Arch Microbiol (2010) 192:499–504
DOI 10.1007/s00203-010-0571-y
SHORT COMMUNICATION
Analysis of expression and regulatory functions of the ribosome-
binding protein TypA in Mycobacterium tuberculosis under stress
conditions
Julia C. Micklinghoff • Mascha Schmidt •
Robert Geffers • Werner Tegge •
Franz-Christoph Bange
Received: 14 December 2009 / Revised: 10 March 2010 / Accepted: 25 March 2010 / Published online: 1 May 2010
Ó Springer-Verlag 2010
Abstract In many bacterial species, the translational
GTPase TypA acts as a global stress- and virulence regulator
and also mediates resistance to the antimicrobial peptide
BPI. On the chromosome of M. tuberculosis, typA is located
next to narGHJI, which plays a role in adaptation of the
pathogen to various environmental conditions. Here, we
show that Mycobacterium tuberculosis is sensitive to P2, a
derivative of BPI. Using a typA mutant of M. tuberculosis,
we found this phenotype to be independent of TypA. We
further tested typA expression in M. tuberculosis under
defined stress conditions, such as oxygen- and nutrient
depletion, low pH, heat shock, antibiotic stress and the
presence of P2, and found that typA expression remains
unaffected by any of these conditions. Analysis of growth
and whole-genome expression revealed similar growth
kinetics and gene expression profiles of the wild type and the
mutant under normal growth conditions as well as under
stress conditions. Our results suggest that in contrast to the
Communicated by Erko Stackebrandt.
Electronic supplementary material The online version of this
article (doi:10.1007/s00203-010-0571-y) contains supplementary
material, which is available to authorized users.
J. C. Micklinghoff  M. Schmidt  F.-C. Bange (&)
Department of Medical Microbiology and Hospital
Epidemiology, Hannover Medical School,
Carl-Neuberg-Straße 1, 30625 Hannover, Germany
e-mail: bange.franz@mh-hannover.de
R. Geffers
Department of Cell Biology, Helmholtz Centre for Infection
Research, 38124 Braunschweig, Germany
W. Tegge
Department of Chemical Biology, Helmholtz Centre
for Infection Research, 38124 Braunschweig, Germany
findings in other bacteria, TypA does not act as a global
stress- and virulence regulator in M. tuberculosis.
Keywords Mycobacterium tuberculosis  typA 
bipA  Rv1165  Regulation  BPI 
Antimicrobial peptide (resistance)
Introduction
The open reading frame Rv1165 from Mycobacterium
tuberculosis H37Rv encodes a protein that shares 48.7%
sequence identity with TypA (also annotated as BipA)
from Escherichia coli. TypA is widely distributed in bac-
teria and is also found in some plants (Scott et al. 2003). It
belongs to the ribosome-binding GTPase superfamily and
has been described as a translational GTPase (Margus et al.
2007). TypA acts as a global stress- and virulence regulator
by selectively influencing the translation of certain proteins
directly at the ribosome. In E. coli, TypA has been shown
to interact with the binding site of the elongation factor G,
and it has been suggested that TypA affects expression of
the global regulator Fis by destabilizing unusually strong
interactions between the 50 untranslated region of fis
mRNA and the ribosome (Owens et al. 2004). In Salmo-
nella typhimurium and Escherichia coli, TypA has been
shown to mediate resistance to P2, a derivative of the
bactericidal/permeability-increasing protein BPI (Barker
et al. 2000; Farris et al. 1998). Other studies have deter-
mined a role of TypA in the regulation of diverse viru-
lence-related mechanisms in pathogenic E. coli, such as
capsule synthesis (Rowe et al. 2000; Grant et al. 2003;
Grant et al. 2003), the expression of genes from different
pathogenicity islands (Grant et al. 2003), flagella-mediated
cell motility and cytoskeletal rearrangements in host
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epithelial cells (Farris et al. 1998). Furthermore, in differ-
ent bacteria, TypA has been found to be involved in the
adaptation to adverse growth conditions, such as low
temperature (Barker et al. 2000; Grant et al. 2001; Pfennig
and Flower 2001; Reva et al. 2006), low pH or sodium
dodecyl sulfate (Kiss et al. 2004).
In M. tuberculosis, typA is located directly downstream
of narGHJI, which encodes a membrane-bound nitrate
reductase. We showed previously that this enzyme plays a
major role in the adaptation of M. tuberculosis to a
changing environment, as it mediates both persistence of
the pathogen under anaerobic conditions and growth on
nitrate as a sole source of nitrogen (Aly et al. 2006; Malm
et al. 2009). As narGHJI and typA are located next to each
other on the chromosome of M. tuberculosis, and given the
function of TypA as a global regulator in other bacteria, we
speculated that TypA has role in adaptation of M. tuber-
culosis to shifting conditions.
In this study, we tested whether typA is co-transcribed
with narGHJI. Furthermore, we determined the effect
of exposure to the antibacterial peptide P2 on growth of
M. tuberculosis and compared it with the effect on an
M. tuberculosis typA deletion mutant. Finally, we looked at
the expression of typA and examined the role of TypA as a
regulatory protein by comparing whole-genome expression
profiles and growth rates of M. tuberculosis H37Rv and the
typA deletion mutant under various stress conditions.
Materials and methods
Strains and growth conditions
Mycobacterium tuberculosis strains were cultured aerobi-
cally at 37°C in Middlebrook 7H9 broth or on Middlebrook
7H10 agar plates (Difco Laboratories, Detroit, MI, USA)
supplemented with 0.5% glycerol, 10% ADS (0.5% bovine
serum albumin fraction V, 0.2% glucose, 140 mM NaCl)
and 0.05% Tween-80 (broth only), unless otherwise stated.
For incubation under stress conditions, cultures were
grown to log phase under normal growth conditions and
then shifted to the respective stress conditions. Anaerobic
incubation: bacteria were cultured in an anaerobic jar
without shaking for 15 h. Incubation at pH5: bacteria were
cultured in 7H9 broth adjusted to pH5 with HCl for 20 min.
Nutrient starvation: bacteria were cultured in PBS for 24 h.
Heat shock: bacteria were incubated at 45°C for 30 min.
Incubation with kanamycin: bacteria were cultured in 7H9
broth supplemented with 50 lg/ml kanamycin. Incubation
with P2: bacteria were incubated in 7H9 broth supple-
mented with casamino acids and 4 lg/ml P2 for 24 h. For
growth experiments, cultures were grown to log phase
under normal growth conditions and then shifted to the
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Arch Microbiol (2010) 192:499–504
respective stress conditions, as described above. Different
dilutions of the cultures were streaked onto 7H10 plates
after 24- and 48-h incubation under stress, and the number
of colony forming units per ml was determined after an
incubation period of 4 weeks.
Generation of deletion mutants in M. tuberculosis
Unmarked deletion mutants were generated by two-step
homologous recombination, as described by Pavelka and
Jacobs (Pavelka and Jacobs 1999). The mutant strain
JM21 contains a 551-bp deletion within typA, and the
mutant strain MS4 contains a 984-bp deletion within the
promoter region of the narGHJI operon. E. coli HB101
was used as a host for molecular cloning. Mutants were
confirmed by PCR and Southern blot analysis (data not
shown).
Determination of the inhibitory effect
of P2 on cell growth
The BACTECTM MGITTM 960 Mycobacterial Detection
System (BD, Sparks, MD, USA) was used to determine the
growth of M. tuberculosis in the presence of P2. The
protocol supplied by BD for antimycobacterial suscepti-
bility testing was used, with the exception that instead of
antibiotics, different concentrations of P2 were added. The
results presented in Fig. 2 represent a single experiment
that is representative of three independent experiments.
The peptide P2 was obtained from Dr. Werner Tegge
(Department of Chemical Biology, Helmholtz Centre for
Infection Research, Braunschweig, Germany).
RNA extraction
Broth cultures were incubated with an equal volume of
5 M GTC buffer (5 M Guanidinium thiocyanate, 0.5%
n-laurylsarcosine, 0.7% sodium citrate, 0.7% b-mercap-
toethanol) at room temperature for 15 min, followed by
centrifugation. The pellet was resuspended in 1 ml trizol
(Invitrogen, Karlsruhe, Germany), and cells were lysed in
a Lysing Matrix B tube (MP Biomedicals, Illkirch,
France) using a Hybaid RiboLyser (Hybaid, Teddington,
UK) at setting 6.0 for 40 s. The tubes were centrifuged,
and the supernatant was mixed with chloroform. RNA
was then isolated from the aqueous phase by the RNeasy
Kit (Qiagen GmbH, Hilden, Germany), according to the
manufacturer’s instructions. The optional DNaseI-digest
was extended to 1 h. A second DNaseI (New England
Biolabs, Ipswich, MA, USA) digest was carried out after
eluting the RNA from the columns for 45 min. Sub-
sequent RNA cleanup was performed with the RNeasy
kit.
Arch Microbiol (2010) 192:499–504
Quantitative real-time PCR
RNA was transcribed into cDNA using the reverse trans-
criptase SuperscriptÒ II and random primers (both Invit-
rogen, Karlsruhe, Germany). First-strand synthesis was
performed according to the Invitrogen protocol. TaqManÒ
realtime-PCR was used for the quantification of typA
mRNA, using a custom gene expression assay (Applied
Biosystems, Foster City, CA, USA). Samples were quan-
tified using a standard curve obtained by serial dilution of a
cDNA standard generated by pooling all cDNAs analyzed.
Reactions were performed in an ABI-Prism 7000 Sequence
Detection System (Applied Biosystems, Foster City, CA,
USA) following the Applied Biosystems protocol. As
normalized results, the quotient of typA mRNA quantity
and sigA mRNA quantity was calculated. Student’s
unpaired t test (two-tailed) was used to assess statistical
significance. P values \0.05 were considered significant.
Microarray analysis
A custom-designed Affymetrix GeneChipÒ M. tuberculosis
Array (Affymetrix, Santa Clara, CA, USA) was used.
cDNA synthesis, fragmentation, labeling, target hybrid-
ization, staining and scanning of the probe array were
performed following the Affymetrix protocol and as
described previously (Malm et al. 2009). Analysis of
microarray data was performed using the Affymetrix
GCOS 1.2 software. Data were normalized using the
RMA (Robust Multi-Array Average) algorithm. Further
downstream analysis was performed using ArrayAssist
(Stratagene, La Jolla, CA, USA).
Results and discussion
typA expression in a narGHJI promoter mutant
The open reading frame Rv1165, annotated as typA, is
located only 22-bp downstream of narI, the last gene of the
narGHJI cluster. This cluster encodes a nitrate reductase
with a role in anaerobic nitrate respiration and in nitrate
assimilation (Weber et al. 2000; Malm et al. 2009). It is
widely accepted that M. tuberculosis has to adapt to an
anaerobic environment of granuloma within infected tissue,
and the NarGHJI-encoded nitrate reductase has been
implicated to play a role in this process (Sohaskey 2008;
Wayne and Sohaskey 2001). To determine the extent of the
narGHJI operon, and whether typA is part of this operon
quantitative real-time PCR was used to compare the
expression levels of typA and narG in M. tuberculosis
Erdmann and MS4, a M. tuberculosis Erdmann mutant
carrying a deletion in the promoter region of the narGHJI
501
operon (Fig. 1). Expression of lpqW, a gene located 98-bp
downstream of typA and not part of the narGHJI operon,
was included as a control. As would be expected, expres-
sion of narG was greatly reduced in the promoter mutant
reaching only 15.1% of the expression level in the wild
type. In contrast, expression of typA and lpqW remained
unaffected when compared to the wild type, showing that
typA is not part of the narGHJI operon.
Effect of the BPI-derived peptide P2 on cell growth
In other bacteria, TypA has been shown to mediate resis-
tance to P2, a peptide derived from the bactericidal/per-
meability-increasing protein BPI, which retains significant
antibacterial activity (Barker et al. 2000; Farris et al. 1998;
Gray and Haseman 1994; Little et al. 1994). BPI is secreted
by neutrophils and selectively exerts bactericidal effects on
gram-negative bacteria due to its ability to bind to the lipid
A region of lipopolysaccharides (LPS) in the outer mem-
brane of the gram-negative cell envelope (Gazzano-Sant-
oro et al. 1992). However, BPI levels have also been shown
to be increased in patients suffering from tuberculosis,
leading to the hypothesis that BPI might have a role in the
host immune response to M. tuberculosis due to the similar
physiochemical properties of LPS and the mycobacterial
(A)
relative expression level (%)
(B) 140 p < 0.05
120 p < 0.05
100
80
60
40
20
0
W
pA
rG
na
ty
lp
Fig. 1 Analysis of the narGHJI operon region. a The narGHJI
operon region on the chromosome of M. tuberculosis. Gene
arrangement (arrows) and region of the promoter deletion in MS4
(box); b Quantitative real-time PCR analysis of narG-, typA- and
lpqW-expression in an M. tuberculosis Erdmann narGHJI promoter
mutant. Data are expressed as relative expression levels of each gene
compared to expression in the wild type (%). Means were calculated
from three independent experiments. As internal standard, relative
expression of lpqW was set to 100%
123
502 Arch Microbiol (2010) 192:499–504

cell wall lipid glycoprotein lipoarabinomannan (LAM;
Juffermans et al. 1998). To determine whether P2 has an
effect on the growth of M. tuberculosis H37Rv and whether
this effect is altered in typA-deficient bacteria, the typA
deletion mutant JM21 was constructed. Growth of the wild
type and the mutant was measured and compared in med-
ium with and without P2, using the BACTECTM MGITTM
960 System. We found that concentrations of 4–64 lg/ml
reduced the growth of M. tuberculosis and the mutant
(Fig. 2). No difference in growth could be observed
between the wild type and the mutant. The mutant even
seemed to be less affected by P2 than the wild type.
However, this was most likely due to an inoculum effect.
These results indicated that in M. tuberculosis, TypA does
not play a role in mediating resistance to P2/BPI.
Expression of typA and role of TypA in transcriptional
regulation
As TypA levels have been found to be elevated in
S. typhimurium in the presence of P2 (Qi et al. 1995) as
well as in the halophytic plant Suaeda salsa under various
(A) 400 0 µg/ml
350 4 µg/ml
16 µg/ml
300
growth index
stress conditions (Wang et al. 2008), we wanted to identify
stress conditions that induce typA expression in M. tubercu-
losis H37Rv. The mRNA level of typA was determined under
different stress conditions that are similarly encountered by
the bacteria in the host during infection, such as a pH level
of 5.0, oxygen- and nutrient depletion and exposure to the
BPI-derivative P2, as well as under antibiotic stress
(incubation with sublethal concentrations of kanamycin),
and after heat shock. TypA expression levels during these
stress conditions were compared to expression during
normal growth conditions (aerobic, log-phase growth in
rich 7H9 broth; Fig. 3). We could determine that typA is
expressed at a significant level even in unstressed bacteria
during normal aerobic growth. However, in contrast to the
findings in other bacteria, in M. tuberculosis, typA
expression remained on the same level during exposure to
P2. Similarly, no significant changes in typA expression
were observed during all other stress conditions examined.
The finding that typA is constitutively expressed during
stress conditions as well as during normal in vitro growth
conditions may indicate that TypA does not play a role as
a regulatory protein during these stress conditions in
M. tuberculosis. However, it may also indicate that in
M. tuberculosis, activity of TypA is not limited to stress
conditions, but might also be present under normal growth
conditions. Therefore, experiments were performed to
determine the regulatory properties of M. tuberculosis

64µg/ml TypA. As TypA is believed to regulate the translation of

250

200
certain proteins directly at the ribosome rather than at the
transcriptional level, proteins that are subjected to regula-
150
tion by TypA cannot be studied by looking at gene
100 expression on mRNA level. A study by Owens and
50

0
90 100 110 120 130 140 150 250

hours 225

200
(B)
expression value

400 0 µg/ml 175

350 4 µg/ml 150

300 16 µg/ml 125

growth index

64 µg/ml 100
250
75
200
50
150
25
100 0
P2
th

0
th

in

ck

50
5.
tio

yc
ow

ow

ho
pH
va

m
gr

gr

ts
na
ar

a
ic

st

0
ka

he
bi
ob

ro
r
ae

ae

90 100 110 120 130 140 150

an

hours
Fig. 2 Growth of M. tuberculosis H37Rv in the presence of P2.
Growth curves as determined by the BACTECTM MGITTM 960
System. a M. tuberculosis H37Rv; b the typA deletion mutant JM21
Fig. 3 Expression of typA under different stress conditions. Expres-
sion values were determined by microarray analysis and processed
using RMA. Means were calculated from four independent
experiments

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co-workers (Owens et al. 2004), however, suggested that
proteins targeted by TypA could be global transcriptional
regulators. Therefore, to determine whether TypA of M.
tuberculosis is involved in the regulation of other tran-
scriptional regulators, microarray analysis was used to
compare whole genome expression profiles of the wild type
and the typA deletion mutant. Again, we looked at condi-
tions that play a role in stress and virulence and determined
expression profiles during oxygen- and nutrient depletion,
pH 5, P2 exposure, heat shock and incubation with suble-
thal concentrations of kanamycin (microarray data are
provided as supplemental material). Only genes that were
regulated at least twofold were considered significant.
Under these conditions, there was no difference in gene
expression between the wild type and the mutant, indicat-
ing that TypA does not, directly or indirectly, influence
transcription in M. tuberculosis. We conclude that under
the conditions studied, TypA does not act as a global
regulator through regulating translation of a transcription
factor. Yet it is still possible that TypA is involved in direct
control of the translation of certain effector proteins at the
ribosome, which could affect bacterial growth. Therefore,
we compared growth of the wild and the mutant under
various stress conditions.
Effect of a typA deletion on cell growth under stress
conditions
In other bacteria, deletion of typA results in reduced growth
under various stress conditions (Pfennig and Flower 2001;
Reva et al. 2006; Kiss et al. 2004). To determine whether a
typA deletion mutant in M. tuberculosis H37Rv has a
growth defect under stress, we compared the growth of
M. tuberculosis and the typA mutant under oxygen- and
nutrient depletion, pH 5, P2 exposure, heat shock and
incubation with sublethal concentrations of kanamycin. No
differences in cell growth or survival could be observed
between the wild type and the mutant under these condi-
tions, as well as under normal in vitro growth conditions
(data not shown). Thus, in summary this study shows that
together with the results obtained by quantitative real-time
PCR and microarray analysis, it appears that TypA of M.
tuberculosis performs a role different to the regulatory
function proposed for other bacteria, as no indications of a
regulatory function under the conditions examined were
found.
Acknowledgments We thank S. Suerbaum for his support. This
work was supported by the Niedersächsische Verein zur Bekämpfung
der Tuberkulose, by the German Research Foundation (DFG) through
the grant SFB 587 to JCM, MS and FCB, and by the state Lower
Saxony through a Georg-Christoph-Lichtenberg Scholarship and a
Wilhelm-Hirte-Foundation Scholarship to JCM.
503
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