlund et al. (52) have diluted stationary E. coli bacteria
105-to 107 -fold into fresh media and measured, in ad-
dition to mass accumulation (OD550), cell sizes and DNA
content per cell, using flow cytometry. They found that a
truly steady state of balanced growth occurs only as long
as the OD550 is kept below 0.01; at higher mass densities,
size and DNA content of the cells began to decrease
gradually, suggesting a shortening of the D-period as a
result of the oxygen limitation that leads to stationary
phase growth. However, mass accumulation still re-
mained exponential up to an OD550 of about 0.8, sug-
gesting that reactions other than those involved in cell
division are not significantly affected until mass densities
much higher than 0.01 (OD550) are reached.
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 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates
Bacterial strain
For physiological studies, E. coli B/r has several ad-
vantages over other E. coli strains. Due to a special
property of its outer cell surface, this strain can be age-
fractionated by the membrane elution technique (67)
and used to measure cell cycle-related parameters.
Helmstetter and Cooper (42) used this strain to measure
the C- and D-periods and deduced from these mea-
surements the relationships between chromosome rep-
lication and the cell division cycle. This strain also (i) has
a lesser tendency for sticking or clumping and snake
formation than many other strains of E. coli, (ii) grows
well in minimal media, and (iii) is free of mutations that
might otherwise influence the growth rate or composi-
tion; for example, its maximum growth rate in Luria-
Bertani medium (3.0 doublings/h) and its growth rate in
glucose minimal medium (1.3 doublings/h) is about 20%
higher than the growth rates of the standard K-12 strains
in these media. For these reasons, E. coli B/r is the
preferred choice for physiological studies and composi-
tion measurements. A disadvantage is that the strain is
genetically incompatible with K-12 strains because of the
B and K restriction systems. B/r mutants deficient in B
restriction or with K-12 restriction and modification
have been used by the authors.
Growth media
As has been discussed in the beginning of this chapter,
the amounts and synthesis rates of the major macro-
molecules (including DNA, rRNA, tRNA, mRNA, pro-
tein) can be plotted as approximately single functions of
growth rate only for a selected choice of growth media.
The media used for the data in Table 2 and Table 3
below include, with increasing growth rate, succinate
minimal medium, glycerol minimal medium, glucose
minimal medium, glucose-amino acids medium, and
glucose-supplemented Luria-Bertani broth (LB medi-
um). The growth rates observed in these media range
between 0.6 and 3.0 doublings/h. As explained above,
exact growth rates in a given medium vary somewhat,
depending on the history of the overnight starter culture
used to inoculate the experimental cultures.
Macromolecular composition
Table 2 lists the amounts of protein, RNA, and DNA
and related physiological parameters for cultures of E.
coli B/r growing exponentially at 37C in different
growth media at rates between 0.6 and 3.0 doublings per
h. The per mass values (top section) represent averages
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obtained from curves drawn as a best fit through indi-
vidually measured points (12). Actual measurements
may deviate by as much as 15% around these curves
(see Fig. 4 in reference 68). Most of this scatter does
not represent random variations in the accuracy of
measurements, but true variations from culture to cul-
ture, reflecting differences in the history of the over-
night starter culture used (see above). The contribution
due to measuring errors was minimized by taking four
to five samples over one to two generations and is
generally less than 5%. Protein and DNA were measured
colorimetrically after appropriate calibration, and RNA
was determined from the A260 of an RNA hydrolysate.
Cell numbers per mass in Table 2 were calculated from
the DNA/cell values as explained in footnote e.
From the per-mass values, protein and RNA per genome,
and protein, RNA, and DNA per cell were calculated.
The sums of the weights of protein, RNA, and DNA are
proportional to the mass in OD460 units and correspond
to 65 to 73% of the dry weight. Lipids, carbohydrates,
soluble metabolites, and salts represent the remaining 27
to 35% of the total dry mass. The relative proportions of
the macromolecules at the different growth rates are il-
lustrated in the composition bar graphs of Fig. 1. The
greatest relative change is found in the RNA sector (in-
crease from 10 to 21%), reflecting the increasing con-
centration of ribosomes at higher growth rates. More
ribosomes are required to support the higher rate of
protein synthesis in rapidly growing cells.
The growth rate-dependent changes in the relative
proportions of DNA, RNA, and protein can be described
by the two ratios: RNA/protein and DNA/protein. With
increasing growth rate, RNA/protein increases and
DNA/protein decreases (Fig. 2a and b). The increasing
RNA/protein ratio reflects the control of ribosome
synthesis (see equation 18 in Table 6 below), and the
decreasing DNA/protein ratio reflects the control of
DNA replication (see legend to Fig. 2j). The RNA/pro-
tein ratio is proportional to the number of ribosomes per
amount of protein and is therefore an approximate
measure for the cytoplasmic ribosome concentration. As
explained above, the growth rate of an exponential cul-
ture is equal to the product of ribosome concentration
times the rate of ribosome function (i.e., the protein
synthesis rate per average ribosome or the ribosome
efficiency; references 5, 8, and 69). Therefore, at a given
growth rate, the protein synthesis rate per ribosome can
be calculated from the RNA/protein ratio. When the
19
Bremer and Dennis
growth rate increases, the rate of ribosome function
approaches a maximum value, corresponding to about
22 amino acids polymerized per second per active ri-
bosome (Fig. 2c, right ordinate scale).
The number of replication origins in a culture was ob-
tained by measuring the amount of DNA that had ac-
cumulated 50 to 80 min after treatment of a culture with
rifampin. Rifampin stops initiation of replication, but
allows the ongoing rounds of replication to go to com-
pletion, so that the number of completed chromosomes
becomes equal to the number of functional origins
present at the time of rifampin addition (12, 45, 47). The
number of replication origins per genome (OG) was then
obtained from the factor increase in the amount of DNA
in a culture after stopping replication initiation by the
addition of rifampin, and by measuring and taking into
account the delay in the action of rifampin (12).
The number of replication origins per genome depends
on the C-period; therefore, the observed values for OG
have been used to calculate the C-period values given in
Table 3. It turned out that the best-fit curve for C as a
function of the growth rate was a simple exponential (i.
e., empirical) function (reference 12; see footnote x), that
has been used to obtain the smoothed values of C in
Table 3. These values have then been used in a reversed
procedure to obtain the smoothed values of OG in Table
2 (see footnote i). Furthermore, we note that the values
calculated for C in Table 3, i.e., from the empirical
function, have been rounded to the nearest full minute,
but for the parameters in Table 2 obtained from C (i.e.,
replication origins and forks), the calculated exact
(nonrounded) values for C were used. This would cause
some deviations in the last decimal point if the number
of replication origins or forks were recalculated from the
rounded C values shown in Table 3.
The initiation mass, expressed as protein per origin, PO
(Table 2; Fig. 2j), is a formal measure for the control of
replication initiation; it has a meaning similar to cell
mass per origin, MO. MO is proportional (factor ln2) to
the initiation mass, defined by Donachie (4) as the
cell mass at the time of initiation, divided by the
number of replication origins at which initiation occurs
(i.e., Mi/Oi). In contrast, MO is the total mass in a given
volume of exponential culture, divided by the number
of copies of oriC present in that volume (Fig. 2j). Both
PO and MO are approximately constant (Table 2, PO = 4
108 amino acids per oriC). The exact growth rate
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dependence of PO (or MO) depends on the strain used
(68). A decreasing initiation mass with increasing
growth rate has been reported for K-12 strains of E. coli
(12, 70, 71).
The time intervals between consecutive initiations of
rounds of replication show little cell-to-cell variation,
whereas the time intervals between consecutive cell di-
visions vary considerably because of (non-Gaussian)
variations in the D-period (29, 72). The tight control on
initiation is presumed to reflect the accumulation of a
hypothetical protein that triggers initiation at a certain
threshold value (73). This putative initiation protein
would be made as an about constant fraction of total
protein synthesis, thereby linking chromosome replica-
tion to protein synthesis. The numbers of replication
origins, termini, and replication forks on the chromo-
some (Table 2) were calculated from the values of the C-
and D-periods (from Table 3, below). These numbers
relate to the extent of chromosome branching as a result
of increasing overlap in rounds of replication as the cells
grow faster (Fig. 1).
Parameters Pertaining to the Macromolecular
Synthesis Rates
The rates of accumulation of protein, RNA, and DNA or
the rate of cell division (or of any other extensive
property, X, of the system) can be calculated by using the
first-order rate equation dX/dt = Xk = X(ln2)/, where
X is the measured amount of the component, is in
doublings per hour, is in minutes, and k = (ln2)/60; the
rate is per minute (for more details, see reference 8).
For DNA, ribosomes, and protein, the rates of synthesis
during periods of balanced growth are essentially equal to
the rates of accumulation since their turnover is negligible
(22, 74). For total RNA, however, the instantaneous
synthesis rate is substantially higher than the accumula-
tion rate because of the instability of mRNA and of spacer
sequences in the primary rRNA and tRNA transcripts.
In Table 3, physiological parameters related to the
macromolecular synthesis rates have been divided into
three groups: parameters pertaining to (i) RNA poly-
merase synthesis and function, (ii) ribosome synthesis
and function, and (iii) DNA synthesis and cell division.
Some of these parameters were observed, and others
were calculated as indicated (see Table 3 footnotes). In
the following the parameters in these three groups and
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 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates

the way they were either measured or calculated are is part of the core enzyme and involved in the main-
discussed. tenance of the conformation of the large ' subunit (76).

For glucose minimal and glucose-amino acids media

RNA Polymerase Synthesis and Function
RNA polymerase concentration
The rate of transcription in the cell depends on the
concentration of RNA polymerase. The fraction of total
protein that is RNA polymerase core enzyme (three
subunits, 2, , and '), p, has been determined after
electrophoretic separation of E. coli proteins (75). Since
the 2 ?subunit is in excess in E. coli (see Table 4), the
amount of core enzyme was assumed to be limited by the
amount of and ' subunit polypeptides. Between 0.6
and 3.0 doublings/h the fraction of RNA polymerase core
enzyme increases from 0.9 to 1.6% of total protein. Re-
cently, it has been established that a further small subunit
with 1.3 and 2.2 doublings/h, the p values would be
about 1.2 and 1.5%, respectively, which may be com-
pared with independently reported p values for a strain
of K12 growing in these two media, equal to 0.5 and
1.8%, respectively (80). The experimental variation in
the higher value (1.8%) observed by those authors (about
30%; Table 1 in reference 80) makes this value consistent
with our somewhat lower value of 1.5%. The p value of
0.5% for glucose minimal medium was associated with a
culture doubling time of 133 min (0.45 doubling/h),
whereas our B/r strain has a doubling time of 45 min
(1.3 doublings/h) in this medium. The dramatic differ-
ence in growth rates in glucose minimal medium makes

Table 4 Stoichiometric content of transcription-translation proteins in E. coli

Molecules (t = 40 min) per:
3 a
Protein Mol wt (10 ) (ai ) (%) (t = 40 min) OD460 (1012) Ribosome Reference(s)
r-Protein 850 13.5 10.2 1.00 48, 74
L7/L12 12 0.81 40.8 4.00 77
EF-Tu 42 5.55 55.1 5.40 78
EF-G 84 1.66 8.2 0.80 78
EF-Ts 31 0.13 1.8 0.18 78
IF1 8 0.04 2.5 0.25 79
IF2 115 0.52 3.1 0.30 79
IF3 20 0.07 2.0 0.20 79
Leu S 100 0.12 0.5 0.05 78
Phe S- 94 0.21 1.0 0.10 78
Lys S 58 0.11 0.8 0.08 78
Arg S 58 0.08 0.6 0.06 78
Gly S 77 0.17 0.9 0.09 78
Val S 106 0.14 0.6 0.06 78
Glu S- 48 0.10 0.9 0.09 78
Ile S 107 0.24 1.0 0.10 78
Phe S- 36 0.11 1.2 0.12 78
Gln S 61 0.11 0.8 0.08 78
Thr S 65 0.09 0.6 0.06 78
RNA polymerase 150 0.52 1.4 0.14 78
RNA polymerase 39 0.37 3.8 0.37 78
RNA polymerase, 375 1.30 1.9 0.19 75
core
a
iSynthesis rate of the protein as a percentage of total protein synthesis rate.

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Bremer and Dennis
the strain difference a major factor in the different values
of p that are observed.
The synthesis of the and ' subunits is transcriptionally
controlled at least in part at the level of termination-
antitermination at an attenuator in front of the rpoB
gene. Under most conditions, this attenuator stops about
80% of the transcripts coming from the upstream L11
and L10 r-protein operons (68, 8186). In the past, it has
been thought that the control of r-protein promoters
might involve the effector nucleotide guanosine tetra-
phosphate, ppGpp (8790), but at least for the r-protein
spc operon it has been shown that its promoter, Pspc, is
constitutive, without specific control (38, 91, 92). The
apparent correlation of the accumulation of spc-mRNA
with the stringently controlled rRNA transcripts is now
assumed to be the result of a control of spc-mRNA
degradation, which depends on the accumulation of r-
protein S8. This protein binds to the spc-mRNA and
inhibits its translation, which, in turn, makes the mRNA
accessible to endonucleolytic cleavage followed by exo-
nucleolytic degradation. Thus, when rRNA synthesis is
inhibited by ppGpp during the stringent response, free
S8 accumulates and causes a reduction in the accumu-
lation of spc-mRNA (for details, see references 25 and
93). We suspect that other r-protein promoters, in-
cluding the promoters for the L11 and L10 operons, are
similarly constitutive. The main regulation of the rpoBC
gene expression might therefore be the readthrough
control at the attenuator, perhaps involving an auto-
regulation by free RNA polymerase and/or cleavage of
the transcript by RNaseIII (86, 87, 94). There is also
translational control of the rpoBC mRNA (9598).
Combining the observed p values with the protein per
cell values of Table 2, the average numbers of core RNA
polymerase molecules per cell, Np, have been calculated
in Table 3 and were found to increase from about 1,800
to 10,200 between growth rates of 0.6 and 3.0 doublings/
h. These values differ somewhat from the values in the
previous edition of this chapter (1,500 to 11,400 at 2.5
doublings/h). The main reason for this is the use of
newer and presumably more accurate values of cells per
mass (Table 2) to calculate the numbers of polymerase
per cell. The previous cell numbers for the different
cultures were determined directly with an electronic
particle counter, whereas the new numbers used here
were obtained indirectly from DNA replication and cell
division data to make all parameter values consistent
with one another (see Cell numbers, above).
22
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The amount of 70, the housekeeping sigma factor that is
employed for most transcription initiation during ex-
ponential phase growth, is present in excess over the
amount of free (and even total) core RNA polymerase
(80, 99).
RNA polymerase activity
The total complement of RNA polymerase enzyme in
the cell can be partitioned into active RNA polymerase
engaged in RNA chain elongation and inactive RNA
polymerase. The inactive polymerase includes promoter-
bound enzyme (especially at mRNA promoters with
long promoter clearance times), DNA-bound polymer-
ase idling during transcription, nonspecifically DNA-
bound polymerase, free holoenzyme, ready to bind to a
promoter; and newly synthesized immature enzyme. The
fraction of enzyme actively engaged in RNA chain
elongation, p, was determined from the total rate of
transcription and the RNA chain elongation rate. Due to
a difference in the chain elongation rates for mRNA and
rRNA (see below), the calculation contains separate
components for mRNA and stable RNA. The calcula-
tions in Table 3 show that p increases with growth rate
(75) and that only 15 to 36% of the RNA polymerase
enzyme is transcriptionally active, i.e., involved in RNA
chain elongation, at any instant.
What is the state of inactive RNA polymerase within the
cell? Experiments with a minicell strain indicate that
most of the (core) RNA polymerase is sequestered with
the DNA (99). This enzyme might either be bound
nonspecifically to DNA (80, 100), or, alternatively, it
might have initiated transcription but is halted at some
pausing site (101, 102), perhaps associating with termi-
nation-antitermination factors (103). It remains unclear
why there is such a large excess of inactive RNA poly-
merase, how the partition between active and inactive
enzyme is maintained, what fraction of the inactive RNA
polymerase is nonspecifically bound to DNA, and to
what extent such nonspecifically bound RNA polymer-
ase equilibrates with free RNA polymerase.
In ppGpp-deficient strains, up to 60% of the total RNA
polymerase is active (69), which has suggested that part
of the inactive polymerase (i.e., 27 to 46% of the total
polymerase) in ppGpp-proficient bacteria is transiently
stalled at ppGpp-dependent pause sites during the syn-
thesis of mRNA (69, 101). Of the remaining inactive
RNA polymerase in ppGpp-deficient cells (i.e., 40% of
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 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates

total), about one half consists of the fractions of total

RNA polymerase that are either free functional or free
nonfunctional (immature) or promoter-bound before
promoter clearance; these three forms have been esti-
mated to represent 5.1, 3.5, and 10.0%, respectively, of
the total polymerase at = 1.0 doubling/h and 7.8, 9.1,
and 1.9%, respectively, at = 2.5 doublings/h (see Table
4 in reference 60). The sum of these fractions is about
19% of total polymerase at either growth rate. The other
half (about 20% of total RNA polymerase) seems to be
involved in ppGpp-independent transcriptional pausing
or might be nonspecifically bound to DNA. As men-
tioned above, the 70 subunit is in excess over free,
functional core enzyme (80, 99), suggesting that essen-
tially all free enzyme is functional holoenzyme.

The total rate of transcription in E. coli is not limited by

the DNA concentration but is limited by the concentra-
tion of free functional RNA polymerase (60). This is
perhaps surprising in view of the fact that only a small
fraction of the polymerase is actively engaged in transcript
elongation at any instant. The condition of excess DNA
was demonstrated in various ways, for example, by using a
mutant bacterial strain with altered DNA replication
control, which results in a lower DNA concentration.
Despite the lower DNA concentration there was no
change in the level of total RNA accumulation, i.e., mainly
of stable RNA (62). This means that the DNA template is
in excess, at least for the transcription of stable RNA.

The concentration of free functional RNA polymerase

depends on the cytoplasmic concentration of total RNA
polymerase (p) and on the concentrations of all pro-
moters in the cell, their kinetic constants, state of re-
pression, associated transcript lengths, and transcription
velocities (for details, see reference 60). The constitutive
promoters of many mRNA genes are saturated at growth
rates above 1.5 doublings/h (92), so that their tran-
scription at higher growth rates is limited by their gene
dosage, rather than by free RNA polymerase.
The basic concepts relating to an excess or limitation in
free RNA polymerase on the transcription from a par-
ticular promoter are illustrated by considering an ide-
alized cell with a given concentration of total RNA
polymerase (2,000 molecules per cell) and a variable
number of mRNA genes (0 to 400 per cell, with a length
of 1,500 nucleotides and typical Vmax and Km values).
From this example, the following relationships become
apparent (Fig. 3). (i) At low DNA concentrations (below
Figure 3 Effect of varying gene concentration on the total rate of RNA
synthesis, the rate of transcription per gene, and the concentration of
free RNA polymerase (from reference 60). The relationships are de-
rived from an idealized cell, where all promoters are identical and the
transcription times of all genes are equal. The volume of the cell is one
unit and concentrations are given as numbers of molecules per cell.
The cell contains 2,000 RNA polymerase molecules, and the number
of promoters per cell [Pt] is varied between 0 and 400 (abscissa in all
panels). Vmax is set at 30 initiations per minute per promoter, and Km
is set at 200 RNA polymerase molecules per cell. All transcripts are
1,500 nucleotides long, and the RNA chain elongation rate is 50 nt/s.
(a) The ordinate is the total steady-state rate of transcription, i = V
[Pt], measured as transcripts/min per cell. (b) The ordinate is the
steady state rate, V, of transcription for one promoter measured as
transcripts/min per promoter, calculated from equation 4 in reference
8. (c) The ordinate is the free RNA polymerase concentration, [Rf],
calculated from equation 7 in reference 60. For panels b and c the
ordinates are shown in log scale to illustrate how V and [Rf] approach
zero as [Pt] increases and the total rate of transcription per cell, i,
approaches its plateau value.

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Bremer and Dennis
50 genes per cell), the increasing concentration of genes
and promoters causes an increasing rate of total tran-
scription (Fig. 3c). The rate of total transcription is then
limited by the total DNA available, and all promoters are
nearly saturated with polymerase and active at near their
maximum rate (Fig. 3b). In addition, most of the total
polymerase is in the form of free RNA polymerase (Fig.
3a). This condition may be described as excess RNA
polymerase. (ii) When the DNA and gene concentrations
are gradually increased to above 200 genes per cell, the
total rate of transcription reaches a plateau (Fig. 3c), but
the free RNA polymerase concentration (Fig. 3a) and the
activity per promoter (Fig. 3b) continue to decrease.
Such conditions, when the total rate of transcription is
limited by the total RNA polymerase, may be described
as excess DNA. Under these conditions, free RNA po-
lymerase is only a small fraction of the total, and the
major fraction of RNA polymerase is either bound to a
promoter or involved in transcript elongation. Excess
DNA appears to be the typical in vivo condition in
bacterial cells.
In this idealized cell (Fig. 3), we have assumed that the
cell volume is constant. In an actual cell, the addition of
extra genes might cause additional crowding; i.e., it
might reduce the available reaction space and thereby
cause some increase in the concentration of free RNA
polymerase.
Partitioning between stable RNA and
mRNA synthesis
The actively transcribing RNA polymerase enzyme can
be further partitioned into the fractions engaged in the
synthesis of stable RNA species (rRNA, tRNA, and their
spacers; s) and of mRNA (m = 1 s). The term s the relaxed response with low levels of ppGpp). When
was originally introduced by Maale and coworkers in
their early studies of the control of ribosome and tRNA
synthesis (104). s reflects the controls of both the rates
of stable RNA and of mRNA synthesis, and the term is
useful in certain formulas related to the control of the
growth rate (e.g., equation 19 in Table 6 below). De-
terminations of s are based on the determinations of
the rate of stable RNA synthesis relative to the total RNA
synthesis rate, rs/rt, and of the chain elongation rates of
mRNA and stable RNA (see formula in footnote e of
Table 3).
The quotient rs/rt was first estimated by Pato and von
Meyenburg in Maale's laboratory from the kinetics of
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radioactive uridine incorporation into RNA after stop-
ping initiation of transcription with rifampin (104).
Because of the instability of some rRNA made in the
presence of rifampin (due to the inhibition of r-protein
mRNA synthesis), this method was not accurate, but the
error could be corrected by a complex extension of the
method of measurements (105107). Later, when cloned
rRNA genes became available as hybridization probes
for rRNA transcripts, determination of rs/rt became
easier and more direct (31, 51). At growth rates between
0.6 and 3.0 doublings/h, rs/rt increases a little over
twofold, from 41 to 90%, implying that the fraction of
mRNA synthesis decreases about sixfold from 59 to 10%
(Table 3, values from reference 31).
Determinations of rs/rt are also needed for finding the
synthesis rates of total RNA and mRNA. The stable
RNA synthesis rate, rs, can be found from the amount of
stable RNA, Rs (using any reference unit, like per mass
or per cell), and the growth rate (see the next section).
The total rate of RNA synthesis, rt, then equals the
quotient rs/(rs/rt), and the mRNA synthesis rate, rm,
equals the difference rt rs. These values were further
used to find the fraction of total RNA polymerase that is
actively engaged in RNA chain elongation at any instant
(see RNA polymerase activity, above).
When rs/rt is plotted as a function of the intracellular
concentration of the effector ppGpp, a unique relation-
ship is obtained, independent of whether concentrations
of ppGpp are varied by using growth media with dif-
ferent nutritional values, or by inducing partial amino
acid starvation in either wild-type (relA+) bacteria
(triggering the stringent response with high levels of
ppGpp) or in relA mutant bacterial strains (triggering
the concentration of ppGpp is close to zero, rs/rt is close
to 1.0 (i.e., almost no mRNA synthesis in relation to
stable RNA synthesis). As the concentration of ppGpp
gradually increases the values of rs/rt gradually decrease;
at high concentrations of ppGpp generated during the
stringent response, the value of rs/rt reaches a minimum
plateau of 0.25 (30). This unique function is surprising,
since rs/rt depends in a complex manner on the controls
of mRNA and stable RNA promoters, and on the con-
centration of free RNA polymerase, as described below.
By combining measurements of transcription from spe-
cific promoters and of DNA replication to find gene
dosages under different growth conditions, it is possible
ASMScience.org/EcoSalPlus
 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates
to determine absolute in vivo activities of specific pro-
moters in transcript initiations per minute per promoter,
and to define the in vivo control of stable RNA and
mRNA promoters in biochemical (Michaelis-Menten)
terms (8, 92). Promoter strength is defined as the
promoter activity (measured in transcripts per unit time
per promoter) per free RNA polymerase concentration
(given in either molar or relative units) under conditions
of low RNA polymerase concentration (so low that it
does not saturate any of the promoters considered).
Mathematically, the promoter strength equals the ratio of
the Michaelis-Menten parameters Vmax and Km (maxi-
mum promoter activity at saturation with RNA poly-
merase and RNA polymerase concentration at half-
maximal activity). If Vmax /Km for a given promoter re-
mains constant under different growth conditions, the
promoter is said to be constitutive. If Vmax and/or Km
changes, it means that the promoter is subject to control.
Control occurs either through the action of a repressor or
activator that binds to the DNA near the promoter, or by
an effector like ppGpp that binds to the RNA polymerase
and thereby changes the promoterpolymerase interac-
tions. As long as the promoter is not saturated with RNA
polymerase, its activity also depends on the free RNA
polymerase concentration as illustrated in Fig. 3.
Of the two tandem promoters of the rRNA (rrn) genes,
transcription from the upstream P1 promoter is inhibited
by the nucleotide effector ppGpp and is controlled,
whereas the downstream P2 promoter is constitutive (for
a review of the controversial issue of rrn P2 control, see
reference 8). However, within the context of the rrn
operon, P2 is subject to promoter occlusion by tran-
scribing RNA polymerase molecules coming from the
upstream P1 promoter and temporarily blocking the
access of RNA polymerase to the downstream P2 (108).
Both rrn P1 and P2 have short promoter clearance times
(= 1/Vmax = 0.52 s) in vivo, so that they are not saturated
with polymerase under most growth conditions; there-
fore, their activities increase with increasing concentra-
tions of free RNA polymerase at increasing growth rates
(8, 92, 108, 109). In contrast, most mRNA promoters,
because of their slow clearance, become saturated with
polymerase so that their activities reach maximum levels
at growth rates above 1.5 doublings/h (92).
The strength of rRNA (and tRNA) promoters is actually
lower than the strength of many mRNA promoters (e.g.,
r-protein mRNA promoters; reference 92). However,
since the mRNA promoters, but not stable RNA
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promoters, become saturated, the stronger mRNA pro-
moters have a higher activity than the stable RNA pro-
moters during growth in nutritionally poor media, but at
the higher concentrations of free RNA polymerase
during growth in rich media, the stable RNA promoters
have a higher activity, despite their lower strength (8,
92). Thus, stable RNA and mRNA promoter controls, as
well as changing free RNA polymerase concentrations,
contribute to the values of s and rs/rt observed under
different growth conditions. The higher concentration of
free RNA polymerase at higher growth rates results both
from an increased concentration of total RNA poly-
merase (Table 3, p) and from the repression of mRNA
genes by exogenous nutrients (60).
The effect of exogenous nutrients on the relative syn-
thesis of mRNA is seen from the following example. In a
culture grown in succinate minimal medium at 0.6
doublings/h, rs/rt equals about 0.4 (Table 3). If the ex-
ogenous succinate is substituted by glycerol, the growth
rate increases to about 1.0 doubling/h and rs/rt increases
by about 20% to 0.5 (Table 3). At the same time the rrn
gene activities increase more than twofold from 4 to 10
initiations/min per rrn gene (Table 3), reflecting both
the control of stable RNA promoters by ppGpp and the
increased free RNA polymerase concentration resulting
from a control of RNA polymerase synthesis (increased
p, Table 3) (60). But the mRNA gene activities also
increase as a result of increased free RNA polymerase
concentration, only somewhat less than the stable RNA
gene activities, which yields the 20% increase in rs/rt.
When, instead of replacing succinate by glycerol, the
growth rate is increased (also to 1.0 doubling/h) by
adding 20 amino acids to the succinate medium, rs/rt
doubles to about 0.8 (Fig. 9 in reference 10), implying a
threefold decrease in the fraction of mRNA synthesis (i.
e., from 0.6 to 0.2). This decreased mRNA synthesis
must reflect the repression of amino acid biosynthetic
enzymes in the amino acid-supplemented medium. As
was explained above, the values in Table 2 and Table 3
for the growth rate of 1.0 doubling/h refer only to cul-
tures grown in glycerol minimal medium, not to succi-
nate-amino acids media that produce the same growth
rate.
In view of the complexity of regulations involving the
values of rs and rm, it is not clear why rs/rt becomes a single
function of ppGpp levels under seemingly all conditions
(i.e., not only during exponential growth at different rates
in the chosen selection of media), and also independent of
25
Bremer and Dennis
the absolute values of rs and rm (30). Understanding of this
phenomenon requires further analysis.
Rates of stable RNA and mRNA synthesis per cell
The rate of stable RNA synthesis per cell (rs) was cal-
culated from the amount of total RNA per cell, deter-
mined from the UV absorbance of RNA hydrolysates
(Table 2), after a small correction for the presence of
mRNA (using the factor fs = fraction of total RNA that is
stable RNA; see Table 1). The value of rs is seen to
increase dramatically with growth rate, which reflects
both the increasing ribosome concentration and the
increasing cell size.
The synthesis rates and amounts of mRNA per cell (rm,
Rm) were found from rs and rs/rt (see above). Both rm and
Rm increase about fourfold in the range of growth rates
considered (Table 3), which mainly reflects the in-
creasing cell size; i.e., rm and Rm per mass remain ap-
proximately constant. A comparison of the values of
total RNA per cell (Table 2) and mRNA per cell (Table
3) shows that the fraction of total RNA that is mRNA (1
fs) decreases from about 2% at a growth rate of 0.6
doubling/h to only 1% at 3.0 doublings/h.
Chain elongation rates of stable RNA and mRNA
Whereas mRNA transcripts elongate at a rate (cm) that
increases somewhat with increasing growth rate and
approaches a maximum value of about 56 nucleotides
per second (56 nt/s) at high growth rates (27, 70, 110),
transcripts of the rrn operons elongate at a rate (cr) that
is nearly constant and equal to about 85 nucleotides (nt)/
s (33, 34, 36, 110, 111). For rrn transcripts in bacteria
grown in glucose-amino acids medium a transcription
time of 60 s has been observed for an rrn operon with a
length of 5,450 bp (32), corresponding to an rRNA chain
elongation rate of 91 nt/s (5,450/60 = 91). This value is
similar to data from other laboratories: 89 nt/s for bac-
teria in glucose amino acids medium, 79 nt/s in glycerol
minimal medium (36), and 78 nt/s in glucose minimal
medium (34). For the range of growth rates between 0.6
and 3.0 doublings/h we have here assumed a constant
value of 85 nt/s for all stable RNA transcription (cs,
Table 3). However, the constancy might not be exact,
because some increase in cr with increasing growth rate
has been reported (33, 36).
The leader regions of rrn transcription units contain
strong Nus factor-dependent antitermination sites (112
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114). As a consequence, transcripts initiated at rrn
promoters are able to transcribe through Rho protein-
dependent transcription terminators and can reverse
transposon-induced polarity (115, 116). Thus, for rRNA
transcripts, pausing or stuttering is minimized by the
antitermination state of the RNA polymerase, which
accounts in part for the increased stable RNA chain
elongation rate (110, 111). When the antitermination
sequences at the rrn promoters are removed, the rrn
transcript elongation rate is reduced, and when these
sequences are inserted at the front of a mRNA promoter,
then the mRNA elongation rate is increased (110). This
antitermination mechanism is essential for efficient rrn
transcription because of the lack of coupled translation
and because of the extensive secondary structures pres-
ent in rRNA transcripts.
Accumulation of the effector ppGpp
The accumulation of ppGpp at different growth rates has
been determined from the A260 of the ppGpp peak after
separation of the bacterial nucleotides by reversed-phase
high-pressure liquid chromatography (31). The role of
ppGpp in the control of stable RNA synthesis has been
controversial since the publication of a proposal that free
or translating ribosomes, rather than ppGpp, regulate
transcription of rrn operons (55, 117, 118). Today
however, the predominant importance of ppGpp in the
control of rRNA synthesis in vitro (119, 120) and during
exponential growth (31, 39, 121) is established and
generally accepted (reviewed in reference 8). During
periods of amino acid insufficiency, ppGpp is derived
from the relA-dependent system and elicits the well-
characterized stringent response (57, 87, 122, 123).
During exponential growth at different rates, the basal
levels of ppGpp are derived mainly from a relA-inde-
pendent system (124126) that involves a product of the
spoT gene (127129). The basal levels of ppGpp listed in
Table 3 are seen to decrease from about 55 to 6 pmol/
OD460 when the exponential growth rate increases from
0.6 to 3.0 doublings/h. These values refer to balanced,
steady-state exponential growth in the selected choice of
media (see above). If growth is limited by the supply of
some amino acid during chemostat growth where the
growth rate is determined by the dilution rate of the
culture (in the original studies of the Maale laboratory
these were called continuous culture experiments; e.g.,
in Fig. 1 and 2 of reference 1), the RelA ppGpp syn-
thetase is presumably activated and produces higher le-
vels of ppGpp (88).
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 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates
The growth medium-dependent control of the SpoT-de-
pendent synthesis of ppGpp, which adjusts the synthesis
of ribosomes during exponential growth and thereby
partly determines the growth rate, is still a mystery (128).
It might involve the GTPase CgtAE that cofractionates
with the 50S ribosomal subunit and interacts with SpoT
(130), or the acyl carrier protein ACP that interacts with
SpoT (131). It has been proposed that this control might
involve a feedback control of the peptide chain elongation
rate (8): whenever cp has a submaximal value, i.e., when it
drops below 22 amino acid residues polymerized per
minute per active ribosome during growth in minimal
media, then the ppGpp synthesis activity of SpoT is
stimulated to reduce the synthesis of ribosomes and
thereby the consumption of amino acids. Such a mecha-
nism would minimize the reduction in cp and save energy
by reducing any excessive synthesis of ribosomes (see
Optimal cell composition for maximal growth).
Ribosome Synthesis and Function
Ribosomal components and their control
The ribosome consists of three species of RNA (16S,
23S, and 5S) and 52 species of protein. The three rRNAs
are processed from a 35S primary transcript derived
from seven unlinked rrn transcription units. The 52
different ribosomal proteins (r-proteins) are encoded by
genes in about 20 different transcription units located at
14 different positions on the E. coli chromosome (13).
During exponential growth at moderate to fast rates,
turnover of ribosomal components is negligible, but
during slow growth, some excess of newly made rRNA
is degraded (54, 132), which contributes to the slight
increase in the proportion of tRNA to rRNA (22, 35,
53).
The primary regulation of ribosome synthesis occurs at
the level of rRNA synthesis; the synthesis of r-proteins is
then adjusted to the accumulating rRNA. Any excessive
accumulation of free r-proteins causes a reduction in the
synthesis and lifetimes of r-protein mRNA (38, 90, 93,
133, 134) and thereby reduces the synthesis of r-protein
(see below).
Each of the seven rRNA (rrn) operons has two tandem
promoters, P1 and P2, from which the rRNA transcripts
are expressed. After correction for position effects on the
E. coli chromosome, all rrn operons are similarly ex-
pressed (reference 32, reviewed in reference 93). The P1
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promoters of all seven rrn operons have the same dis-
criminator sequence, GCGC, bordering similar TA-
TAAT 10 regions (93, 135, 136). Upstream of each of
these P1 promoters are three binding sites for the pro-
tein factor Fis; the binding of Fis to these sites stimulates
expression from P1. In addition, P1 is negatively con-
trolled by ppGpp, which binds to the interface of the
and ' subunits of the RNA polymerase (59, 137, 138)
near the active site (139). In the presence of the adaptor
protein DksA (140), this binding of ppGpp to RNA
polymerase reduces the strength of the P1 promoter
directly, and also indirectly by reducing expression of Fis
(8). The rrn promoter strength is given as Vmax/Km for
the RNA polymerase promoter interaction (see Parti-
tioning between stable RNA and mRNA synthesis,
above)
In the absence of both ppGpp and Fis, the (isolated) P1
and P2 promoters have about equal activities that only
increase with increasing free RNA polymerase concen-
tration. Their activities then increase in parallel with
increasing growth rate, and, before they become satu-
rated, also in parallel with the activity of constitutive
mRNA promoters (see Partitioning between stable
RNA and mRNA synthesis, above). Under such con-
ditions the strength of the rrn promoters remains con-
stant at different growth rates, i.e., P1 and P2 are both
constitutive (108, 141).
In the absence of ppGpp, but the presence of Fis, the
strength of P1 still remains constant, but due to Fis,
about twofold higher than the strength of the P2 pro-
moter, and the activities of P1 and P2 also increase in
parallel (i.e., by the same factor) with increasing growth
rate, again reflecting only the increasing concentration of
free RNA polymerase (141).
r-Protein synthesis
A measure used for the synthesis of r-protein is the
proportion of total protein that is r-protein, r. Values of
r have been determined (i) directly from the protein
content of ribosomes after separation from other proteins
by sucrose gradient centrifugation (5, 48), and (ii) indi-
rectly from the RNA-to-protein ratio (see footnote m in
Table 3). Both methods give similar values. The pro-
portion of r-protein increases from about 8% of total
protein at a growth rate of 0.6 doubling/h to 23% at 3.0
doublings/h (Table 3). The mRNAs of r-protein operons
contain internal elements that control their elongation,
27
Bremer and Dennis
translation and lifetime. The regulatory r-proteins spe-
cific to each operon that are not rapidly incorporated into
assembling ribosomes bind to these elements, which are
often structural mimics of their binding sites on rRNA,
and may cause transcript attenuation or rapid degrada-
tion of the entire mRNA; this has been called retro-
regulation (reviewed in reference 93). In this manner, r-
protein synthesis is adjusted and matched to rRNA
synthesis.
Ribosomes and tRNA per cell
Given that r-protein matches rRNA and that rRNA and
tRNA are synthesized in a nearly constant proportion,
corresponding to about nine tRNA molecules per 70S
ribosome (22, 35, 58, 142; see Cell growth-related
parameters, above), the numbers of ribosomes (Nr) and
of tRNA molecules per cell (Nt) were calculated from the
total amount of RNA per cell (RC; Table 2) after sub-
traction of the small amount of mRNA (only 1 to 2% of
the total RNA; see above). In the growth range consid-
ered, the average number of ribosomes per cell (Nr) is
seen to increase about 10-fold from 8,000 to 73,000
(Table 3). This reflects both an increasing ribosome
concentration (ribosomes per OD460 unit of cell mass, or
per total amount of protein) and the increasing cell size
(OD460 units or protein per cell). For achieving rapid
growth, only the ribosome concentration is relevant.
Ribosome activity
The fraction of ribosomes engaged in peptide chain
elongation at any instant, r, has been estimated from
the ribosome content of polysomes and was found to be
about 80% and independent of the growth rate (40).
Here we have assumed a slightly higher value of 85%
(from Table 2 in reference 25), based on the assumption
that the maximum value for the rate of peptide chain
elongation in vivo is 22 amino acid residues per second
(see the next section). The relative constancy of r may
only be approximate, mainly indicating that the fraction
of inactive ribosomes (the difference 1 r) is relatively
small, probably between 10 and 20%, which includes
both mature free 30S and 50S particles ready to initiate
translation, and immature particles. Since the assembly
and maturation of ribosomes takes about 5 min (143), it
appears that, at least in fast-growing bacteria, immature
subunits in the final stages of assembly constitute the
major portion of the inactive ribosomes.
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Peptide chain elongation rate
The average peptide chain elongation rate, cp, has been
estimated directly from (i) pulse-labeling kinetics of
nascent polypeptides of given length (41, 144), and (ii)
the first appearance of -galactosidase activity after in-
duction (144147) and indirectly (iii) from calculations
based on the RNA-to-protein ratio under the assump-
tion that 85% of the ribosomes are active at any instant.
All three methods give similar values; the values in Table
3 were calculated from the RNA-to-protein ratio. These
measurements indicate that the average peptide chain
elongation rate increases with growth rate from about 13
amino acid residues per second at 0.6 doubling/h to a
maximum value of 22 amino acid residues per second at
growth rates above 1.5 doublings/h.
The chain elongation rate of -galactosidase in serovar
Typhimurium has also been measured at different
growth rates between 0.5 and 1.9 doublings/h by mea-
suring the time from the entry of labeled threonine into
the pool of threonyl-tRNA until the label appears in the
N-terminal threonine residues in the free, completed -
galactosidase tetramers (148). With this method, the
chain growth rate was found to be 16 to 20 amino acids
per second at 37C, and independent of the growth rate.
It is not clear why the chain growth rate in those ex-
periments did not increase with the growth rate.
What limits the peptide chain elongation rate? The an-
swer to this question is still controversial. During fast
growth, i.e., in media supplemented with all 20 amino
acids, the peptide chain elongation rate was found to
have essentially the same, apparently maximal value of
22 amino acid residues polymerized per second (Table
3). We assume, therefore, that ribosomes are saturated
with substrates (elongation factor Tu-GTP-aminoacyl-
tRNA ternary complex) under such conditions, implying
that the peptide chain elongation rate is limited by
structural rearrangements within the ribosome (149,
150), the rate of peptide bond formation, or ribosome
translocation on the mRNA. Furthermore, we assume
that the submaximal chain elongation rates during slow
growth are probably limited by the smaller size of the
amino acid pools and reduced tRNA charging (i.e., by
the ratio of charged to uncharged tRNA [151]) and to
some extent by the types of codons being employed.
An alternative interpretation of the growth-medium-
dependent changes in the rate of ribosome function has
been proposed in a theoretical study by Elf and
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 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates
Ehrenberg (152), who suggest that in any particular cell
at any given time, only one amino acid can be limiting,
so that the cell then undergoes a type of temporary
single amino acid deprivation, with ribosomes halted at
the codons for the limiting amino acid. This temporary
pause in protein synthesis should allow a maximal
charging of the tRNAs for all other amino acids, while
the depletion of the pool for the limiting amino acid
causes a derepression of the corresponding biosynthetic
operons. The ensuing amino acid synthesis allows the
ribosomes in that particular cell to resume function until
some other amino acid becomes limiting. For the whole
culture, this kind of stuttering ribosome function
produces an average reduction in cp.
The question, whether it is possible to deplete the pools
for more than one amino acid, has been addressed in
Stents's laboratory by Broda (153), who found that on
simultaneous starvation for arginine and histidine, nei-
ther amino acid could be detected in the pool. That
study also found that, in a shift-down from an amino
acid-supplemented to a minimal medium, both isoleu-
cine and valine pools were depleted. In addition, we note
that stuttering ribosomes cause premature terminations
of polypeptides, e.g., during single amino acid depriva-
tion (154), or during treatment with ribosome inhibitors
(like puromycin) that induce the heat shock response
(155). Since premature termination of peptides has not
been observed during growth of bacteria in minimal
media, we suggest that such long translational pauses do
not occur under these conditions, and that several tRNA
species may be submaximally charged in a given cell at
the same time.
In this context, an unanswered question is how poly-
peptides elongate in single cells, especially during growth
in minimal media, when the average value of cp is re-
duced below the maximum. For example, one could ask
whether there is always a small fraction of cells in which
all peptide chain elongation is temporarily halted, be-
cause the pool for a limiting amino acid is totally (but
temporarily) depleted, or whether such a situation never
arises. As far as we are aware, experiments addressing
this issue have not been done.
Another explanation for the reduced ribosome function
at low growth rates has been proposed by Ehrenberg and
Kurland (156) and Lovmar and Ehrenberg (157), based
on their maximum fitness theory (see Optimal cell
composition for maximal growth, below). According to
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that theory, the peptide chain elongation rate during
exponential growth in different media is not limited by
amino acids or tRNA charging, but by the concentration
of ternary complex substrates, i.e., the complex of elon-
gation factor Tu (EF-Tu), GTP, and aminoacyl-tRNA.
Furthermore, based on in vitro measurements of peptide
chain elongation rates and in vivo measurements of dif-
fusion rates, the maximum in vivo chain elongation rate
was calculated to be 50 (rather than 22) amino acids per
second, implying that in vivo ribosomes are never satu-
rated with substrates, even at the highest growth rates. In
support of this idea, Kruger et al. (158) have observed that
the in vivo translation rate of the GAA by tRNAGlu
lacking the 5-methylaminomethyl modification at U34
was 47 codons per second compared with a rate of 18
codons per second for the fully modified tRNA. In con-
trast the translation rate of the GAG codon by the un-
modified tRNA was only 1.9 codons per second
compared with a rate of 7.7 codons per second for the
fully modified tRNA.
After a nutritional shift-up, the peptide chain elongation
rate increases to the postshift steady-state rate, appar-
ently within the first minute (28), well before there is any
significant additional accumulation of the macromolec-
ular components of the ternary complex (i.e., EF-Tu and
tRNA). Therefore, we suggest that the reduced peptide
chain elongation during growth in minimal media re-
flects the reduced sizes of several, if not most, amino acid
pools, which causes a reduced tRNA charging (159) and
a more or less evenly reduced rate of ribosome function
without significant stuttering. In amino acid-supple-
mented media the increased amino acid pools, in turn,
are assumed to produce a maximal charging of tRNA
and thereby a maximum rate of ribosome function.
It has been reported that ppGpp-EF-Tu complexes bind
to ribosomes where they reduce the peptide chain
growth rate and increase the fidelity of proofreading by
inhibiting the binding of the ternary complex substrate
and the formation of peptide bonds (160). Since the
ppGpp levels are increased in slow-growing bacteria and
decrease rapidly after a nutritional shift-up, this effect
might contribute to the reduced ribosome function
during growth in minimal media and to its rapid in-
crease in the presence of exogenous amino acids. How-
ever, it is unlikely that the low basal levels of ppGpp
during exponential growth have this effect, since the
peptide chain elongation rate is apparently unaffected in
a ribosome control mutant with 10-fold reduced levels of
29
Bremer and Dennis
ppGpp (161). Moreover Sorensen at al. (162) have
shown that ppGpp does not affect the peptide chain
elongation rate.
The utilization of codons in the genes of E. coli is not
random (163165). It is conceivable that, in poor media,
the greater proportion or abundance of mRNAs with
hard-to-read codons might contribute to the reduced
average peptide chain elongation rate. Genes expressed
from strong promoters tend to contain a higher pro-
portion of major codons, whereas genes expressed from
weak promoters contain a lower proportion of major
codons. With some exceptions, major codons are rec-
ognized by abundant tRNA species and minor codons
are recognized by rare tRNA species (166). However, the
concentration of minor tRNAs is probably not limiting
the rate of peptide chain elongation. Instead, each co-
doncognate tRNA pair probably has a specific transit
time for progressing through the A and P sites of the
ribosome that depends on the physical-chemical nature
of the tRNAmRNA (anticodoncodon) interaction; the
limiting step is the initial codon-dependent interaction
of the ternary complex and the deposition of the tRNA
into the A site of the ribosome (165). As an example, the
GAA and GAG glutamic acid codons are both recog-
nized by the abundant tRNAGlu2, but decoding of the
prevalent GAA triplet involving strict Watson-Crick
base pairing occurs threefold more rapidly than the
decoding of the GAG triplet involving wobble base
pairing (147, 167, 168).
Ribosomal RNA gene dosage and activity
Most of the seven rrn genes on the E.coli chromosome
map near the chromosomal origin of replication (13).
The number of rrn genes per average cell (Nrrn) in an
exponential culture is much greater than 7, ranging from
12 to 38, depending on the extent of chromosome
branching (see Fig. 1). From the number of rrn genes
and the rate of rRNA synthesis, the average rate of ini-
tiation of rRNA chains at each rrn gene (irrn) was cal-
culated and was found to increase with increasing
growth rate from 4 transcripts per minute to 68 tran-
scripts per minute per gene (Table 3).
During the cell cycle the number of rrn genes per cell
doubles through a series of incremental steps occurring
each time an rrn transcription unit is replicated. Since in
a given cell new rounds of replication are initiated
synchronously at all origins and the replication forks
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move with about equal speed (see DNA replication and
cell division, below), and since 5 of the 7 rrn genes are
clustered near oriC, the number of rrn genes per cell is
expected to almost double in a series of closely spaced
steps shortly after a new round of replication is initiated.
Despite this near doubling of rrn genes, measurements
of DNA and RNA synthesis rates in age-fractionated
cultures indicate that replication of the rrn genes causes
no abrupt or concomitant increase in the rate of rRNA
synthesis (169). Instead, as cells progress through the
division cycle, the rate of rRNA synthesis increases by a
factor of 2 in a slow, uniform, and continuous manner,
without perturbation, regardless of the cell age at which
the rrn genes are replicated (22, 169). This implies that
the rate of transcript initiation at rrn genes decreases
nearly twofold when the rrn genes located near the or-
igin of replication are duplicated. During growth in rich
media, when 90% of all transcription comes from stable
RNA genes (Table 3: rs/rt at = 3.0), initiation of rep-
lication occurs at eight origins (Fig. 1), so that 40 (i.e.,
8 5) rrn genes double to 80 within a short interval of
time without causing any noticeable change the rate of
total rRNA synthesis in the cell. Thus, the copy number
of rrn genes does not limit the rate of rRNA synthesis
during the cell cycle.
This conclusion has been further corroborated by the
observation that the rRNA synthesis rate is not reduced
in bacteria with a mutational defect in the control of
chromosome replication that leads to a 40% reduction in
the concentration of all genes, including rrn genes (62).
Furthermore, up to three rrn genes may be deleted from
the E. coli chromosome without much change in the
growth rate, again suggesting that the rrn gene dosage
does not normally limit the rate of rRNA synthesis
during exponential growth (22).
The reason for the rrn gene dosage independence of total
rRNA synthesis is assumed to be the condition of DNA
excess in the bacteria (see discussion of Fig. 3 above in
connection with the RNA polymerase activity). The
replication of long transcription units with strong pro-
moters such as rrn operons (i.e., increase in the DNA
concentration) under the condition of DNA excess is
expected to cause a corresponding reduction in the con-
centration of free RNA polymerase such that the initia-
tion frequency at all unsaturated promoters is reduced.
These observations indicate that an average rate of 68
initiations per minute per rrn operon at the maximum
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 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates
growth rate of 3.0 doublings/h (Table 3) involves a
fluctuation in the range between 45 and 90 initiations
per minute per rrn gene during the cell cycle (actually
the fluctuation is somewhat less because rRNA genes are
not all clustered at exactly the same map position). A
rate of 90 initiations per minute implies that the in vivo
time for the formation of the open promoter complex at
the rrn promoters must be less than 1 second (for details
about reactions and rate constants involved in rrn
transcript initiation, see reference 8).
Translation frequency of mRNA
The average mRNAs made during growth in rich media
at higher growth rates are more crowded with ribosomes
than mRNAs made at low growth rates; i.e., the average
spacing of ribosomes on the average mRNA, dr, de-
creases from 142 nucleotides to 69 nucleotides in the
range of growth rates considered (Table 3). Here again,
there is no indication that mRNA is a limiting factor for
protein synthesis. Immediately after a nutritional shift-
up the concentration of mRNA decreases temporarily
because the increased rate of rRNA synthesis occurs
partly at the expense of the synthesis of mRNAs that are
repressed in the more nutritious postshift medium (28);
at the same time, the protein synthesis rate increases due
to an increased rate of peptide chain elongation (51, 107,
170). The greater spacing of ribosomes on mRNAs made
during slow growth could potentially cause some mRNA
instability or premature termination of transcription
(polarity), but whether this is indeed the case has not
been established.
The rate of initiation of translation of lacZ mRNA has
been found to be nearly independent of the growth rate,
which has suggested that the concentration of free,
translation-ready ribosomes is approximately constant
(25). This means that the decreasing value of dr during
fast growth in rich media reflects the different compo-
sition of mRNA; i.e., mRNAs made predominantly in
rich media (including mRNA for r-proteins) have ap-
parently stronger ribosome binding sites than the aver-
age mRNAs made predominantly during slow growth in
minimal media (including the mRNAs for amino acid
biosynthesis). An average mRNA molecule made at a
growth rate of 0.6 doublings/h is translated about once
every 4 seconds, whereas an average mRNA molecule
made at a growth rate of 3.0 doublings/h is translated
four times faster, about once every second (see Table 2 in
reference 25).
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Component proteins of the
transcription-translation apparatus
The protein composition of the ribosome is essentially
invariant with the growth rate, and each of the 52 dif-
ferent r-proteins is present in one copy per 70S particle
(74, 171). The only exception to this is protein L7/L12,
which is present in four copies per ribosome (77). This
implies that the synthesis rate of each r-protein is strictly
coordinate with the synthesis of rRNA and also tRNA at
growth rates above 0.6 /h. At slower growth rates there
appears to be a slight excess in the synthesis rate of
stable RNA (54, 132, 172); the excess rRNA is rapidly
degraded, whereas the tRNA accumulates.
The synthesis rates of other components of the tran-
scription-translation apparatus also appear to be subject
to growth medium-dependent regulation (73, 78, 79,
173175). These components include translation initia-
tion and elongation factors, the subunits of RNA poly-
merase, and the aminoacyl-tRNA synthetases (Table 4).
From the available data it seems clear that the concen-
tration of all these components increases with growth
rate, but the increases might not beand, for at least
some of the proteins such as RNA polymerase, are not
strictly parallel with the increase in ribosome (and r-
protein) concentration. Table 4 lists the proteins that
have been examined in this context and gives the i
values for each (i.e., the synthesis rate of the protein as a
percent of the total protein synthesis rate) at a growth
rate of 1.5 doublings/h ( = 40 min). In addition, the
numbers of molecules of each protein per unit of mass
and per ribosome are also indicated. In compiling the
information in this table we had to reinterpret or ex-
trapolate some of the original measurements in the cited
references. If the synthesis of these proteins were strictly
coordinate with synthesis of ribosomes, the listed
number of molecules per ribosome would remain con-
stant and not change with changes in the steady-state
growth rate. It is surprising, considering the recent
emphasis on systems biology, that there has not been a
systematic and comprehensive proteomic effort to
measure the relative or absolute amounts of individual E.
coli proteins even at a single growth rate.
The r-protein operons also encode the genes specifying
the , , ', and subunits of RNA polymerase and the
protein synthesis elongation factors Tu, Ts, and G (for a
review, see reference 176). There is a second gene for Tu
on the E. coli chromosome that is not in an r-protein
operon; presumably two copies of this gene are required
31
Bremer and Dennis
to produce the six molecules of Tu per ribosome at high
growth rates. The and ' RNA polymerase subunit
genes, although cotranscribed with the L10 and L12 r-
protein genes, are regulated somewhat independently by
a transcription attenuator and RNaseIII processing site
located between the upstream r-protein genes and the
downstream RNA polymerase genes (see RNA poly-
merase synthesis and function, above).
The elongation factor Tu is required for the GTPase-
dependent deposition of aminoacylated tRNA into the A
site of the translating ribosome. The charging level of
tRNA is about 75 to 90% (159). There are between two
and three tRNA molecules bound to each translating
ribosome (177, 178). The six copies of Tu are available
for ternary complex formation with GTP and the re-
maining aminoacylated tRNAs. The concentration of the
ternary complex required to initiate the process of
amino acid addition on the translating ribosome is thus
maximized.
It has been reported that during periods of amino acid
insufficiency the synthesis of r-protein, like that of rRNA
and tRNA, is subject to stringent regulation at the level
of transcription (8789). Many of the genes that are
cotranscribed with r-protein genes appeared also to be
stringently regulated, but the regulation is likely to be at
the level of mRNA degradation (see below). These in-
clude the genes encoding the elongation factors G, Tu,
and Ts (179181). In contrast, the genes encoding the ,
', and subunits of RNA polymerase were not found to
be stringently regulated (179). The regulation of tran-
scription of the and ' genes is mediated by control at
the attenuator in the L10 (rplJL rpoBC) operon (94).
With respect to aminoacyl-tRNA synthetases, the data
on their stringent regulation were equivocal (179).
The stringent regulation of r-protein genes has come
under question after a thorough investigation of the
control of the promoter of the spc operon, Pspc. This
investigation showed that the promoter is constitutive.
At low growth rates, its rate of transcript initiation first
increases with increasing growth rate due to the in-
creasing concentration of free RNA polymerase and then
approaches a constant level of about 25 transcript in-
itiations per minute per promoter at growth rates above
1.5 doublings/h, reflecting saturation with RNA poly-
merase (92). The previously observed apparent stringent
control of the amount of spc mRNA can now be ex-
plained as the result of a regulation of spc mRNA decay
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in response to ppGpp-dependent changes in rRNA
synthesis (25, 38).
Synthesis and function of tRNA
There are about nine tRNA molecules per ribosome in
exponentially growing E. coli, and this ratio shows little
variation for growth rates above 0.6 doubling/h (Table
3). Since the peptide chain elongation rate approaches 22
amino acids per second, each tRNA is required to cycle
through the ribosome on average about two times per
second. Ikemura (166) has quantitated over 70% of the
total tRNA population into 26 separate species, at least
one for each amino acid except for proline and cysteine.
For each of these 18 different amino acids there is at
least one major tRNA isoacceptor, which is present at a
molar ratio of 0.15 to 0.60 copy per ribosome. The
aminoacyl-tRNA synthetases are present at about 0.1
copy per ribosome; each synthetase molecule is therefore
required to aminoacylate about 10 molecules of its
cognate tRNA every second (= 1 cognate tRNA per
second per ribosome) to sustain protein synthesis at a
rate of about 20 amino acids per second per ribosome.
DNA Replication and Cell Division
Chromosome replication and segregation
The C-period is the time interval required for the rep-
lication forks to move from the origin (oriC, at 84.6 min
on the E. coli genetic map) to the terminus (terA-F,
multiple sites centered near 36 min on the genetic map;
182, 183). Pulse-labeling of the terminus in cells with
synchronized replication has indicated that both repli-
cation forks created at every initiation event move with
equal speed (0.6 to 1.2 kb/min for wild-type strains)
clockwise and counterclockwise, respectively, with very
little variation from cell to cell (184, 185). In addition,
the time intervals between consecutive replications of
any given section of the chromosome are very constant
and equal to the mass doubling time (72, 186, 187). This
suggests that both the times between consecutive in-
itiations of rounds of replication and the replication
velocities themselves are constant within an exponen-
tially growing cell population.
The C-period was first measured by Helmstetter and
Cooper (42) in exponential cultures that were age frac-
tionated. Because of the considerable variability from cell
to cell in the duration of the D-period (see below), the
initiation age, termination age, and interdivision inter-
vals all vary (179). This makes the determination of C
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 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates
and D from age-fractionated or synchronous cultures
somewhat inaccurate. The C-period has also been
measured in exponential cultures without age fraction-
ation from (i) the relative frequencies of genes at given
map locations (equation 9, Table 5 below; reference 37),
(ii) the increase in DNA after stopping initiation of
replication (65, 188190), and (iii) flow cytometry (44,
47). Cooper and Helmstetter (3) estimated that the C-
period was constant (41 min) for growth rates above 1
doubling/h and increased in proportion to at lower
growth rates. Measurements of the increase in DNA
after a stop in initiation of replication (12, 188) suggest
that the C-period decreases gradually with increasing
growth rate, from about 67 min at 0.6 doubling/h to 33
min in bacteria growing at 3.0 doublings/h (Table 3). It
is not known what causes this increase in the average
speed of the replication forks at higher growth rates.
The D-period is the time between termination of a
round of replication and the following cell division when
the replicated chromosomes are segregated. Helmstetter
and Cooper (42) estimated the average D to be about 22
min for growth rates above 1 doubling/h and to increase
as a constant fraction of the doubling time for growth
rates below 1 doubling/h. The average D-period has also
been determined in exponential cultures again without
age fractionation from the increase in the cell number
after a replication stop by thymine starvation (46). Cells
that do not terminate replication due to an experimen-
tally induced replication stop will not divide, whereas
cells that have already terminated and have made ter-
mination protein (i.e., cells in the D-period) divide once
(191). These experiments suggest that the average D-
period decreases from about 30 min during slow growth
(0.67 doubling/h) to about 22 min during rapid growth
(3.0 doublings/h).
Average C- and D-intervals have also been obtained by
the method of flow cytometry, which measures the dis-
tribution of the amounts of DNA (labeled with a fluo-
rescent dye) per cell in exponential cell populations
(frequency of cells as a function of DNA per individual
cell). In this manner, values of C equal to 42 min and D
equal to 22 to 24 min were found for E. coli B/r during
balanced growth at a 27-min doubling time (47). For a
similar doubling time (30 min), our data in Table 3 show
C = 44 min and D = 24 min, i.e., about the same values.
However, a recent investigation based on a new method
to evaluate flow cytometric data (precise determina-
tions of C and D periods), Michelsen et al. (44)
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reported C = 39 min and D = 13 min for a 30-min
generation time. On average, the C-period measured in
that work was concluded to be constant, equal to about
40 min, at generation times below 60 min; above 60 min
it increased linearly with increasing generation time. The
D-periods showed a similar behavior, with an average
value approximately half the average value of C. Those
findings seem to support the conclusions of Helmstetter
and Cooper of constant C- and D-periods mentioned
above (42). However, due to the considerable scatter in
those data (two- to fivefold variations in the values for a
given generation time) our gradually changing values of
C and D in Table 3 obtained with different methods are
generally consistent with the flow cytometric measure-
ments.
At a given doubling time, the average C- and D-periods
determine the average initiation age, ai, and the average
termination age, at (Fig. 1; see Table 5 below). De-
pending on the values of the three parameters, C,D, and
, rounds of replication may be initiated at the begin-
ning, in the middle, or near the end of the cell cycle (Fig.
1). If initiation occurs on average at the beginning of the
cell cycle, it means that initiation actually occurs shortly
before division in some cells of the population and
shortly after in others.
In thymine-requiring bacteria, where the DNA replica-
tion velocity can be altered by changing the thymine
concentration in the growth medium, the C-period, and
thus the initiation age, can be experimentally changed.
This has no effect on cell growth rate or on the control of
replication initiation. When the C-period is extended,
the time of cell division, which occurs C plus D min after
initiation, is delayed. As a consequence, cells are larger
than normal, but their ribosome concentration (Nr/P) is
unaltered (62).
The control of replication initiation depends on the
amount of protein per oriC, PO. Changes in PO (e.g., by
mutation) do not affect the initiation age (192). This ap-
parent paradox reflects the fact that a change in the ini-
tiation time (without a change in C and D) causes an equal
change in the time of division so that the initiation age
remains unaltered, but the greater PO, the larger the cells.
Chromosome segregation and cell division
The D-period is the time between termination of a
round of replication and the following cell division.
33
Bremer and Dennis

Table 5 Equations relating the cell composition in exponential cultures to basic cell cycle parameters
Parameter Symbol Equation Equation no. Reference(s)
Protein/cell PC PC = PO OC = PO 2(C+D)/ 1 4
(C+D)/
RNA/cell RC RC = K(PO/cp)(1/) 2 where K = (nucl/rib) ln2/[s (lt) 2 7
r 60]
DNA/cell GC GC = [/(C ln2)] [2(C+D)/2D/] 3 3
Mass/cell MC MC = k1. PC+k2.RC+k3.GC, 4 7
where:
k1 = 1.35 1018 OD460 units per amino acid residue
k2 = 4.061018 OD460 units per RNA nucleotide residue
k3 = 3.01 1011 OD460 units per genome equivalent of DNA

Peptide chain elongation cp cp = K'/[(R/P) ] 5 5, 48

r-protein/total protein r r = (R/P) [(aa/ribosome) s (1 t)/(nucl./rib)] 6 5, 48
Origins/cell OC OC = 2(C+D)/ 7 27, 49
D/
Termini/cell TC TC = 2 8 27, 49
No. of gene X/cell XC XC = 2[C(1m)+D]/ where: 9 27, 49
m = map location of gene X relative to location of oriC
= (m+ 16)/50 for map locations (m) between 0 and 36 min
= (84 m)/50 for map locations between 36 and 84 min
= (m 84)/50 for map locations between 84 and 100 min
Replication forks/cell FC FC = 2 [2(C+D/2D/] 10 27, 49
Origins/genome OG OG = (C/) ln2/(l 2 C/
) 11 27, 49
No. of gene X/genome XG XG = OG 2mC/ 12 27, 49
Initiation age i i = 1 + n-( C + D)/, where n is the next lower integer value of 13 3
[(C+ D)/]; i.e., n = int[(C+D)/]

Termination age t t = 1D/ 14 3

Origins/cell at initiation Oi Oi = 2n; for a definition of n see Equation 13 15 3
Cell mass after division Md Md = Mc/(2 ln2) 16 50
i
Cell mass at initiation Mi Mi = Md 2 17 50
a
See Tables 1 and 2 for definitions.

Recent advances in cell imaging coupled with genetic
and biochemical measurement have begun to reveal a
complex mechanism whereby cells sense when chro-
mosome replication and segregation are completed and
where to form the plane for the upcoming cell division
(reference 193 and references therein). At the core of
this mechanism is the formation of a ring structure
composed of the tubulin homolog FtsZ, near the mid-
point of the cell at about the time that replication is
complete. The positioning of the Z ring is determined by
two separate systems. The Min system is composed of
three proteins that cycle between the poles of the cell
and prevent the assembly of the FtsZ ring. As a conse-
quence of Min cycling, the Z ring is directed to the
midpoint of the cell where the concentration of the Min
proteins is lowest. The second system, nucleoid occlu-
sion, is mediated by the SlmA protein in E. coli. This
protein binds to sites on the chromosome and prevents
FtsZ from forming a ring that would lead to guillo-
tining of the partially replicated chromosome. When
chromosome replication is completed and the nucleoids
begin to separate the Z ring can then form at the cell
midpoint region that has been vacated. Once the Z ring
is formed, the linear recruitment of a host of additional
membrane-associated proteins that are essential for cell
division begins. The earliest proteins added to the
complex contribute to stabilizing the ring, whereas the
later additions function in septum formation. The
temporal controls of these complex processes are not
well understood.

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 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates
Variability of the D-period
The cell division is believed to require the action of a
protein synthesized at the time of termination of repli-
cation (191). During the D-interval the completed
chromosome structures (Fig. 1) are segregated and a cell
constriction is formed near the center of the elongated
bacterium, which gradually deepens until it separates the
two daughter cells. The time until the onset of con-
striction after of a round of replication is completed
varies from cell to cell, but minimally it takes about
8 min, whereas the time between the onset of constric-
tion and final cell separation (T-period) has a constant
duration of about 9 min (43, 46, 63, 194).
To visualize the variability of D in different cells of an
exponential culture, one may consider a subpopulation
of cells which have just terminated a round of replica-
tion. Although no simple method exists to obtain cells
synchronized with respect to their last termination of
replication, the kinetics of cell divisions in such a hy-
pothetical culture can be derived indirectly from (i)
studies of cultures synchronized with respect to their last
cell division; (ii) cultures in which replication has been
retarded or stopped by partial or full thymine starvation,
respectively; and (iii) the cell size distribution (including
cells showing a constriction) of an exponential culture,
using electron microscopic data (43, 46, 63, 194). If the
relative cell number in such a (hypothetical) cell popu-
lation is set equal to 1.0 at the beginning of the D-period
(t = 0), then this number doubles to 2.0 when all cells
have ended the D-period. For the first 17 min no divi-
sions are expected to occur in this culture (i.e., the first
constrictions form at t = 8 min, followed 9 min later by
the first divisions; see above), so that the relative cell
number remains constant at 1.0 until t = 17 (D0 =
minimum D-period). At this time the rate of division
abruptly increases from zero to a value given by 1/avg,
where avg is the average length of time after D0 until
division occurs. The rate of cell division then decreases
exponentially, (1/e)-fold for every avg period passed after
t = 17. The average length of the D-period, Davg, equals
the sum, D0 + avg. For a culture growing in glucose
minimal medium at 1.3 doublings/h, the average D-pe-
riod was found to be 29 min (reference 46; i.e., some-
what longer than the value of 26 min obtained by
interpolation from the values of D in Table 3), so that
avg = 12 min (= 29 17) for that particular culture.
At t = Davg, 37% (= 1/e = 0.37) of the cells have not yet
divided, and 63% have divided, so that the relative cell
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number in the hypothetical culture synchronized at
termination of replication (set at t = 0) has increased to
1.63 at t = 29 min, i.e., after an average D-period has
elapsed. During the next three avg periods the fraction of
undivided cells decreases to 14, 5, and 1%, respectively
(= 1/en; n = number of avg periods elapsed after t =17).
In other words, with an average D-period of 29 min, it
takes 65 min (17 + 4avg) until 99% of all cells in the
initial population have ended their D-period and di-
vided.
These observations suggest that the D-period has three
phases: (i) a period of about 8 min preparing for the
formation of the constriction; (ii) a period of variable
length during which constriction begins with a constant
probability per unit of time (= 1/avg); and (iii) a T-
period of about 9 min, during which the constriction is
completed and the two daughter cells are separated. The
first period would correspond to the assembly and sta-
bilization of the FtsZ ring on the inner surface of the
cytoplasmic membrane, the second to the addition and
activation of several transmembrane proteins that have
domains in the periplasmic space and function to build
the septum, and the third to the building and completion
of the septum and the separation of the daughter cells
(reference 193 and references therein).
It might be argued that this stochastic behavior in the
cell division process is based on a model that might not
be correct. However, the variability of D was established
from observed data which showed a clear exponential
decrease in the number of remaining undivided cells
after a replication stop (see Fig. 5c in reference 46). It is
not known what might cause this stochastic variation in
the time until the onset of constriction. The variation is
independent of the growth of the cells during the D-
period; i.e., when the rate of mass accumulation was
varied after a replication stop, either increased by adding
amino acids to the minimal medium, or reduced by the
addition of sodium azide; the resulting changes in cell
growth had no significant effect on the pattern of cell
divisions during the D-period (194).
Variable cell cycle
The exponential fluctuation in the length of the D-pe-
riod affects a number of cell cycle-related parameters,
including (i) the size of the newborn daughter cells at the
time of division, (ii) the age at which the newborn
daughter cells initiate the next round of replication (i.e.,
35
Bremer and Dennis
the age at which the critical initiation mass is reached),
and (iii) the duration of the subsequent cell cycle (29).
The time between two divisions, i.e., the duration of a
particular cell cycle, is affected by the variations of D
both at the end of the preceding cycle and also at the end
of the current cycle. This produces a symmetrical (non-
Gaussian) frequency distribution of cell cycle periods
(frequency as a function of cell cycle duration) with a
sharp peak at the average doubling time and two ex-
ponentially decreasing trails extending to either side of
the peak, determined by avg as defined above (see Fig.
12e in reference 43). The integral of this distribution can
be directly observed as the gradually increasing cell
number in a synchronous culture (e.g., Fig. 2 of refer-
ence 43). In such synchronous cultures where all cells
have divided at t = 0, the next division occurs over a
wide range of times, some within minutes after t = 0,
others long after the average doubling time. One might
ask: how can the time between two divisions last only a
few minutes when the doubling time of the culture is 45
min and the average D-period is 29 min, as in the ex-
ample above?
The answer to this paradox is seen as follows: with an
average D-period of 29 min, the actual D-period for a
few cells (i.e., 1%) may last 65 min or longer (see above).
Since rounds of chromosome replication in cells with a
45-minute mass doubling time are initiated and termi-
nated in regular intervals of 45 min, it means that during
a 65-minute D-period (in that 1% of the cells), a next
round of replication must have terminated 20 min be-
fore the previous D-period ends at t = 65; this results in
an overlap between the previous D-period and the newly
initiated D-periods. If this nested D-period for the
next cycle happens to be short (e.g., minimally 17 min),
then the daughter cells from a division occurring after a
long D-period might divide again immediately thereaf-
ter, if not even a few minutes earlier. In such (rare) cases
the large cells before division would contain three con-
strictions: the center one leading to the first division at t
= 0 and the other two producing the next divisions that
follow shortly thereafter.
When the D-period is extended in a particular cell (i.e.,
division is delayed), the mass of the daughter cells is
proportionately increased, the age (in minutes after di-
vision) at which the daughter cells initiate the next
round of DNA replication is proportionately reduced
and the cell cycle in the daughters is likely to be
36
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shortened (depending on the same stochastic variation
in the length of the daughter cell D-period). Similarly,
when the D-period is shortened in a particular cell (i.e.,
division occurs earlier than the average), the mass of the
daughter cells is proportionately decreased, the age (in
minutes after division) at which the daughter cells ini-
tiate the next round of DNA replication is proportion-
ately extended, and the cell cycle in the daughters is
likely to be extended (depending on the same stochastic
variation in the length of the daughter cell D-period).
This implies that any particularly long cell cycles are
followed by shorter-than-average cell cycles and vice
versa. This also produces the perhaps unexpected feature
of synchronous cultures that the large extent of asyn-
chrony observed for the first division after the syn-
chronous division at zero time (see above) does not lead
to a further increase in the extent of asynchrony during
the following cell cycles.
Macromolecular Composition during Growth at
Different Temperatures
Between 20C and 37C the growth rate of E. coli B/r in
glucose minimal medium linearly increases about
threefold from 0.4 to 1.3 doublings/h; above 38C the
growth rate begins to decrease again (34). In this tem-
perature range, protein and RNA per mass (PM, RM), the
fraction of total protein that is RNA polymerase (p),
and the fractions of total RNA synthesis corresponding
to stable RNA and mRNA synthesis (rs/rt, rm/rt) remain
constant (34). At least in the medium range between 25
and 37C, the peptide and RNA chain elongation rates
increase in proportion to the growth rate (34). The C
period also changes with temperature in proportion to
the doubling time (i.e., the ratio C/ is constant; refer-
ences 192 and 195), which implies identical replication
fork patterns and gene dosages at different temperatures.
Thus, in this medium range, the chain elongation rates
for DNA, RNA, and polypeptides have about equal
temperature coefficients. In the absence of further reg-
ulation, this results in a temperature independence of the
macromolecular cell composition. During the first 30
min after a temperature upshift, extensive temporary
changes in the macromolecular synthesis rates have been
observed (e.g., reference 34) and a new RNA polymerase
sigma subunit is induced (196). These temporary per-
turbations constitute the heat- or cold-shock response
and reflect active regulation and adjustment to the
postshift temperature. At higher temperatures, between
38 and 42C, more complex changes occur, including
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 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates

protein degradation, which causes a reduction in the
growth rate (197).
MATHEMATICAL DESCRIPTION OF CELL
COMPOSITION AND GROWTH
Cell Composition as a Function of the Culture
Doubling Time
A number of equations have been reported that describe
the macromolecular composition of an average cell in an
exponential culture as a function of the culture doubling
time and five additional parameters: the C- and D-peri-
ods, protein per origin (PO), ribosome activity (r), and
peptide chain elongation rate (cp) (see History, above)
(22, 28, 198, 199). These equations, reproduced in Table
5, follow from the definitions of their constituent para-
meters without special, simplifying assumptions or hy-
potheses. The equations are useful for work dealing with
the cell composition and were used for the calculation of
many of the parameters in Table 3. Additional equations
in Table 5 can be used to calculate the copy number of
genes per cell or per genome equivalent of DNA as a
function of their map location. These latter equations are
needed to find the concentrations of particular genes in a
bacterial culture, which is necessary for the determina-
tion of their absolute transcriptional activities. A
knowledge of absolute gene activities, in turn, allows one
to define the transcriptional control of the gene in bio-
chemical (Michaelis-Menten) terms and thereby distin-
guish the specific promoter control from changes in gene
activity due to changes in the free RNA polymerase
concentration (8). Moreover, this distinction helps to
identify effectors involved in the promoter-specific con-
trol occurring under different growth conditions.
Age Distribution and the Concept of the
Average Cell
The age of a given cell, a, is defined as the time elapsed
since the last division relative to the mass or culture
doubling time . Thus, a newly born cell has the age zero
and when that cell is about to divide again after
minutes it has the age 1.0. If all cells would divide reg-
ularly in exactly the same intervals, equal to one mass
doubling time, then the oldest cells in the population
would have the age 1.0 and the age distribution would
end abruptly at age 1.0. The age distribution from such a
hypothetical exponential culture (without a spread in
generation times) is called ideal age distribution and is
given by the formula n(a) = ln2 21a for ages between 0
and 1.0 and n(a) = 0 for a >1, where n(a) is the differ-
ential frequency (dN/da) of cells in the population
having the age a. The formula shows that, in an expo-
nential, nonsynchronous population, zero-age cells are
twice as frequent as cells of the age 1.0, because every
dividing cell gives rise to two newly born cells of age
zero. However, due to the spread of generation times
reflecting the variability of the D-period (see above), the
ideal age distribution does not occur in reality. Rather,
some cells divide before reaching the age 1.0 and others
divide later at an age greater than 1.0 (29, 46). Conse-
quently, the actual age distribution deviates from the
ideal age distribution by not abruptly ending at age 1.0,
but rather by gradually decreasing to zero at ages above
1.0 (for details, see Fig. 5 in reference 29).
The averagenumber of a component per cell has to be
distinguished from the number of that component per
average cell. The first is obtained as the number of the
component per unit volume of culture, divided by the
number of cells in that volume, whereas the latter refers
to a particular average cell. This is a cell at an average
age, defined by the fact that 50% of all cells in the
population are younger and 50% are older. Because
young cells are more frequent than old cells in an ex-
ponential population, this average cell has an age of 0.41,
rather than 0.50. The number of any component in the
average cell is always an integer; for example, the
number of chromosome replication origins in the aver-
age cell is equal to 2, 4, or 8, depending on the growth
rate (see Fig. 1). In contrast, the average number of
origins per cell (given by equation 7 in Table 5) may
have any noninteger value, such as 2.7 for a growth rate
of 1.0 doubling/h (Table 2), implying that some cells in
the population have four origins, while others have only
two. The average numbers of components per cell are
calculated from the formulas in Table 5.
To calculate a subpopulation average for a certain age
range, the equation for the ideal age distribution (200)
has been used in the past, with integration over different
age intervals. For example, the Cooper-Helmstetter
equation and Donachie's equation for the average
amounts of DNA and protein, respectively, per cell
(equations 3 and 1 of Table 5) were originally derived
under the assumption of an ideal age distribution.
However, a reexamination of these equations has indi-
cated that they are independent of any assumptions,
including the assumption of an ideal age distribution
and of synchronous initiation at all origins in the cell at a

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Bremer and Dennis
given initiation age (27, 50). The formulas in Table 5
give correct values irrespective of the age distribution. In
fact, the cell cycle variability has no effect on the average
cell composition.
The conclusion that the cell cycle variability has no effect
on the composition and growth parameters has been
disputed by Alberghina and Mariani (201). These au-
thors have not distinguished the doubling time, , de-
fined as cell number doubling time in an exponential
culture (equal to the mass doubling time), from the
average interdivision interval, denoted by bar. The
latter may be determined from growth curves of syn-
chronous cultures (43) and depends on the particular
subpopulation of cells for which individual division in-
tervals are measured. Although synchronous cells have
the same division age, they are generally somewhat out
of phase with respect to their replication cycles. This
means that a zero-age population may contain a large
number of subpopulations in which the cells are in step,
only in different phases, with respect to their last round
of chromosome replication. Each of these subpopula-
tions (with different phase relationships between their
replication age and division age) would give a different
synchronous growth curve and a different average in-
terdivision interval despite an equal mass doubling time
(29). For these reasons, and bar (and, similarly, C and
C bar and D and D bar) must be distinguished in theory
(7, 27); only with C,D, and are the relationships in
Table 5 strictly valid. However, the extent of variability
in the cell cycle is such that the differences between
and bar, etc., amount to only a few percent (29) and, in
practice, are negligible.
Cell Composition at a Defined Cell Age
In some instances the cell composition at certain cell
ages becomes important; in particular, at the time of cell
division (either shortly before or shortly after) or at the
time of initiation of chromosome replication. In these
cases, it is also not necessary to use the age distribution
formula. Instead, the following relationships can be used.
(i) The average amount of a component in the sub-
population of zero-age cells (immediately after division)
is 1/(2ln2) times the average amount of that component
in the population as a whole (obtained as described
above). Correspondingly, the factor to obtain the average
amount of the component in the cells immediately be-
fore division has twice that value, i.e., 1/(ln2). (ii) The
amount of protein (or cell mass) per replication origin in
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a cell at the time of initiation (initiation mass defined
by Donachie [96]) is PO/ln2 (or MO/ln2), where PO (or
MO) is the total protein (or cell mass) divided by the
total number of chromosomal replication origins in a
unit volume of exponential culture. (Table 2 shows only
PO, but MO may be calculated from the data in the table.)
For the mathematical relationships dealing with the age
distribution, see references 27, 29, and 202.
Control of the Growth Rate
In previous editions of this chapter, we treated the
growth rate as a given parameter to describe the
Modulation of chemical composition and other para-
meters of the cell by growth rate. Since we no longer
assume the growth rate to be an independent variable,
we have now altered the title to Modulation ...at dif-
ferent exponential growth rates. This might not seem to
be much different, but the word by growth rate implies
a causal relationship, whereas at different growth rates
only means a description without further implications,
not even that of a correlation between growth rate and
composition or parameter values. Therefore, instead of
asking how the growth rate determines the macromo-
lecular composition, we may ask the reverse question:
how does the composition determine the growth rate?
Or, more accurately, how do the three factors (i) genetic
background of the particular strain used, (ii) initial
conditions (the history as discussed in the introduction),
and (iii) the nutritional environment determine the cell
composition and the growth rate? In the following sec-
tion this question is discussed.
Parameters limiting the bacterial growth rate
Only a few physiological parameters in a bacterium limit
cell growth, whereas most physiological parameters and
reaction rates are not directly growth limiting. The DNA
concentration is not growth limiting. Maale and
Kjeldgaard (2, 199) discussed the idea that the amount of
protein per DNA is constant and that the amount of
RNA per DNA increases in direct proportion to the
growth rate. These relationships seemed to suggest a
limitation by DNA, such that DNA limits mRNA syn-
thesis and mRNA limits protein synthesis. This would
result in a constant ratio of protein to DNA. Maale
argued that the apparent proportionality of the ratio of
RNA to DNA reflected the control of growth (199).
Similarly, Koch (203) and Daneo-Moore and Schock-
man (204) used rate constants of RNA synthesis per
DNA in models for the control of RNA synthesis or
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 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates
growth. However, a DNA replication (initiation) mutant
that has a reduced DNA concentration and therefore an
increased ratio of RNA to DNA at all growth rates
(because the denominator, DNA, is reduced) has an
unaltered growth rate (62, 64, 192). The reduced DNA
concentration is the result of an increased initiation
mass (protein per oriC, PO). This mutant shows that
RNA synthesis and growth are not normally limited by
the concentration of DNA; in the mutant, the ratio of
RNA to DNA is no longer exactly proportional to the
growth rate. Moreover, the amount of protein per DNA
is generally not exactly constant, even in wild-type cells
(see Table 2 and Fig. 2b).
In any living cell where protein turnover is negligible the
ribosome concentration (measured as number of ribo-
somes per amount of protein, Nr/P) and the protein
synthesis rate per average ribosome [er = dP/dt)/Nr] are
growth limiting. The product of these two factors is
equal to the rate of protein synthesis per amount of
protein [(dP/dt)/P] because the number of ribosomes,
Nr, cancels in the product. The rate per amount of any
stable component in the cell defines the exponential
growth function; i.e., the value of (equation 18, Table
6). Equation 18 is identical to the one used by Schleif (5)
to evaluate the RNA-to-protein ratio as a function of
growth rate (see also equation 5, Table 5). In his case,
however, the growth rate, , was the independent vari-
able and R/P was the dependent variable. By making
the dependent parameter, we have here exchanged the
roles of the two variables.
A given ribosome concentration results from the regu-
lation of ribosome synthesis, which in turn involves the
regulation of RNA polymerase synthesis and function,
Table 6 Basic parameters determining the bacterial growth
rate
Parameter Symbol Equation b
Equation no.
Growth rate = (60/ln2) (Nr/P) 18
(doublings/h) er, where the ribo-
some efficiency, er =
r cp
Growth rate = (60/K) [s p 19
(doublings/h) p r cs cp]0.5,
where K = ln2
[(nucl/prib)(aa/pol)/
(lft)]0.5
a
cs and cp in these equations should be expressed as rates per minute to
obtain the growth rate in doublings/hour. For definitions, see Table 1.
(Equation 19 is from reference 9.)
b
For a definition or explanation of symbols, see Tables 1 and 3.
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and the regulation of rRNA genes. These additional
concepts have been taken into account in the more
complex growth equation 19 (Table 6), which contains
six factors: RNA polymerase concentration (p), RNA
polymerase activity (p), partitioning of active RNA
polymerase into stable RNA and mRNA-synthesizing
enzyme (s), ribosome activity (r), and the chain
elongation rates for stable RNA and polypeptides (cs,
cp). As was discussed in the beginning (see Growth rate
dependency of the macromolecular composition of E.
coli), the particular values of these growth-limiting
parameters, and thus the growth rate of a given culture,
depend not only on the composition of the growth
medium, but also on the history of the stationary starter
culture that is used to inoculate an experimental culture.
For any growth equation to be meaningful, the para-
meters must be constant in time. For example, in equa-
tion 18 (Table 6), both the ribosome concentration and
activity must be constant during the exponential growth
phase of a culture. If they changed in time growth would
not be exponential. In the preceding discussion (see
Observed cell composition of E. coli B/r), the constancy
of physiological parameters during exponential growth
was implicit in the definition of exponential growth, but
if one asks for the conditions that lead to exponential
growth, then one has to explain why these parameters
have certain constant values under given conditions.
Equations such as those in Table 6 only identify growth-
limiting parameters and predict their effect on the growth
rate, but they do not explain the mechanisms involved in
their control. Further insights into these questions are
found by observing the changes of macromolecular
synthesis rates when bacteria are shifted to a different
nutritional environment.
Establishment of exponential growth after
medium shifts
The parameter values that determine the growth of a
bacterial culture under balanced conditions (meaning
that all exogenous nutrients are at saturating con-
centrations) are reset within minutes after bacteria
enter a new, more nutritious environment, hours before
a new steady-state of exponential growth and macro-
molecular composition is reached. It had already been
observed 40 years ago that a key element in the adap-
tation to a new growth medium is the control of the
stable rRNA and tRNA synthesis rate (2, 105). For ex-
ample, in a shift from a minimal to an amino acid-
39
Bremer and Dennis
supplemented medium, stable RNA (Rs) in the culture
begins abruptly, within a minute after the time of the
shift, to accumulate at the new, higher exponential rate
that is to become the final postshift steady-state growth
rate () of the bacteria in the new medium (2, 28, 51,
198). Mathematically, this can be expressed as a stepwise
increase in the quotient [(dRs/dt)/Rs], which defines the
exponential growth function. Since rRNA is synthesized
as a constant fraction of stable RNA (see Cell growth-
related parameters, above) and r-protein matches rRNA
(74, 93), the faster exponential accumulation of stable
RNA after the shift-up produces an equally faster ex-
ponential accumulation of ribosomes and an exponen-
tially increasing rate of protein synthesis (28, 51, 198).
In addition to the stepwise increase in the rate of ribo-
some synthesis, the rate of ribosome function (er) and
the synthesis of RNA polymerase as a proportion of the
total protein synthesis rate (p; see Table 3) also increase
stepwise to their final values at the time of the medium
shift (51). The RNA polymerase subunit genes are co-
transcribed with ribosomal protein genes, and at lower
growth rates in minimal media, the number of RNA
polymerase molecules per ribosome remains about
constant (0.22 to 0.24; Table 3). However, at increasing
growth rates in amino acid-supplemented media, the
number of RNA polymerase molecules per ribosome
becomes increasingly reduced (see Table 3).
As explained above, the bacterial growth rate equals the
product of ribosome concentration and function, Nr/P er
(equation 18, Table 6). The value of er increases rapidly
to its final value, but Nr/P approaches its final value only
gradually over several hours of postshift growth, even
though its final value is already determined at the time of
the shift by the changed ratio of the stable RNA and
protein synthesis rates (28). Since the rate per amount of
stable RNA synthesis [(dRs/dt)/Rs] determines the final
growth rate, any accompanying increase in the rate of
ribosome function, er, reduces the final ribosome (and
tRNA) concentration, Nr/P, so that the product Nr/P er
remains the same, i.e., equal to (dRs/dt)/Rs.
How bacterial growth and the macromolecular compo-
sition respond to the opposite situation, when the nu-
tritional environment suddenly deteriorates, is less well
known. A shift-down from an amino acid-supplemented
to a minimal medium produces an instant growth lag
that may last several hours (205). This lag is caused by a
disappearance of isoleucine and valine from the cellular
40
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amino acid pools and can be prevented when these two
amino acids are kept in the growth medium (153). As far
as we are aware, neither the cause for this amino acid
deficiency, nor the control of macromolecular synthesis
rates after a shift-down in the presence of these two
amino acids has been investigated. In the latter case,
stable RNA synthesis might decrease immediately to its
lower exponential rate, without any lag.
Control of growth and macromolecular
composition after medium shifts
The relationships between macromolecular composition
of bacteria and growth rate and the effects of medium
shifts were described in mathematical terms by Maale
and coworkers nearly 50 years ago (1, 205), and those
earlier analyses were later extended (e.g., reference 7), as
described in History, above. In the following, new
results and data on the control of rRNA synthesis are
applied to those earlier analyses.
Based on studies of the control of rrn promoter activities
(reviewed in reference 107), the initial stimulation of
stable RNA synthesis after a nutritional shift-up can now
be explained as follows. The exogenous nutrients added
to the growth medium cause a shift of RNA polymerase
molecules in the bacteria from mRNA to stable RNA
genes in two ways: (i) they repress mRNA genes for the
biosynthesis of these nutrients, which increases the
concentration of free RNA polymerase. (ii) They in-
crease the bacterial amino acid pools and tRNA charg-
ing, which increases the rate of ribosome function (er).
This causes a reduced accumulation of the effector
ppGpp (11), which increases the strength of stable RNA
promoters (11). The increases in free RNA polymerase
and in the strength of stable RNA promoters produce
the initial stepwise increase in the stable RNA synthesis
rate, i.e., in the value of (dRs/dt)/Rs.
The subsequent exponential accumulation of stable RNA
at the new rate [i.e., the maintenance of (dRs/dt)/Rs at a
constant level] involves the control of RNA polymerase
synthesis (9). The synthesis rate of ribosomes depends
on a mutual relationship between ribosomes and RNA
polymerase: the rate of ribosome synthesis is propor-
tional (factor a) to the number of RNA polymerase
molecules, and the rate of RNA polymerase synthesis is
proportional (factor b) to the number of ribosomes. The
two factors reflect the controls of rRNA synthesis and of
RNA polymerase synthesis, respectively (each factor is a
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 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates
product of several component parameters). If a and b
remain constant in time, then both ribosomes and RNA
polymerase increase exponentially at a rate equal to the
square root of the product ab [ = (60/ln2)ab; equation
19 of Table 6, which contains the component parameters
of a and b: s, p, r, p, cs, cp, defined in Table 3].
If a and b increase stepwise by the same factor f, the
theory predicts that stable RNA and ribosomes should
immediately begin to increase exponentially at an f-fold
increased rate and produce an f-fold increased growth
rate, as observed after a nutritional shift-up. From the
data in Table 3, we have calculated a and b for different
growth rates, using the formulas given in reference 150.
We then considered a preshift culture growing at =
0.6 doubling/h (the lowest growth rate in Table 3,
approximated by growth in succinate minimal medi-
um), to find out what would happen if this culture were
shifted to a number of hypothetical media with higher
nutritional contents, producing growth rates of 1.0, 1.5,
2.0, 2.5, and 3.0 doublings/h (i.e., the higher growth
rates shown in Table 3). For each of these five (hypo-
thetical) shifts, the factor changes in a and b (fa and fb)
were calculated from the data in Table 3. For shifts to
media producing 1.0 and 1.5 doublings/h, fa and fb were,
indeed, the same and equal to f, the factor increase in
the growth rate, i.e., f = (1.0/0.6) = 1.7 and f = (1.5/
0.6) = 2.8, respectively. This means that the ratio of
RNA polymerase molecules to ribosomes remains un-
changed under these conditions (equal to about 0.23;
see Table 3), and that the initial controls in the syn-
thesis of stable RNA and RNA polymerase suffice to
explain the observed constancy of (dRs/dt)/Rs during
postshift growth.
For shifts to increasingly more nutritious (i.e., amino
acid-supplemented) growth media that produce growth
rates between 2.0 and 3.0 doublings/h, fa is increasingly
greater than f and fb is correspondingly smaller than f,
implying that the ratio of RNA polymerase molecules to
ribosomes decreases during postshift growth. In these
cases a cannot immediately increase to its final value,
because that would cause an overshoot in the expo-
nential accumulation of stable RNA, but later fa must
become greater than f to compensate for the lower
value of fb, reflecting a lesser accumulation of RNA
polymerase.
A major component factor defining a is the distribution
of transcribing RNA polymerase between stable RNA
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and mRNA genes, measured by the parameter s
(fraction of active polymerase engaged in stable RNA
synthesis; see Table 3). In shifts from succinate to glu-
cose-amino acids medium, s approaches its final value
gradually after the shift (51), i.e., with increasing post-
shift time, relatively more RNA polymerase molecules
transcribe stable RNA genes and relatively less tran-
scribe mRNA genes. This gradual redistribution of RNA
polymerase between mRNA and stable RNA genes
keeps (dRs/dt)/Rs constant during postshift growth when
fa > fb.
The gradual rise in s during postshift growth is as-
sumed to be caused, at least in part, by a saturation of
mRNA promoters, which occurs when the concentration
of total RNA polymerase increases during growth in rich
media (92). The saturation of mRNA promoters raises
the concentration of free RNA polymerase, which sti-
mulates transcription from unsaturated stable RNA
promoters (8). Changes in the abundance of ppGpp or
in the ppGpp-dependent accumulation of Fis (a factor
stimulating the strength of stable RNA promoters) are
likely to participate in this adjustment. However, the
levels of ppGpp during growth in media supplemented
with all amino acids are so low that the strength of rrn
promoters is near maximal (108, 141). Therefore, post-
shift adjustments in the rate of stable RNA synthesis
result mainly from an increasing concentration of free
RNA polymerase.
Optimal Cell Composition for Maximal Growth
Two attempts have been made in the past to explain the
changing cell composition in different nutritional en-
vironments as an expression of an optimization principle
that allows the cell to achieve a maximum growth rate
under given conditions.
The first such proposal was made by Maale (199), who
pointed out that the protein synthesis rate per average
ribosome in bacteria should be constant and maximal
under most growth conditions and that this constancy is
economically advantageous for the cell. Since ribosomes
are more expensive than their substrates, they should
always work at their maximum rate and therefore be
saturated with substrates. Thus, higher growth rates can
only be achieved by increasing the ribosome concen-
tration, since the rate of protein synthesis per ribosome
is already maximal. The increase in r in proportion to
the increasing growth rate can then be understood as a
41
Bremer and Dennis
consequence of the constant and maximal rate of ribo-
some function.
A different proposal was made by Ehrenberg and Kur-
land (156) based on their maximal fitness theory. That
theory predicts that an optimal utilization of nutrients
would be achieved if both the substrates for ribosome
function and the ribosome concentration would increase
with increasing growth rate (substrates for the ribosome
are the different elongation factor Tu-aminoacyl tRNA-
GTP ternary complexes). This can be understood as
follows. At high ribosome concentrations, the substrate
pools, even at saturating concentrations, would represent
only a small fraction of the total mass of the protein-
synthesizing system. At low ribosome concentrations the
same pool would constitute a greater fraction of the total
system mass, and the cost required to produce that
substrate pool would no longer be negligible. The cells
would then save resources and energy by reducing the
concentration of substrates, especially when this con-
centration is above the Km for substrate binding, such
that substantial reductions in substrate concentrations
can be compensated for by only small increases in ri-
bosome number. These predictions were refined by
Lovmar and Ehrenberg (157), who combined new data
on the peptide chain elongation rate measured in vitro
under optimal conditions with measurements of in vivo
diffusion rates within the bacterial cell. From those data,
they calculated a Vmax value of cp in vivo equal to 50
amino acid residues per second (about twice the ob-
served maximum of 22 amino acid residues per second
in Table 3) and concluded that the substrate con-
centrations for ribosome function are always limiting in
vivo, so that even at the highest observed growth rates, cp
has only reached its half-maximal value.
How are these proposals supported by observations? If cp
were always kept constant and maximal as proposed by
Maale (199), then the ribosome concentration should
extrapolate to zero at zero growth rate, i.e., bacteria in
nutritionally very poor media or in stationary phase
should have a near-zero number ribosomes. Actually the
ribosome concentration, as approximated by the RNA/
protein ratio, is seen to extrapolate to a positive value at
= 0 (Fig. 2a). This indicates submaximal values of cp
and an overproduction of ribosomes during growth in
poor media.
The reduction in cp at low growth rates (Table 3) is
consistent with the maximal fitness theory. However, the
42
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value of cp reaches a maximum plateau at growth rates
above 1.5 doublings/h (Table 3), in contrast to the pre-
diction from the maximal fitness theory that cp should
continue to increase with increasing growth rate. Fur-
thermore, after a nutritional shift-up, cp increases im-
mediately (stepwise) close or equal to its final postshift
value, but the concentrations of the macromolecular
component requirements for the ternary complex
(tRNA, elongation factors, charging enzymes) are ex-
pected to increase only gradually over several genera-
tions to their final values (reference 28; see Peptide
chain elongation rate, above). For these and other
reasons we suggest that (i) the value of 22 amino acid
residues polymerized per second per active ribosome
represents the Vmax value for cp under in vivo conditions;
and (ii) the lower cp values observed at lower growth
rates result from reduced levels of tRNA charging in
bacteria grown in the absence of exogenous amino acids.
The value of 22 amino acids per second is an average
over all codons being translated; the step times for in-
dividual codons may vary from this average (147, 167,
168).
It might seem plausible that during growth in nutri-
tionally poor media the biosynthesis of amino acids
cannot keep up with the demand for protein synthesis
and would thus cause the submaximal charging of
tRNA and reduction in cp. However, it would be equally
plausible if the bacteria had evolved an alternative
strategy to keep cp maximal without the need for in-
creased amino acid biosynthesis, by appropriately
down-regulating the synthesis of ribosomes and thereby
reducing the ribosome concentration (Nr/P). This would
lead to the same product (Nr/P) r cp, and thus to the
same growth rate (equation 18 in Table 6) and rate of
amino acid consumption. In other words, the rate of
amino acid biosynthesis does not have to become a
limiting factor to keep the ribosome function maximal
if the ribosome concentration could be sufficiently
lowered.
This again raises the question: why do bacteria over-
produce ribosomes and reduce cp during growth in poor
media instead of down-regulating ribosome synthesis to
keep cp maximal, as postulated by Maale? As an alter-
native to Maale's proposal and to the maximal fitness
theory, we propose that the evolutionary selection for an
overproduction of ribosomes and their substrates at low
growth rates lies in the advantage for the bacteria to
rapidly adapt to changing growth conditions; i.e., to
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 Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates
restart protein synthesis and growth immediately when
conditions improve; for example, when they are coming
from a nutritionally poor medium, like lake water in
nature, into a nutritionally rich environment, like an
animal intestine. During evolution the advantage of
being able to rapidly resume fast growth under improved
conditions might have outweighed the greater expense
caused by the overproduction of ribosomes and sub-
strates during growth in nutritionally poor media. This
might reflect the fact that under natural conditions en-
teric bacteria are subject to frequent changes in their
nutritional environment.
This raises a new question: how do the bacteria control
the overproduction of ribosomes during slow growth?
As was explained above, the bacteria adjust the syn-
thesis of ribosomes during exponential growth by
controlling the ppGpp synthesis activity of the spoT
gene product (128); the mechanism of this control is
still not understood. During growth in minimal media,
the basal levels of ppGpp are so high, that the P1
promoters of the rrn genes are nearly turned off (141).
However, rRNA synthesis still continues from the
constitutive P2 promoters that are not subject to a
control by ppGpp. As a result, when intracellular levels
of ppGpp continue to increase, the fraction of the total
RNA synthesis that is stable RNA (rs/rt) reaches a 25%
lowest value (31), and the 25% residual rRNA and
tRNA transcripts originate at the P2 promoters of stable
RNA genes (108, 206). Even during the most severe
repression of rRNA synthesis, during the stringent re-
sponse to amino acid starvation, 25% of the residual
RNA synthesis is rRNA and tRNA transcribed from the
P2 promoters (30). Thus, the presence of an additional
constitutive P2 promoter for stable RNA genes may
have evolved to guarantee that ribosome synthesis is
never completely turned off during growth in a nutri-
tionally poor environment.
The ReC subunit
The ReC subunit is placed appropriately to interact with
the chi sequence in a region where mutations are
known to alter chi recognition. As the separated strands
pass through the protein, they emerge in the proximity
of the single nuclease domain located in RecB. The
existence of a single nuclease domain with access to
either the 3-ended or the 5-ended strand explains the
observation that one or the other strand is preferentially
cleaved.
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Cheese is milk that has grown upit is preeminently the food
of manthe older it grows the more manly it becomes, and in
the last stages of senility it almost requires a room to itself.
EDWARD BUNYAN (-), The Epicures Companion
The proposed mechanism of action. RecBCD nuclease
involves preferential interaction of the 3-ended strand
with the nuclease domain of RecB until that strand is
retained in the complex, by RecC binding chi, allowing
preferential access of the 5 end to the nuclease (Fig. 10).
Finally the nuclease domain of RecB interacts with RecA
and facilitates loading of RecA on the ssDNA generated
by degradation of the 5-ended strand following chi
recognition (59, 247, 391).
The RecD subunit. The RecD subunit must also play a
role in the nuclease activity of the complex, although it
does not contain the nuclease active site, as the RecBC
enzyme has very little nuclease activity (13, 198, 199).
1. In vitro, RecF is not needed for the promotion of
RecA action on DNA gaps, and it weakly accelerates a
reaction that is already efficiently catalyzed by RecO
and RecR. Therefore, why does the inactivation of
recF, recO, or recR confer a similar phenotype?
2. Is the action of RecF, as a suppressor of the anti-RecA
protein RecX, significant in vivo, and if so, under
which conditions?
3. Among presynaptic enzymes, the function of RecN
remains a mystery, even though structural and bio-
chemical data are now available. Is it because this
protein does not exert a defined enzymatic activity,
but rather plays a structural role of holding DNA
molecules together?
4. Then, RecN would become crucial only when re-
combination initiation is slowed down for some
reason as exemplified by the use of a secondary re-
combination pathway. Along with RecN, RecG is one
of the less understood recombination proteins. Dec-
ades of studies have failed to identify a cognate re-
solvase enzyme, indicating that it is unlikely that
RecG acts in concert with a Holliday junction en-
donuclease. Therefore, how does a protein that cat-
alyzes only branch migration resolve a Holliday
junction?
Although recombination between sister chromatids
might be facilitated by the proximity of the two ho-
mologous DNA molecules behind replication forks, it is
more difficult to visualize how following -irradiation,
43
Bremer and Dennis
RecA can repair tens of DNA DSBs, none of which may
originally be close to its homologous intact sequence.
Another mystery is the precise role of the long and still-
growing list of enzymes that control RecA actions.
bulletted list entry
another bulletted list entry with some more to
create a turned line line line line line line
third bulletted list entry
Although recombination between sister chromatids
might be facilitated by the proximity of the two ho-
mologous DNA molecules behind replication forks, it
is more difficult to visualize how following -irradia-
tion, RecA can repair tens of DNA DSBs, none of
which may originally be close to its homologous intact
sequence.
equation DNA 2c488X12 0 13. Bachmann, B. 1990. Linkage map of Escherichia coli K-12, edi-
Another mystery is the precise role of the long and still-
growing list of enzymes that control RecA actions.
Unnumbered List item 1
Unnumbered List item 2
Unnumbered List item 3
ACKNOWLEDGMENTS
This work was supported by the National Science Foundation.
The opinions, findings, and conclusions expressed in this publi-
cation and those cited are those of the authors and do not necessarily
reflect the views of the National Science Foundation.
No potential conflicts of interest relevant to this review were
reported.
ADDENDUM The development of new powerful technologies such
as single-molecule techniques in vitro and high spatial and temporal
resolution microscopy in vivo might help to answer these questions.
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