A conserved RpoS-dependent small RNA controls the synthesis of major porin OmpD.

Abstract

A remarkable feature of many small non-coding RNAs (sRNAs) of Escherichia coli and Salmonella is their accumulation in the stationary phase of bacterial growth. Several stress response regulators and sigma factors have been reported to direct the transcription of stationary phase-specific sRNAs, but a widely conserved sRNA gene that is controlled by the major stationary phase and stress sigma factor, σ(S) (RpoS), has remained elusive. We have studied in Salmonella the conserved SdsR sRNA, previously known as RyeB, one of the most abundant stationary phase-specific sRNAs in E. coli. Alignments of the sdsR promoter region and genetic analysis strongly suggest that this sRNA gene is selectively transcribed by σ(S). We show that SdsR down-regulates the synthesis of the major Salmonella porin OmpD by Hfq-dependent base pairing; SdsR thus represents the fourth sRNA to regulate this major outer membrane porin. Similar to the InvR, MicC and RybB sRNAs, SdsR recognizes the ompD mRNA in the coding sequence, suggesting that this mRNA may be primarily targeted downstream of the start codon. The SdsR-binding site in ompD was localized by 3'-RACE, an experimental approach that promises to be of use in predicting other sRNA-target interactions in bacteria.

Full text

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A conserved RpoS-dependent small RNA controls
the synthesis of major porin OmpD
Kathrin S. Fro ¨hlich1, Kai Papenfort1, Allison A. Berger2and Jo ¨rg Vogel1,*
1RNA Biology Group, Institute for Molecular Infection Biology, University of Wu ¨rzburg, Josef-Schneider-Strasse
2, D-97080 Wu ¨rzburg and2Max Planck Institute for Infection Biology, Charite ´platz 1, D-10117 Berlin, Germany
Received August 26, 2011; Revised November 8, 2011; Accepted November 10, 2011
ABSTRACT
A remarkable feature of many small non-coding
RNAs (sRNAs) of Escherichia coli and Salmonella
is their accumulation in the stationary phase of bac-
terial growth. Several stress response regulators
and sigma factors have been reported to direct the
transcription of stationary phase-specific sRNAs,
but a widely conserved sRNA gene that is controlled
by the major stationary phase and stress sigma
factor, pS(RpoS), has remained elusive. We have
studied in Salmonella the conserved SdsR sRNA,
previously known as RyeB, one of the most
abundantstationary phase-specific
E. coli. Alignments of the sdsR promoter region
and genetic analysis strongly suggest that this
sRNAgeneisselectively
We show that SdsR down-regulates the synthesis
ofthemajorSalmonella
Hfq-dependent base pairing; SdsR thus represents
the fourth sRNA to regulate this major outer
membrane porin. Similar to the InvR, MicC and
RybB sRNAs, SdsR recognizes the ompD mRNA in
the coding sequence, suggesting that this mRNA
may be primarily targeted downstream of the start
codon. The SdsR-binding site in ompD was localized
by30-RACE,anexperimental
promises to be of use in predicting other sRNA–
target interactions in bacteria.
sRNAs in
transcribedby
pS.
porinOmpDby
approachthat
INTRODUCTION
10 years ago, when several pioneering screens discovered
a plethora of small non-coding RNAs (sRNAs) in
Escherichia coli, it was noted with surprise that many of
these sRNAs accumulated in the stationary rather than
exponential phase of growth (1–5). This observation was
inremarkablecontrast
rate-independent expression of house-keeping RNAs and
to thecommongrowth
cis-encoded antisense RNAs of plasmids and phages (6–8)
and fueled speculation that the new sRNAs belong to
defined stress regulons. That is, stationary phase was in-
creasingly being understood as a growth phase wherein
bacteria prepare themselves for hard times, up-regulating
numerous stress response regulators including the alter-
native sigma factor, sS. Encoded by the rpoS gene in
E. coli (9), sSis a major stress sigma factor in many
enterobacteria (10–12). Its activity sharply increases not
only as cells cease to grow, but also under a variety of
other stress conditions such as heat or osmotic shock
(13,14). The combined results of gene fusion and global
transcriptome studies have suggested that sSdetermines,
directly or indirectly, the expression of ?10% of all
protein-encoding E. coli genes, many of which have key
functions in stress survival (15,16).
A decade has passed and several of these differentially
expressed sRNAs have indeed been assigned to various
important regulons of E. coli and Salmonella. Examples
include the RyhB and IsrE sRNAs as members of the
iron-responsive Fur regulon (17–19); MicA and RybB,
which are activated by the envelope stress sigma factor,
sE(20–25); CyaR, whose transcription is governed by
the cAMP–CRP complex (26–28); ArcZ and FnrS,
which respond to oxygen availability via the ArcA/B or
Fnr systems (29–31); and MgrR, which is a member of the
Mg2+-responsive PhoP/Q regulon (32). The strict regula-
tion of these sRNAs by their cognate transcription factors
is often accompanied by the presence of an optimal
binding site in the sRNA promoter region, and in fact
some of these sRNAs are the most highly regulated
genes in their respective regulons (20).
It has been speculated that every major transcriptional
regulon contains at least one conserved sRNA gene (33).
Regarding the sSregulon, several sRNAs have been
reported to accumulate in stationary phase and to show
greatly diminished expression in an rpoS deletion strain, as
one would expect for an sS-transcribed sRNA. However,
none of them is widely conserved: the 105nt GadY sRNA,
which acts to stabilize the oppositely encoded gadX
mRNA (34), is found in a few E. coli strains only and
*To whom correspondence should be addressed. Tel: +49 931 3182575; Fax: +49 931 3182578; Email: joerg.vogel@uni-wuerzburg.de
Published online 17 December 2011Nucleic Acids Research, 2012, Vol. 40, No. 8 3623–3640
doi:10.1093/nar/gkr1156
? The Author(s) 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Page 2

IsrE (?100nt), a homologue of the widely conserved
RyhB sRNA (iron starvation), is specific to Salmonella
(18). Thus, a sS-dependent sRNA which is conserved
throughout the enterobacterial clade had yet to be
identified from among the nearly 100 sRNA species cur-
rently known in E. coli and Salmonella.
Here, we report the characterization of Salmonella SdsR
(sigma S-dependent sRNA), a conserved sRNA that is a
member of the sSregulon. SdsR was originally reported
under the name RyeB as an abundant, stationary phase-
specific, ?100nt sRNA transcribed from the pphA-yebY
intergenic region of E. coli (1,2). Its expression was shown
to be inversely correlated with that of SraC (a.k.a. RyeA,
Tpke79, IS091), a ?250nt sRNA that is transcribed from
the opposite strand (1–5). The extensive antisense comple-
mentarity of SdsR and SraC suggests mutual processing
by the double strand-specific endoribonuclease, RNase III
(2). Whether such co-processing would be of any function-
al relevance has remained unresolved, as have the cellular
functions of SdsR and SraC.
In contrast with the poor conservation of the sraC
sequence, sdsRgenes can
enterobacteria (35), as though sdsR and not sraC has
been maintained by selection. In addition, SdsR has re-
peatedly been pulled down with the sRNA chaperone Hfq
in both E. coli and Salmonella (1,36–38). This Hfq rela-
tionshipsuggeststhat SdsR
transcriptional regulator of gene expression because
Hfq-associated sRNAs commonly regulate trans-encoded
target mRNAs by short base pairing interactions, result-
ing in the repression or activation of targets at the levels of
translation, RNA stability or both (39).
The mRNA targets of Hfq-associated sRNAs, as
inferred from the global Hfq coIP data, encode proteins
that have functions in many cellular pathways (36,38,40).
One such prominent target is the rpoS mRNA itself,
whoseexpression isdirectly
Hfq-binding sRNAs (DsrA, RprA, ArcZ) and repressed
by OxyS (31,41–45). Another prominent group of targets
are mRNAs of proteins with envelope localization; almost
a third of all characterized E. coli/Salmonella sRNAs
repress porins and outer membrane proteins (OMPs)
(46–48). Although many of these sRNAs accumulate in
stationary phase, none of them is known to depend on sS.
This article presents evidence for a novel link between
sS, Hfq and sRNAs, revealing SdsR as an sS-dependent
repressor of porin synthesis in Salmonella. Protein analysis
of a Salmonella strain over-expressing SdsR detected
down-regulation of OmpD, a very abundant OMP in
several enterobacteria (49). The ompD mRNA has been
a hotspot for regulation by Hfq-associated sRNAs, and
was recently shown to be repressed by the conserved RybB
and MicC sRNAs, as well as the virulence region-specific
InvR sRNA (50–54). Using an experimental 30-RACE
approach for target site prediction, followed by mutation-
al analysis, we have determined that SdsR represses the
ompD mRNA by means of a short stretch of complemen-
tarity in the coding sequence, yet at a different site than
each of the other three repressors. In light of the many
stress conditions that activate sS, we hypothesize that
bepredicted inmany
functions asa post-
activatedbyseveral
SdsR contributes to the post-transcriptional control of
ompD under such conditions.
MATERIALS AND METHODS
DNA/RNA oligonucleotides and plasmids
Sequences of all oligonucleotides employed in this study
are listed in Supplementary Table S1. All plasmids used in
this study are summarized in Supplementary Table S2.
Plasmid pBAD-SdsR (pKP19-8) was constructed as
pBAD-RybB in ref. (21), but using primers JVO-0902/
JVO-0903 to amplify the complete sdsR gene from its tran-
scriptional start site to 30bp downstream of the termin-
ator T-stretch from genomic DNA. The same insert was
used for the pZE12-luc-derived pPL-SdsR (pKF68-3) fol-
lowing the cloning strategy described in ref. (55). For
plasmids expressing different SdsR variants from the
pLLacO promoter, pKF68-3 served as a template for
PCR amplification with the primer pairs JVO-7159/
pLLacO C (pPL-SdsR +7; pKF97-1), JVO-7161/pLLacO
C (pPL-SdsR +19; pKF99-1) and JVO-7163/JVO-4731
(pPL-SdsR*; pKF101-26) and self-ligation was carried
out asin ref.(56).
(pKF105-1)was constructed
PCR-product of JVO-7224/pLLacO C on pFS135. A
single nucleotide exchange (C46G) was inserted in the
ompD complementation plasmid (insert: 493bp upstream
of the translational start site to 89bp after the stop codon)
with primers JVO-7225/JVO-7328 to obtain pKF109-1.
For the sdsR/sraC, complementation plasmid psdsR
(pVP203-1), a fragment carrying both sdsR and sraC
(comprising 198bp upstream of the transcriptional start
site of SdsR to 353bp downstream of the terminator
T-stretch), wasamplified
JVO-0052 and genomic DNA; the product was treated
with XbaI/XhoI and ligated
digested low-copy version of pZE12 in which the origin
had been swapped with pSC101* [pVP003; (57)]. To
exchange the cytosine at position ?13 in the sdsR
promoter, pVP203-1 served as the template for PCR amp-
lification with the primer pairs JVO-4262/JVO-4265
(psdsR C-13G; pKF106-2), JVO-4263/JVO-4265 (psdsR
C-13A; pKF107-1)andJVO-4264/JVO-4265
C-13T; pKF108-3), and the resulting DNA fragments
were self-ligated. Competent E. coli TOP10 were used
for all cloning purposes.
Similarly,
by
pPL-SdsR-TMA
self-ligation ofa
usingprimers JVO-0051/
intoan equivalently
(psdsR
Bacterial strains
A complete list of bacterial strains employed in this study
is provided in Supplementary Table S3. The Salmonella
enterica serovar Typhimurium strain SL1344 (JVS-0007),
the E. coli MC4100 derivative relA+ (JVS-5105) and the
attenuated Shigella flexneri BS176 (JVS-0012) are referred
to as wild-type strains and were used for mutant construc-
tion. Phage P22 or P1 transduction (using standard proto-
cols) was employed to transfer each single chromosomal
modification to a fresh Salmonella or E. coli wild-type
background, respectively, as well as to obtain strains
carrying multiple mutations. Single mutant derivatives
were constructed by the ?Red recombinase one-step
3624Nucleic Acids Research, 2012,Vol.40, No. 8

Page 3

inactivation method using pKD4 as the template.
Toeliminate theKanR
mutants, cells were transformed with the FLP recombin-
ase expression plasmid pCP20 (58). Mutant susceptibility
to kanamycin and loss of the temperature-sensitive FLP
expression plasmid were tested. Briefly, for JVS-9251
(SL1344 ?PsraC) and JVS-9312 (MC4100 ?PsraC),
Salmonella or E. coli wild-type cells carrying the pKD46
helper plasmid were transformed with the DNA fragment
to be integrated, amplified from pKD4, using JVO-7742/
JVO-7743 (Salmonella) or JVO-7859/JVO-7860 (E. coli),
and insertion of the KanRmarker gene was verified by
PCR using JVO-0051/JVO-0902 or JVO-1043/JVO-2390,
respectively. The single copy transcriptional sdsR-lacZ
fusion was constructed as in ref. (59). The sdsR mutant
strain (deletion of the sdsR gene downstream of nucleotide
+6 with respect to the transcriptional start site) was
obtained by the ?Red recombinase protocol using a
DNA fragment generated
(JVO-6533/JVO-6534) on pKD4. Obtained clones were
tested by PCR (JVO-0052/JVO-0903) and transformed
with pCP20. Upon verifying loss of the KanRcassette,
mutants were transformed in the presence of pCP20 with
pKG136 and colonies were screened for the integration of
lacZ-Y downstream of the sdsR promoter (JVO-0052/
pMC874-lac). To construct the chromosomal single-
nucleotide exchange in ompD and an isogenic WT strain,
SL1344 ?ompD (JVS-0735) carrying pKD46 was trans-
formed with a PCR product from JVO-5738/JVO-5739
using pKF109-1 or pVP42-3, respectively, as templates.
Integration of the CmRcassette-containing fragment
upstream of the complete ompD gene, including its own
promoter, was verifiedby
JVO-2192. P22 lysates of these strains were used to trans-
duce JVS-8827 (SL1344 ?sdsR) to obtain JVS-9155
(SL1344 CmR::ompD*DsdsR) and JVS-9154 (SL1344
CmR::ompDDsdsR), respectively. Similarly, chromosomal
integration of the constitutive PLtet-O1promoter upstream
of the Salmonella ompD gene was constructed by
?Red-mediated recombination using a DNA fragment
generated by PCR amplification (JVO-2191/JVO-2192)
on pVP192-1 as template. Transformants were screened
by PCR (JVO-0802 and JVO-2192), and a P22 lysate of
a positive clone was used to transduced JVS-1574 (to
obtain JVS-9488), JVS-8726 (to obtain JVS-9491) and
JVS-8798 (to obtain JVS-9655).
cassetteof
?Red-derived
byPCRamplification
PCRusing JVO-0802/
Bacterial growth conditions
Cells were grown aerobically in Luria Broth (LB) medium
at 37?C unless stated otherwise. A final concentration of
0.2% L-arabinose was added to cultures to induce expres-
sion from pBAD-derived plasmids. Where appropriate,
liquid and solid media were supplemented with antibiotics
at the following concentrations: 100mg/ml ampicillin,
50mg/mlkanamycin,20mg/ml
15mg/ml tetracycline. To apply osmotic shock, cells were
cultured at 37?C in M9 minimal medium [1? M9 salts,
2mM MgSO4, 0.1mM CaCl2, 0.4% (v/v) glycerol, supple-
mented with thiamine (0.5mg/ml), L-histidine (40mg/ml)
and cas-amino acids (0.2%)] to an OD600 of 0.3.
chloramphenicol and
Cultures were split and NaCl was added to one batch at
a final concentration of 0.3M. To apply heat shock, cells
were grown in LB at 30?C to an OD600of 0.3. Cultures
were split and growth was continued at either 30?C or
44?C. Stringent response was induced by addition of
serine hydroxamate (SHX; final concentration: 100mM)
to cells during exponential growth (OD600 of 0.15) in
Nutrient Broth (Difco, #234000) supplemented with
0.75mM L-serine. Envelope stress was induced by poly-
myxin B (final concentration: 5mg/ml) in cells grown in LB
to early stationary phase (OD600of 1.5).
In vitro RNA synthesis and determination of SdsR in vivo
copy number
For synthesis of the SdsR in vitro transcript, ?200ng of a
DNAfragmentamplified
(employing primer pair JVO-7023/JVO-7025) served as
template in a T7 transcription reaction using the
Megascript kit (Ambion). The correct size and integrity
of the RNA were confirmed on a denaturing polyacryl-
amide gel. To estimate the in vivo abundance of SdsR
(copy number per cell), total RNA corresponding to 0.5
OD of wild-type Salmonella were compared to serial dilu-
tions of the SdsR in vitro transcript (0.5/1/2.5/5/10 and
20ng) by northern blot and hybridization with an SdsR
riboprobe.
fromSalmonella gDNA
Protein sample analysis
To prepare total protein samples, cells were collected
by centrifugation (2min; 16000g; 4?C) and resuspended
in 1? SLB (Fermentas) to a final concentration of 0.01
OD/ml. For 11% SDS–PAGE, 0.1 OD were loaded per
lane and gels were stained overnight with Coomassie
Blue. To analyse protein levels by western blot, 0.02 OD
(detection of OmpD-GFP fusion proteins and OMPs) or
0.1 OD (detection of RpoS and ribosomal protein S1)
were loaded per lane and resolved by SDS–PAGE, after
which proteins were transferred to PVDF membranes as
described in ref. (57). GFP fusion proteins and RpoS were
detected using commercially available antibodies directed
against GFP (1:5000; mouse; Roche) and RpoS (1:1000;
mouse; #W0009, Neoclone), repectively. The major
OMPs, and ribosomal protein S1, were detected using
an antiserum recognizing
rabbit; provided by R. Misra) and an anti-S1 antibody
(1:5000; rabbit; provided by M. Springer), respectively.
Anti-mouse oranti-rabbit
conjugated with horseradish peroxidase (1:10000; GE
Healthcare) were used in all cases. Signals were visualized
using the Western Lightning reagent (PerkinElmer) and an
ImageQuant LAS 4000 CCD camera (GE Healthcare).
OmpC/F/D/A (1:20000;
secondaryantibodies
RNA isolation and northern blot analysis
Total bacterial RNA was isolated from culture aliquots
using TRIzol reagent (Invitrogen) and analysed by
northern blot as previously described (55). Briefly, for
sRNA and mRNA detection, 5 or 10mg of total RNA
was resolved on 4–6%/7 M urea polyacrylamide gels
andelectroblotted.Membranes
with gene-specific 50-end-labelled DNA-oligonucleotides
werehybridized
Nucleic Acids Research,2012, Vol.40, No. 83625

Page 4

or
Roti-Hybri-Quick hybridization solution (Roth) and
washed in three subsequent steps with SSC wash buffers
supplemented with 0.1% SDS (5?/1?/0.5? SSC or
2?/1?/0.5?, respectively). Signals were determined on a
Typhoon FLA 7000 phosphorimager (GE Healthcare)
and band intensities quantified with AIDA software
(Raytest, Germany). SraC, SdsR and its variants,
the TMA chimera, ompD mRNA and 5S rRNA,
were detected by the 50-end-labelled oligonucleotides
JVO-2390,JVO-1032, JVO-0396,
JVO-0322, respectively. Riboprobes were synthesized by
T7-mediated in vitro transcription of ?200ng of template
DNA (amplified on gDNA with JVO-7692/JVO-7693 or
JVO-0902/JVO-0997 for osmY mRNA or SdsR sRNA,
respectively) in the presence of
MAXIscript kit (Ambion).
riboprobesat42?Cor65?C,respectively,in
JVO-4314 and
32P-a-UTP with the
30-RACE
30-RACE experiments were carried out following the
protocols in refs. (5) and (52) with a few modifications.
Briefly, 7.5mgoftotal
dephosphorylated with 10U of calf intestine alkaline
phosphatase (CIP; #M0290, New England Biolabs) in
the presence of 1? NEB buffer 3 in a total volume of
25ml at 37?C for 1h. Following P:C:I extraction, the
RNA was precipitated from the aqueous phase together
with 250pmol of RNA adapter E1 and 15mg GlycoBlue
(#AM9515, Ambion) using three volumes of 30:1 etha-
nol:sodium acetate (pH 6.5) mix. For ligation of the
RNA linker, the pellet was resuspended in H2O and
allowed to dissolve for 10min at 65?C, after which a
20ml reaction containing 20U T4 RNA ligase (#M0204,
New England Biolabs), 1? T4 RNA ligase buffer, 10%
(v/v) DMSO and 10U SUPERaseIn RNase Inhibitor
(#AM2694, Ambion) was incubated at 16?C overnight.
TheligatedRNA was
precipitated with three volumes of 30:1 ethanol:sodium
acetate (pH 6.5) mix. The RNA was reverse transcribed
for 5min at 50?C and 60min at 55?C in the presence of
adapter E1-specific oligo E3RACE (39pmol) using 200U
SuperscriptIIIreversetranscriptase
Invitrogen) in a 20ml reaction mix containing 1? FS
buffer, 2mMdNTPs,
SUPERaseIn RNase Inhibitor. Template RNA was
digested by RNase H. To identify ompD-specific frag-
ments, 1ml aliquots of the RT reaction were used as tem-
plates in a PCR reaction with 1mM each linker-specific
primer, E3RACE and gene-specific primer, JVO-2678
(binds at the transcriptional start of ompD mRNA),
1.25U Taq-DNApolymerase
ThermoPol buffer and 1.5mM dNTPs. Cycling conditions
were as follows: 95?C for 5min; 35 cycles of 95?C for 40s,
58?C for 40s, and 72?C for 50s and 72?C for 5min). The
PCR products were resolved on 3.5% agarose gels.
Selected bands were purified and subcloned using the
TOPO TA Cloning Kit (pCR 2.1-TOPO, Invitrogen) as
recommended by the manufacturer. Inserts of obtained
clones were amplified by PCR (M13fwd/M13rev) and
analysed by sequencing.
DNA-free RNAwas
P:C:I-extractedand
(#18080-093,
5mMDTT and10U
(#M0267,NEB),1?
b-galactosidase assay
Levels of b-galactosidase expressed from the single-copy
transcriptional sdsR-lacZ
(JVS-9145) fusions were assayed from three biological rep-
licates as follows: at selected timepoints, cells were collected
by centrifugation (2min, 16000g, 4?C) and resuspended in
Z-Buffer to a final concentration of 1 OD/ml. After the
addition of 0.15 vol. equiv. chloroform and 0.1 vol.
equiv. 0.1% SDS, samples were vigorously vortexed for
15s and stored on ice. In a microtiter plate, 200ml of
each cell lysate were mixed with 40ml ONPG (40mg/ml)
and the absorbances at OD405and OD600determined at
28?C over time (0–45min) with a Victor3plate reader
(1420 Multilabel Counter,
b-galactosidase levels were determined at timepoints at
which the absorbance of o-nitrophenol (OD405) increased
linearly with time and was within the linear response range
of the detector. Absorbance at OD600was measured to
control for the amounts of cell debris.
(JVS-8717)or osmY-lacZ
PerkinElmer).Relative
Sequence retrieval and alignments
Information for sequence alignments was collected using
BlastN searches(http://www.ncbi.nlm.nih.gov/sutils/
genom_table.cgi) of the following genome sequences (ac-
cession numbers are given in parentheses): Salmonella
typhimurium LT2 (NC_003197), Salmonella typhi Ty2
(NC_004631),Citrobacter
(NC_009792), E. coli K12 (NC_000913), S. flexneri 2a
str 301 (NC_004337), Enterobacter Sp.638 (NC_009436),
Cronobacter turicensis z30232 (NC_013282), Klebsiella
pneumoniae 342 (NC_011283), Serratia proteamaculans
568 (NC_009832), Yersinia pestis KIM (NC_005088),
Yersiniaenteroliticasubsp.
(NC_008800), Dickeya dadantii Ech 703 (NC_012880),
Pantoea ananatis LMG 20103 (NC_013956), Sodalis
glossinidiusstr.‘morsitans’
pyrifoliae Ep1/96 (NC_003197), Photorhabdus luminescens
subsp. laumondii TT01 (NC_005126) and Xenorhabdus
nematophila ATCC 19061 (NC_014228). Alignments
were made using MultAlin (http://multalin.toulouse.inra
.fr/multalin/multalin.html).
koseri ATCC BAA-895
enterolitica8081
(NC_007712),Erwinia
RESULTS
Expression of SdsR but not SraC is conserved in
diverse enterobacteria
The sdsR gene was originally discovered in E. coli down-
stream of the conserved yebY gene on the minus strand of
the chromosome, and had predicted homologues in
several other species. Here, inspection of available
genomesequencesidentified
g-proteobacteria, representative examples of which are
shown in Figure 1A. In contrast, the sraC gene encoded
on the plus strand was only found in a subset of these
organisms (Figure 1A, Supplementary Figure S1).
To determine how expression patterns of the sdsR-sraC
locus compared among related bacteria, we probed RNA
samples of E. coli, Salmonella and Shigella grown in rich
sdsRgenesin many
3626 Nucleic Acids Research, 2012,Vol.40, No. 8

Page 5

medium from exponential to stationary phase (Figure 1B).
The three species uniformly showed a highly stationary
phase-specific accumulation of full-length SdsR and a
faint, ?70nt processing product (30-end of the sRNA),
consistent with previous observations in E. coli (1,2). In
contrast, probing of SraC yielded a mosaic picture: the
SraC species of Shigella and Salmonella were ?35nt
longer than their E. coli counterpart, and their expression
peaked much earlier during growth. The difference in
RNA size reflects the fact that the mapped ?10 box of
the sraC promoter element in E. coli (5) is located ?35bp
further upstream in Shigella and Salmonella, while the
30-end is conserved (Supplementary Figure S1). All in
all, there is considerable variability in both the sequence
and the expression of SraC, in contrast with the uniform-
ity of SdsR, which argues that SdsR is the primary sRNA
expressed by this locus.
SdsR is abundant and transcribed by pS
A previous RNomics (cDNA shot-gun cloning) approach
revealed SdsR to be among the most abundant sRNAs in
stationary phase E. coli cells (2). Here, we have quantita-
tively corroborated this finding by determining the in vivo
copy number of SdsR at various stages of growth in
Salmonella. To this end, cellular levels of SdsR RNA
from different growth phases were compared on a
northern blot along with an in vitro-synthesized SdsR
transcript (Figure 1C). This analysis shows that SdsR
RNA levels increase almost 30-fold from exponential to
stationary phase and that when accumulation plateaus
about 3h after the cells reach an OD600of 2.0 (early sta-
tionary phase) the sRNA is present in approximately 300
copies/cell.
Given the high stationary phase specificity of sdsR
expression, we hypothesized that the sdsR promoter is
AB
C
Figure 1. (A) Genomic context of the sdsR gene in various Enterobacteria. Synteny analysis of the sdsR/sraC genes in various enterobacterial species
revealed partial conservation of the locus. In all cases sdsR is positioned downstream of yebY or homologues; the flanking gene at the 30-end is
variable. Distances to flanking genes are indicated in bp. STM: Salmonella typhimurium; STY: Salmonella typhi; CKO: Citrobacter koseri; ECO:
Escherichia coli; SFL: Shigella flexneri; ENT: Enterobacter; CTU: Cronobacter turicensis; KPN: Klebsiella pneumoniae; SPR: Serratia proteamaculans;
YPE: Yersinia pestis; YEN: Yersinia enterolitica; DDA: Dickeya dadantii; PAN: Pantoea ananatis; SGL: Sodalis glossinidius; EPY: Erwinia pyrifoliae;
PLU: Photorhabdus luminescens; XNE: Xenorhabdus nematophila. (B) Expression levels of SdsR and SraC sRNAs in Salmonella, E. coli and Shigella.
Northern blot analysis of total RNA isolated from wild-type Salmonella (JVS-1574), E. coli (JVS-5105) and Shigella (JVS-0012) cells grown to OD600
of 0.5, 1.0, 2.0 and 3h after cells had reached an OD600of 2.0. SdsR and SraC sRNAs were detected by radio-labelled oligo probes directed against
the conserved sRNA sequences. 5S rRNA levels were determined to confirm equal loading. (C) SdsR copy number over growth. SdsR levels at
various timepoints (OD600of 0.5, 1.0, 2.0 and 2, 3, 4 or 6h after cells had reached an OD600of 2.0) in wild-type Salmonella were compared by
northern blotting to signals of in vitro transcribed SdsR in indicated amounts. SdsR was detected using a riboprobe; probing for 5S RNA confirmed
equal loading. Expression of RpoS at different growth stages was determined by western blotting. Detection of ribosomal protein S1 was used as
loading control.
Nucleic Acids Research,2012, Vol.40, No. 83627