Bacterial Transcriptomics What Is Beyond The RNA Horiz-Ome

The regulation of bacterial transcription has been a topic
of interest for several decades; the transcriptional regu-

latory circuit of the lac operon was described as early as
1959 (REF.1). Bacterial genes can be arranged in operons,
which are groups of contiguous genes regulated by a com-
mon operator
2
. Bacterial transcription is carried out by
a single RNA polymerase (RNAP) holoenzyme complex
that consists of a core enzymatic machine and a -factor.
This RNAP holoenzyme binds to different -factors that
recognize different promoters and so control specific sets
of genes
3
. Different bacteria have different numbers of
-factors, although most have at least a member of the
housekeeping -factor family (
70
)
4
. Transcription fac-
tors are additional regulatory factors, although they are
not necessarily part of the holoenzyme. Many transcrip-
tion factors can either promote or repress transcription,
depending on the promoter. Escherichia coli encodes 314
transcription factors, of which 35% are activators, 43%
are repressors and 22% are dual regulators
5
. Transcription
is terminated through two different mechanisms: Rho-
dependent and Rho-independent termination
6
. Finally, the
initiation of protein translation requires (in most cases) a
ShineDalgarno motif, which is a short sequence close to
the start codon that recruits the ribosome to themRNA
7
.
In addition to this basic regulatory machinery, vari-
ous other proteins and regulatory elements increase
the complexity of the events leading from DNA to pro-
tein: RNAP-associated proteins affect the processivity
of RNAP
8
; internal promoters within operons
9
, small
RNAs (sRNAs)
10
and riboswitches (RNAs that regulate
their own gene activity)
11
affect transcription and transla-
tion; additional transcription termination regulates the
termination process
6
; and non-canonical ribosome bind-
ing motifs
12
and leaderless mRNAs that are translated
13

affect protein translation. However, many of the exam-
ples of these processes have been considered as oddities
and, until recently, the general view outside the field of
microbiology was that bacterial transcription is simple
and well understood.
Over the past 10years, regulation of gene expres-
sion in bacteria has come back into the spotlight, with
many discoveries being made owing to the combination
of classic genetics and biochemical assays with high-
throughput technologies. The large amount of data col-
lected has revealed that many of the oddities may instead
be the rule. For instance, sRNAs account in some cases
for 1020% of the bacterial RNA products and may have
an important regulatory role
14
, and riboswitches affect
gene expression upon metabolite binding
15
. The new evi-
dence suggests that the definition of the operon should
be redefined, although the new information makes that a
challenge. For example, 20% of all Bacillus subtilis genes
in polycistronic operons are transcribed from more than
one promoter
16
. Similarly, almost 6% of the polycistronic
operons contain an internal read-through terminator, at
which partial continuation of the transcription occurs
17
.
*Centre for Genomic
Regulation, Universitat
Pompeu Fabra, Av. Dr.
Aiguader 88, 08003
Barcelona, Spain.
ICREA (Instituci Catalana de

Recerca i Estudis Avanats),
Passeig Llus Companys, 23,
08010 Barcelona, Spain.
Present address: Harvard

Medical School, 77 Louis
Pasteur Avenue, Boston,
Massachusetts 02115, USA.
||
These authors contributed
equally to this work.
Correspondence to L.S.
e-mail: luis.serrano@crg.eu
doi:10.1038/nrmicro2620
Bacterial transcriptomics: what is
beyond the RNA horiz-ome?
Marc Gell*
||
, Eva Yus
||
, Maria Lluch-Senar* and Luis Serrano*
Abstract | Over the past 3years, bacterial transcriptomics has undergone a massive revolution.
Increased sequencing capacity and novel tools have made it possible to explore the bacterial
transcriptome to an unprecedented depth, which has revealed that the transcriptome is more
complex and dynamic than expected. Alternative transcripts within operons challenge the
classic operon definition, and many small RNAs involved in the regulation of transcription,
translation and pathogenesis have been discovered. Furthermore, mRNAs may localize to
specific areas in the cell, and the spatial organization and dynamics of the chromosome have
been shown to be important for transcription. Epigenetic modifications of DNA also affect
transcription, and RNA processing affects translation. Therefore, transcription in bacteria
resembles that in eukaryotes in terms of complexity more closely than was previously thought.
Here we will discuss the contribution of omics approaches to these discoveries as well as the
possible impact that they are expected to have in the future.
REVI EWS
658 | SEPTEMBER 2011 | VOLUME 9 www.nature.com/reviews/micro
2011 Macmillan Publishers Limited. All rights reserved
Transcriptome
The complete set of RNA
molecules produced in a cell.
DNA microarrays
Technology used to carry out
measurements of a large
number of transcript levels
simultaneously. Microarrays
consist of a series of
microscopic spots of DNA
oligonucleotides targeting
specific sequences. These
probes hybridize to the target
species (usually cDNA).
Probetarget hybridization is
quantified to determine the
abundance of nucleic acid
sequences in the sample.
Tiling arrays
A subtype of DNA microarray
chips. Tiling arrays differ
according to the nature of the
probes. Probe sequences are
tiled and cover the entire
genome. They are used for
whole-transcriptome profiling.
All of these discoveries have benefited from the tech-
nical revolution of the so-called omics disciplines. Gene
expression studies have clearly been at the forefront of
this revolution, and modern techniques have provided
a high-resolution view of various aspects of transcriptome
organization. What is now on the horizon? How can
we integrate the newly acquired knowledge? In this
Review, we summarize the quantum leap that bacterial
transcriptomics has taken over the past several years and
suggest areas where the new technologies could have a
majorimpact.
A technical revolution
Recent years have witnessed a revolution in the field
of bacterial transcriptomics (BOX1; FIG.1a). Pioneering
RNomics studies used cDNA synthesis from RNA sam-
ples, cloning and Sanger sequencing
18
, but the transcrip-
tomics revolution started when the development of DNA
microarrays provided a tool to globally quantify gene
expression
19
. Several years later, technological break-
throughs allowed the design of microarrays containing
high-density tiled probes (termed tiling arrays) that cover
the entire genome of an organism or contiguous regions
of it, and such tiling arrays provided the first compre-
hensive transcriptome map for E.coli
20
and several other
bacterial species
2123
. However, tiling experiments have
only recently been able to deliver strict strand-specificity
and high-resolution cDNA mapping
21
through the use
of actinomycin D in the reverse transcription reaction
to inhibit DNA polymerase activity
24
(FIG.1b). The tiling
arrays also revealed that most transcripts are present in
levels that are just above background noise
25
. Thus, this
system has a far from ideal signal-to-noise ratio
26
and
cannot offer single-based resolution of transcription
start sites (TSSs) (FIG.1c).
In 2008, RNA-seq was introduced, whichinvolves
deep sequencing of cDNA generated from RNA prepa-
rations
27,28
. This technology has overcome some of the
drawbacks of tiling arrays: it provides single-base reso-
lution, a better signal-to-noise ratio owing to a reduced
background and a higher dynamic range
29
. RNA-seq
allows the detection of various transcriptional features,
including the 5 end of all RNAs
30,31
, and TSSs can
be detected by selecting for primary RNA transcripts
that have a 5-triphosphate (processed RNAs have a
5-monophosphate)
32
. The current sensitivity of ultra-
sequencing makes it possible to study unculturable
bacteria or bacteria that cannot be isolated (such as endo-
symbionts), and has allowed the transcriptomes of micro-
bial communities (metatranscriptomes) to be explored,
such as those of communities found in marine samples
(see below)
33,34
. However, metatranscriptomics technol-
ogy is not yet standardized and several issues remain to
be solved, such as the unequal coverage of genes, which
could result in apparent premature transcriptional
termination for genes with low expression (FIG.1c).
The operon, a concept under revision
Genes in bacterial genomes are organized into oper-
ons, which are defined as functional genomic units that
contain multiple genes under the control of a single
promoter. However, this definition no longer stands in
various aspects (FIG.2). It was thought that this organiza-
tion leads to an equal level of expression for all genes, but
uneven gene expression within operons was observed
several years ago
35
, and recent genome-wide transcrip-
tomics studies have revealed that in many bacterial spe-
cies, consecutive genes within operons do not have the
same expression level, leading to operon polarity
21,36

(FIG.2a). Operon polarity could be explained by the pres-
ence of internal transcription terminators, by the activity
of sRNAs or by riboswitches within operons that block or
activate transcription in response to metabolite binding
(see below)
3739
. The combinatorial effect of internal pro-
moters and terminators would result in the production
of different transcripts, thereby adding plasticity to the
operon. For instance, mapping transcriptional initiation
sites in Helicobacter pylori revealed 337 operons, but the
detection of internal TSSs added 192 additional cistrons
40

(FIG.2b), whereas in Mycoplasma pneumoniae, 341 refer-
ence operons could be divided into 447 smaller suboper-
ons
21
(FIG.2b). Furthermore, transcription initiation may
be coupled to termination in some cases. In the E.coli
galactose operon, two promoters are separated by 5 bp.
Transcription driven from the first promoter is termi-
nated earlier than that from the second, suggesting that
transcription termination could depend on transcrip-
tion initiation
41
, possibly as a result of different assem-
blies of the RNA transcriptional machinery as dictated
by the promoter. Transcription elongation factors such
as NusA, NusG or GreA can determine the processivity
Box 1 | Computational resources to process transcriptomics data
Computational approaches have dramatically changed with the evolution of
transcriptomics. In 2004, the first DNA ultrasequencing platforms enabled the
simultaneous reading of several hundred thousand DNA fragments with a read length
greater than 100 bases. Today, they produce over 200 million 75100base reads, and
the large throughput of modern sequencing platforms has resulted in them gradually
replacing the arraybased technologies. RNAseq
28
is probably one of the most complex
nextgeneration applications.
The first step after the data have been acquired is to align sequences to a reference
genome, which can be done using a growing number of software packages
154
. The
choice of alignment algorithm is strongly influenced by both the experiment in
question and the details of the sequencing technology used. These algorithms have to
cope with unprecedented amounts of data and specific errors associated with the
specific platform (for example, a tendency for insertion or deletion errors to occur in
homopolymer reads, or an increase in the likelihood of sequence errors towards the end
of the read). Alignment programs use heuristic techniques to quickly identify a small set
of locations in the reference sequence where the best mapping is most likely to be
found. Within this set of putative mapping locations, slower and more accurate
algorithms are used to map the reads. The first tools used hashbased alignment
methods: examples include SOAP
155
, MAQ
156
and ELAND (Illumina, unpublished).
A second generation of tools has been developed on the basis of the BurrowsWheeler
transform (BWT): examples include BOWTIE
157
, BWA
158
and SOAP2 (REF.159). BWT
implementations are much faster than their hashbased counterparts at the same
sensitivity level.
The second step is to represent and analyse the data. Different tools are available, but
some of the most widely used and powerful are within the Bioconductor project: the
ShortRead package provides functionality for the import, quality assurance, visualization
and basic manipulation (such as pileup calculation and read property examination) of
shortread DNA sequences. In addition, there are packages that address more specific
aspects, such as identifying differentially expressed genes (DEGseq) or providing
alternative basecalling algorithms (Rolexa).
REVI EWS
NATURE REVIEWS | MICROBIOLOGY VOLUME 9 | SEPTEMBER 2011 | 659
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Deep sequencing
New sequencing technologies
that make use of massive
parallelization of the
sequencing process. Data
provided by these new
sequencers consist of a large
number of reads (millions) in
each run but with a short read
length (of a few hundred
bases).
Cistrons
Segments of DNA that have
the information to produce a
polypeptide chain.
of the polymerase
42
. Moreover, other proteins (such as
Spx and Dsk), the 6S sRNA or even small molecules
(including (p)ppGpp or NTPs) can bind to the initial
RNAP complex independently of DNA and change the
properties of the holoenzyme, although binding depends
on environmental conditions
42
. It is tempting to imag-
ine that promoters could engage various transcription
complexes that could produce different RNA outputs.
Almost half of the polycistronic operons in M.pneumo-
niae show natural polarity and transcriptional attenu-
ation (optional use of terminator signals)
21
(FIG. 2a).
Transcriptome mapping data for M.pneumoniae have
revealed that, within an operon, transcription attenua-
tion generally coincides with the stop codon
21
. Possibly,
as a result of transcriptionaltranslational coupling, the
ribosome induces transcriptional termination in stop
codons by an as-yet-unknown mechanism. Indeed,the
-subunit of M. pneumoniae RNAP associates with
the ribosomal protein RpsD, suggesting a crosstalk
between the transcription and translation machineries
43
.
Furthermore, the first ribosome bound to the mRNA
cooperates with the RNAP in transcription elongation
44
.
These data not only support a coupling between tran-
scription and translation but also offer an explanation
for transcriptional attenuation based on an increased
probability of RNAP release if the ribosome dissociates
when it encounters a stop codon. The complexity of
bacterial operons can thus be compared to that of alter-
native splicing in eukaryotes. However, the regulatory
mechanisms of this alternative transcription remain to
be unveiled. Deep sequencing technologies now allow
TSSs to be identified to a single-base precision, therefore
making it possible to map all internal promoters within
an operon
32
. However, lacking still is a technology that
could identify the termination site with the same preci-
sion, although it is expected that long reads provided by
single-molecule DNA sequencing
45,46
could cover that
gap. This knowledge is essential to understand the rules
governing RNAP release and transcriptional termina-
tion at internal operon sites. Combining ultrasequenc-
ing and chromatin immunoprecipitation followed by
sequencing (ChIPseq) to target all proteins associated
with RNAP could also offer insight into which transcrip-
tional complexes are assembled at each promoter, as well
Figure 1 | Methodological transcriptomics tool kit. a | Various methods that have been recently developed to measure
different transcriptome features by mapping transcripts, antisense transcripts, transcription start sites (TSSs) and
protein-binding sites. b | Strand specificity in RNA-seq studies. Second-strand cDNA synthesis during reverse transcription
reactions can by prevented with actinomycin D (bottom panel). c | Comparison of the advantages of transcriptome
mapping based on either deep sequencing (RNA-seq) or tiling arrays. These are representative loci of the Mycoplasma
pneumoniae chromosome; the gene name is indicated above the dotted line. ChIPchip, chromatin immunoprecipitation
followed by microarray; RNAP, RNA polymerase.
REVI EWS
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as the relationship between the assembly of these and
the processivity and recognition of transcriptional ter-
mination signals. Native elongating transcript sequenc-
ing (NET-seq) is a new method that visualizes RNAs
invivo as they are produced
47
. This technique utilizes
the high stability of theRNAPDNARNA complex
that allows it to be affinity purified without crosslink-
ing, so that bound RNAs can be subsequently submit-
ted to deep sequencing. This method could be applied
to bacteria, allowing nascent transcripts to be identified
and both transcription elongation and RNAP pausing
to be monitored.
Regulations at the operon level
Like eukaryotic promoters, bacterial promoters can be
regulated by more than one signal or transcription fac-
tor
48
. In Caulobacter crescentus, 6% of the genes are tran-
scribed from multiple TSSs
22
, indicating that various
Figure 2 | Recent discoveries in bacterial transcriptomics. a | Intra-operon decaying expression. Almost half of the
consecutive genes within operons in Mycoplasma pneumoniae show staircase behaviour
21
. In general, the steps tend to
occur in the proximity of the stop codons at the end of every gene. b | Alternative transcripts in operons. Three alternative
transcripts are present in the cag25cag18 primary operon in Helicobacter pylori
40
. c | Abundance of antisense RNA. The
number of antisense RNAs in various species as a percentage of the number of open reading frames (ORFs). d | Leaderless
mRNAs. Transcripts starting at the translation initiation codon are preferentially bound by the 70S ribosome, whereas
those with a ShineDalgarno sequence are preferentially recognized by the 30S ribosomal subunit. e | mRNA intrinsic
regulation. A lysine riboswitch is located upstream of a lysine transporter gene (lmo0795)
60
. In the presence of lysine
lmo0795, transcription is prevented by a Rho-independent terminator. Functional elements present on mRNA molecules
are depicted. f | Overlapping untranslated regions (UTRs). The lmo0649 mRNA (red) can be transcribed from two
promoters. Transcription from the distal promoter generates a long UTR that overlaps with the coding region of lmo0648
(blue)
60
. g | Chromosome structure. Bacterial chromatin exhibits different degrees of compaction and supercoiling
depending on the growth phase (it is more compacted in the exponential phase compared to the stationary phase) and
on the transcription status (not shown). h | Epigenetic modifications. Methylation (Me) states of bases (in this case a
guanosine (G)) that affect transcription can be inherited. Binding of a repressor to an unmethylated promoter prevents
transcription of the repressor protein. Perturbations that lead to promoter methylation prevent the repressor from binding
to the promoter; this methylated state is inherited and a new stable state is created. TSS, transcription start site.
REVI EWS
Stationary phase
A stage of bacterial growth in
which the growth rate slows as
a result of nutrient depletion
and accumulation of toxic
products.
activators and/or repressors can regulate transcription.
Thus, an operon could respond to two or more different
inputs, enabling higher regulatory plasticity and respon-
siveness to stimuli. In some cases, converging signals
regulate the promoter by cooperatively binding to the
DNA, thus increasing the robustness of the response
49
.
Cooperative binding can result in either bimodal or all-
or-none expression and can turn on gene expression after
an input has crossed the threshold required for activa-
tion
48
; an example of this is the famous phage switch in
E.coli
50
. Complex regulatory mechanisms can likewise be
observed when there are bidirectional promoters, which
are also common in eukaryotes; 66% of divergent gene
pairs in yeast
51
and 10% of all promoters in the human
genome show such regulation
52
. Depending on the overlap
between the two promoters, they might be co-expressed
53
,
co-expressed with a particular hierarchy
54
or anti-regulated
on each strand. They might also share transcription fac-
tor target sites, which would be consistent with the fact
that divergent genes tend to have related functions in
higher eukaryotes
55
. To achieve a global view of such
complex regulatory control in bacteria, all transcription
factor binding sites will have to be mapped and all TSSs
precisely determined.
In the case of the gene that encodes Rns, a transcrip-
tional regulator from the AraC family, the binding site
for activators is located downstream of the core pro-
moter but, surprisingly, Rns activates its own expres-
sion by binding both upstream and downstream of the
promoter
56
in a manner that is reminiscent of enhancers
in downstream regions in eukaryotes. A recent study on
E.coli that examined 600 combinations of promoter and
coding regions from various genes found evidence that
the regulatory information stored in the coding regions
of genes strongly affects gene expression levels, indicating
that this could be a more general phenomenon
57
.
DNA looping, which was discovered in the ara and
gal operons and subsequently found in other bacterial
regulatory regions
58
, is used in some bacteria to regulate
transcription. In several E.coli promoters, including the
hdeABp promoter, H-NS represses RNAP complexes
carrying the
70
subunit. However,
38
RNAP com-
plexesare not repressed. Atomic force microscopy and
other techniques revealed that DNA traps
70
, but not
38
,
by completely wrapping it in cooperation with H-NS
59
.
The increase in resolution of experiments that address
the three-dimensional (3D) structure of chromatin (that
is, chromosome conformation capture (3C) experiments),
in combination with ChIPseq experiments, will allow
a complete analysis of all DNA looping regions and
open the way to a comprehensive study of their role in
transcriptional regulation.
sRNAs galore
Although it was known that bacteria express sRNAs of
50500 bases, the sheer abundance of such sRNAs, and
especially antisense RNAs (some of which are thought
to have regulatory roles), was unexpected
21,40,60,61
(FIG.2c).
These RNAs can work in cis by targeting a gene within
the same locus
62
or in trans
63,64
by targeting loci elsewhere
in the genome. sRNAs are involved in regulating various
processes, such as transcriptional interference, transcrip-
tional activation, translational control and regulation of
mRNA half-life
65
. sRNAs may be a part of ribonucleo-
protein complexes (and thus modulate protein activity,
as is the case for 4.5S RNA and transfer mRNA (tmRNA;
also known as SsrA)
66
), sequester proteins (as exempli-
fied by 6S RNA, which binds RNAP and induces changes
in gene expression at the transition to the stationary
phase
67
) or directly regulate mRNA translation and/or
stability
68
. sRNAs could also indirectly regulate tran-
scription of neighbouring genes by promoting changes
in DNA supercoiling
69,70
as a result of transcription of the
sRNA gene. For example, in M. pneumoniae, genes with
overlapping antisense transcripts have lower expression
levels
21
. Trans-encoded sRNAs typically interact with
multiple mRNAs
71
, as these sRNAs contact their target
mRNAs in discontinuous patches. Thus, a single RNA
can globally modulate particular physiological responses
and networks in a manner similar to a transcription
factor but at the post-transcriptional level
7275
and with
varying degrees of stringency and outcomes
76
.
The high number of sRNAs indicates that they must
have an important role in bacterial physiology. Unveiling
the functions of these sRNAs in gene regulation should
be addressed by high-throughput experiments after
overexpression or knockout of trans sRNAs or after gene
silencing of cis RNAs by complementary antisenseRNA.
It has been suggested that the number of antisense
transcripts roughly anticorrelates with the genome size
77

based on the finding that in the E.coli genome, which
contains ~4,000 genes, antisense RNAs represent 2.4%
of the total number of genes, whereas in the M.pneumo-
niae genome, which is much smaller than the genomes
of most other bacteria and contains only 689 protein-
coding genes, including only a handful of transcription
factors
21
, ~12% of the genes encode antisense RNAs.
Such a disproportionately large antisense RNA popu-
lation could be the biological consequence of genome
reduction, with transcriptional regulation taken over
by RNA-mediated control. However, the use of ultrase-
quencing technology in E.coli has led to the percentage
of antisense RNAs being revised upward from 2.4% to
20% of the genes
78
. Similarly, in H.pylori, 27% of genes
encode for antisense RNAs
32
, and manual examination
of M. pneumoniae transcripts raised the total to close
to 20%. These data suggest that the proportion of anti-
sense RNA could be ~1020% of the genes in bacteria,
and that the small numbers previously observed were
a result of low-sensitivity detection technologies. These
numbers are similar to those found in certain eukaryotes
(with ~20% in humans
79
and plants
80
) and slightly higher
than in yeast (7%)
51
. Recent metatranscriptomic stud-
ies of complex samples containing thousands of bacte-
rial sequences have also corroborated the abundance of
sRNAs within bacterial communities; for example, over
40,000 new sRNAs were identified in a singlestudy
33
.
Aside from the challenge of determining the function
of most of these sRNAs, there are also technical issues
that remain to be solved. For example, the heterogene-
ity that arises from the diverse protocols and sampling
methods used in the different laboratories leads to an
REVI EWS
overall poor reproducibility in sRNA determination;
the results of independent transcriptomics studies in
Burkholderia cenocepacia did not overlap
81
, and stud-
ies in Salmonella enterica subsp. enterica serovar Typhi
and S.enterica subsp. enterica serovar Typhimurium
detected only partial overlap, with an overlap of 42 out
of 82 annotated sRNAs in one data set
82
and of 5 out of
52 in another data set
83
. Strand specificity needs to be
ensured when sequencing RNA transcripts, as artefac-
tual sRNAs could be found opposite to coding genes
24
.
Thus, complementary high-throughput approaches
21,84

or validation by either quantitative PCR or RACE
85
are
required to confirm all newly discovered sRNAs before
starting functional studies. These technical issues not-
withstanding, the use of omics technologies has allowed
the discovery of conserved sRNAs that could not be
identified by genome sequence analyses
86
, as well as
new mechanisms, such as the binding of the 6S RNA to
RNAP to produce short RNA products
87
.
Deep sequencing in combination with pulldowns of
Hfq proteins in bacteria have identified sRNAs as the
major target of these proteins
83
. Complete catalogues of
sRNAs from several species will allow the identification
of conserved sRNAs, which could point to functionality,
whereas anticorrelated expression patterns could suggest
a mechanism of regulation of mRNA transcription by
sRNAs and identify new targets for functionalstudies.
sRNAs and small peptides
Some sRNAs are multifunctional as they encode func-
tional peptides. Probably the most paradigmatic exam-
ple of this is RNAIII in Staphylococcus aureus, which was
one of the first cis-acting RNAs to be described and is
involved in quorum sensing. RNAIII encodes a 26-amino-
acid peptide that may be involved in biofilm integrity
88
.
Other cases have been documented in E.coli
89,90
and
in Listeria monocytogenes
60
. For example, SgrS causes
translational repression when pre-annealed with ptsG
in E.coli
91
. Interestingly, the 5 region of SgrS contains a
43-amino-acid open reading frame (ORF) termed sgrT,
which is translated during glucose-phosphate stress.
Downregulation of ptsG mRNA does not require SgrT,
and SgrT by itself has no effect on ptsG mRNA stability.
However, cells expressing SgrT alone have a defective
glucose uptake, even when they exhibit nearly wild-type
levels of PtsG. Together, these data suggest that SgrS is
a bifunctional sRNA that encodes physiologically redun-
dant but mechanistically distinct functions that contrib-
ute to the same stress response
90
. This list is expectedto
expand substantially as small peptides are difficult
to detect using standard proteomics approaches and
require appropriate isolation and fractionation techniques
that specifically enrich small proteins
92
. The combination
of deep sequencing, translation in the three phases of the
identified sRNAs and the guided search for all ORFs with
a translation initiation codon using mass spectrometry
could reveal a new layer of regulation in bacteria.
RNA processing
It is generally assumed that post-transcriptional regula-
tion of RNA in eukaryotes is extremely important and
complex, whereas it is either minimal or non-existent in
bacteria. Thus, nascent mRNA in bacteria is thought to
be bound to ribosomes and degraded after some min-
utes. However, recent studies have shown the existence
of mechanisms that affect mRNA stability in bacteria
(FIG.3a), as well as different types of RNA processing.
RNA degradation is usually triggered by an endonucleo-
lytic cleavage, followed by a rapid exonucleolytic activity.
Different ribonucleases (RNases) are responsible for the
first step, depending on the species, and some proteins
can even cleave their own mRNA and thereby autoregu-
late their expression
93
. Several factors affect the stability
of a given mRNA, including RNA-binding proteins and
sRNA
94
. For example, removal of the 5-triphosphate by
the enzyme RppH triggers mRNA degradation in E.coli
95
.
Furthermore, RNA stability is species dependent and can
vary depending on the environmental conditions
96
.
There are several examples of RNA processing in
which internal cleavage occurs in a regulated manner
97
.
It is expected that differential identification of primary
RNAs with a 5-triphosphate and processed RNAs with
a 5-monophosphate
32
will help to identify invivo mRNA
processing sites at the genome level and under different
conditions. Contrary to the common view, polyadenyla-
tion (addition of untemplated adenosine residues to
the 3 end of transcripts) is also present in bacteria and
cellular organelles of bacterial origin (TABLE1). In fact
bacteria perform two types of polyadenylation: classic
poly adenylation catalysed by poly(A) polymerase I, which
adds exclusively adenosines, and addition of a mixture of
different bases with about 50% adenosines
98
catalysed by
polynucleotide phosphorylase (PNPase). Genome-wide
studies using poly(T) oligo nucleotides to enrich these
transcripts indicated that around 0.010.2% of the tran-
scripts have poly(A) tails, but this low number could be
related to the fact that they are degraded very rapidly
99
;
in B.subtilis, around 20% of the total RNA was found to
be polyadenylated
100
and a recent genome-wide study of
polyadenylated RNA in E.coli revealed that around 72%
of all RNAs that contain a Rho-independent termination
signal are polyadenylated
101
. Thus, polyadenylation is a
common modification and, moreover, can vary with the
growth phases
102
. Several possible roles for polyadenyla-
tion in bacteria have been suggested; it was reported that
polyadenylation targets transcripts for degradation by the
degradosome
103
and that polyadenylation is involved in
quality control for transcriptional and processing errors
104
.
Furthermore, RNA editing can alter the genetic
information by mutation, deletion or insertion of a base.
This process has been well documented for eukaryotic
endosymbionts, in which RNA editing is promoted
by guide RNA, which is another type of sRNA. In the
mitochondrion of Trypanosoma brucei, for instance,
many mRNAs cannot be translated owing to multiple
frameshifts; in this case, guide RNAs serve as templates
for correcting such mistakes at the mRNA level with the
aid of ribonucleoprotein complexes
105,106
.
Finally, RNA splicing has been reported in endo-
symbionts
107
and bacteriophages
108
, indicating that this
feature is also not exclusive to eukaryotes (TABLE1). These
findings highlight the ancient role of RNA and ribozymes
REVI EWS
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and provide insight into putative functions of some of
the still uncharacterized sRNAs. Group II self-splicing
introns are large catalytic molecules that are considered
to be related to the progenitor of spliceosome introns.
They were initially identified in the organelles of lower
eukaryotes and plants and are also present in bacteria,
although their frequency is unknown
109
. Genome-wide
RNA-seq analysis could be used to obtain a global view
of all of these post-transcriptional processes, although
this would involve a modification of existing standard
mapping methods to allow for the identification of
added poly-base tails that are not present in the genome
or of the fusion of non-contiguous DNA sequences.
Leaderless mRNAs and regulation by UTRs
The ShineDalgarno ribosomal recognition sequence
was long considered to be essential for ribosome
attachment and efficient translation of mRNA. The lat-
est transcriptomics studies have now demonstrated that
several RNA transcripts are leaderless: that is, that these
transcripts start with one start codon (FIG.2d) or have
non-canonical ribosome-binding sites
110
. The mecha-
nism by which leaderless transcripts with no or a very
short 5 untranslated region (UTR) could be recog-
nized by the ribosome has long been unclear. However,
in E.coli, it was shown that the ATG initiation codon
can become the ribosome-binding sequence
111
and that
leaderless transcripts can bind 70S ribosomes rather than
30S ribosomal subunits, suggesting a different pathway
of translation for these mRNA
112
. Genome-wide tran-
scriptional studies have revealed that there are 26 leader-
less RNAs in H.pylori
40
and 25 in S.Typhimurium
82
.
These transcripts may be preferentially translated
when bulk mRNAs cannot be translated, inducing a
Figure 3 | Perspectives in transcriptomics. a | RNA modifications. There is growing evidence that RNA in bacteria can
undergo a number of post-transcriptional modifications that alter its stability and functionality. New ultrasequencing
methods coupled with classical biochemical approaches should allow comprehensive studies on these modifications and
reveal their function. b | Spatial organization of transcription and translation. The colocalization in space of functionally
related genes could drive the assembly of newly synthesized proteins into complexes
143
. The implementation of new
methodologies, such as chromosome conformation capture (3C) in bacteria
129
, will make it possible to obtain a
three-dimensional view of bacterial chromatin. c | Full-length transcripts. New sequencing technologies could provide
operon-long reads. The assembly-free sequencing of transcripts will allow the characterization of alternative transcript
termination and provide the chance to study the relationship between transcription initiation and termination. d | RNA
protein interactions. The identification of RNA-binding proteins will be another means of gaining insight into the roles of
newly discovered RNAs. e | RNA secondary structure. High-throughput RNA structure determination should increase our
understanding of how non-coding RNAs, such as untranslated regions and small RNAs (sRNAs), function. f | Single-cell
transcriptomes. A substantial percentage of the transcripts are in the stochastic range. Studies in individual bacteria will
provide new biological insights into population heterogeneity and regulation. RNAP, RNA polymerase.
REVI EWS
non-transcriptional shift between proteins produced
in non-optimal conditions, such as starvation or station-
ary phase, when 70S monosomes prevail
113
. Consistent
with this, in E.coli, leaderless mRNAs are poorly trans-
lated during the exponential phase and are only translated
when genes containing a canonical ribosome binding site
are not translated
114
; this exemplifies another regulatory
mechanism at the level of translation.
Transcriptome studies have also revealed that many
mRNAs contain a 5 UTR of up to several hundred bases
(the average length in M.pneumoniae is ~60nucleo-
tides)
21
. These regions may be regulating translation
through secondary structure. Some of these long UTRs
encode proteins or peptides; a classic example is the
E.coli trp operon leader peptide, which controls pre-
mature transcriptional termination depending on
tryptophan availability
115
. Alternatively, they can also
contain riboswitches, which are regulatory regions that
affect mRNA stability and translation efficiency
40,60
.
Riboswitches are regulated by binding small molecules
that act as environmental signals. Several classes of
riboswitches that could regulate translation in response
to metabolites have been described
17
(FIG.2e). For exam-
ple, the S-adenosylmethionine (SAM)-III riboswitch is
a SAM-binding element that is found in the 5 UTR of
the metK gene, which encodes the SAM synthetase, in
Lactobacillus spp.
116
. SAM binding to SAM-III results
in sequestration of part of the ribosome binding site
sequence of the metK mRNA, thus inhibiting ribo-
some binding and leading to repression of translation
initiation
116,117
. Recent experiments have revealed that
SAM-III is a reversible riboswitch that can differentially
regulate the transcript at various stages of the lifetime of
the transcript, thus allowing the cell to respond rapidly
to SAM fluctuations by regulating metK expression
118
.
There are other roles for UTRs. For instance, mogR
mRNA in L.monocytogenes can be transcribed from two
different promoters. Transcription from the first pro-
moter produces a long isoform that is induced only in the
stationary phase and generates a long 5 UTR that over-
laps with three genes. This 5 UTR binds to the mRNA
of the overlapping genes, leading to their degradation
60

(FIG.2f).
The abundance of leaderless mRNAs, as well as
the presence of mRNAs with long 5 UTRs, suggests the
existence of a hidden and complex layer of regulation
by the UTRs. Determination of the 5 end of all mRNAs
could elucidate different mechanisms of translational
regulation and guide further studies. As determining
the structure of RNA is key to understanding its func-
tion, another promising technique is the use of RNases
to determine the specific secondary structures of RNAs,
which can be performed genome-wide. This allows base-
paired regions within RNAs to be identified, revealing
potential riboswitches and other relevant structures.
This method has been successfully applied to yeast
119

and mouse transcriptomes
120
and is valuable for both
UTRs andsRNAs.
Bacterial DNA is more than a random bundle
Like eukaryotes, bacteria contain nucleoid proteins that
bind DNA in a sequence-specific or non-sequence-
specific manner, thereby promoting nucleoid compac-
tion and domain formation and varying gene expression
levels in response to environmental perturbations
108
.
E.coli contains around 450 nucleoid structure domains,
which have an average length of around 10 kbp, with
variable boundaries and a random distribution along the
chromosome
121,122
. Nucleoid compaction and domain
formation of DNA in bacteria are expected to have an
effect on transcriptional regulation
14,123
. Because nucleoid
structure can be altered by changes in DNA supercoil-
ing or changes in expression of histone-like proteins,
it has been proposed that nucleoid reorganization and
dynamics in E.coli could assume the role of a transcrip-
tion factor (REF.124). For example, changes in DNA
supercoiling in E.coli induced by osmotic stress result
in major changes in gene expression
118
.
Table 1 | Bacterial transcriptome features that recall eukaryotic complexity
Feature Present in eukaryotes? Present in bacteria?
Small RNAs Yes Yes
Complex promoter regulation Yes, eukaryotes have many transcription factors
and other proteins
Yes, but simpler than in eukaryotes
Alternative transcripts Yes Yes, alternative promoters and terminators are
present
RNA processing Yes Yes, but mainly related to the regulation of
degradation, with few examples of specific processing
RNA splicing Yes Yes, but mainly restricted to organelles (mitochondria
and chloroplasts), tRNAs and ribosomal RNAs, and
there are few examples
Polyadenylation Yes, polyadenylation stabilizes RNA Yes, polyadenylation destabilizes RNA
Localized translation Yes, many mRNAs are transported to specific sites
where they are translated
Yes, recent data show that mRNAs in bacteria do
not diffuse and are translated on specific nucleoid
localizations
Epigenetic modifications Yes Yes, but little information available
Impact of chromatin and nucleoid
structure on transcriptional regulation
Yes, supercoiling and chromatin domains have a
major impact on transcription
Yes, several examples have been found
REVI EWS
Occupancy studies in E.coli have shown the presence
of regions in the chromosome with contiguous protein
binding of around 1.6 kbp in length, some of which cor-
respond to active transcriptional regions, such as the
ribosomal RNA operon
125
. Interestingly, most E. coli or
Salmonella spp. genes are expressed at a low level on
average, the mRNA/DNA ratio is less than one
103
and
would thus have little or no impact on nucleoid structure
(FIG.2g). In agreement with this observation, only very
few loci have a high concentration of RNAP, as observed
by electron microscopy
126
. When cells are in the station-
ary phase, the transcription factories disassemble, RNAP
is distributed throughout the chromosome and the 3D
structure becomes less compact (FIG. 2g). However,
other regions with contiguous protein occupancy
correspond to transcriptionally silent areas and could be
involved in establishing chromatin domains
125
. Locally,
chromatin domains induced by RNAP supercoiling can
be consolidated if the encoded protein is a membrane
protein
127,128
. This is owing to the fact that insertion of
a protein into the membrane, or periplasm, during co-
transcriptional translation will tether its mRNA to the
cell membrane, and this will enhance topological effects
on the DNA
127,128
. Once established, chromatin domains
can induce a type of topological memory generating lin-
eages with different expression states, thereby creating
cellular subpopulations that have the same genome but
are primed to respond to various environments
128
.
High-throughput techniques have been applied to the
determination of long-range DNA interactions and 3D
DNA structure invivo in eukaryotes: namely 3C
129
andthe
later variations circularized chromosome conformation
capture (4C), carbon-copy chromosome conforma-
tion capture (5C)
130
and Hi-C
131
. These techniques rely
on crosslinking, restriction digestion and re-ligation of
DNA regions that are close in space but not in the pri-
mary sequence
132
. They have been applied to regions of
the human genome
133,134
and in a genome-wide study in
yeast
135
. Similar studies in bacteria would help to unveil
the structure and dynamics of the bacterial chromosome,
as well as its role in transcription regulation.
Epigenetics: memory of the bacterial nucleoid
Until recently, methylation of bacterial DNA was con-
sidered to be used primarily to distinguish foreign DNA.
However, DNA methylation has important roles in bac-
teria, such as during chromosome partitioning, DNA
replication, DNA repair, timing of transposition and
conjugation
136
. In bacteria, cytosine can be methylated at
N4 (m4C) or N5 (m5C) and adenine can be methylated
at N6 (m6A). It has been suggested that m4C is related
to epigenetic transcriptional regulation
136
, and several
studies have revealed that individual bacterial cells can
have a particular DNA methylation status that changes
transcription of some operons in a specific, heritable
manner
137,138
. This occurs through a competition for
methylation sites between a methylase and the protein
that recognizes the unmethylated state (FIG.2h); binding
of the protein that recognizes the unmethylated state will
prevent access of the cognate methylase to the site, which
affects transcription of the downstream genes. This effect
can be enhanced if the gene downstream encodes for the
protein that binds the unmethylated site and if its tran-
scription requires a non-methylated status. If changes
in environmental conditions result in degradation or
inhibition of the protein protecting the methylation site,
then the site will be methylated, blocking further tran-
scription; this status will be inherited by the subsequent
generations. A typical example is the pap pilin phase
variation
125
. The pap operon has two leucine-responsive
protein (Lrp) binding sites. Binding of Lrp to the proxi-
mal site blocks transcription and permits methylation of
the distal site, which prevents Lrp from binding there. If
poly(A) polymerase I is expressed, it dimerizes with Lrp
and binds at the distal site, allowing methylation of the
proximal site and therefore transcription of the operon.
It is estimated that the methylation status of only a small
fraction of the approximately 20,000 methylated sites in
the E.coli chromosome can change when subjected to
different conditions (for a review, see REF.136).
Although DNA methylation is the best-known mech-
anism involved in epigenetic regulation in bacteria, for-
mation of a possible feedback loop by other mechanisms
also permits inheritance of epigenetic states
139
. The latest
ultrasequencing technologies that sequence single DNA
molecules in real time have made it possible to deter-
mine which bases are methylated, as these bases induce
a delay in the polymerization process
140
. This opens the
possibility of complete genome-wide analyses of DNA
methylation at adenine and cytosine, which could be
determined under various environmental perturbations
and thus provide insights into the role of epigenetics in
transcriptional regulation
140
.
mRNA localization in bacteria
Organisms from all three domains of life can local-
ize proteins to specific regions within a cell, either by
targeting the protein with a signal that directs it to its
destination or by locating the mRNA that encodes the
protein to the appropriate region. Both mechanisms
operate in eukaryotes, whereas only the first mecha-
nism has been reported in bacteria
141
. Originally, track-
ing single RNA molecules in E.coli showed localized
motion consistent with Brownian motion of a polymer
bound to the DNA and free diffusion
142
, but recent stud-
ies are challenging the notion that mRNA in bacteria is
randomly distributed. Analyses of mRNA localization
and diffusion in C. crescentus and E.coli have shown
that mRNA diffuses two orders of magnitude less than
would be expected in solution and that it restricts ribo-
somal mobility
143
. As a consequence, translation could
be spatially organized by using the chromosome layout
as a template (FIG.3b) and proteins encoded by neigh-
bouring genes could be produced in close proximity.
Therefore, spatial co-expression could promote imme-
diate protein interactions and macromolecular complex
formation
143
. If this proves to be a general phenomenon,
it would justify the existence of operons encoding pro-
tein complexes, as well as of chromosomal 3D domains
harbouring genes involved in related functions. Such
3D clusters that include genes that are co-regulated
by a given transcription factor have been described in
REVI EWS
mammals, in which the term transcriptional factories
has been proposed
144
.
Thus, the influence of chromatin structure on gene
expression and on the organization of RNA synthesis in
space is not an exclusive feature of eukaryotes. Recently,
it was found that certain mRNAs in E.coli can migrate
to the destination of their encoded proteins. This process
is controlled by sequences within the RNA transmem-
brane-coding sequence. Although the molecular mecha-
nism is unknown, the data suggest an active transport
of the mRNA to its translation location, as occurs in
eukaryotes
145
. Other studies have shown that complexes
involved in mRNA degradation show specific localization
(for a recent review, see REF.146).
If applied to all RNAs in a bacterium, technologies
that allow the visualization of single RNA molecules in
the cell
147,148
could elucidate whether mRNA localiza-
tion is a widespread phenomenon. Determining dis-
tinct RNA localizations could allow the identification of
specific sequences that guide this localization, thereby
opening a new researcharea.
Perspectives
Future directions should involve integrating our knowl-
edge of transcription with the physiology of the micro-
organism. Therefore, data should be complemented
with other levels of regulation. Technology based on
single-molecule sequencing
46
could represent the next
important advance in transcriptomics, as it should be
capable of delivering reads with lengths up to tens of
thousands of bases, allowing full-length polycistronic
transcripts to be sequenced and providing direct
association of promoters and terminators (FIG. 3c).
Currently, transcriptomes are constructed by using
short-read libraries in combination with TSS detection
methods
30,40
; this approach can provide information on
internal promoters but not on terminators, and neither
can it provide a complete list of the alternative tran-
scripts that compose an operon. Genome-wide map-
pings with long reads will not only identify the different
5UTRs and 3UTRs but will also deconvolve the exact
molecular nature of the operon by sequencing full-
length transcripts. Additionally, technologies based on
single-molecule sequencing will substantially enhance
the speed at which data are acquired. Current Illumina
and Roche technologies measure the incorporated
bases by stopping the sequencing reaction. By contrast,
new single-cell sequencing technologies can monitor
base incorporation in real time, thereby decreasing
sequencing run length from days tohours
46
.
The number of known sRNAs is increasing every day,
but the functions of many of these remain unclear. New
high-throughput methods for detecting RNAprotein
interactions are expected to reveal important biological
insights into RNA function (FIG.3d) and the regulation
of protein activity by RNAs. Despite the important role
that the 3D structure of RNA can have in its function,
relatively few RNA structures have been determined
experimentally. For instance, in several trans-acting
sRNAs, only a part of the sequence is involved in base
pairing with the target. Based on a 3D structure, it is pos-
sible to hypothesize which residues are available to base
pair. Parallel analysis of RNA structure (PARS), a new
method for studying RNA secondary structure genome-
wide, obtains information about the secondary structure
of RNA by treating RNA with structure-specific enzymes
and then subjecting the fragments to deep sequenc-
ing. PARS was initially developed for mRNAs, but the
method could also be used on bacterial sRNA fractions
and even entire transcriptomes
119
(FIG.3e).
Transcript levels for the majority of the RNAs are
generally low (the abundance of mRNA for genes with
more than 100 protein copies per cell ranges from 0.05
to 5 mRNAs per cell
149
). This, together with the fact that
transcription is a stochastic process (as it deals with
very few sites in a large chromosome, and transcription
factors are bound nonspecifically to DNA and diffuse
along DNA for most of the time
150
) results in large tem-
poral variations among cells
149
(FIG.3f). Gene expres-
sion hetero geneity within individual cells is essential
for various processes
151
. The single-cell transcriptomics
data that are needed to understand this stochasticity in
eukaryotes are already available
152
, so a major challenge
is to provide similar single-cell data sets for bacteria.
In addition to these future technical prospects,
integration of data that combine different sources
of information is needed to provide a global picture of
transcription.
As mentioned above, chromatin organization and epi-
genetics in bacteria could play a major part in transcrip-
tion and translation. Determining the role of genomic
architecture and dynamics, together with understand-
ing the function of the massive amount of sRNAs,
presentsthe next important challenge in deciphering
the mechanisms of regulation of gene expression atthe
transcriptional and translational levels. Therefore,
the evidence that non-canonical DNA-binding proteins
could have a role in shaping the nucleoid architecture
that links metabolism and transcription is highly relevant.
In this scenario
153
, obtaining a global overview of DNA-
binding proteins, and disentangling their effects on tran-
scription and metabolism, would greatly enhance the
design of the multilayer models required to understand
the complexity of a biologicalsystem.
Note added in proof
Recently, a paper describing single-cell RNA analysis in
bacteria was published
160
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Acknowledgements
We would like to thank M.Isalan and B.Lehner for critical
reading of the manuscript. This work was supported by the
Consolider programme of the Spanish Ministry of Research,
the Fundacin Marcelino Botn and the European Research
Council.
Competing interests statement
The authors declare no competing financial interests.
FURTHER INFORMATION
Authors homepage: http://serrano.crg.es
Bioconductor: http://www.bioconductor.org
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The regulation of bacterial transcription has been a topic

of interest for several decades; the transcriptional regu-

ICREA (Instituci Catalana de

Present address: Harvard

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