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J Bacteriol. Mar 2007; 189(6): 2376–2391.
Published online Dec 28, 2006. doi:  10.1128/JB.01439-06
PMCID: PMC1899396

Extensive and Genome-Wide Changes in the Transcription Profile of Staphylococcus aureus Induced by Modulating the Transcription of the Cell Wall Synthesis Gene murF[down-pointing small open triangle]


A murF conditional mutant was used to evaluate the effect of suboptimal transcription of this gene on the transcriptome of the methicillin-resistant Staphylococcus aureus strain COL. The mutant was grown in the presence of optimal and suboptimal concentrations of the inducer, and the relative levels of transcription of genes were evaluated genome wide with an Affymetrix DNA microarray that included all open reading frames (ORFs) as well as intergenic sequences derived from four sequenced S. aureus strains. Using a sensitivity threshold value of 1.5, suboptimal expression of murF altered the transcription of a surprisingly large number of genes, i.e., 668 out of the 2,740 ORFs (close to one-fourth of all ORFs), of the genome of S. aureus strain COL. The genes with altered transcription were distributed evenly around the S. aureus chromosome, and groups of genes involved with distinct metabolic functions responded in unique and operon-specific manners to modulation in murF transcription. For instance, all genes belonging to the isd operon and all but 2 of the 35 genes of prophage L54a were down-regulated, whereas all but one of the 21 members of the vraSR regulon and most of the 79 virulence-related genes (those for fibronectin binding proteins A and B, clumping factor A, gamma hemolysin, enterotoxin B, etc.) were up-regulated in cells with suboptimal expression of murF. Most importantly, the majority of these altered gene expression profiles were reversible by resupplying the optimal concentration of IPTG (isopropyl-β-d-thiogalactopyranoside) to the culture. The observations suggest the coordinate regulation of a large sector of the S. aureus transcriptome in response to a disturbance in cell wall synthesis.

Transcriptomic approaches are powerful tools for the identification of groups of genes with similar expression patterns and for the establishment of putative regulatory relations between them. Using microarray technology, recent reports have described extensive changes in gene expression profiles when Staphylococcus aureus was challenged by different cell wall inhibitors such as oxacillin, bacitracin, d-cycloserine, and vancomycin (12, 19, 20, 34). The group of genes whose transcription was altered during treatment with antibiotics that target different steps in cell wall synthesis was proposed to represent members of a cell wall stimulon responding to a specific form of stress in a coordinate manner.

The large number and diversity of genes that respond to cell wall inhibitors by altered transcription suggest that many functions of the bacterial cell are connected through regulatory loops to one or more steps in cell wall synthesis which may also serve as control points of metabolism. Such a control should also be operational in the absence of antibiotic treatment, and we searched for a system in which the existence of such a regulatory system could be experimentally demonstrated.

The method of choice became available with the isolation of an S. aureus murF conditional mutant in which the rate of transcription of murF depended on the concentration of the IPTG (isopropyl-β-d-thiogalactopyranoside) inducer in the medium (30). The MurF ligase catalyzes the last cytoplasmic step in the synthesis of the peptidoglycan precursor: it adds the d-alanyl-d-alanine dipeptide to the UDP-linked MurNAc-tripeptide, thus forming the peptidoglycan building block, the UDP-linked MurNAc pentapeptide. The assembly of this peptidoglycan building block is essential for all the cell wall synthesis reactions that take place on the outer surface of the plasma membrane.

The biochemical consequences of suboptimal transcription of murF include reduction in the amounts of UDP-linked MurNAc-pentapeptide in the cell wall precursor pool and accumulation of a tripeptide precursor which incorporates into the peptidoglycan in the form of un-cross-linked monomers. Bacteria with suboptimal murF transcription also grow slower and have reduced beta-lactam antibiotic resistance (30). In fact, the murF gene had been previously added to the wide list of auxiliary genes that are essential for the optimal expression of methicillin resistance in S. aureus (6, 31).

We used custom-made DNA microarray chips to profile the effect of suboptimal transcription of murF on gene expression in the methicillin-resistant S. aureus (MRSA) strain COL, including the reversibility of the effects.


Bacterial strains and growth conditions.

Staphylococcus aureus strain COL and its derivative, the murF conditional mutant COLspacmurF (30), were used for all the experiments described in this paper. The cells were grown at 37°C with aeration in tryptic soy broth (TSB) or tryptic soy agar (Difco Laboratories, Detroit, MI). The growth medium of the conditional mutant strain COLspacmurF was supplemented with ITPG (100 μM) (Sigma, St. Louis, MO), erythromycin (10 μg/ml) (Sigma), and chloramphenicol (10 μg/ml) (Sigma) unless otherwise described.

Growth conditions used in the microarray analysis.

COLspacmurF was grown overnight in TSB supplemented with erythromycin, chloramphenicol, and 100 μM IPTG. The overnight culture was washed twice to remove the antibiotics and then back diluted to an optical density at 620 nm (OD620) of 0.01 into fresh prewarmed medium supplemented with 100 μM IPTG. The culture was incubated until the OD620 reached 0.7 (mid-log phase). A 10-ml portion of the culture was used for RNA preparation (condition A of the experiment).

The culture grown with the optimal IPTG concentration was back diluted to an OD620 of 0.01 into fresh prewarmed TSB supplemented with a suboptimal concentration of IPTG (2.5 μM) and was incubated until the OD620 reached 0.7. A 10-ml portion of this culture was used for RNA preparation (condition B of the experiment). IPTG was added to the culture grown in condition B, to the optimal concentration (100 μM). After 30 min of incubation, 10 ml of this culture was used for RNA preparation (condition C of the experiment).

Sampling and RNA preparation.

At the time of sampling, a double volume of RNAprotect Bacteria reagent (QIAGEN, Hilden, Germany) was added to the culture. The mixture was incubated for 5 min at room temperature, the cells were centrifuged for 10 min at 3,500 rpm and 4°C, and the pellets were frozen in dry ice and kept at −70°C. Total RNA was extracted as described previously (31). The RNA quality and quantity was determined by agarose gel electrophoresis and by measuring the absorbance at 260 and 280 nm. Each of the cultures grown in conditions A, B, and C was repeated three times.

DNase I purification of total RNA.

Total RNA from all samples were treated with DNase I by using the RNase-free DNase set protocol and RNeasy minikit protocol (QIAGEN, Valencia, CA). Total RNA was quantitated using the NanoDrop spectrophotometer (Nanodrop Technologies, Wilmington, DE).

The protocols for staphylococcal genomic chip design, Staphylococcus aureus array probe selection, Affymetrix probe preparation and array processing, data quality control, and statistical analysis are described in the supplemental material.

Microarray validation by real-time reverse transcription-PCR (RT-PCR).

Total RNA preparation was performed exactly as described for the microarray assay. Residual DNA was removed from the samples by performing two on-column DNase digestion steps with RNase-free DNase (QIAGEN). cDNA was synthesized with TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA) and random hexamers. Specific primers for the genes tested and for 16S rRNA, used as endogenous control, were designed using Primer Express software (Applied Biosystems) and are listed in Table S1 in the supplemental material. Real-time PCR was performed with the ABI PRISM 7000 sequence detection system (Applied Biosystems) and SYBR green technology. The three biological replicates were analyzed in duplicate and normalized against 16S rRNA gene expression. Each assay included samples for the three conditions and standard curves, both for the target gene and for the 16S rRNA control gene. For all experiments, the amounts of target and control genes were determined from the appropriate standard curve. In order to obtain a normalized relative gene expression value, the amount of target gene for each condition was divided by the respective amount of control gene.

Determination of oxacillin MIC.

COLspacmurF cultures were plated on tryptic soy agar with 100 μM and 2.5 μM IPTG and incubated overnight at 37°C. Oxacillin Etests (AB Biodisk, Solna, Sweden) were used to determine the MICs.

In vivo autolysis assay.

Triton X-100-stimulated autolysis was determined as described previously (5). COL cultures were grown to an OD620 of 0.7. COLspacmurF cultures were grown exactly as described for the microarray assay conditions A, B, and C.

Preparation of autolytic extracts.

Crude autolytic extracts were prepared as described previously (28). Bacterial cultures were grown as described for the microarray assay conditions A, B, and C. The bacterial pellets were frozen to −70°C and extracted with 4% sodium dodecyl sulfate (SDS) at room temperature or with 3 M LiCl and 0.1% Triton X-100 at 4°C, and the supernatants were used as autolytic extracts.

Crude cell wall preparation.

Bacteria were grown as described for the microarray assay conditions A and B, chilled rapidly, harvested by centrifugation, and boiled in 8% SDS for 30 min. After cooling to room temperature, the samples were washed with water until all traces of SDS were removed and were lyophilized.

In vitro autolysis assay.

Crude cell walls were suspended in 50 mM Tris-Cl (pH 7.5) to an initial OD620 of 0.5 and incubated with 10 μg/ml of each LiCl autolytic extract. Lysis was measured as the decrease in OD620 during incubation at 37°C.

Zymogen profiles.

Crude cell walls were incorporated in polyacrylamide resolving gels (10% acrylamide-0.2% bisacrylamide) at a final concentration of 1 mg/ml. The SDS-extracted supernatants were separated by the technique of Laemmli (13) at a constant current of 20 mA at 4°C. The lytic effect of the enzymes was visualized as described previously (29). After a washing step in water, the gels were incubated at 37°C in zymogen buffer for 1 h.

Metabolic pattern determination.

Strain COL was grown to an OD620 of 0.7, and COLspacmurF cultures were grown exactly as described above for microarray assay conditions A and B. The cells were washed twice and resuspended in the same volume of buffer (0.15 M NaCl, 0.1 mM NaPO4, pH 7.2). Biolog GP2 microplates (Biolog, Hayward, CA) were inoculated with 150 μl of the cell suspension in each well and incubated overnight.


The structure and biosynthesis of the bacterial peptidoglycan are the targets of some of the most powerful antimicrobial agents. Inhibition of these targets not only causes interference with bacterial growth but also invokes in the cells rapid changes in the pattern of transcription of a large number of genes (11, 22, 35), suggesting the existence of complex regulatory loops which link a wide variety of steps in metabolism to some steps in cell wall biosynthesis.

In all these studies with antibacterial agents, the altered gene expression profiles are the direct or indirect consequences of inhibition of enzymes involved in peptidoglycan synthesis. Furthermore, in the case of oxacillin, not one but several enzymes (penicillin binding proteins) are affected. The same is true for vancomycin and d-cycloserine, each of which inhibits at least two cell wall synthesis enzymes. Also, depending on the dose used, these inhibitions can also cause irreversible effects leading to loss of viability and/or lysis. Therefore, at least some of the regulatory abnormalities observed in antibiotic-treated cells may be related to delayed indirect effects on bacterial cell structure metabolism.

The availability of a mutant with a conditional mutation in the cell wall synthetic gene murF offered an opportunity to explore these regulatory circuits by a novel method that does not involve the use of antimicrobial agents. In the mutant COLspacmurF, constructed in the background of the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL (30), transcription of the murF gene is under the control of an IPTG-inducible promoter, which allowed us to compare the transcriptomes of S. aureus grown with optimal and suboptimal transcription of a key determinant of cell wall precursor synthesis.

Experimental design.

The experimental design consisted of growing COLspacmurF under optimal (100 μM) (condition A) and suboptimal (2.5 μM) (condition B) concentrations of the inducer for six generations (mass doubling times). In order to test the reversibility of transcriptional changes, a portion of the culture grown with the suboptimal IPTG concentration was exposed for a single generation time to optimal levels of the inducer (condition C). Cells were harvested at the same optical density in the mid-exponential phase of growth, RNA was prepared, and the number and nature of genes which underwent a change in transcriptional pattern were evaluated using a custom-made Affymetrix DNA microarray.

The impact of the decreased transcription of the murF gene has been previously described in detail for several concentrations of inducer (30). For the purposes of this study, only two IPTG concentrations were used. Cultures grown in 100 μM IPTG (condition A) showed properties which were similar to those of the parental strain COL, in which murF is under the control of its native promoter. Cultures grown at the suboptimal concentration of 2.5 μM IPTG (condition B) showed extensive changes in various properties, which included a reduced growth rate, a reduction in the cellular amounts of the cell wall precursor pentapeptide to about 50% of the normal level, and an increase in the concentration of the abnormal cell wall precursor tripeptide, which also incorporated as an unsubstituted monomer into the peptidoglycan of the cell wall. Cultures grown with 2.5 μM IPTG also showed decreased and heterogeneous resistance to oxacillin (Fig. 1a to c).

FIG. 1.
Phenotypic characterization of strain COL and the COLspacmurF conditional mutant grown with the optimal IPTG concentration (100 μM) (condition A) and a suboptimal IPTG concentration (2.5 μM) (condition B). (a) Growth curves were monitored ...

Changes in the transcriptional profile of cells grown in suboptimal concentrations of IPTG.

The reduced rate of transcription of murF caused alterations in a surprisingly large number of genes. With a threshold value set at 1.5, as many as 668 genes were affected (393 were up-regulated and 275 were down-regulated), out of the total 2,740 described open reading frames (ORFs) for S. aureus strain COL. With a threshold value set at 2.0, the total number of genes affected was 316 (203 overexpressed and 113 repressed). With a threshold set at 5.0, the number of genes affected was reduced to 59 (44 up-regulated and 15 down-regulated). The genes involved were homogeneously distributed along the entire chromosome (see Fig. S1 in the supplemental material).

All 668 genetic determinants identified in the microarray experiment using the lower threshold value are listed in Table S2 in the supplemental material.

The surprisingly large number of genes, representing almost one-fourth of all the genetic determinants, that responded to the murF stress provides a striking view of the scope and complexity of the regulatory circuitry interconnecting functionally diverse aspects of the S. aureus cell to a single cell wall synthesis function. Most importantly, the majority of transcriptional changes were reversible even after a relatively short time of restoring suboptimal murF transcription to normal levels.

Activation of the cell wall stimulon.

In pioneering studies, Utaida and colleagues (34) and Kuroda and colleagues (12) described extensive and unique changes in the gene expression profile of S. aureus strains after exposure to inhibitory concentrations of cell wall targeting antibiotics. In the study by Utaida et al. a derivative of S. aureus strain 8325 was exposed to oxacillin, d-cycloserine, and bacitracin at concentrations that reduced growth to 70% of the control value (34). A total of 163 genes showed altered transcription. In the study by Kuroda et al., the MRSA strain N315 was exposed to 10 times the MIC of vancomycin for 10 min, which caused altered expression of 169 genes. It was proposed that genetic determinants whose expression was altered under these conditions belonged to a coordinately regulated “cell wall stimulon” (34).

More recently, McAleese and colleagues tested the number and nature of genes with altered expression induced by brief (10-min) treatment of the MRSA strain JH1 with 1 or 8 times the MIC for vancomycin (19). The same authors also observed changes in the transcription of a large number of genes (224) when cultures of strain JH1 and its isogenic vancomycin-resistant derivative JH9 were compared by microarray analysis during growth in drug-free medium (19). Most recently, McCollum and colleagues described altered transcription of 109 genes when the methicillin-susceptible S. aureus strain Newman was treated with 10 times the MIC of vancomycin for 10 min (20).

A substantial proportion of the determinants that responded to the murF stress were similar to genes that show altered expression after treatment with wall targeted antibiotics. Table Table11 shows a selection of genes that showed altered transcription both during murF stress and also in at least two of the published studies in which antibiotic treatment was used to induce the cell wall stimulon. The match between genes detected in the murF transcriptional study and any of the other studies was between 40 and 62%. However, the direction of gene expression (up- versus down-regulation) and the degree of change were not necessarily the same in the studies compared. McAleese et al. proposed a “core” cell wall stimulon composed of 15 genes that were commonly observed with altered expression in each one of the four previous studies that used antibiotic treatment for the induction (19). Table Table22 shows that 11 of these 15 genes were also up-regulated in the murF system.

Genes whose expression was altered in conditional mutant COLspacmurF when grown in the presence of a subinhibitory inducer concentration (condition B) and which also showed differential expression for at least other two of the specific conditions tested ...
Genes belonging to the “core” cell wall stimulon and cell wall synthesis-related genes which show altered expression in this and the other studies (12, 19, 20, 34)

The most obvious differences between the natures of genes that responded to the murF stress and the ones that were up-regulated during treatment with the cell wall inhibitors were a group of genetic determinants that included cell wall synthesis genes such as pbpB, murA, murZ, fmtA, atl, which appear to be up-regulated in most of the studies using antibiotics but remained unchanged during the suboptimal murF expression (Table (Table2).2). The lack of transcriptional changes in these determinants during murF stress fits the radically different types of perturbation of wall synthesis in the antibiotic-treated bacteria: the cells are apparently trying to compensate for cell wall synthesis enzymes that are inactivated by the antibiotics, while in the murF system the synthetic apparatus of cell walls remains intact.

Expansion of the cell wall stimulon.

Table S3 in the supplemental material lists genes of the cell wall stimulon identified in the murF system in the order of their chromosomal location. Also shown in that table are the corresponding genes detected by Utaida et al. (34) and Kuroda et al. (12). This exhibit allows one to expand the list of genes that belong to the cell wall stimulon by the addition of new genes localized in the direct vicinity of previously identified cell wall stimulon determinants. The proposed new members of the cell wall stimulon belonged to operons predicted by an algorithm developed at TIGR (http://www.tigr.org/tigr-scripts/operons/pairs.cgi?taxon_id=40141) which detects conserved gene pairs in two or more bacterial genomes and estimates a probability for the genes to belong to the same operon (see Table S3 in the supplemental material). It seems that genetic determinants which are transcribed in the same mRNA molecule will tend to respond to the induced stress in a similar manner, either up- or down-regulated, and also will follow the same pattern of reversibility upon readdition of IPTG.

Changes in expression of the vraSR regulon.

Kuroda and colleagues identified within the cell wall stimulon a group of 46 determinants in which altered transcription depended on the presence of an intact vraSR, a two-component sensory/regulatory system of S. aureus (12). As many as 21 of the members of the vraSR regulon also responded to the decreased transcription of murF, and all but two of these genes were up-regulated (Table (Table3).3). Except for opuD, ctpA, SACOL1956, and SACOL2519, the altered transcription was reversible in the rest of the determinants.

Members of the VraRS regulon (12) which were found to be differentially expressed when the COLspacmurF conditional mutant was grown with a suboptimal inducer concentration (condition B)

Changes in the expression of sortase-dependent surface-anchored proteins and proteins involved with iron metabolism.

The effect of murF stress on the transcription of surface-attached proteins suggests functional specificity. Out of the 20 previously described sortase-dependent S. aureus proteins (17, 18, 25), the genetic determinants of 11 were differentially expressed under the suboptimal expression of murF. Of these, 10 of the genetic determinants were overexpressed in condition B, while one gene, isdC, was repressed (Fig. (Fig.2a2a).

FIG. 2.
(a) Twenty sortase-anchored surface proteins were identified in the S. aureus COL genome. All the listed surface proteins are anchored through a sortase A-dependent mechanism, except for IsdC, which is sortase B dependent. Determinants which were found ...

This differential response appears to correspond to the two kinds of sortases used for anchoring these proteins. Each one of the 10 overexpressed proteins is attached to the cell wall at the lipid 2 level by a sortase A-dependent process (26, 33). Overexpression of the 10 sortase A-dependent genes may be a response by the cells compensating for the shortage of normal, pentapeptide cell wall precursors which are replaced in the condition B cells by the abnormal tripeptide precursor which does not carry the oligoglycine branches needed for the attachment of these proteins (32).

In contrast, the protein product of the solitary gene isdC, the expression of which was repressed, is anchored to the cell wall by a sortase B-dependent reaction in which the acceptors are in the polymerized cell wall and are equipped with the oligoglycine branches (16, 18).

A unique pattern of response to the perturbation of wall synthesis is also apparent in Fig. 2b to d, which shows that the determinants isdC through -G and srtB are transcribed from a single promoter and respond in a uniform manner (by down-regulation and reversibility) to the suboptimal transcription of murF. No changes were observed in the transcription of isdA and isdB, each of which is transcribed from a separate promoter and in the opposite direction (Fig. (Fig.2b)2b) (18).

Several of the genes of the S. aureus isd locus (iron-surface determinant) are involved in iron acquisition (15). Interestingly, the transcription of over 30 additional genes involved in iron metabolism of S. aureus were also found to be down-regulated under the condition of suboptimal transcription of murF (Table (Table4;4; genes marked with asterisks in Table S2 in the supplemental material).

Genes related to iron scavenging and metabolism whose expression was found to be altered when COLspacmurF was grown with suboptimal IPTG (condition B)a

Repression of the ica operon and the pur operon.

Figure S2a in the supplemental material shows the rapid and reversible overexpression of icaR, the repressor of the ica operon, in response to modulation of murF transcription. The pattern of changes suggests that the primary responder to the reduced transcription of murF was the icaR repressor, the overexpression of which presumably was responsible for the repression of the rest of the operon, which has been associated with the capacity of S. aureus to form biofilms (4).

Figure S2b in the supplemental material shows that the entire purine biosynthetic pathway underwent powerful and reversible repression under the conditions when cell wall synthesis was perturbed and slowed down in the murF system. Previous studies showed that the reduced transcription of murF results in a thinning of the bacterial cell wall due to a reduced incorporation of peptidoglycan (30). It is interesting that a recent study with S. aureus strains selected for reduced susceptibility to vancomycin showed exactly the opposite pattern of transcription of the purine biosynthesis operon: the entire operon was overexpressed in the bacteria with reduced vancomycin susceptibility, which showed a substantially increased thickness of the cell wall. It has been proposed that the increased transcription of the purine pathway in this system reflects the increased needs of the vancomycin-resistant bacteria for ATP, due to the excess cell wall material produced (21).

Changes in the transcription of a mobile element.

S. aureus strain COL carries a chromosomally located prophage, L54a, which until now had been found to be specific to this strain (8). Interestingly, 33 of the 72 genetic determinants of the prophage showed decreased transcription under the conditions of cell wall stress induced by suboptimal transcription of murF (see Fig. S3 in the supplemental material). Each of these changes was reversible upon readdition of the optimal concentration of the IPTG inducer to the medium. The only exceptions to this uniform pattern of repression were two genetic determinants, ORFs SACOL0328 and SACOL0332, which showed overexpression in the bacteria growing with the suboptimal IPTG concentration. It appears, therefore, that the perturbation of cell wall synthesis caused reinforcement of the lysogenic state of the prophage for most of the phage genetic determinants. The two exceptional genes showing overexpression may represent repressors.

The uniform down-regulation of all but two of the 35 prophage-affected determinants shows a striking contrast with recent reports that documented activation of the SOS system and induction of lysogenic phages in bacteria treated with cell wall-active antibiotics (1, 14).

Changes in the expression of virulence-related determinants.

Modulation of murF expression also had a powerful and selective effect on the transcription of 79 virulence-related genes. The 55 genes overexpressed in cultures grown with suboptimal transcription of murF included determinants of several pathogenicity islands and such important virulence determinants as fibronectin binding proteins A and B, clumping factor A, enterotoxin B, etc. On the other hand, expression of several genes, such as sek, sei, etc., on the pathogenicity island υSa1 showed reversible repression in bacteria growing with suboptimal transcription of murF (Table (Table5).5). The surprisingly large number of virulence related determinants that were up-regulated during murF stress suggests perhaps a more enhanced “defensive” posture by the bacteria during a compromised state of synthetic capacities.

Virulence-related genes found to be differentially expressed in the COLspacmurF conditional mutant when grown with suboptimal inducer concentrations (condition B)a

Repression of autolytic activity.

Figure Figure3a3a shows the responses of several known regulators of autolytic activity to the reduced rate of cell wall synthesis in the S. aureus conditional mutant cultivated at suboptimal concentrations of IPTG. It may be seen that the lrgA and -B genes, encoding a putative antiholin (10), and the genes cidC and cidB, proposed to encode a regulator of hydrolase activity (24), are overexpressed, while the genetic determinant of lytM is repressed, under conditions of murF stress. These regulatory changes would be expected to result in lower concentrations of autolytic enzymes and lower autolytic activity in the bacteria.

FIG. 3.
(a) Absolute values of expression for the genes related to the autolysis process described for S. aureus. For details, see the legend to Fig. Fig.2d.2d. (b) Zymogen assays. Cell wall extracted from strain COL (left panel) and from COLspacmurF ...

Follow-up experiments were done in an attempt to confirm these expectations by direct determination of the relative amounts of autolysins, using the zymogen assay and comparison of the relative activities of autolysins in lithium chloride extracts. In addition, the proneness of cells to Triton X-100-induced autolysis was also determined.

Bacteria grown in the presence of optimal expression of murF (condition A) and bacteria grown with reduced murF transcription (condition B) were used in these experiments. Figure Figure3b3b shows that crude autolytic extracts prepared from cells grown in condition B (lane 4) showed substantially less-intensive autolytic bands in the zymogen extract irrespective of whether the cell wall substrate incorporated into the gel was derived from the parental strain COL or from the bacteria grown in condition B. As a comparison we used autolytic extracts from the parental strain COL (lanes 1 and 2), bacteria grown in condition A (lane 3), and bacteria which received the optimal concentration of IPTG (condition C) (lane 5). Suggestive evidence for a somewhat increased intensity of the autolytic bands may be apparent in lane 5.

Figure Figure3c3c shows the substantially decreased lysis of cells grown under condition B in the Triton X-100 autolysis assay, and Fig. Fig.3d3d compares the relative activities of lithium chloride extracts prepared from bacteria grown in condition A, B, or C. Also included are extracts prepared from the parental strain COL. Decreased activity of the lithium chloride extractable autolysins is apparent in bacteria grown under condition B, and evidence for increased autolytic activity is apparent in the extracts derived from the condition C cells.

Validation of the microarray assays.

A selected group of 10 genetic determinants showing extensive up- or down-regulation during murF stress were chosen to test the validity of the microarray data using real-time RT-PCR. Table S4 in the supplemental material shows the results, which confirm both the degree and direction as well as reversibility of the transcriptional changes observed in the microarray system.

Operon-specific changes.

Examination of the nature of genes with modified transcription in the murF system allows several interesting conclusions. The transcriptional changes appear to be operon specific. In fact, by applying this principle it was possible to enlarge the number of genes that belong to a “cell wall stimulon” by over 80 new members (see Table S3 in the supplemental material). Operon-specific responses were also striking in the case of genes belonging to the ica group of determinants and the purine synthesis operon (see Fig. S2 in the supplemental material) and genes involved with iron metabolism (Table (Table4),4), all of which were down-regulated during murF stress.

Changes in intergenic regions.

The Affymetrix microarrays used in these experiments included all intergenic sequences derived from ORF clusters of the four genomes. In 55 of these cases we noted that the expression of intergenic sequences underwent extensive and reversible alterations in response to the modulation in murF expression. In none of these cases was there an observable change in the expression in either one of the two ORFs directly neighboring the particular intergenic sequence (see Table S5 in the supplemental material). The nature of these intergenic sequences is not known. Whether or not some of these represent RNAs with possible regulatory roles is currently under investigation (9, 23).

Altered fermentation patterns in bacteria grown with suboptimally expressed murF.

The transcriptional changes left their mark on the physiology and biochemical capacity of the bacteria also. The most striking documentation of the extent of metabolic shifts precipitated by the slowdown in the transcription of murF was the demonstration that bacteria grown under conditions of optimal or suboptimal murF transcription had different fermentation capacities as shown by the Biolog assay (Fig. (Fig.4).4). Bacteria grown in the presence of the optimal IPTG concentration showed the capacity to ferment eight distinct carbon sources (N-acetyl-d-glucosamine, α-d-glucose, d-mannose, d-trehalose, d-lactic acid methyl ester, l-lactic acid, sucrose, and glycerol). The same bacteria grown under conditions of suboptimal murF expression could utilize the same eight carbon sources but also acquired the additional capacity to ferment nine other carbon sources. Besides the compounds normally used by strain COL (and by the conditional mutant grown with optimal expression of murF), bacteria grown with suboptimal expression of murF were also able to metabolize dextrin, maltose, α-hydroxybutyric acid, adenosine, 2′-deoxyadenosine, inosine, thymidine, uridine, and pyruvic acid methyl ester. These expanded fermentation patterns are consistent with the findings of the microarray data, which indicate that in bacteria grown at suboptimal inducer concentrations, there is an up-regulation of genes which code for anaerobic stress-related enzymes (pyruvate formate lyase and formate acetyltransferase), enzymes of nitrogen metabolism (urease and nitrite and nitrate reductases), and enzymes in the intermediary metabolism of pyrimidines and purines. The striking activation of the anaerobic stress pathway, including overexpression of SACOL0204 and SACOL0205 (genes pflB and pflA), encoding homologues of formate acetyltransferase and pyrurate/formate lyase which catalyze the nonoxidative transformation of pyruvate to acetyl coenzyme A and formate, corresponds to the typical response of a bacterium to oxygen-limiting conditions (27). These two genes were also overexpressed when S. aureus 8325 was challenged with hydrogen peroxide, i.e., during oxidative stress (2). Besides the pflBA genes, the murF stress also led to the activation of genes related to fermentative metabolism such as ldh, fruK, and lactate permease and formate/nitrite transporter protein genes.

FIG. 4.
Biolog plate assay performed for strain COL (a) and mutant COLspacmurF (b) grown with the optimal inducer concentration (condition A) and mutant COLspacmurF grown with suboptimal inducer concentration (condition B) (c).

These observations indicate that S. aureus is responding to the deficit of murF transcription in a way similar to what was described for oxidative stress. Yet the cultures used in the experiments with the murF conditional mutant were vigorously aerated throughout the course of the studies. Also, the key components of the SOS system, such as lexA, uvrA, uvrB, and recA (3, 7), remained unchanged during murF stress. Therefore, the activation of the anaerobic pathway by limiting the source of oxygen is excluded as an explanation. The mechanism of this major shift of energy metabolism is not clear at the present time.

It has been postulated (35) that the cell wall stimulon senses a low-molecular-weight peptidoglycan metabolite. This metabolite could be sensed at the cytoplasmic, membrane, or cell wall level. In fact, in the murF mutant the abnormal tripeptide structure should be present in large amounts in these three cellular compartments. Therefore, it is conceivable that the metabolite involved is either the tripeptide or the concentration of the normal pentapeptide cell wall precursor.

It is widely agreed that in bacteria the primary control of metabolism occurs at the level of transcription. The wide range of determinants responding to the murF stress is a striking demonstration of the multitude and complexity of transcription level links that connect a large number of diverse cellular functions to a single step in cell wall synthesis in a reversible manner. The alterations in the S. aureus transcriptome described in this study may be a model for the type of control that allows bacteria to rapidly adapt to changing conditions in the nutritional or physical chemical environment.

Supplementary Material

[Supplemental material]


Partial support for this study was provided by a grant from the U.S. Public Health Service (RO1AI045738) awarded to Alexander Tomasz and by contract POCI/BIA-MIC/58416/2004 (Fundação para a Ciência e a Tecnologia, Portugal) awarded to H. de Lencastre. R. G. Sobral was supported by grants SFRH/BD/3138/2000 and 022/BI/2005 (Fundação para a Ciência e a Tecnologia, Portugal) and was also the recipient of a short-term fellowship from Fundação Calouste Gulbenkian (78401).

We are grateful to Sandro Pereira for his assistance in sample preparation for real-time RT-PCR and for scientific discussions. We are indebted to the members of “Núcleo de Infecções Sexualmente Transmissíveis—Unidade de Chlamydia-Neisseria” of the Instituto Nacional de Saúde Ricardo Jorge, Lisboa, Portugal, for technical help with the real-time RT-PCR technique, especially for the support and availability of Joao Paulo Gomes and Alexandra Nunes.


[down-pointing small open triangle]Published ahead of print on 28 December 2006.

Supplemental material for this article may be found at http://jb.asm.org/.


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