JOURNAL OF BACTERIOLOGY, Mar. 2008, p. 1956–1965
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 190, No. 6
Variation in the Group B Streptococcus CsrRS Regulon and Effects
Sheng-Mei Jiang,1,2Nadeeza Ishmael,3Julie Dunning Hotopp,3Manuela Puliti,4Luciana Tissi,4
Nikhil Kumar,3Michael J. Cieslewicz,1,2Herve ´ Tettelin,3and Michael R. Wessels1,2*
Division of Infectious Diseases, Children’s Hospital Boston,1and Harvard Medical School,2Boston, Massachusetts 02115;
The J. Craig Venter Institute, Rockville, Maryland 208503; and Microbiology Section, Department of
Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia, Italy4
Received 16 October 2007/Accepted 8 January 2008
CsrRS (or CovRS) is a two-component regulatory system that controls expression of multiple virulence
factors in the important human pathogen group B Streptococcus (GBS). We now report global gene expression
studies in GBS strains 2603V/R and 515 and their isogenic csrR and csrS mutants. Together with data reported
previously for strain NEM316, the results reveal a conserved 39-gene CsrRS regulon. In vitro phosphorylation-
dependent binding of recombinant CsrR to promoter regions of both positively and negatively regulated genes
suggests that direct binding of CsrR can mediate activation as well as repression of target gene expression.
Distinct patterns of gene regulation in csrR versus csrS mutants in strain 2603V/R compared to 515 were
associated with different hierarchies of relative virulence of wild-type, csrR, and csrS mutants in murine models
of systemic infection and septic arthritis. We conclude that CsrRS regulates a core group of genes including
important virulence factors in diverse strains of GBS but also displays marked variability in the repertoire of
regulated genes and in the relative effects of CsrS signaling on CsrR-mediated gene regulation. Such variation
is likely to play an important role in strain-specific adaptation of GBS to particular host environments and
pathogenic potential in susceptible hosts.
Many bacterial species utilize two-component systems
(TCS) as a means to regulate gene expression in response to
signals from the environment (1, 27). While several variations
exist, the basic model of a TCS consists of a sensor histidine
kinase that usually is positioned at the cell surface or periplas-
mic space to interact with external stimuli. Contact with an
appropriate stimulus triggers a conformational change in the
sensor protein that alters the autokinase activity of its cyto-
plasmic domain. Subsequent transfer of the phosphate group
from the sensor to a cognate regulator component, in turn,
modulates the activity of the regulator as a transcriptional
repressor or activator of one or more target genes. Coordinate
regulation of gene expression in response to environmental
cues may be especially important for an organism like group B
Streptococcus (GBS [S. agalactiae]) that exists in a commensal
relationship with its human host as an asymptomatic colonizer
of the genital and gastrointestinal tracts but has the potential
to cause invasive infection during pregnancy and childbirth, in
the colonized infant during the first weeks of life, or in the
setting of concomitant chronic illness or advanced age (7, 26).
Regulated changes in expression of virulence factors and met-
abolic pathways enhance the organism’s adaptation to survive
in the varied niches encountered in its existence as a commen-
sal or as an invasive pathogen.
In keeping with the adaptation of GBS to diverse host en-
vironments, the genome sequences of GBS strains 2603V/R
(hereafter referred to as 2603) and NEM316 revealed 17 and
20 predicted TCS, respectively (9, 30). Of these, the CsrRS (or
CovRS) system has been investigated most thoroughly. Two
independent studies reported that inactivation of csrR or csrRS
resulted in increased expression of the cyl operon encoding the
GBS ?-hemolysin/cytolysin and a corresponding increase in
hemolytic activity as well as a marked decrease in expression of
cfb and its product, CAMP factor, that enhances the hemolytic
activity of staphylococcal sphingomyelinase (12, 15). Both
groups demonstrated, as well, that csrR mutants were attenu-
ated in rodent models of GBS infection, a finding that sup-
ported the importance of CsrRS in pathogenesis.
Transcriptional profiling experiments using genomic mac-
roarrays found evidence that CsrRS influenced expression of
more than 100 genes in the type III GBS strain NEM316 (15).
In contrast to the orthologous CsrRS (CovRS) system in Strep-
tococcus pyogenes strain MGAS5005, in which CsrR is reported
to act chiefly as a repressor, studies in GBS revealed similar
numbers of activated and repressed genes (10, 15).
These initial studies provided important insights into the
function of the CsrRS system as a global regulator of GBS
gene expression that is likely to play a critical role in patho-
genesis of GBS infection. However, many key features of this
important regulatory system remain obscure. While the genes
encoding CsrRS are highly conserved, the repertoires of genes
regulated by the system appear not to be identical among GBS
strains. For example, the cps operon that directs capsular poly-
saccharide biosynthesis was regulated to a modest degree in
NEM316 (15). In contrast, measurement of transcripts of cpsE
and of type-specific capsular polysaccharide revealed no sig-
nificant difference between csrR mutants and their respective
* Corresponding author. Mailing address: Division of Infectious
Diseases, Children’s Hospital Boston, 300 Longwood Ave., Boston,
MA 02115. Phone: (617) 919-2900. Fax: (617) 730-0254. E-mail:
† Supplemental material for this article may be found at http://jb
?Published ahead of print on 18 January 2008.
wild-type parent strains in either 2603 or 515 (12). A compar-
ison of csrS and csrR mutants in strains 2603 and 515 revealed
similar but less extreme changes in expression of three regu-
lated genes in csrS mutants compared to csrR mutants, but
whether both CsrS and CsrR have similar relative effects on
expression of the entire repertoire of regulated genes has not
been investigated previously (12). Finally, Lamy et al. demon-
strated binding of the CsrR protein to the promoter regions of
three genes whose expression is repressed by CsrR (15). How-
ever, it remains to be determined whether genes for which
expression is activated by CsrR also are regulated by direct
binding of CsrR or rather by repression of intermediate regu-
In the present investigation, we used genomic microarrays to
perform transcriptional profiling of csrR and csrS mutants in
the background of strains 2603 and 515. We compared the
repertoire of CsrRS-regulated genes in these strains with that
described previously for strain NEM316 to determine the ex-
tent of conservation and diversity of the CsrRS regulon among
GBS strains. We found considerable diversity in the CsrRS
regulons and evidence for both activation and repression of
target genes. Direct binding studies using recombinant CsrR
revealed binding to both positively and negatively regulated
promoters. Finally, we found that divergent patterns of regu-
lation in csrS and csrR mutants were associated with strain-
specific alterations in virulence. The results imply that variabil-
ity in the CsrRS regulon may contribute to adaptation of
particular GBS strains to specific host niches.
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. GBS strains used in this
study included type Ia strain 515 (32) and type V strain 2603 (2603V/R) (30) and
their derivative ?csrR and ?csrS mutants (12). GBS was grown in liquid culture
in Todd-Hewitt broth (THB; Difco), on trypticase soy agar (TSA) supplemented
with 5% defibrinated sheep blood (PML Microbiologicals), or on Todd-Hewitt
agar supplemented with antibiotics and 5% defibrinated sheep blood. Escherichia
coli DH5? and E. coli M15(pREP4) were grown in Luria-Bertani broth or on
Luria-Bertani agar. When appropriate, antibiotics were added at the following
concentrations: ampicillin, 100 ?g/ml; and kanamycin, 25 ?g/ml for E. coli. GBS
was grown without shaking in liquid culture. E. coli was grown with shaking at
37°C. Plasmid pGEM-T (Promega) was used for the direct cloning of PCR
products; plasmid pQE30 was used for the expression of recombinant His6-
tagged CsrR (Qiagen).
RNA isolation. GBS strains grown overnight on TSA blood agar plates were
inoculated in 10 ml THB broth and collected by centrifugation (3,200 ? g, 5 min)
at mid-exponential-phase growth (optical density at 650 nm of 0.3). The pellet
was resuspended in 0.5 ml 0.9% NaCl and 1 ml RNA Protect buffer (Qiagen) and
kept at room temperature for 5 min. After centrifugation, the bacterial pellet was
treated for 15 min at 37°C with 100 U mutanolysin (Sigma) and 15 mg/ml
lysozyme (Sigma) in Tris-EDTA buffer, pH 8.0, in a final volume of 100 ?l. Total
bacterial RNA was then isolated using an RNeasy mini kit (Qiagen) accord-
ing to the manufacturer’s instructions. RNA samples were treated with
DNase I (Invitrogen) for 30 min at 37°C to remove any contaminating DNA.
The RNA concentration was adjusted to 100 ng/?l, and samples were stored
at ?80°C until use.
Microarray fabrication. GBS amplicon microarrays were prepared as de-
scribed previously (30) using DNA fragments of the annotated open reading
frames (ORFs) from GBS strain 2603 (30). Additional primer pairs were de-
signed to replace unresponsive 2603 amplicons and to include additional ORFs
from strain A909, a serotype Ia strain (29). PCR primer pairs were designed with
Primer3 (23) and locally developed Perl scripts. DNA fragments were amplified
with these primers using a final concentration of 1? AmpliTaq buffer (Applied
Biosystems, Blanchburg, NJ), 2.5 mM MgCl2, 0.8 mM deoxynucleoside triphos-
phate (dNTP) mix (Applied Biosystems), 1.25 U AmpliTaq DNA polymerase
(Applied Biosystems), 0.15 ?M each primer, and 20 ng/?l DNA with denatur-
ation at 95°C for 5 min followed by amplification with 35 cycles at 95°C for 45 s,
55°C for 45 s, and 72°C for 45 s and a final elongation step at 72°C for 10 min.
Amplicons were purified using Montage 96 well SEQ plates (Millipore, Billerica,
MA) and spotted onto UltraGAPS aminosilane-coated glass slides (Corning,
Corning, NY) using 50% dimethyl sulfoxide as the spotting buffer. Amplicons
were bound to the slides by UV cross-linking at 25,000 ?J/cm2. Printed slides
were stored until use in a benchtop desiccator. The final array contains amplicons
for 2,086 genes from strain 2603 and 206 genes from strain A909, representing
96.4% and 97.0% of the annotated ORFs in the 2603 and A909 strains, respec-
tively. The slide design has been deposited in ArrayExpress as A-TIGR-25,
A-TIGR-26, and A-TIGR-27.
Probe labeling and hybridization. Total RNA (2 ?g) from each sample was
reverse transcribed into single-stranded cDNA using 1? first strand buffer (In-
vitrogen), 10 mM dithiothreitol (DTT), 6 ?g random hexamers (Invitrogen), 0.5
mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.3 mM dTTP, 0.2 mM aminoallyl-
dUTP (Invitrogen), and 400 U SuperScript II reverse transcription (RT) enzyme
(Invitrogen). Cy dyes were chemically coupled to the incorporated aminoallyl-
dUTP using Cy3- or Cy5-NHS-ester fluorescent dyes (Amersham-Pharmacia,
Slides were prehybridized in 8% goat serum or 1% bovine serum in 5? SSC
(1? SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% sodium dodecyl
sulfate (SDS) for 60 min at 42°C, washed in water and then isopropanol, and
dried by centrifugation (20). Cy dye-labeled probes from mutant and wild-type
strain RNAs were resuspended in hybridization buffer containing 50% formam-
ide, 5? SSC, 0.1% SDS, and 0.6 ?g/?l salmon sperm DNA (Applied Biosystems)
and hybridized to the microarray slide at 42°C for 16 to 20 h in a sealed,
humidified chamber (Corning) (20). Following hybridization, slides were sequen-
tially washed in 2? SSC and 0.1% SDS for 10 min at 55°C, 0.1? SSC and 0.1%
SDS for 10 min at room temperature, 0.1? SSC for 10 min at room temperature,
and deionized water for 5 min at room temperature and then dried by centrif-
ugation (20). RNA was separately isolated twice, and probes were prepared (see
above) and hybridized to the array, resulting in two biological replicates for each
experimental condition. Technical replication consisted of (i) ?3-fold spotted
replication on a single slide; (ii) hybridization of each RNA sample ?4 or more
times, including two dye-swap replicates; and (iii) duplicate or triplicate ampli-
cons for a subset of genes. This level of replication resulted in ?28 data points
for each gene per experimental condition.
Scanning and image analysis. Slides were scanned using an Axon 4000B
microarray scanner (Axon Instruments, Union City, CA) at 10-?m resolution.
Data were saved as two independent 16-bit TIFF files corresponding to the Cy3
and Cy5 channels and were analyzed using TIGR Spotfinder to assess relative
expression levels (24). Data from TIGR Spotfinder were stored in MAD, a
relational database designed to effectively capture microarray data (24). The data
from this study have been deposited in ArrayExpress (E-TIGR-131).
Data normalization and analysis. To adjust for differences in labeling and
detection efficiencies of the fluorescent labels, the data were normalized using
the MIDAS software tool (24). A low-intensity filter (100 K) was used to elim-
inate background fluorescence; data were normalized with iterative log mean
centering (3). Ratios were calculated as the log2(mutant/wild-type) for all spots
with fluorescence above background. The mean and standard deviation were
calculated from all of the log ratios for a given gene across all technical and
biological replicates using local Perl scripts.
Identification of regulated genes potentially organized in operons. Chromo-
somal DNA sequences flanking CsrRS-regulated genes were inspected to iden-
tify potential operons on the basis of gene orientation and the presence of
predicted Rho-independent terminators of transcription. Genes were added to
the final list of regulated genes displayed in Table S1 in the supplemental
material if they completed putative CsrRS-regulated operons but did not meet
the threshold value to be classified as up- or down-regulated in the CsrR and/or
CsrS mutant(s) or had fewer than 18 hybridization data values.
Search for potential promoter motifs upstream of regulated genes/operons.
Intergenic regions upstream of CsrRS-regulated single genes or operons were
searched for the presence of conserved motifs using AlignAce (http://atlas.med
.harvard.edu/) and BEST (4). BEST (Binding-site Estimation Suite of Tools)
runs, optimizes, and compares the results of four motif-finding programs: Align-
Ace, BioProspector, CONSENSUS, and MEME. Intergenic regions were sub-
mitted to the programs as follows: (i) all up- and down-regulated genes from
each experiment, (ii) all up-regulated genes from each experiment, and (iii) all
down-regulated genes from each experiment. Overrepresented motifs were iden-
tified and used to search the entire genome sequence of strains 2603 and 515. For
each motif, the frequency of occurrence and location with regard to adjacent
genes/operons were determined, as well as the extent of CsrRS regulation of
VOL. 190, 2008DIVERSITY OF THE GROUP B STREPTOCOCCAL CsrRS REGULON 1957
qRT-PCR. For real-time quantitative RT-PCR (qRT-PCR), cDNA was gen-
erated using 2 ?g of total RNA, 2.5 ng random hexamers (Invitrogen), 0.5 mM
dNTP mix, and 200 U SuperScript III reverse transcriptase (Invitrogen) in a
20-?l reaction mixture with RT at 25°C for 10 min followed by 50°C for 50 min
and termination at 85°C for 5 min. Gene-specific primers (Operon Technologies)
were designed to amplify ?150-bp fragments and to be 100% identical in both
2603 and 515 using Primer3 (23). Transcript levels were quantified using the
Quantitect SYBR green PCR kit (Qiagen). Briefly, cDNA (2 ?l of a 1:100
dilution of the above mixture) was used as template in a reaction containing 1?
QuantiTect SYBR green mix (Qiagen) and gene-specific primers (see Table S2
in the supplemental material). The standard curve for each transcript was gen-
erated using a serial dilution of 515 csrS cDNA and gene-specific primers for
SAG_0944. Reactions were run on an iCycler iQ (Bio-Rad). The reactions were
denatured at 95°C for 15 min followed by amplification with 45 cycles at 94°C for
15 s, 55°C for 30 s, and 72°C for 30 s. Reactions were followed by a melt curve
analysis with a disassociation step at 95°C for 1 min and 55°C for 1 min plus
0.5°C/cycle for 80 cycles.
Microarray analysis. A curated set of regulated data (see Table S1 in the
supplemental material) was assembled from (i) genes ?2-fold regulated in 2603
or 515 with n ? 18 and genes more than 2-fold regulated in NEM316; (ii) genes
?2-fold regulated in at least two strains with n ? 10; and (iii) manual curation
to add likely genes cotranscribed with those in the previous two conditions. The
role category composition of this data set was compared to that of the population
of amplicons on the microarray using Fisher’s exact test with a Bonferroni
stepdown correction for multiple experiments implemented in TIGR MeV (24).
A P value of ?10?4was considered significant.
This subset and the complete data set were clustered using various algorithms
in the TIGR MeV software to facilitate analysis of the data. Support trees were
constructed using Euclidean distance and average linkage with resampling of
experiment and sample trees by bootstrapping. K-means clustering was per-
formed using Euclidean distance and calculated means. Ten clusters were se-
lected and converged after eight iterations. Clustering of the subset and the
complete data set resulted in similar conclusions.
Expression and purification of His6-CsrR. Plasmid vector pQE-30 was used
for GBS CsrR protein overexpression in E. coli. A 687-bp PCR-generated
BamHI/HindIII DNA fragment corresponding to the CsrR coding sequence was
amplified using specific primers #880 and #881 (see Table S2 in the supplemen-
tal material) and then cloned between the BamHI and HindIII sites of pQE-30
to give pQE30CsrR. The resulting clone was first transformed into E. coli DH5?
for amplification and then isolated and introduced into the E. coli M15 bearing
the plasmid encoding the lac repressor, pREP-4. The transformants were inoc-
ulated into LB medium containing both ampicillin (100 ?l/ml) and kanamycin
(25 ?g/ml) with 500 ?l of the overnight culture and grown at 37°C until A600?
0.5 to 0.7. Expression of His6-CsrR was induced by the addition of isopropyl-?-
D-thiogalactopyranoside (IPTG) to 1 mM and then followed by 3 h of incubation
at 37°C. Cells were lysed by sonication, and insoluble cell debris was removed by
centrifugation (20,800 ? g, 20 min). The cell lysate was then passed over an
Ni2?-nitrilotriacetic acid agarose column for purification according to the man-
ufacturer’s instructions (Qiagen). His6-CsrR was suspended in 50% glycerol and
stored at ?20°C. The concentration of purified His6-CsrR was estimated by
comparison with a bovine serum albumin standard using the bicinchoninic acid
EMSA. For electrophoretic mobility-shift assays (EMSA), pairs of specific
oligonucleotide primers were used for the PCR amplification of 250- to 305-bp
DNA fragments representing the potential promoter regions of target genes cyl
(?-hemolysin), cfb (CAMP factor), and scpB (C5a peptidase) (see Table S2 in the
supplemental material). The purified PCR products were end labeled with ?-[S-
32P]dATP in the presence of T4 polynucleotide kinase using the Promega gel
shift assay system kit. Labeled probes were purified from free nucleotides on a
G-25 spin column (Amersham). As a negative control, a similar size DNA
fragment was also amplified from the promoter region of the capsule synthesis
(cps) locus. His6-CsrR was serially diluted and added to 10 ng of probe DNA in
binding buffer [20 mM Tris (pH 7.5), 1 mM CaCl2, 1 mM DTT, 10 ?g/ml
poly(dI-dC) and 100 ?g/ml bovine serum albumin] in a total volume of 10 ?l.
DNA and protein were incubated for 15 min at room temperature. The reaction
products were mixed with 2 ?l of 50% glycerol and loaded onto a 5% Tris-
borate-EDTA (TBE) polyacrylamide gel (Bio-Rad; TBE is 90 mM Tris-borate,
2 mM EDTA [pH 8.0]). After loading samples, the gel was run at room tem-
perature in 0.5? TBE buffer at 350 V for 15 to 20 min and then dried and
exposed to X-ray film. In some experiments, purified His6-CsrR was subjected to
in vitro phosphorylation before addition to the assay mixture: 10 ?g His6-CsrR
protein was incubated for 90 min at 37°C with 32 mM acetyl phosphate in freshly
made phosphorylation buffer (20 mM NaH2PO4, pH 8.0, 10 mM MgCl2, 1 mM
DTT) in a total volume of 100 ?l.
Mouse virulence studies. Adult outbred male CD-1 mice were obtained from
Charles River Breeding Laboratories (Calco, Italy). The animals were 6 to 8
weeks of age at the beginning of each experiment. GBS strains were grown
overnight at 37°C in THB (Oxoid, Ltd., Basingstoke, England), washed, and
diluted in RPMI 1640 medium (Gibco Life Technologies, Milan, Italy). The
inoculum size was confirmed by quantitative cultures. Mice were inoculated
intravenously via the tail vein with different infecting doses of GBS in a volume
of 0.5 ml. Control mice were injected by the same route with 0.5 ml of RPMI
1640 medium. Mortality was recorded at 24-h intervals for 30 days. The 50%
lethal dose (LD50) was calculated by the method of Reed and Muench (22).
GBS-infected mice were evaluated for signs of arthritis and mortality. After
challenge, mice were examined daily by two independent observers for 1 month
to evaluate the presence of joint inflammation. Arthritis was defined as visible
erythema and/or swelling of at least one joint. Clinical severity of arthritis was
graded on a scale of 0 to 3 for each paw, according to changes in erythema and
swelling (0 ? no change; 1 point ? mild swelling and/or erythema; 2 points ?
moderate swelling and erythema; 3 points ? marked swelling, erythema, and/or
ankylosis). Thus, a mouse could have a maximum score of 12. The arthritis index
(mean ? standard deviation) was calculated by dividing the total score (cumu-
lative value of all paws) by the number of animals in each experimental group.
Microarray data accession numbers. The slide design for this study has been
deposited in ArrayExpress as A-TIGR-25, A-TIGR-26, and A-TIGR-27. The
microarray data from this study have been deposited in ArrayExpress (E-
To investigate the function of the CsrRS TCS in GBS, we
previously constructed mutants in the background of GBS type
Ia strain 515 and type V strain 2603 (12). A nonpolar inacti-
vating mutation was introduced into csrR (515?csrR or
2603?csrR) or csrS (515?csrS or 2603?csrS). Analysis of ex-
pression patterns of a limited number of genes revealed in-
creased expression of cylE (?-hemolysin/cytolysin) and scpB
(C5a peptidase) and reduced expression of cfb (CAMP factor)
in the csrR mutants in both strain backgrounds and similar,
though less extreme, changes in the csrS mutants (12). In the
present study, we used GBS genomic microarrays as a more
comprehensive means to investigate genome-wide changes in
gene expression that result from inactivation of csrR or csrS in
the same two GBS strain backgrounds.
Inactivation of csrR or of csrS was associated with altered
expression of a large number of genes in both strain back-
grounds (Table 1). Using as a threshold a twofold change in
gene expression between the mutant and wild type, we found
evidence of CsrRS regulation of 134 genes in strain 2603 and
TABLE 1. Numbers of regulated genes in csrR and csrS mutants
of GBS strain 2603 or 515 relative to their respective
wild-type parent strainsa
Mutant genotypeRegulation pattern
No. of genes in
aThe threshold is a twofold change in transcript abundance.
1958JIANG ET AL. J. BACTERIOL.
80 genes in strain 515. One significant difference between the
2603 and 515 strains was the presence of 12 IS1381 transposase
subunits found to be up-regulated in 2603?csrR. A glycosyl-
transferase (SAG_1548, SAG_1551) in the up-regulated and
cotranscribed SAG_1548 to -1555 is disrupted by an IS1381
transposase (SAG_1549, SAG_1550). Although the IS1381
transposase genes are on the opposite strand relative to the
cotranscribed unit, the amplicon-based microarray queries
both the transcript and its reverse complement. Therefore,
up-regulation of genes SAG_1548 to -1555 results in what is
likely to be artifactual “up-regulation” of the inserted trans-
posase. Further, the high nucleotide identity of all the trans-
posases results in positive results for all chromosomal loca-
tions. Therefore, the IS1381 transposases were removed from
subsequent analyses, as it is unlikely that this apparent up-
regulation is biologically relevant.
Bioinformatics software was used to search for putative
CsrR-binding motifs associated with CsrRS-regulated individ-
ual genes and operons in the genome sequences of strains 2603
and 515. Among a number of candidate motifs, we failed to
identify any that were both overrepresented in intergenic re-
gions and preferentially located upstream of CsrRS-regulated
genes. Therefore, this analysis did not support the earlier sug-
gestion of a distinct CsrR binding sequence (15) but suggested
rather that CsrR recognizes regions of DNA that are not
readily identified by a canonical nucleotide sequence motif.
Validation of microarray results by qRT-PCR. To confirm
the changes in transcript abundance observed by microarray
hybridization, we performed qRT-PCR for a subset of regu-
lated genes using RNA samples from mutant and wild-type
strains. Nine genes were selected for qRT-PCR testing using
RNA samples from 515?csrR, 515?csrS, 2603?csrR, and
2603?csrS, and their respective wild-type parent strains. The
change (fold) in gene expression by qRT-PCR correlated well
with those calculated from the microarray experiments in each
of the mutant strains (R2? 0.94; see Fig. S1 and Table S3 in
the supplemental material).
Characterization of the CsrRS regulon in GBS strains 2603
and 515. Transcriptional analysis using genomic microarrays
confirmed the previously reported regulation of known or pu-
tative virulence factors including the cyl operon (SAG_0662 to
-0673) encoding the GBS ?-hemolysin/cytolysin, scpB (SAG_
1236; C5a peptidase), and cfb (SAG_2043, CAMP factor), as
well as a second gene transcriptionally linked to cfb (SAG_
2042) that is predicted to encode a rhodanese-like protein of
unknown function (see Table S1 in the supplemental material).
A second locus encoding a predicted protein with 67% amino
acid identity to C5a peptidase was also regulated by CsrRS
(SAG_0416). As reported previously, and in contrast to the
earlier analysis of GBS strain NEM316, we did not find a
consistent pattern of regulation of the cps capsular polysaccha-
ride synthetic operon in strain 2603. In 515?csrR, there was a
trend of down-regulation of the cps operon, but this trend only
reached the twofold threshold for cps1aJ (SAL_1283) and
cps1aH (SAL_1285) among the 16 genes in the cps operon.
Regulated genes in one or both strains encoded proteins
involved in a wide range of cell functions including known or
predicted virulence factors, transporters of amino acids, pep-
tides, sugars, and metals, and proteins that mediate adaptation
to environmental stresses (see Table S1 in the supplemental
material). Among the various functional categories of regu-
lated genes, transporters of amino acids, peptides, and amines
were most significantly overrepresented in the study (P ?
10?7), followed by transport and binding proteins (P ? 10?5)
and pathogenesis genes (P ? 10?5). We found evidence for
regulation of expression of several proteins that are predicted
to be secreted or surface associated, including SAG_0297
(aminopeptidase C), SAG_1002 (putative protease), SAG_
1890 (putative endopeptidase O), and two operons predicted
to encode membrane proteins (SAG_0364 to -365 and SAG_
0798 to -799).
Genes encoding proteins involved in transport of a variety of
substrates constituted a large group of regulated genes. These
included the oppA1-F operon (SAG_0148 to -0152), encoding
an oligopeptide ABC transporter that has been implicated in
modulating the attachment of GBS to host cells and the adc
operon (SAG_0154 to -0156), homologs of which encode a
zinc/manganese transporter in Streptococcus pneumoniae and a
manganese acquisition and homeostasis system in Streptococ-
cus gordonii (6, 16, 25). Also regulated by CsrRS are homologs
of a system involved in iron transport (SAG_1007–1010).
In keeping with the inferred role of CsrRS in adaptation to
changing environments, the system regulates several stress re-
sponse mechanisms in GBS. These included homologs of
AphC and AphF (SAG_1833 to -1834), two components of the
alkyl hydroperoxide reductase of S. pyogenes. In that species,
the alkyl hydroperoxide reductase system contributes to scav-
enging endogenous hydrogen peroxide and has been linked to
virulence in a murine infection model (2). Homologs of the
enterococcal and lactococcal general stress protein Gls24
(SAG_1135 and SAG_1137) were also regulated by CsrRS in
GBS. Gls24 has been implicated in stress response and viru-
lence in Enterococcus faecalis (18, 28). The GBS CsrRS also
regulates expression of two separate operons predicted to en-
code components of a glycine/betaine osmoregulation system
(SAG_1796 to -1797 and SAG_0241 to -0244), a system that
mediates adaptation to osmotic stress in Bacillus subtilis and
Lactococcus lactis (13, 14, 19, 31). Although their role in GBS
is undefined, expression of two predicted transcriptional reg-
ulators is also controlled by CsrRS: SAG_0712 encoding a
putative regulator of the OmpR family and SAG_0938 encod-
ing a predicted GntR family transcriptional regulator (11, 27).
Comparison of the CsrRS regulons in three different GBS
strains. The availability of genome-wide transcriptional profil-
ing data in strains 2603 and 515 together with the previously
described results in strain NEM316 provided the opportunity
to compare the CsrRS regulons in independent GBS isolates
representing the three most important capsular serotypes in
human infection, types Ia (strain 515), III (strain NEM316),
and V (strain 2603). This analysis revealed a core group of 39
genes whose expression was changed as a result of inactivation
of csrR and/or csrS in all three strain backgrounds (Fig. 1 and
Table 2). Two-way comparisons showed further overlap in the
repertoire of CsrRS-regulated genes, with 16 genes regulated
in both 2603 and 515, 18 in 2603 and NEM316, and 3 in 515
and NEM316. For each of the three strains, certain CsrRS-
regulated genes were regulated in only one strain background.
A higher number of uniquely regulated genes were identified
in NEM316, but this may be a result of the differences in array
platform and mutant design between the two studies. These
VOL. 190, 2008 DIVERSITY OF THE GROUP B STREPTOCOCCAL CsrRS REGULON 1959
combined results suggest that CsrRS regulates a conserved
core group of genes in multiple GBS strains, including those
coding for the important virulence factors ?-hemolysin, C5a
peptidase, and CAMP factor, as well as a large repertoire of
genes whose regulation varies among GBS isolates.
Differential effects on gene regulation of inactivating CsrR
versus CsrS. Mutation of csrR in strain 2603 was associated
with increased expression of 94 genes and reduced expression
of 13 genes. This pattern suggests that CsrR acts predomi-
nantly as a transcriptional repressor but that it can also activate
gene transcription, directly or indirectly. Inactivation of csrS
resulted in expression changes of a smaller number of genes
with 36 genes up-regulated in 2603?csrS and 18 genes down-
regulated (Table 1). For 27 genes, we observed altered expres-
sion in both 2603?csrR and in 2603?csrS (Fig. 2). In 23 of these
27 genes, gene expression was up-regulated in both 2603?csrR
and 2603?csrS. For most regulated genes, mutation of csrR
produced a greater effect than did mutation of csrS (see Fig. S2
in the supplemental material). We observed a change in ex-
pression in 2603?csrR but not in 2603?csrS for 80 genes (Fig.
2). For the majority of these genes, the change in expression in
2603?csrS was in the same direction as that in 2603?csrR, but
it did not reach the twofold threshold. For two linked genes,
SAG_1706 (hypothetical protein) and SAG_1707 (putative
drug resistance transporter), expression was increased in
2603?csrR and decreased in 2603?csrS.
While the total number of CsrRS-regulated genes was some-
what lower in strain 515 than in 2603 (Table 1), we observed a
similar pattern with respect to the relative effects of mutation
in csrR versus csrS. That is, the predominant pattern was a
greater effect on target gene expression in 515?csrR than in
515?csrS, but for several genes, the effect was greater in
515?csrS than in 515?csrR (Fig. 3).
CsrR binds directly to promoter regions of both activated
and repressed genes. An earlier investigation of GBS strain
NEM316 used DNase I protection and EMSA to demonstrate
direct binding of recombinant CsrR to a DNA segment up-
stream of the cyl operon (15). DNase I protection experiments
also suggested direct binding to the promoter regions of two
other genes whose expression, like that of the cyl operon, is
repressed by CsrRS. To further characterize the interaction of
CsrR with regulated promoters, we expressed CsrR as a His6
fusion in Escherichia coli and purified the recombinant protein
by Ni2?-affinity chromatography. His6-CsrR was used in
EMSA with DNA probes from strain 2603 that corresponded
to the promoter regions of the cyl operon and scpB (C5a
peptidase), two genes whose expression is repressed by CsrRS;
and cfb (CAMP factor), a gene whose expression is activated
by CsrRS. For all three promoters, band shifts were observed
in the presence of His6-CsrR, indicating direct binding of CsrR
to DNA sequences upstream of both CsrRS-repressed and
TABLE 2. Conserved genes of the CsrRS regulon in three
LocusGene product name or function
expression (CsrR or
SAG_0201 Oxidoreductase, putative
SAG_0267 Conserved hypothetical protein
SAG_0297 Aminopeptidase C
SAG_0371 Hypothetical protein
SAG_0416 Serine peptidase, S8/S53 family
SAG_0662 CylX protein
SAG_0663 CylD protein
SAG_0664 CylG protein
SAG_0665 Acyl carrier protein AcpC
SAG_0666 CylZ protein
SAG_0667 CylA protein
SAG_0668 CylB protein
SAG_0669 CylE protein
SAG_0670 CylF protein
SAG_0671 CylI protein
SAG_0672 CylJ protein
SAG_0673 CylK protein
SAG_0712 DNA-binding response
SAG_0770 Membrane protein, putative
SAG_0771 Cell wall surface anchor family
SAG_1002 Protease, putative
SAG_1050 Ribonucleotide reductase,
SAG_1206 Conserved domain protein
SAG_1548 Glycosyl transferase, group 2
family protein, interruption C
SAG_1551 Glycosyl transferase, group 2
family protein, interruption N
SAG_1552 Conserved hypothetical protein
SAG_1553 Hypothetical protein
SAG_1554 Hypothetical protein
SAG_1641 YaeC family protein
SAG_1642 ABC transporter, substrate-
SAG_1643 Glutamine amidotransferase,
SAG_1706 Conserved hypothetical protein
SAG_1890 Endopeptidase O
SAG_1908 Hypothetical protein
SAG_2042 Rhodanese-like domain protein
SAG_2043 CAMP factor
SAG_2063 Pathogenicity protein, putative
10.95 7.21 2.81
FIG. 1. Conserved and strain-specific gene regulation by CsrRS.
Numbers within the circle corresponding to each GBS strain represent
the number of genes that are CsrRS regulated in one, two, or all three
strains. The number of regulated genes shown here is lower than that
in Table 1 and elsewhere in the text because this analysis only included
genes for which expression data were evaluable in all three strain
1960JIANG ET AL. J. BACTERIOL.
FIG. 2. Differential regulation of gene expression in strain 2603?csrR compared to 2603?csrS. Pairs of bars represent the change (fold) in gene
expression relative to wild type in strains 2603?csrR (filled bars) and 2603?csrS (open bars). Genes are grouped into those with altered expression
in both 2603?csrR and 2603?csrS (A), in 2603?csrS only (B), or in 2603?csrR only (C). Putative operons are indicated by arrows below the
CsrRS-activated genes (Fig. 4). No shift was observed after
incubation of His6-CsrR with a DNA segment corresponding
to the promoter region of the cps operon of strain 2603, a
result that is consistent with the absence of CsrRS regulation
of this locus in strain 2603 and that serves as a negative control
for the specificity of CsrR binding to regulated promoters.
Specificity of the binding interaction for each of the regulated
promoters was also supported by competition with excess un-
labeled probe, but not with excess unlabeled cps promoter
sequences. These results indicate that CsrR binds directly to
both positively and negatively regulated promoter sequences.
Phosphorylation of CsrR increases its affinity for promoter
DNA. Signaling through TCS typically is transduced by phos-
phorylation or dephosphorylation of the regulator component
in response to interaction of the sensor with an environmental
stimulus. In keeping with this general model, the phosphory-
lation state of the regulator has been shown to change its
affinity for target DNA sequences in several TCS in other
species, including the homologous CsrRS system in Streptococ-
cus pyogenes (5, 8, 17). To test the importance of CsrR phos-
phorylation for gene regulation in GBS, we incubated His6-
CsrR with acetyl phosphate to phosphorylate the CsrR protein.
EMSA using a probe for the cyl operon promoter revealed a
minor increase in binding affinity (approximately twofold) for
the phosphorylated compared to unphosphorylated His6-CsrR
(Fig. 5). This modest effect of phosphorylation at the cyl pro-
moter is consistent with the results of Lamy et al., who found
no significant effect of phosphorylation on binding of CsrR to
the cyl promoter (15). In contrast, phosphorylation had a
marked effect on binding of CsrR to the scpB or cfb promoters,
increasing binding affinity by approximately eightfold. Phos-
phorylation also appeared to enhance the formation of higher-
molecular-size complexes, a result that suggests phosphoryla-
tion may promote oligomerization of CsrR. These results
indicate that phosphorylation increases binding of CsrR to
regulated promoters and that individual target promoters dif-
fer in their relative affinities for the phosphorylated versus
unphosphorylated regulator protein.
Inactivation of CsrR or CsrS has strain-specific differential
effects on virulence. The microarray analysis revealed both
qualitative and quantitative differences in the relative effects
on gene regulation of mutating csrR or csrS in the background
of strain 2603 compared to that in 515 (Fig. 2 and 3 and Table
1). Furthermore, opposite regulatory effects were observed for
2603?csrS and 2603?csrR (e.g., with SAG_1706 to -1707),
whereas such divergent effects were not observed in the back-
FIG. 3. Differential regulation of gene expression in strain 515?csrR compared to 515?csrS. Pairs of bars represent the change (fold) in gene
expression relative to wild type in strains 515?csrR (filled bars) and 515?csrS (open bars). Genes are grouped into those with altered expression
in both 515?csrR and 515?csrS (A), in 515?csrS only (B), or in 515?csrR only (C). Putative operons are indicated by arrows below the horizontal
1962JIANG ET AL. J. BACTERIOL.
ground of strain 515. To investigate whether such differential
effects of CsrR compared to CsrS might be reflected in the
relative pathogenic potential of the mutant strains, we tested
the virulence of csrR and csrS mutants and wild-type strains
2603 and 515 in a murine model of systemic infection and
septic arthritis. Mice were challenged intravenously with vari-
ous doses of GBS and observed for development of signs of
arthritis and for mortality. Wild-type strain 515 was the most
virulent in these studies, with an LD50of 7.2 ? 104. The LD50
for 515?csrR was 320-fold higher at 2.3 ? 107, whereas
515?csrS had an intermediate level of virulence (LD50of 8.5 ?
106). This hierarchy of relative virulence is the same as that
reported previously for these strains in a murine intraperito-
neal challenge model (12). Similarly, strain 2603?csrR was
attenuated in virulence (no deaths at challenge doses up to 108
CFU) relative to wild-type strain 2603 (LD50of 2.8 ? 107
CFU), as reported previously for the intraperitoneal challenge
model (12). In striking contrast, strain 2603?csrS was more
virulent (LD50of 2.4 ? 106CFU) than wild-type 2603. This
virulence hierarchy was reflected not only in the relative le-
thality of the three strains in the 2603 background but also in
severity of arthritis, whether scored by number of affected
joints or by clinical severity index (Fig. 6).
These results demonstrate that differential patterns of reg-
ulation by CsrRS in strains 2603 and 515 are associated with
striking differences in the overall relative virulence of csrS
mutants in the two strain backgrounds. In particular, we ob-
served increased expression of certain genes in 2603?csrS, but
not in 2603?csrR or in either mutant in the 515 background. Of
these, possible virulence genes include SAG_1135 and SAG_
1137 that encode homologs of Gls24, a stress response protein
shown to contribute to virulence in experimental enterococcal
infection (18, 28). A similar pattern of regulation was noted for
SAG_1796 and SAG_1797, which are predicted to encode a
glycine/betaine osmoregulation system implicated in adapta-
tion to osmotic stress in other species (13, 14, 19, 31). Differ-
ential CsrS-dependent regulation of these loci in the 2603
strain background may account for the unexpectedly high vir-
ulence of strain 2603?csrS.
FIG. 4. Binding of purified His6-CsrR to promoter regions of
CsrRS-regulated genes. EMSA were performed using32P-labeled DNA
fragments corresponding to the promoter region of the indicated gene or
operon and purified His6-CsrR protein. Lanes contain32P-labeled pro-
moter sequence only (lane 1),32P-labeled promoter sequence plus His6-
CsrR protein (lane 2),32P-labeled promoter sequence plus His6-CsrR
protein plus excess unlabeled promoter sequence (lane 3), or32P-labeled
promoter sequence plus His6-CsrR protein plus excess negative con-
trol promoter sequence (from the cpsA promoter in panels A to C and
from the recA promoter in panel D). Band shifts indicating CsrR
binding are observed for promoter regions of genes whose expression
is up-regulated (cfb, CAMP factor) or down-regulated (cyl operon,
?-hemolysin; and scpB, C5a peptidase), but not for the promoter of the
unregulated cps operon encoding capsular polysaccharide biosynthetic
FIG. 5. Phosphorylation of CsrR enhances binding to regulated
promoters. EMSA were performed using32P-labeled DNA fragments
corresponding to the promoter region of the indicated gene or operon
and increasing concentrations of purified His6-CsrR protein without
prior treatment (untreated) or after incubation with acetyl phosphate
(phosphorylated). Band shifts indicating binding of CsrR were ob-
served at an approximately twofold lower concentration of CsrR after
phosphorylation for the cyl operon promoter, but at approximately
eightfold lower concentration after phosphorylation for the cfb and
FIG. 6. Relative virulence of wild-type strain 2603, 2603?csrS, or
2603?csrR in a murine arthritis model. The values shown represent the
clinical arthritis index after intravenous challenge with 1 ? 107CFU of
the indicated GBS strain. Data represent means ? standard deviations
for three independent experiments, each using 10 animals per exper-
VOL. 190, 2008DIVERSITY OF THE GROUP B STREPTOCOCCAL CsrRS REGULON 1963
Results of the present investigation provide several new in-
sights into the CsrRS TCS in GBS and its potential functions
during infection. Transcriptional profiling studies using
genomic microarrays yielded a comprehensive picture of global
gene regulation by CsrRS in GBS strains 2603 and 515 in
addition to that reported previously for strain NEM316. We
found evidence for an extensive regulon in all three strains,
including genes that encode products known or predicted to
enhance bacterial survival under varied conditions and/or to
contribute to pathogenicity in the human or animal host. Mi-
croarray hybridization experiments and qRT-PCR confirmed
CsrRS regulation of three major virulence determinants iden-
tified in earlier studies: ?-hemolysin, CAMP factor, and C5a-
peptidase. There was also evidence of regulation of several
predicted surface or secreted proteins of unknown function
that may participate in GBS adherence or in modification of
the local host environment.
The largest functional class of CsrRS-regulated genes en-
coded transport systems for various small molecules including
peptides, amino acids, sugars, and metals. This broad regula-
tion of small molecule transporters is consistent with the pos-
tulated role of CsrRS in mediating adaptation of GBS to varied
environmental and nutritional circumstances encountered
within the colonized or infected host. We also found evidence
for CsrRS regulation of stress response systems such as the
OpuA betaine uptake osmoprotection system. Adaptation to
osmotic stress may be especially important for GBS survival at
mucosal sites in which fluid and solute shifts result in changing
osmotic conditions. Similarly, CsrRS appears to regulate ex-
pression of genes encoding alkyl hydroperoxide reductase, an
enzyme implicated in resistance to endogenous hydrogen per-
oxide stress in S. pyogenes. Such resistance may be adaptive
during interaction of GBS with host phagocytes or under other
circumstances of oxidative stress.
Comparison of the repertoire of genes regulated by CsrRS
in three different GBS strains suggests that there is a “core
regulon” of genes that are regulated by CsrRS in multiple
strains, but that there is substantial diversity in the remainder
of the regulon. For the three strains studied to date, the core
regulon consists of 39 genes, including the virulence factors
described in earlier reports. Thirty-seven genes show evidence
of CsrRS regulation in two of the three strains, while 155 genes
are regulated in only one strain. Such variation in CsrRS regu-
lons is likely to be a reflection of the overall genomic variability
in this species (29). Heterogeneity in gene regulation among
individual isolates may account, in part, for differences in ad-
aptation to specific host environments and for the pathogenic
potential of particular strains. The molecular basis for variabil-
ity in CsrRS repertoire remains to be determined and may
involve multiple factors, including strain-specific variation in
promoter sequences of regulated genes and the presence or
absence of additional interacting regulators.
By characterizing changes in gene regulation in csrS mutants
as well as csrR mutants, we uncovered another level of com-
plexity in CsrRS-mediated gene regulation. For most regulated
genes, we observed a similar trend in gene expression in the
csrS mutant as in the csrR mutant, but the change in gene
expression was of a lower magnitude in the csrS mutant. How-
ever, in a significant minority of cases, mutation in csrS, but not
in csrR, resulted in a change in gene expression, or the degree
of change in expression was greater in the csrS mutant than in
the csrR mutant. In two cases, we observed divergent effects on
gene regulation in the csrS compared to csrR mutants. The
most common pattern of regulation—a greater effect by inac-
tivating CsrR than CsrS—is consistent with the basic TCS
model: removal of the transcriptional regulator has a maximal
effect, usually by derepressing target gene transcription. Inac-
tivation of the sensor may have a similar, but often lesser, effect
by preventing signaling that activates or inactivates the regu-
lator. However, other patterns of responses are also possible.
An equivalent effect of inactivating CsrS might be expected for
genes whose promoter regions bind phospho-CsrR with much
higher affinity than the unphosphorylated CsrR, assuming
CsrR phosphorylation is dependent on CsrS signaling. In
agreement with this formulation, we found heterogeneity in
the importance of CsrR phosphorylation in determining the
relative binding affinity of CsrR for different target promoters
in vitro. Alternatively, or in addition, differential effects could
be mediated by interaction of either CsrS or CsrR with other
transcriptional regulators with various specificities for CsrRS-
regulated genes. For example, evidence has been presented
that the serine/threonine kinase Stk1 interacts with CsrR to
regulate expression of ?-hemolysin and CAMP factor in GBS
Strain-to-strain variation in the CsrRS regulon and in the
relative regulatory effects of the sensor and regulator compo-
nents implies that GBS strains vary not only in gene content
but also in their dynamic capacity to adapt to changing envi-
ronments in the host. A particular repertoire of CsrRS gene
regulation may confer a survival advantage or pathogenic po-
tential in a specific microenvironment. This conclusion is sup-
ported by our finding that the virulence of csrR and csrS mu-
tants relative to their respective parent strains differs in strains
2603 and 515. Thus, an individual strain may be better adapted
for survival in a particular host site such as the bovine mam-
mary gland or the human gastrointestinal or genitourinary
tract. In this way, the species as a whole has enhanced adap-
tation to varied host niches. Together, these results provide
further evidence that CsrRS serves as a global regulatory sys-
tem in GBS that functions in both conserved and variable ways
to enhance adaptation of this important pathogen for survival
in the host.
We thank Dennis L. Kasper for helpful advice.
This work was supported in part by NIH grant AI59502.
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