 |
INTRODUCTION |
Staphylococcus aureus is
an important human pathogen. Its pathogenesis is very complex and
probably involves the synthesis of cell wall-associated adhesins and
the secretion of extracellular toxins with damaging effects on host
cells, including those of the immune system (48).
Nevertheless, even the ability of S. aureus to survive
suboptimal growth conditions within the host should be a significant
property which contributes to the virulence of this organism and is
closely connected with the expression of stress genes (14).
In the gram-positive bacterium Bacillus subtilis, the
alternative sigma factor 
B regulates a large number of
general stress genes (2, 7, 9, 47, 61; A. Petersohn
et al., submitted for publication). Some of these genes are involved in
the protection of DNA, membranes, and proteins against oxidative
damage, which might represent an important component within the stress
response of glucose-starved cells (4, 21, 55). Moreover,
B-dependent proteins contribute to survival under
extreme environmental conditions such as heat or osmotic stress,
repeated freezing and thawing, and acid or alkaline shock of starving
B. subtilis (23, 63). In summary, the
B regulon is expected to provide multiple stress
resistance to starving B. subtilis cells in anticipation of
future stress (26, 60).
A similar physiological role has been postulated for the RpoS
(S) regulon in Escherichia coli and
Salmonella enterica serovar Typhimurium (27, 39).
In this context, it is interesting that orthologues of the
B-dependent genes like katE, dps,
opuE, and osmC in B. subtilis are
regulated by RpoS in E. coli (4, 20, 39, 57, 62). Since rpoS mutants of gram-negative pathogens show
significantly reduced virulence (45, 51, 66), it has been
suggested that in pathogenic gram-positive bacteria, the
B regulon also has a function in the ability of bacteria
to interact with host defense mechanisms and persist during infection.
Over the last few years, B was identified in the
gram-positive pathogens S. aureus (34, 67),
Mycobacterium tuberculosis (16), and
Listeria monocytogenes (6, 65). As expected, S. aureus and L. monocytogenes
B mutant cells showed diminished stress tolerance
compared with wild-type cells (10, 35, 44, 65). Recent
results concerning the involvement of B in the virulence
of these bacteria do not support the idea that B plays a
significant role in infection processes (10, 35, 44).
However, the question arises of whether the infection models analyzed
until now really reflect the natural situation in the host.
In order to elucidate the function of B in the
pathogenesis of S. aureus, it is necessary to know the genes
which are under the control of this alternative sigma factor. Until
now, only a few proteins have been identified that belong to the
B regulon in S. aureus, among them
asp23 and coa (25, 35, 43). It has
also been demonstrated that the transcription of sar,
encoding a global regulator which controls the synthesis of a variety
of extracellular and cell surface proteins involved in the pathogenesis of S. aureus, is partly regulated by B
(17, 41). Therefore, it was very surprising that a
sigB mutation is associated with an enhanced SarA level
(13). The overproduction of alpha-hemolysin, thermonuclease,
and some other extracellular proteins might be the consequence of the
up-regulation of SarA in the mutant (13, 35). The role of
B in the regulation of SarA remains obscure and needs to
be further analyzed.
The discovery and functional characterization of new
B-dependent proteins should improve our understanding of
the physiological role of the B regulon in S. aureus. High-resolution two-dimensional (2-D) protein gel
electrophoresis is an excellent technique for visualizing a very large
set of proteins synthesized by a bacterial cell. Looking for proteins
that are no longer induced in a regulatory mutant is a good strategy
with which to define the structure of regulons. In this study, we used
2-D protein gel electrophoresis and N-terminal sequencing of proteins
to detect new members of the B regulon to get a more
comprehensive view of the physiological role of the general stress
response of S. aureus.
| |
MATERIALS AND METHODS |
Bacterial strains and growth conditions.
The S. aureus strains used in this study were wild-type S. aureus COL and the isogenic sigB mutant
(35). S. aureus strains were cultivated in LB
(53) or in a synthetic medium described earlier
(25). Heat stress conditions were provoked as follows. Cells
were cultivated in LB to an optical density at 540 nm of 0.5 and
transferred to 48°C. The time of the shift was regarded as zero.
Samples were taken during exponential growth immediately prior to the
shift or at the time indicated in the relevant figure legends.
Preparative 2-D gel electrophoresis and N-terminal
microsequencing.
For preparation of cell extracts, bacteria were
grown in the synthetic medium mentioned above. At an optical density at
500 nm of 1.0, cells were harvested by centrifugation of 50 ml of the
culture, washed twice with Tris-EDTA buffer, and resuspended in Tris
containing 2 mM phenylmethylsulfonyl fluoride. After incubation for 10 min on ice with lysostaphin (50 µg/ml), cells were disrupted using a
French press. The lysate was centrifuged (10 min, 10,000 rpm [Heraeus
12148]) at 4°C; the supernatant fluid was stored frozen. Preparative
2-D gel electrophoresis and N-terminal microsequencing of proteins were
carried out as described earlier (56) by using immobilized
ptt gradients of 4 to 7 and 3 to 10. For microsequencing, the
Coomassie-stained protein spots were cut from several 2-D gels and the
collected gel pieces were concentrated as previously described
(50, 54). The proteins or peptides generated by treatment
with cyanogen bromide were blotted onto a polyvinylidene difluoride
membrane, stained, and sequenced as previously described (56).
Analysis of transcription.
Total RNA of the S. aureus strains was isolated from exponentially growing or stressed
cells by the acid phenol method described by Majumdar et al.
(40) with modifications described previously (25,
61).
Northern blot analyses were carried out as described earlier
(64). Chemiluminescent signals were detected by the
Lumi-Imager from Boehringer Mannheim and analyzed by using the program
LumiAnalyst (Boehringer Mannheim).
The specific RNA probes were prepared by in vitro translation with T7
polymerase and with the appropriate PCR fragments as templates. The PCR
fragments were generated by using chromosomal DNA of S. aureus COL which was purified with a chromosomal DNA isolation kit
in accordance with the protocol of the manufacturer (Promega) and the
oligonucleotides listed in Table 1.
The oligonucleotides complementary to the C-terminal region of the
genes contain the T7 recognition sequence (29) at the 5' end
(25).
Sequence analyses.
Preliminary sequence data was obtained
from The Institute for Genomic Research (TIGR) through the website at
http://www.tigr.org. Database searches were carried out using the BLAST
program (3).
| |
RESULTS |
Identification of proteins belonging to the B
regulon on 2-D protein gels.
First, we looked for conditions that
allowed induction of B-dependent stress proteins only in
the wild type. Because B is active in cells growing in a
synthetic medium (25), the protein synthesis patterns of
exponentially growing cells of S. aureus COL and its
isogenic sigB mutant cultivated in a synthetic medium were
compared. This allowed us to identify 27 proteins belonging to the
B regulon (Fig. 1A and B).
These proteins, designated Csb (controlled by sigma B), were not or
hardly detectable in the sigB mutant and might be under the
positive control of B. The N-terminal sequences of 18 of
these proteins were determined, and they are listed in Table
2. By using the uncompleted DNA sequence
of S. aureus COL kindly provided by TIGR (updated May 1999 and August 1999), we were able to find the open reading frames coding
for the majority of the proteins (Table 2). A protein database search
was done with the deduced amino acid sequences of the newly identified
B-dependent genes (Table
3).
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FIG. 1.
B-dependent proteins of S. aureus. (A) 2-D pattern of cytoplasmic proteins from S. aureus COL and its isogenic sigB mutant. The proteins
from 100 (A) or 500 (B) µg of crude cell extract of exponentially
growing cells were separated by preparative 2-D polyacrylamide gel
electrophoresis. Proteins were stained with silver nitrate (A) or
Coomassie blue (B). The protein spots identified are indicated by
arrows. Comparison of the protein synthesis patterns of wild-type and
sigB mutant S. aureus COL grown in synthetic
medium allowed the identification of proteins belonging to the
B regulon. These proteins, designated Csb (controlled by
sigma B), were not or hardly detectable in the mutant strain. (B)
Sectors of 2-D gels covering the region where SarA (Csb35) is located.
Cytoplasmic protein extracts of wild-type S. aureus COL and
of its isogenic sigB mutant grown in synthetic medium were
separated.
|
|
Only three proteins, Csb3, Csb9, and Csb35, have been described in
S. aureus so far. Whereas Csb3 and Csb9 are identical to so
far hypothetical proteins of S. aureus (8, 38),
the N-terminal sequence of Csb35 resembles regulatory protein SarA of
S. aureus (12) (Fig. 1B; Table 3). The
transcription of the sar locus was already reported to be
partly controlled by B (17, 41). In our
experiments, we showed that the amount of SarA is diminished in a
sigB mutant (Fig. 1B). Unfortunately, the function of Csb3
and Csb9 is not known. However, it is interesting that Csb3 is similar
to YfkM in B. subtilis, which is also regulated by
B (46).
The putative functions of nine of the newly identified proteins were
derived from similarities to known proteins of other organisms.
However, we did not confirm the physiological function of any of these
proteins by experiments. Among them are three with similarities to
various dehydrogenases: Csb22, Csb24, and Csb28 (Table 3).
Interestingly, Csb24 and Csb28 shared similarities with proteins
described to be B dependent in B. subtilis
(46, 47) (Table 3).
Csb10 resembles various ATP-binding cassette transport (ABC
transporter) proteins, and Csb29 is very similar to
B-dependent BmrU in B. subtilis.
Proteins encoded by the bmrRU operon in B. subtilis, such as Bmr (transporter) and BmrR (regulator), are
known to be responsible for the drug resistance (1).
However, BmrU itself did not show any significant similarities to any
known proteins in the database and its function remains a matter of speculation.
For the Csb4 protein, we observed weak similarities to YckG of B. subtilis (22), which might encode a
hexulose-6-phosphate synthase. Proteins Csb7, Csb8, Csb12, and Csb19
did not display significant similarities to proteins with known
functions in the database. Open reading frames coding for proteins Csb5
and Csb13 could not be identified in the databases, and their N termini showed no similarities to known proteins.
Promoter characterization and transcriptional analyses of the newly
identified B-dependent genes.
In B. subtilis, the recognition sequences for the
B-containing RNA polymerase are strongly conserved and a
consensus of all of the B-dependent promoter sequences
currently available was derived: GTTTaa and GGG(A/T)A(A/T) for the 
35
and 10 regions, respectively, which are separated by 13 to 15 nucleotides (47). The known B-dependent
promoters in S. aureus are very similar to the consensus sequence in B. subtilis (17, 25, 34, 67).
Recently, we have shown that the B promoter of
asp23 is recognized by EB in B. subtilis (25). Therefore, we used the consensus of
B. subtilis to search for B-dependent
promoters in front of the identified genes. As a result, we could find
similar promoter structures immediately upstream of the translational
start codon of 15 of these genes (Table 2).
For transcriptional analyses, we selected 11 genes and in all cases we
confirmed their B dependency by Northern blots. These
results implied that the transcription of the newly identified genes
really depends on B-containing RNA polymerase in vivo.
In all cases, the synthesis of the B-dependent
transcripts was heat inducible in complex medium and the induction
failed in the sigB mutant. Furthermore, we can distinguish between genes controlled solely by B and genes regulated
in a more complex way.
Only csb7, csb9, and csb16 are
transcribed solely by B under the conditions tested so
far (Fig. 2). For the genes
csb7 and csb16, we detected one monocistronic
transcript, respectively. Both genes were heat inducible. In the case
of csb9, two main heat-inducible transcripts were found
(0.73 and 2.4 kb), which are synthesized in a
B-dependent manner. While the 0.73-kb transcript is a
monocistronic transcript of csb9, the 2.4-kb transcript
contains, in addition to the csb9 message, the mRNA of
the open reading frame downstream of csb9, whose product
(Csb9-1) is very similar to ManA (mannose-6-phosphate isomerase) in
B. subtilis (49) (Table
4).
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FIG. 2.
Northern blot analyses of solely
B-dependent genes. RNA was isolated from S. aureus COL and its isogenic sigB mutant growing in LB
at 37°C (lanes co) and at various times after a shift to 48°C. The
membrane was hybridized with digoxigenin-labeled RNA probes for the
respective genes. Relevant transcripts are indicated. Schematic
representations of the gene loci based on sequences of S. aureus COL (TIGR, unpublished data) are shown (PB,
B-dependent promoter). The broken lines represent the
RNA probes used in the experiments whose results are shown. The operon
structure of the csb9 locus was verified by using an RNA
probe specific for csb9-1.
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TABLE 4.
Similarities of new B-dependent S. aureus proteins whose genes are cotranscribed with the newly
detected genes, forming operons
|
|
Transcription of csb4, csb10, csb12,
csb22, csb28, csb29, csb33,
and csb35 (sar) is only partly regulated by
B (Fig. 3A and B).
Transcription still occurred in the mutant, indicating that, in
addition to B, a second sigma factor is involved.
However, with the exception of csb22, in all cases, the
main contribution to transcription was that of B. Only a
low basal level of transcription was found in the sigB mutant which is not heat induced.
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FIG. 3.
Northern blot analyses of genes partly regulated by
B. RNA was isolated from S. aureus COL and
its isogenic sigB mutant growing in LB at 37°C (lanes co)
and at various times after a shift to 48°C. The membrane was
hybridized with digoxigenin-labeled RNA probes for the respective
genes. Relevant transcripts are indicated. Schematic representations of
the gene loci based on sequences of S. aureus COL (TIGR,
unpublished data) are shown (PB, B-dependent
promoter; PA, A-dependent promoter). The
broken lines represent the RNA probe used in the experiments whose
results are shown. (A) B-dependent genes
monocistronically transcribed. (B) B-dependent operons.
We verified the operon structures of the csb10 and
csb22 loci by using additional probes (csb10-1,
csb10-4, and csb22-1). MM, synthetic medium.
|
|
According to the sizes of their transcripts, csb12,
csb28, csb29, and csb35
(sar) seem to be monocistronically transcribed by
B (Fig. 3A). In contrast, the detected
B-dependent transcripts of the genes
csb4, csb10, csb22, and
csb33 should contain additional messages of open reading
frames located downstream of the genes (Fig. 3B). These data
indicate that additional genes belong to the B regulon
whose products are very similar to subunits of ABC transporters (Csb10-1 and Csb10-4); to NifS (Csb10-2) and NifU (Csb10-3) in B. subtilis (36); to nhaC in S. carnosus, which might encode an Na+/H+
antiporter protein; and to YckF in B. subtilis (Csb4-1).
Transcriptional regulation of two different clp-like
genes in S. aureus.
Recently, we have started a second
approach in order to identify B-dependent genes in
S. aureus by looking for genes homologous to
B-dependent genes of B. subtilis. By this
approach, we found at least two genes homologous to clpC in
B. subtilis. The corresponding gene products show 44%
(Clp2161) and 69% (Clp2392) identity to B. subtilis
ClpC. Both proteins contain two nucleotide binding regions that are
highly conserved among the Clp ATPases and are separated by a spacer of
60 amino acids in Clp2161 and 67 amino acids in Clp2392. The lengths of
the spacers are typical for ClpC proteins (58). The gene
order of the clp2392 operon supports the assumption that
clp2393 encodes a ClpC protein. The other ClpC-related
protein, Clp2161, also shows similarities to ClpE in Lactococcus
lactis (54% identical amino acids); however, there is no putative
zinc binding domain typical for ClpE proteins.
In Northern blot experiments for clp2161, we detected one
heat-inducible transcript of about 2.3 kb, corresponding to the size of
the proposed open reading frame only in the wild type and not in the
sigB mutant. We found a promoter sequence very similar to
the consensus of promoters recognized by B in front of
the gene (GTTTTA N14 TGGAAA).
Computer analysis predicted the presence of a terminator
structure at the end of the putative gene (Fig.
4). In contrast, the transcription of
clp2392 did not appear to be influenced by a mutation in
sigB. We found a 4.5-kb heat-inducible transcript probably
containing four genes homologous to ctsR, yacH,
yacI, and clpC of B. subtilis
(32, 33). Upstream of ctsR, we found a
promoter region possibly recognized by the vegetative sigma
factor A. Furthermore, three CtsR boxes were
localized around the promoter region.
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FIG. 4.
Northern blot analyses of clpC-homologous
genes in S. aureus. RNA was isolated from S. aureus COL and its isogenic sigB mutant growing in LB
at 37°C (lanes co) and at various times after a shift to 48°C. The
membrane was hybridized with digoxigenin-labeled RNA probes for
clp on contig 2161 (236099...21954) and contig
2392 (2866...1966). Relevant transcripts are indicated. Schematic
representations of the gene loci based on sequences of S. aureus COL (TIGR, unpublished data) are shown (PB,
B-dependent promoter). The broken lines represent the
RNA probes used in the experiments whose results are shown. We used
additional probes to find out which genes are cotranscribed with
clp2392.
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|
| |
DISCUSSION |
Our data on the B-dependent general stress regulon
in B. subtilis suggested that this stress response
might fulfill a physiological role similar to that of the
S-dependent response in E. coli and
other gram-negative bacteria, thus providing nonspecific, multiple, and
general stress resistance to nongrowing cells (26, 27, 39,
63). Moreover, some S-dependent genes control
virulence genes of gram-negative bacteria (66). Therefore,
it was tempting to speculate that B is also involved in
the control of virulence gene expression in gram-positive bacteria.
This suggestion was supported by the finding that B
initiates transcription at one of the three promoters of
sarA encoding one of the important regulators of
virulence-associated genes in S. aureus (41).
Only a few genes under B-control have been identified so
far; among them is that for alkaline shock protein Asp23. However, the
identification of Asp23 as a B-dependent protein did not
make any progress in the functional analysis of the regulon since
nothing was known about the function of Asp23 itself (25, 35,
37). In order to obtain a more comprehensive picture of the role
of B in stress or starvation survival in general,
including evidence for the suggested function of the B
regulon in virulence control, new members of the general stress regulon
in S. aureus should be identified. The putative functions of
these new proteins should allow a preliminary prediction of the
physiological role of the entire regulon. In the present study; we
showed that the synthesis of about 27 proteins visible on 2-D gels of
crude protein extracts of S. aureus COL are under the positive control of alternative sigma factor B. Eighteen
of these proteins were identified by N-terminal sequencing, and the
open reading frames coding for the proteins were localized on the
uncompleted DNA sequence of S. aureus COL kindly
provided by TIGR (updated May 1999 and August 1999). By transcriptional analyses of these new genes, about eight additional
B-dependent genes were found which are cotranscribed
with the newly detected genes, forming operons. Comparison of all of
the newly identified genes with the B. subtilis genome
showed that 20 of them are homologous to B. subtilis genes,
only 7 of which are known to be regulated by B (Tables 3
and 4). The proteins belonging to the B regulon in
S. aureus but not in B. subtilis may provide
evidence about additional functions of the regulon in gram-positive
pathogens. Moreover, two of the genes (csb12 and
csb22) in S. aureus did not have any orthologues
in B. subtilis. These genes are particularly interesting
because they may form a reservoir of B-dependent genes
whose products could interact in a specific manner with the host.
Unfortunately, the function of these genes is still unknown.
For most of the genes described so far, at least a second sigma factor,
in addition to B, is involved in gene expression.
Obviously, the corresponding proteins seem to be necessary even under
conditions under which B is not active. However,
particularly in response to heat shock, the B-dependent
promoter seemed to be the strongest one in all of the cases tested so
far. This complex regulation was also described for many
B-dependent proteins in B. subtilis (2,
19, 31, 55). Only csb7, csb9, and
csb16 seem to be transcribed solely by B
under the conditions tested so far.
The newly identified genes allow first conclusions on the physiological
role of the B regulon in S. aureus.
Protection of starved B. subtilis cells against oxidative
damage could be the most essential component of
B-mediated stress resistance (21). In
accordance with this, a sigB mutant of S. aureus
showed increased sensitivity to hydrogen peroxide (10, 35).
Furthermore, maintenance of intracellular redox balance under stress
and starvation might be very important and requires the reduction of
oxidized biological molecules by using NAD(P)H. Therefore, a sufficient
level of NAD(P)H seems to be a prerequisite for the cell to face
oxidative stress. Among the newly identified genes, three are possible
dehydrogenases which could be involved in the generation of reduction
equivalents like NAD(P)H and FADH. In B. subtilis, the
nifS gene product might contribute to NAD biosynthesis by
generating the Fe-S clusters required for NadA activity
(59). Interestingly, among the newly identified S. aureus genes there is one nifS-homologous gene.
Prokaryotic, as well as eukaryotic, organisms possess multidrug
resistance efflux transporters whose expression is induced by various
structurally divergent compounds such as antibiotics, inhibitors, and
other toxic substances. In B. subtilis, the bmrUR operon, which encodes proteins that may contribute to resistance to
multidrug compounds, is regulated by B. Very recently,
it was shown that B is activated in mycobacterial cells
after exposure to rifampin, streptomycin, and cycloserine
(42). In S. aureus, methicillin resistance is
widespreaded; however, the mechanism of this phenomen is not fully
understood. Interestingly, it was reported that the sigB
mutant of S. aureus COL showed a drastic reduction in
methicillin resistance (15). Among the newly identified
B-dependent proteins of S. aureus, we found
proteins with significant similarities to
Na+/H+ antiporters or ABC transporters. The
Na+/H+ antiporters are widely distributed in
cell membranes from bacteria to mammals. The antiporters play important
roles in the Na+ cycle across the cytoplasmic membrane of
all living cells. In bacteria, the antiporter extrudes Na+
or Li+ in exchange for H+. Besides their
function in antibiotic resistance, they may play a role in (i) the
establishment of an electrochemical potential of Na+ across
the cytoplasmic membrane, (ii) the extrusion of Na+ and
Li+, (iii) intracellular pH regulation under alkaline
conditions, and (iv) cell volume regulation (11, 28).
Besides the identification of
B-dependent genes by proteomics, we started a second
approach. In B. subtilis, we know of more than 150 genes
belonging to the regulon (Petersohn et al., submitted for publication);
among them are Clp proteins with essential functions in stress
resistance (24, 30).
Recently, ClpC was shown to be involved in the virulence of L. monocytogenes (52). Therefore, we looked for
clp-homologous genes in the S. aureus genome
sequence. As a result, we localized two open reading
frames possibly encoding ClpC proteins. It is a remarkable finding that
at least one clpC-homologous gene seemed to be
controlled solely by alternative stress sigma factor B
and the other is probably controlled by the global regulator of class
III general stress genes CtsR; however, experimental evidence for this
is still lacking (18, 33). The Clp proteins are involved in
several physiological processes, such as proteolysis, stress tolerance,
competence, cell division, and virulence. The presence of at least two
clpC-homologous genes in S. aureus
implies an essential role of the protein in the physiology of this organism.
The identification of new members of the B regulon is a
preliminary but essential step toward a more comprehensive
understanding of the role of this large regulon in stress adaptation
and virulence. No evidence has previously been presented for a role of
B in the infection process and virulence (10,
44). Analysis of the effects of promising mutations in individual
B-dependent genes on stress adaptation and infection is
another approach to the problem of whether and to what extent
B-dependent proteins contribute to survival within
the host. These studies will provide an essential contribution to the
understanding of the cell physiology of S. aureus.
We are indebted to P. Bednarski for critical reading the
manuscript. We are very grateful to Ines Kullik for providing S. aureus strain COL 
sigB and Markus Bischoff for
fruitful discussion. Elke Krüger and Ulf Gerth are acknowledged
for a helpful discussion of Clp proteins. Furthermore, we thank Renate
Gloger for excellent technical assistance. Preliminary sequence data
was obtained from TIGR through the website at http://www.tigr.org.
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B Regulon in
Staphylococcus aureus
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Abstract
Introduction
