Research Article
15 November 2006

Reporter Metabolite Analysis of Transcriptional Profiles of a Staphylococcus aureus Strain with Normal Phenotype and Its Isogenic hemB Mutant Displaying the Small-Colony-Variant Phenotype


In this study, full-genome DNA microarrays based on the sequence of Staphylococcus aureus N315 were used to compare the transcriptome of a clinical S. aureus strain with a normal phenotype to that of its isogenic mutant with a stable small-colony-variant (SCV) phenotype (hemB::ermB). In addition to standard statistical analyses, systems biology advances were applied to identify reporter metabolites and to achieve a more detailed survey of genome-wide expression differences between the hemB mutant and its parental strain. Genes of enzymes involved in glycolytic and fermentative pathways were found to be up-regulated in the hemB mutant. Furthermore, our analyses allowed identification of additional differences between the normal-phenotype S. aureus and the SCV, most of which were related to metabolism. Profound differences were identified especially in purine biosynthesis as well as in arginine and proline metabolism. Of particular interest, a hypothetical gene of the Crp/Fnr family (SA2424) that is part of the arginine-deiminase (AD) pathway, whose homologue in Streptococcus suis is assumed to be involved in intracellular persistence, showed significantly increased transcription in the hemB mutant. The hemB mutant potentially uses the up-regulated AD pathway to produce ATP or (through ammonia production) to counteract the acidic environment that prevails intracellularly. Moreover, genes involved in capsular polysaccharide and cell wall synthesis were found to be significantly up-regulated in the hemB mutant and therefore potentially responsible for the changed cell morphology of SCVs. In conclusion, the identified differences may be responsible for the SCV phenotype and its association with chronic and persistent infections.
The opportunistic pathogen Staphylococcus aureus is one of the major causes of nosocomial and community-acquired diseases that may range from superficial skin infections to life-threatening systemic infections and toxicoses (15). The ability of this species to cause such a wide spectrum of disease and to adapt to changing conditions is conferred by an impressive arsenal of pathogenicity and virulence factors that are globally regulated (3).
S. aureus may have an intrinsic ability for resisting treatment with antimicrobial agents that extends beyond what are now considered classical mechanisms of drug resistance (24). The discovery and characterization of a naturally occurring subpopulation of S. aureus, designated small-colony variants (SCVs), and their association with chronic and persistent infections have provided new insight into the understanding of the pathogenesis of S. aureus (26). Several studies showed that SCVs, in contrast to their normal-phenotype parental strain progenitors, can be internalized by and persist within nonprofessional phagocytes (34-36). The capacity of SCVs to persist intracellularly and to hide within host cells can be regarded as a strategy of the bacteria for survival within the host and an additional strategy to evade antibiotic challenge and host defenses (26).
Clinical (i.e., genetically undefined) SCVs are frequently auxotrophic for hemin or menadione, two compounds involved in the synthesis of the electron carriers cytochrome and menaquinone, respectively, and exhibit a high rate of reversion to a normal, large-colony form. The genetic nature of the observed auxotrophies and the instability of the auxotrophic phenotype remain to be determined. To create a genetically and phenotypically stable SCV, a hemB-knockout mutant was created by allelic exchange (36). Genetically defined S. aureus hemB mutants have been compared with SCVs recovered from clinical specimens and have proved to exhibit the major characteristics of the SCV phenotype of clinical strains: slow growth, decreased pigment formation, resistance to aminoglycosides, low coagulase activity, and reduced hemolytic activity (1, 29, 30, 34, 36).
To provide a more complete analysis of SCV phenotypes and to gain a clearer insight into physiological changes that lead to in vivo antibiotic resistance and persistence, SCV mutants that reproduce the SCV phenotype were compared to their parental strain by various approaches. By application of a high-resolution two-dimensional protein gel electrophoresis technique coupled with matrix-assisted laser desorption ionization-time of flight mass spectrometry, proteins involved in the glycolytic pathway and in fermentation pathways were found to be induced in an exponentially growing hemB mutant compared to its wild-type parental strain (12). Again compared to the parent strain, phenotype microarray analysis of over 1,500 phenotypes revealed that a hemB mutant was defective in utilizing a variety of carbon sources including tricarboxylic acid (TCA) cycle intermediates and compounds that generate ATP via electron transport (37). Furthermore, hexose phosphates and other carbohydrates that provide ATP in the absence of electron transport stimulated growth of the hemB mutant compared to its wild-type parental precursor strain. Finally, based on a subgenomic DNA microarray analysis (i.e., 460 genes), it has been suggested that SigB might play a role in the expression of the SCV phenotype (19).
Despite these recent analyses of SCV phenotype and insights into the physiological differences between the normal phenotype and the SCV, we are still lacking an understanding of the signaling and regulatory mechanisms underlying the expression of the SCV phenotype of S. aureus. It is anticipated that identification of differences between the normal phenotype and SCV phenotype might provide clues into this circuitry. Here, genome-wide techniques offer unprecedented potential for identification of undiscovered phenotypic differences as these techniques screen on a system-wide level. In none of the previous studies was a complete analysis of genes differentially expressed in SCV and normal-phenotype S. aureus performed.
In this study, a comparative, genome-wide transcriptome analysis of an S. aureus hemB mutant displaying the clinical SCV phenotype versus the wild-type parental strain with normal phenotype was conducted. First, we employed a standard statistical analysis of the transcription data. Second, we harnessed the potential of recent systems biology advances to analyze the simple but notoriously overwhelming transcriptome data. We employed a recent genome-scale reconstruction of the S. aureus metabolic network (7) and a novel pathway-driven computational algorithm (20) to further extract metabolism-related transcriptional differences between the mutant and the parental strain.


Bacterial strains and growth conditions.

S. aureus wild-type strain A22223I, recovered from a fistula tract of a patient with chronic osteomyelitis and most recently used in a Caenorhabditis elegans infection model (30), and its isogenic hemB mutant (A22223I hemB::ermB), displaying the small-colony-variant phenotype, were used for all experiments. The strain A22223I was selected as the parent strain for the construction of the hemB mutant, because the strain was recovered in parallel with an S. aureus isolate displaying the SCV phenotype. As this hemin-auxotrophic SCV isolate revealed an unstable phenotype, exhibiting a high rate of reversion to the large-colony form, the isogenic parent strain A22223I was used to construct a genetically defined and stable SCV phenotype. This hemB mutant was constructed by allelic replacement with an ermB cassette-inactivated hemB gene, as previously described (34, 36). Furthermore, a complemented mutant with restored normal phenotype was constructed as previously reported (36). To keep the strains in a low passage number, strains were taken from stored small aliquots. Overnight cultures in tryptic soy broth (TSB) medium (Becton Dickinson GmbH, Heidelberg, Germany) were inoculated with a single colony. In precultures (prior to RNA isolation), the hemB mutant was grown in TSB supplemented with erythromycin at a concentration of 2.5 μg/ml.

RNA isolation.

For RNA isolation, shaking flasks (100 ml TSB in 500-ml flasks as used in a previous study [12]) were inoculated to an optical density at 578 nm of 0.05 into fresh TSB medium at 37°C and 160 rpm by using overnight cultures (15). Samples were collected and processed at least in triplicate to analyze at least three RNA samples for each strain and time point. Cells of the parent strain were harvested after 150, 270, 375, 480, and 600 min and cells of the hemB mutant after 240, 330, 390, 495, and 600 min, to provide bacteria in the same growth phase (Fig. 1) and as performed in a previous study (12). A volume of 10 ml of a bacterial suspension of the parent strain was immediately mixed with 10 ml of RNAprotect (QIAGEN, Hilden, Germany), vortexed for 5 s, incubated for 5 min at room temperature, and pelleted by centrifugation for 10 min at 4,000 × g. To compensate for the difference in cell number, 10 10-ml-volume suspensions of the hemB mutant were pelleted by centrifugation (10 min at 4,000 × g). Each pellet was immediately resuspended in 1 ml of RNAprotect (QIAGEN). Then, the 10 hemB mutant suspensions were pooled, vortexed for 5 s, incubated for 5 min at room temperature, and harvested by centrifugation (10 min at 4,000 × g). The pooled bacterial pellets were resuspended in 1 ml RNApro solution (Qbiogene, Heidelberg, Germany) and purified on a Matrix E column (Qbiogene). Cells were separated by mechanical lysis using a FastPrep Instrument (Qbiogene): once at 30 s, attitude of disruption 6.5, 30 s on ice, and once at 30 s, attitude 6.5. Further RNA purification was performed using the RNeasy Mini Kit (QIAGEN) according to the manufacturer's recommendations. Contaminating DNA in the RNA preparations was removed using DNase as described by the manufacturer (QIAGEN).
The RNA quality and quantity were determined by measurement of the absorbance at 260 and 280 nm (Eppendorf BioPhotometer; Hamburg, Germany) and agarose gel electrophoresis (intact rRNA bands). Purified RNA was stored at −70°C. Independent samples of RNA were used for each time point on separate microarrays.

cDNA synthesis, labeling, and microarray hybridization.

S. aureus N315 microarrays were purchased from Scienion (Scienion AG, Berlin, Germany) and were produced by spotting 2,338 PCR products (of the 2,593 open reading frames [ORFs] of the annotated genome of S. aureus N315 [reference identification: NC_002745]) on a glass slide (details about the microarrays can be found at ). Each open reading frame was present in duplicate on the microarray. cDNA was synthesized from mRNA as recommended by the manufacturer of the microarray: RNA (12 μg unless otherwise indicated) from either A22223I or its hemB mutant was mixed with 1 μg of random hexamer primer (Invitrogen, Karlsruhe, Germany), 1 μl of RNase OUT (Invitrogen), and RNase-free water up to a volume of 10 μl. The samples were denatured at 70°C for 10 min and then cooled on ice for 1 min. Labeled cDNA was synthesized by mixing the denatured RNA with 200 U of Superscript III reverse transcriptase (Invitrogen), Cy3- and Cy5-dUTP, deoxynucleoside triphosphate mix, and appropriate buffer in a reaction mix according to the manufacturer's protocol (Scienion). The mixture was incubated for 10 min at 25°C, followed by incubation at 47°C for 60 min. Two hundred units of Superscript III reverse transcriptase (Invitrogen) was added again, followed by a further incubation for 40 min at 47°C. The reaction was stopped by adding 5 μl of 500 mM EDTA. Then, the mixture was incubated for 15 min at 65°C after NaOH (5 μl, 1 M) was added to hydrolyze the RNA. The sample was neutralized with 12.5 μl of Tris-HCl (1 M, pH 7.5), and the resulting cDNA was purified using a QIAquick PCR purification kit (QIAGEN). The volumes of the labeled cDNA solutions were reduced to 3 μl by using a SpeedVac (Thermo Electron Corp., Waltham, MA). The Cy3- and Cy5-labeled cDNA solutions were mixed, resuspended in 49 μl of prewarmed (48°C) hybridization solution (Scienion), and incubated for 5 min at 48°C. The mixture of labeled products was denatured for 2 min at 95°C. The combined samples were hybridized to the S. aureus N315 microarrays for 72 h at 48°C. The slides were washed according to the manufacturer's protocol and stored at −70°C. For each of the five time points, at least three DNA microarrays plus one dye-switch experiment (to check cDNA synthesis and labeling) were analyzed.

Data analysis.

The hybridized microarrays were scanned with a GMS418 array scanner (Affymetrix, Santa Clara, CA). A geometric raster was laid over the resulting microarray picture to distinguish the signals from the background. After localization of single spots, the spot intensities and the global background were calculated.
The hybridization patterns and intensities were quantitatively analyzed using the Imagene 6 software (BioDiscovery, El Segundo, CA). The replicates were averaged, and the spots identified by Imagene 6 as flawed were omitted. The data set was normalized by application of the LOWESS algorithm. The data from Imagene 6 were exported into Expressionist (GeneData, Basel, Switzerland) and Excel (Microsoft Corporation, Redmond, WA) software for further analysis as, e.g., identification of microarrays that present technical outliers. The expression intensities of one single array, resulting from multiple scans with different gains, were averaged. In a next step, the averaged intensity values of all arrays for each time point as well as for all time points combined were used for t tests. Genes with a change of <0.4- or ≥2.0-fold were characterized as having significantly differing amounts of transcripts based on t tests with a P value cutoff of at least 0.05. Gene functions were assigned to the respective accession numbers and annotations as compiled on DOGAN, a web page for S. aureus N315 ( ). Fisher's exact test was used to decide whether functional annotations show a tendency of over- or underrepresentation in a candidate gene list compared to all measured and annotated items.
The microarray data were also analyzed by a recently developed algorithm that uses the topology of an organism's metabolic network to uncover underlying metabolism-related transcriptional regulation (20). This algorithm first converts a genome-scale metabolic network (we employed the recently reconstructed genome-scale metabolic network of S. aureus N315 [7]) into a bipartite metabolic graph. In this graph, each metabolite node is then scored based on the normalized transcriptional response of its neighboring enzymes. Using the genes' P values as inputs to score the enzyme nodes, the algorithm identifies so-called reporter metabolites, designating metabolites around which the most significant transcriptional changes occur. The mapping of transcription data onto a metabolic network, which underlies the employed algorithm, allows identifying spots (so-called reporter metabolites) around which significant regulation occurs and thus assists in carving out metabolism-related insight from the microarray data.

Validation of array data by real-time PCR.

To determine the validity of the array data, selected transcriptional changes obtained with the microarray analysis were compared with those from quantitative real-time PCR. For a list of the genes and primer sequences used for the real-time PCR analysis, see Table S1 in the supplemental material. The real-time PCR was performed by using the iCycler (Bio-Rad Laboratories GmbH, Munich, Germany) with a QuantiTect reverse transcription kit (QIAGEN) and the DyNAmo HS SYBR Green qPCR Kit (Finnzymes Oy, Espoo, Finland). Reaction mixtures were initially incubated for 15 min at 95°C, followed by 40 cycles of 15 s at 95°C, 30 s at 55.0°C, and 30 s at 72°C. PCR efficiencies were derived from standard curve slopes in the iCycler software v. 3.0a (Bio-Rad Laboratories). The expression rates were calculated using Gene Expression Analysis for iCycler iQ Real-Time PCR Detection System v1.10 (Bio-Rad Laboratories). Melting-curve analysis was also performed to evaluate PCR specificity and resulted in single, primer-specific melting temperatures.


SCVs recovered from clinical specimens are frequently auxotrophic for hemin. For S. aureus, in vitro gentamicin-selected SCVs are found that carry mutations in the hem operon, which encodes enzymes required for hemin biosynthesis (28). Furthermore, an Escherichia coli SCV has been isolated from a patient and has been shown to carry a hemB mutation that renders the bacterium defective in hemin biosynthesis (27). In this study, a stable genetically defined S. aureus SCV hemB mutant was employed to serve as a model organism for the transcriptional changes that occur upon loss of hemin biosynthesis. The model organism was used to limit the uncertainty associated with using an undefined genetic background and reversible phenotypes found in genetically undefined clinical SCVs and gentamicin-induced SCVs (36). Past studies using defined S. aureus hemB mutants, in part in comparison with clinical SCVs, had previously shown that this organism confers the major features of the SCV phenotype (1, 9, 12, 29, 30, 34, 36, 37).

Transcriptional differences between the S. aureus hemB mutant and its parent strain.

In this study, the hemB mutant displaying the SCV phenotype was applied on a full-genome microarray to get a complete view of the transcriptional profile of SCVs. More specifically, we compared the expression levels of the S. aureus hemB mutant and its parent strain at five time points, corresponding to different growth phases: lag phase, early exponential phase, mid-exponential phase, late exponential phase, and stationary phase (Fig. 1).
To verify the microarray data, quantitative real-time reverse transcription-PCR studies were performed for selected genes of the S. aureus parent strain, its hemB mutant, and its complemented mutant. In fact, the results of quantitative real-time reverse transcription-PCR analyses of selected transcripts were found to be in excellent accordance with the microarray analysis. This was true for the pooled time points (Table 1) as well as for the single time points (data not shown).
With the standard statistical analysis of the acquired genome-wide transcription data, when values from different growth phases were pooled, 170 genes were found to be significantly changed when the wild-type and the hemB-disrupted strains were compared. Compared to the parent strain, 48 of these genes were significantly down-regulated and 122 genes were significantly up-regulated in the hemB mutant. All significantly differently expressed genes in the combined analysis of all values from all growth phases are listed in Table 2. Data from each phase of growth are listed in Tables S2 to S6 in the supplemental material. To reveal functional groups of genes that are in general differentially expressed in the hemB mutant compared to the parent strain, we performed Fisher's exact test using the classification from the DOGAN web page (results are shown in Table 3).
As demonstrated in past studies, the phenotype of the hemB mutant is characterized by a significantly reduced growth rate and an excretion of lactate instead of acetate, both giving indication of metabolic differences between the mutant and its parental strain. In fact, recent proteomic and phenotypic microarray studies underlined the significance of metabolic differences, such as the defect of the hemB mutant in utilization of a variety of carbon sources, including TCA cycle intermediates and compounds that ultimately generate ATP via electron transport. Furthermore, hexose phosphates and other carbohydrates that provide ATP in the absence of electron transport were found to stimulate growth of the hemB mutant (12, 37). Thus, besides the standard statistical analysis we put a special focus on metabolism-related transcriptional changes by using a recently developed algorithm from systems biology (20).
Before using this algorithm, we first extracted the genes from the microarray data that are related to metabolic functions. For this, we employed the recently reconstructed S. aureus metabolic network (7), leaving us with approximately 560 genes that encode enzymes where substrates and products are known. In a next step, we used the computational method of Patil and Nielsen (20) to computationally map the transcriptional changes of this set of genes onto the metabolic network (cf. Materials and Methods). Thus, by this linking of transcription data with metabolism (in contrast to an otherwise isolated analysis of single genes) the transcriptional changes in the hemB mutant (compared to its wild-type strain) were considered in a metabolic context. In fact, this approach enables a condensation of transcriptional data to a number of metabolites around which substantial transcriptional changes occur. Patil and Nielsen called these metabolites “reporter metabolites” (20) as they mark spots in the metabolism around which regulation occurs most likely in order to either increase, decrease, or redirect a metabolic flux. In a broader view, combined with information on the submetabolisms in which these metabolites occur, areas of significant regulatory action can be identified.
We first determined the reporter metabolites from P values obtained when the microarrays from all the different time points were combined. One of the top-scoring reporter metabolites that were identified in this combined analysis was 5-aminolevulinate, an intermediate in the synthesis of the electron transporter heme. It is synthesized from glutamate-1-semialdehyde 2,1-aminomutase (encoded by hemL, SA1491) and further metabolized by aminolevulinic acid dehydratase (encoded by hemB, SA1492). The appearance of 5-aminolevulinate might be explained by the following two possibilities: (i) the up-regulation of hemL is an effect of the hemB knockout on expression from a distal promoter and, thus, most likely from the promoter of the hem operon (hemAXCDBL), or (ii) it is an effect resulting from the erm promoter: only hemL (SA1491) and hemB (SA1492) were found to be up-regulated whereas none of the other hem genes that belong to the hem operon (hemAXCD) showed a changed transcription profile. This led to the assumption that the change in transcription level is an effect of the erm promoter, which is indeed located upstream of hemL and upstream of the part of hemB spotted on the microarray. Therefore, it is assumed that the observed changes are an effect of the erm promoter. In any case, the occurrence of 5-aminolevulinate as a reporter metabolite provides evidence that the employed pathway-driven analysis of microarray data is able to uncover areas of regulatory action.
In the combined analysis of all time points, it was further found that most of the significant reporter metabolites (P < 0.05) cluster in three distinct metabolic regions: central carbon metabolism, arginine and proline metabolism, and purine synthesis (see Fig. S1 in the supplemental material). Below, we will focus our discussion on these areas.

Arginine and proline metabolism.

The mutation in hemB led to an identification of l-arginine, l-arginine (extracellular), and l-ornithine (extracellular) as reporter metabolites, which are all involved in the arginine-deiminase (AD) pathway. The gene for the arginine/ornithine antiporter, arcD (SA2426), showed a 16.29-fold-higher expression in the combined analysis compared to the parent strain (Table 2). This reflects a significant difference between the hemB mutant and its parental strain. Similar changes were found for arcA (arginine deiminase, SA2428, 4.96-fold up-regulated) and arcB (ornithine transcarbamoylase, SA2427, 4.24-fold up-regulated).
The hemB mutant may regulate the AD pathway through a Crp/Fnr family transcriptional regulator. Fnr family regulators have previously been linked to AD system regulation (16, 17, 31), and they have proven to be responsible for anaerobic gene regulation in many gram-negative and some gram-positive bacteria (6, 12, 31). Particularly in Streptococcus suis, it has been shown that the AD system is induced under microaerophilic and anaerobic conditions (6). In accordance with the observations in S. suis, the S. aureus gene SA2424 product, a hypothetical protein similar to the transcription regulator from the Crp/Fnr family, was up-regulated in the hemB mutant (9.18-fold, pooled values for all five time points) in comparison with the parent strain, and it might play an important role in the regulation of the AD pathway.
It was demonstrated that the AD system can be considered a system that protects streptococci against acidic stress (2, 4), a trait that would correlate with the ability of SCVs to persist intracellularly (36). It may be assumed that the up-regulated AD system allows S. aureus SCVs to counteract (through ammonia production) the intracellular acidic environment. However, it is also conceivable that the hemB mutant uses this pathway to produce ATP as was hypothesized in previous studies (7, 12).
Also from the arginine and proline metabolism, another set of reporter metabolites was identified (l-4-hydroxyglutamate semialdehyde, l-glutamate 5-semialdehyde, N 2-succinyl-l-glutamate, and N 2-succinyl-l-glutamate 5-semialdehyde). The genes whose products act on these metabolites are rocA (SA2341) and rocD (SA0181), which are both down-regulated in the hemB mutant compared to its wild-type progenitor strain. From the analysis of data collected from each phase of bacterial growth (Fig. 2), significant changes are evident only at the second through the fifth time point. These genes are involved in arginine catabolism, where arginine is cleaved by arginase to give ornithine, which is converted to glutamate semialdehyde by ornithine aminotransferase (rocD gene product). Finally, the conversion of glutamate semialdehyde to glutamate is catalyzed by the rocA gene product. The roc pathway is generally considered an aerobic pathway for the metabolism of l-arginine that terminates in l-glutamate (17, 18). Potentially, the down-regulation of rocA and rocD prevents a drain of ornithine towards glutamate, which in turn would result in a lower efficiency of the AD pathway to counteract acidic conditions or provide ATP.

Central carbon metabolism.

We identified a large number of metabolites from central carbon metabolism as reporter metabolites. More specifically, these metabolites belong to the terminal part of glycolysis, glycerol metabolism, pyruvate metabolism, and acetate metabolism and to the TCA cycle (Fig. 2 and 3).
The significant changes in glycolysis on one hand were caused by an up-regulation of an operon consisting of SA0727, SA0728, SA0730, and SA0731 and on the other hand by a significant down-regulation of genes encoding two sets of isoenzymes (SA1510 [homologous to SA0727] and SA2204 [homologous to SA0730]) (Fig. 3).
Genes encoding products responsible for the metabolism of glycerol showed significantly increased expression levels in the hemB mutant (Fig. 3): glycerate kinase (SA2220, 3.25-fold), glycerol diffusional facilitator (glpF, SA1140, 5.58-fold), and aerobic glycerol-3-phosphate dehydrogenase (glpD, SA1142, 5.45-fold). glpD is known to be induced by glycerol and repressed by glucose (14). Consistent with the model for expression of glycerol-3-phosphate dehydrogenase for Bacillus subtilis (22), the glycerol uptake operon antiterminator regulatory protein (glpP, SA1139, 2.1-fold) was also up-regulated in the hemB mutant compared to the parental strain. While a regulatory linkage in B. subtilis between antitermination by GlpP and the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was described, phosphoenolpyruvate-protein phosphatase (ptsI, SA0935), one of the two major proteins of the PTS, was found to be down-regulated in the hemB mutant compared to its parental strain (0.59-fold). Of interest, a murine model revealed that an S. aureus ΔptsI mutant had a 10-fold-higher 50% lethal dose than its virulent wild-type strain did (13).
Pyruvate as well as intra- and extracellular lactate also occurred as a reporter metabolite (Fig. 3). Here, significant changes occurred in the expression of both lactate dehydrogenases, which catalyze the reversible NAD-dependent interconversion of pyruvate to l-lactate, representing the final step in anaerobic glycolysis of lactic acid bacteria. The gene for the anaerobic l-lactate dehydrogenase (lctE, SA0232) revealed a dramatically increased expression (40.16-fold up-regulated in the hemB mutant in comparison of pooled values), while the aerobic l-lactate dehydrogenase (SA2395) was down-regulated. This observation of an anaerobic metabolism occurring in the hemB mutant is in line with earlier indications that principally fermentative pathways are activated in the hemB mutant (7). The massive up-regulation of the anaerobic l-lactate dehydrogenase in the hemB mutant is obviously required for terminal oxidation of NADH. Simultaneously, the gene encoding a lactate importer (lctP, SA0106) showed a reduced expression level in the mutant, the reason for which remains elusive.
In contrast to the parental strain, the hemB mutant shows a lower level of acetate utilization (12). Accordingly, transcriptional differences in acetate metabolism were found: with the exception of the pyruvate oxidase (pox, SA2327), genes from pathways leading to acetate were significantly down-regulated in the hemB mutant (Fig. 3). Pyruvate oxidase utilizes Pi to produce acetyl-P (oxygen dependent), which in turn produces ATP and acetate. The up-regulation of pyruvate oxidase can be considered a clear benefit for the energy-starved hemB mutant.
Despite the increased expression of genes belonging to lower glycolysis and the pathway leading to lactate (Fig. 3), the gene responsible for converting phosphoenolpyruvate to pyruvate (pyruvate kinase, pykA, SA1520) (bridging these two aforementioned segments of increased expression) did not change its expression level at all. Here, it is conceivable that the significantly increased expression of several PTSs (among which is ptsG [SA2326], the PTS responsible for glucose uptake) takes over the conversion of phosphoenolpyruvate to pyruvate.
In the TCA cycle, several reporter metabolites occurred as a result of a decreased expression of aconitate hydratase (citB, SA1184, 0.34-fold), isocitrate dehydrogenase (citC, SA1517, 0.39-fold), and citrate synthase (citZ, SA1518, 0.42-fold). In addition, intermediate compounds produced by the pyruvate dehydrogenase and the oxoglutarate dehydrogenase complexes were identified as reporter metabolites (Fig. 2 and 3). The genes particularly contributing to this were pdhBCD (SA0944 to SA0946, 0.66-, 0.34-, and 0.50-fold changes, respectively), odhAB (dihydrolipoamide succinyltransferase, SA1244, 0.20-fold), and 2-oxoglutarate dehydrogenase E1 (SA1245, 0.26-fold). Interestingly, these differences were significant only at the time points 2 through 5, representing early-exponential through stationary growth (Fig. 2).

Purine biosynthesis.

Our analysis identified several intermediates from the purine synthesis as reporter metabolites (see Fig. S1 in the supplemental material; Fig. 2). The differently regulated genes related to these metabolites were the genes of the pur operon (comprising purCDEFHKLMNQ) (SA0918, SA0926, SA1048, SA0922, SA0925, SA0917, SA0921, SA0923, SA0924, and SA0920), folD (FolD bifunctional protein, SA0915), and fhs (formyltetrahydrofolate synthetase, SA1553). All genes showed a decreased expression profile in the hemB mutant compared to the parent strain (Table 2). The genes folD and fhs strictly belong to the biosynthesis of folate coenzymes, which are, however, required for purine synthesis. The analysis of the different growth phases showed that the reported genes are down-regulated only up to mid-log phase (time points 1 to 3, Fig. 2), while at the late exponential and stationary phases of growth no difference between the hemB mutant and its parental strain was detected. Fisher's exact test confirmed these findings for pooled values and lag through logarithmic phase as given in Table 3.
In B. subtilis, a regulator protein, PurR (S. aureus homologue SA0454), was found to repress purA (adenylosuccinate synthase, S. aureus homologue SA0016), glyA (serine hydroxymethyl transferase, S. aureus homologue SA1915), folD, and the complete pur operson (8). In Lactococcus lactis, an activator homologous to the B. subtilis purR repressor was identified (11). We found that expression of staphylococcal purR was only slightly elevated in the hemB mutant in contrast to its wild-type progenitor strain (1.45-fold). The reason for the significant down-regulation of purine synthesis remains unknown.

Membrane bioenergetics.

The phenotype of hemin-auxotroph SCVs is likely linked to disruption of electron transport (25, 36). Consistent with this idea, all of the genes involved in membrane bioenergetics were found to be overrepresented in the combined analysis and in the stationary growth phase if analyzed on its own. Affected genes were the NADH dehydrogenase subunit (ndhF, SA0411, 5.0-fold), cytochrome d ubiquinol oxidase subunit II homologue (SA0937, 2.13-fold), hypothetical protein similar to ferredoxin oxidoreductase beta subunit (SA1132, 2.34-fold), cytochrome d ubiquinol oxidase subunit I homologue (SA0937, 2.14-fold), hypothetical protein similar to thioredoxin reductase (SA1311, 2.12-fold), and ferredoxin (fer, SA1315, 2.31-fold).

Cell division and cell wall synthesis.

S. aureus SCVs show impaired cell separation and incomplete or multiple cell walls (10). Genes involved in these processes were found to be up-regulated in the hemB mutant, for example, pbpA (penicillin-binding protein 1, SA1024, 2.4-fold), llm (lipophilic protein affecting bacterial lysis rate and methicillin resistance, SA0702, 2.2-fold), pbp2 (penicillin-binding protein 2, SA1283, 2.4-fold), and varS (two-component sensor histidine kinase, SA1701, 4.09-fold). VarS has been described as a sensor critical for the control of penicillin-binding proteins (38). This protein may play a major role in the alteration of expression of genes involved in cell wall synthesis and cell division and, therefore, may contribute to the reduced susceptibility of the hemB mutant to antimicrobial agents.

CPs and adhesions.

The role of the S. aureus capsule in the pathogenesis of staphylococcal infections has been investigated in several animal models of infection. Of interest, we found that the genes for capsular polysaccharide (CP) synthesis, capA, capB, capC, capD, capE, capF, and capG (SA0144 to SA0150), were up-regulated in the hemB mutant compared to the parent strain, especially at the lag phase and early logarithmic phase. The group of genes for “adaptation to atypical conditions,” which includes the genes encoding proteins responsible for capsular polysaccharide synthesis, were found to be overrepresented at lag phase and early logarithmic phase and also if values for all growth phases were pooled (Table 3). These findings correlate with a previous study demonstrating growth-dependent expression of CPs (23). While CPs have been shown to enhance virulence in animal models of staphylococcal pathogenesis, antithetically they have been found to reduce an early step in infection, bacterial adherence (32, 33). However, our microarray experiments showed that when expression of genes for CPs was reduced, expression of genes for adhesions was increased (e.g., fibrinogen-binding protein A [clfA, SA0742, 4.74-fold up-regulated in hemB mutant at early logarithmic growth phase] and clumping factor B [clfB, SA2423, 2.96-fold up-regulated in hemB mutant at logarithmic growth phase]). Since adhesion to host cells represents the precursor step to internalization for the bacteria within host cells (5, 21), it may be assumed that up-regulation of genes coding for adhesions may increase the hemB mutant's ability to invade and persist intracellularly.
In summary, while previously detected single genomic traits of the S. aureus hemB mutant were confirmed by this approach, this full-genome microarray also offered a more complete genomic analysis of the hemB mutant and provided insight into the expression profile. Profound differences were identified especially in the purine biosynthesis as well as in the arginine and proline metabolism. Of particular interest, a hypothetical gene of the Crp/Fnr family (SA2424), being part of the AD pathway, whose homologue in Streptococcus suis is assumed to be involved in intracellular persistence, showed a significantly increased transcription in the hemB mutant. The hemB mutant potentially uses the up-regulated AD pathway to produce ATP or (through ammonia production) to counteract the acidic environment that prevails intracellularly. The metabolic rearrangements may be responsible for the association of SCVs with chronic and persistent infections. Furthermore, genes involved in capsular polysaccharide and cell wall synthesis were found to be significantly up-regulated in the hemB mutant and thus potentially responsible for the changed cell morphology of SCVs and its consequences. Further work, however, is necessary to decipher the regulatory program of the SCV phenotype.
FIG. 1.
FIG. 1. Growth of the S. aureus A22223I parental wild-type strain (⧫) and its isogenic hemB mutant (▪) in TSB (with standard deviations). The times of sampling for transcriptional analysis are indicated by the symbols on the growth curves. Time points for RNA isolation were selected as previously described (12). Optical density was measured at 578 nm.
FIG. 2.
FIG. 2. Clustered top-scoring reporter metabolites (P values < 0.05) which occurred at more than one time point. Black boxes denote time points when the respective metabolite that occurred was identified as significant. CoA, coenzyme A; FAD, flavin adenine dinucleotide; FADH2, reduced flavin adenine dinucleotide.
FIG. 3.
FIG. 3. Illustration of significant transcriptional changes in the context of metabolic pathways of central carbon metabolism based on the combined analysis of all time points. Metabolites in gray circles denote reporter metabolites (P values < 0.05). Gene names are given in italics. A gene's down-regulation in the hemB mutant compared to the parental strain is indicated by a red box around the gene locus (based on the S. aureus N315 sequence), and a gene's up-regulation is indicated by a green box. Dark colors denote strong down- and up-regulation. PEP, phosphoenolpyruvate; FAD, flavin adenine dinucleotide; FADH2, reduced flavin adenine dinucleotide.
TABLE 1. Validation of array data by real-time PCRa
Gene identifierGeneQuantitative real-time RT-PCRb result (relative difference in expression change)  Expression difference of the hemB mutant analyses compared to the wild type using microarraysc
  hemB mutantComplemented mutantWild type 
SA0150Capsular polysaccharide synthesis enzyme Cap5G29.91.11.0Sig. up-reg.
SA0232l-Lactate dehydrogenase21.11.01.2Sig. up-reg.
SA0742Fibrinogen-binding protein A494.612.61.0Sig. up-reg.
SA0922Phosphoribosylpyrophosphate amidotransferase PurF1.024.310.6Sig. down-reg.
SA1141Glycerol kinase40.83.61.0Sig. up-reg.
SA1142Aerobic glycerol-3-phosphate dehydrogenase128.01.02.4Sig. up-reg.
SA2206Immunoglobulin G-binding protein SBI1.04.38.3Sig. down-reg.
SA2424Hypothetical protein, similar to transcription regulator Crp/Fnr family protein163.11.01.3Sig. up-reg.
SA2426Arginine/ornithine antiporter891.01.01.3Sig. up-reg.
SA2428Arginine deiminase989.02.11.0Sig. up-reg.
Transcription rates of selected genes for the S. aureus hemB mutant in comparison to the wild type and the complemented mutant for pooled time points. The relative differences in expression change were calculated using Gene Expression Analysis for iCycler iQ Real-Time PCR Detection System v1.10 (Bio-Rad). The lowest expression was set to 1.0. gyrA (SA0006) was used as a housekeeping gene for relative quantification.
RT-PCR, reverse transcription-PCR.
See Table 2. Abbreviations: sig. up-reg., significantly up-regulated; sig down-reg., significantly down-regulated.
TABLE 2. Genes differently expressed in the S. aureus hemB mutant for combined values over the whole time course (0 to 10 h)
Function group and ORF (N315)P value by t testaFold changebDescription
Adaptation to atypical conditions   
    SA01470.003.16Capsular polysaccharide synthesis Cap5D
    SA01440.003.60Capsular polysaccharide synthesis Cap5A
    SA15490.002.64Hypothetical protein similar to serine proteinase Do heat shock protein HtrA
    SA01450.002.65Capsular polysaccharide synthesis Cap5B
    SA24940.003.05Cold shock protein
    SA10960.002.23Heat shock protein
    SA01460.002.89Capsular polysaccharide synthesis Cap8C
    SA23360.002.30ATP-dependent Clp proteinase chain ClpL
    SA01480.012.60Capsular polysaccharide synthesis Cap8E
    SA01500.032.04Capsular polysaccharide synthesis Cap5G
    SA01490.032.14Capsular polysaccharide synthesis Cap5F
Antibiotic production   
    SA01730.012.19Hypothetical protein similar to surfactin synthetase
Cell wall   
    SA16910.003.52Hypothetical protein similar to protein 1A/1B
    SA12830.002.14Penicillin-binding protein 2
    SA10240.002.32Penicillin-binding protein 1
Membrane bioenergetics (electron transport chain and ATP synthase)   
    SA04110.005.00NADH dehydrogenase subunit
    SA02110.000.10Hypothetical protein similar to NADH-dependent dehydrogenase
    SA02100.000.13Hypothetical protein similar to NADH-dependent dehydrogenase
    SA03660.000.38Alkyl hydroperoxide reductase C
    SA09380.012.13Cytochrome d ubiquinol oxidase subunit II homologue
    SA11320.022.34Hypothetical protein similar to ferredoxin oxidoreductase beta subunit
    SA09370.022.14Cytochrome d ubiquinol oxidase subunit I homologue
    SA13110.022.12Hypothetical protein similar to thioredoxin reductase homologue
Metabolism of amino acids and related molecules   
    SA24280.004.96Arginine deiminase
    SA23410.000.381-Pyrroline-5-carboxylate dehydrogenase
    SA11650.000.39Threonine synthase
    SA11660.000.39Homoserine kinase homologue
    SA04190.012.13Cystathionine gamma-synthase
    SA04160.012.05Hypothetical protein similar to carboxylesterase
    SA24270.024.24Ornithine transcarbamoylase
    SA23890.032.06Truncated hypothetical protein similar to metalloproteinase Mpr precursor
Metabolism of carbohydrates and related molecules   
    SA11420.005.46Aerobic glycerol-3-phosphate dehydrogenase
    SA09450.000.35Dihydrolipoamide S-acetyltransferase component of pyruvate dehydrogenase complex E2
    SA12450.000.202-Oxoglutarate dehydrogenase E1
    SA20080.003.40Alpha-acetolactate synthase
    SA07300.002.38Phosphoglycerate mutase
    SA11840.000.34Aconitate hydratase
    SA12440.000.26Dihydrolipoamide succinyltransferase
    SA11410.002.57Glycerol kinase
    SA07280.002.43Phosphoglycerate kinase
    SA20070.002.68Hypothetical protein similar to alpha-acetolactate decarboxylase
    SA07290.002.16Triosephosphate isomerase
    SA01820.000.36Hypothetical protein similar to indole-3-pyruvate decarboxylase
    SA15530.000.33Formyltetrahydrofolate synthetase
    SA10890.000.38SucD succinyl coenzyme A synthetase alpha chain
    SA23270.002.27Hypothetical protein similar to pyruvate oxidase
    SA15540.000.30Acetyl coenzyme A synthetase
    SA02320.0040.16l-Lactate dehydrogenase
    SA02190.012.22Formate acetyltransferase activating enzyme
    SA06540.022.40Fructose 1-phosphate kinase
    SA15170.020.39Isocitrate dehydrogenase
Metabolism of coenzymes and prosthetic groups   
    SA14920.002.06Delta-aminolevulinic acid dehydratase
Metabolism of nucleotides and nucleic acids   
    SA09230.000.22Phosphoribosylformylglycinamidine cycloligase PurM
    SA09240.000.23Phosphoribosylglycinamide formyltransferase
    SA09210.000.24Phosphoribosylformylglycinamidine synthetase PurL
    SA09220.000.26Phosphoribosylpyrophosphate amidotransferase PurF
    SA09160.000.28Hypothetical protein similar to phosphoribosylaminoimidazole carboxylase PurE
    SA09250.000.27Bifunctional purine biosynthesis PurH
    SA09200.000.34Phosphoribosylformylglycinamidine synthase II
    SA22970.002.63Hypothetical protein similar to GTP-pyrophosphokinase
    SA03730.000.32Xanthine phosphoribosyltransferase
    SA09180.010.39Phosphoribosylaminoimidazolesuccinocarboxamide synthetase homologue
Metabolism of phosphate   
    SA23010.002.27Hypothetical protein similar to alkaline phosphatase
No similarity   
    SA22210.003.60Hypothetical protein
    SA14760.005.66Hypothetical protein
    SA20110.004.10Hypothetical protein
    SA22720.002.05Hypothetical protein
    SA17740.002.29(Bacteriophage phiN315) hypothetical protein
    SA05360.003.20Hypothetical protein
    SA20910.000.26Hypothetical protein
    SA17730.002.26(Bacteriophage phiN315) hypothetical protein
    SA17720.002.48(Bacteriophage phiN315) hypothetical protein
    SA17030.003.22Hypothetical protein
    SA05350.012.47Hypothetical protein
    SA08850.022.12Hypothetical protein
    SA21130.032.25Hypothetical protein
    SAS0140.032.22Hypothetical protein
    SA22680.042.16Hypothetical protein
Pathogenic factors (toxins and colonization factors)   
    SA05870.000.13Lipoprotein streptococcal adhesin PsaA
    SA24300.002.37Zinc metalloproteinase aureolysin
    SA22060.000.29Immunoglobulin G-binding protein SBI
    SA03900.003.81(Pathogenicity island SaPIn2) 14
    SA20970.012.68Hypothetical protein similar to secretory antigen precursor SsaA
    SA07420.012.14Fibrinogen-binding protein A
    SA23560.022.12Immunodominant antigen A
Phage-related functions   
    SA17960.006.74(Bacteriophage phiN315) hypothetical protein
    SA17820.002.86(Bacteriophage phiN315) hypothetical protein
    SA17850.002.04(Bacteriophage phiN315) hypothetical protein
    SA17830.012.38(Bacteriophage phiN315) hypothetical protein
    SA17880.022.03(Bacteriophage phiN315) hypothetical protein
    SA17750.022.31(Bacteriophage phiN315) hypothetical protein
    SA17970.022.12(Bacteriophage phiN315) hypothetical protein
Protein folding   
    SA16590.002.89Peptidyl-prolyl cis /trans isomerase homologue
RNA modification   
    SA17130.022.10RNA methyltransferase homologue
    SA18850.022.13Hypothetical protein similar to RNA helicase
RNA synthesis   
    SA24240.009.18Hypothetical protein similar to transcription regulator Crp/Fnr family protein
    SA20920.000.20Hypothetical protein similar to transcription regulator
    SA19490.004.15Lytic regulatory protein truncated with Tn554
    SA19560.003.35Lytic regulatory protein truncated with Tn554
    SA17000.004.09Two-component response regulator
    SA24290.003.61Hypothetical protein similar to arginine repressor
    SA01080.000.30Staphylococcal accessory regulator A
    SA22960.002.71Hypothetical protein similar to transcriptional regulator MerR family
    SA21030.002.20Hypothetical protein similar to divergon expression attenuator LytR
    SA11390.002.11Glycerol uptake operon regulatory protein
    SA06530.002.82Hypothetical protein similar to repressor of fructose operon
    SA19610.002.26Hypothetical protein similar to transcription antiterminator BglG family
    SA24180.012.18Hypothetical protein similar to response regulator
    SA01870.010.39Hypothetical protein similar to transcription regulator
    SA22950.032.65Gluconate operon transcriptional repressor
    SA22870.032.06Staphylococcal accessory regulator A
Sensors (signal transduction)   
    SA17010.004.09Two-component sensor histidine kinase
    SA16530.002.03Signal transduction protein
    SA24170.012.15Hypothetical protein similar to sensor histidine kinase
Similar to unknown proteins   
    SA01750.005.39Conserved hypothetical protein
    SA17020.003.31Conserved hypothetical protein
    SA04120.005.20Conserved hypothetical protein
    SA02130.000.23Conserved hypothetical protein
    SA22200.003.26Conserved hypothetical protein
    SA04130.002.83Conserved hypothetical protein
    SA02120.000.14Conserved hypothetical protein
    SA05880.000.13Conserved hypothetical protein
    SA23290.003.96Conserved hypothetical protein
    SA17120.003.19Conserved hypothetical protein
    SA09080.003.66Conserved hypothetical protein
    SA21460.002.72TcaA protein
    SA09190.000.31Conserved hypothetical protein
    SA07250.002.75Conserved hypothetical protein
    SA10210.002.33Conserved hypothetical protein
    SA10310.002.67Conserved hypothetical protein
    SA10320.002.17Conserved hypothetical protein
    SA08240.002.29Conserved hypothetical protein
    SA14330.010.37Conserved hypothetical protein
    SA03010.010.40Conserved hypothetical protein
    SA09620.012.08Conserved hypothetical protein
    SA10330.022.38Conserved hypothetical protein
    SA24810.022.23Conserved hypothetical protein
    SA04150.022.16Conserved hypothetical protein
    SA22980.032.01Conserved hypothetical protein
    SA01740.042.01Conserved hypothetical protein
    SA10710.042.02Conserved hypothetical protein
Transport/binding proteins and lipoproteins   
    SA24260.0016.29Arginine/ornithine antiporter
    SA05410.004.96Hypothetical protein similar to amino acid transporter
    SA11400.005.59Glycerol uptake facilitator
    SA19600.003.85PTS mannitol-specific IIBC component
    SA02930.003.77Hypothetical protein similar to formate transporter NirC
    SA21560.003.72Permease LctP homologue
    SA05890.000.11Hypothetical protein similar to ABC transporter ATP-binding protein
    SA23030.000.21Hypothetical protein similar to membrane-spanning protein
    SA02080.000.13Maltose/maltodextrin transport permease homologue
    SA23020.000.31Hypothetical protein similar to ABC transporter
    SA08480.000.35Oligopeptide transport system ATP-binding OppF homologue
    SA21670.002.69PTS sucrose-specific component
    SA02070.000.21Hypothetical protein similar to maltose/maltodextrin-binding protein
    SA08490.000.35Hypothetical protein similar to binding protein OppA
    SA04320.002.52PTS enzyme II, phosphoenolpyruvate dependent, trehalose specific
    SA02090.000.20Maltose/maltodextrin transport permease homologue
    SA02060.000.20Multiple sugar-binding transport protein
    SA06400.002.01Hypothetical protein similar to ABC transporter required for expression of cytochrome bd
    SA01720.002.19Hypothetical protein similar to membrane protein LmrP
    SA01860.000.38Hypothetical protein similar to phosphotransferase enzyme II
    SA06390.012.04Hypothetical protein similar to ABC transporter required for expression of cytochrome bd
    SA05310.012.20Proline/betaine transporter homologue
    SA04170.012.15Hypothetical protein similar to sodium-dependent transporter
    SA06550.012.25Fructose-specific permease
    SA03740.010.39Xanthine permease
    SA20530.012.06Glucose uptake protein homologue
    SA24340.022.31Fructose phosphotransferase system enzyme homologue
Only ORFs with a P value by t test of <0.05 have been listed.
Only ORFs with a fold change of <0.4 or ≥2.0 for the hemB mutant versus the parent strain have been listed.
TABLE 3. Results of Fisher's exact test showing overrepresented groups of genes in the hemB mutant at different growth phases
Time when group is overrepresented and group nameGroup size (total size = 2,322)Selection group sizeSelection sizeP valuea
Pooled values of the whole time course  337 
    Metabolism of carbohydrates and related molecules13334 0.00
    Membrane bioenergetics (electron transport chain and ATP synthase)5417 0.00
    Transport/binding proteins and lipoproteins25653 0.00
    Metabolism of nucleotides and nucleic acids7418 0.02
    Phage-related functions4311 0.04
    Adaptation to atypical conditions4411 0.04
Lag phase  279 
    Protein synthesis8525 0.00
    Metabolism of nucleotides and nucleic acids7422 0.00
    Metabolism of carbohydrates and related molecules13327 0.00
    Adaptation to atypical conditions4412 0.00
Early logarithmic phase  171 
    Metabolism of carbohydrates and related molecules13327 0.00
    Transport/binding proteins and lipoproteins25634 0.00
    Adaptation to atypical conditions447 0.04
Logarithmic phase  113 
    Metabolism of carbohydrates and related molecules13319 0.00
    Metabolism of nucleotides and nucleic acids7410 0.00
    Transport/binding proteins and lipoproteins25621 0.01
Early stationary growth phase  189 
    Membrane bioenergetics (electron transport chain and ATP synthase)5413 0.00
    Metabolism of carbohydrates and related molecules13320 0.00
    Transport/binding proteins and lipoproteins25631 0.01
Stationary growth phase  46 
    Membrane bioenergetics (electron transport chain and ATP synthase)548 0.00
    Protein synthesis857 0.00
    Metabolism of carbohydrates and related molecules1336 0.04
Only groups that are overrepresented are listed.


We sincerely thank Daniela Kuhn for excellent technical assistance. We are indebted to the invaluable help of Kiran Patil (DTU, Lyngby, Denmark) with the computational algorithm.
This work was supported in part by grants from BMBF (Pathogenomic Network) to C.V.E., K.B., and G.P.; from the Deutsche Forschungsgemeinschaft (EI 247/7-1) to C.V.; and from the National Institutes of Health (AI42072) to R.P.

Supplemental Material

File (fig__s1__seggewiss_et_al__supplemental_material_.eps)
File (supplemental_material__tables_s1_s6_.doc)
ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.


Baumert, N., C. von Eiff, F. Schaaff, G. Peters, R. A. Proctor, and H. G. Sahl. 2002. Physiology and antibiotic susceptibility of Staphylococcus aureus small colony variants. Microb. Drug Resist. 8 : 253-260.
Casiano-Colón, A., and R. E. Marquis. 1988. Role of the arginine deiminase system in protecting oral bacteria and an enzymatic basis for acid tolerance. Appl. Environ. Microbiol. 54 : 1318-1324.
Cheung, A. L., A. S. Bayer, G. Zhang, H. Gresham, and Y. Q. Xiong. 2004. Regulation of virulence determinants in vitro and in vivo in Staphylococcus aureus. FEMS Immunol. Med. Microbiol. 40 : 1-9.
Degnan, B. A., M. C. Fontaine, A. H. Doebereiner, J. J. Lee, P. Mastroeni, G. Dougan, J. A. Goodacre, and M. A. Kehoe. 2000. Characterization of an isogenic mutant of Streptococcus pyogenes Manfredo lacking the ability to make streptococcal acid glycoprotein. Infect. Immun. 68 : 2441-2448.
Dziewanowska, K., J. M. Patti, C. F. Deobald, K. W. Bayles, W. R. Trumble, and G. A. Bohach. 1999. Fibronectin binding protein and host cell tyrosine kinase are required for internalization of Staphylococcus aureus by epithelial cells. Infect. Immun. 67 : 4673-4678.
Gruening, P., M. Fulde, P. Valentin-Weigand, and R. Goethe. 2006. Structure, regulation, and putative function of the arginine deiminase system of Streptococcus suis. J. Bacteriol. 188 : 361-369.
Heinemann, M., A. Kummel, R. Ruinatscha, and S. Panke. 2005. In silico genome-scale reconstruction and validation of the Staphylococcus aureus metabolic network. Biotechnol. Bioeng. 92 : 850-864.
Johansen, L. E., P. Nygaard, C. Lassen, Y. Agerso, and H. H. Saxild. 2003. Definition of a second Bacillus subtilis pur regulon comprising the pur and xpt-pbuX operons plus pbuG, nupG (yxjA), and pbuE (ydhL). J. Bacteriol. 185 : 5200-5209.
Jonsson, I. M., C. von Eiff, R. A. Proctor, G. Peters, C. Ryden, and A. Tarkowski. 2003. Virulence of a hemB mutant displaying the phenotype of a Staphylococcus aureus small colony variant in a murine model of septic arthritis. Microb. Pathog. 34 : 73-79.
Kahl, B. C., G. Belling, R. Reichelt, M. Herrmann, R. A. Proctor, and G. Peters. 2003. Thymidine-dependent small-colony variants of Staphylococcus aureus exhibit gross morphological and ultrastructural changes consistent with impaired cell separation. J. Clin. Microbiol. 41 : 410-413.
Kilstrup, M., and J. Martinussen. 1998. A transcriptional activator, homologous to the Bacillus subtilis PurR repressor, is required for expression of purine biosynthetic genes in Lactococcus lactis. J. Bacteriol. 180 : 3907-3916.
Kohler, C., C. von Eiff, G. Peters, R. A. Proctor, M. Hecker, and S. Engelmann. 2003. Physiological characterization of a heme-deficient mutant of Staphylococcus aureus by a proteomic approach. J. Bacteriol. 185 : 6928-6937.
Kok, M., G. Bron, B. Erni, and S. Mukhija. 2003. Effect of enzyme I of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) on virulence in a murine model. Microbiology 149 : 2645-2652.
Lascelles, J. 1978. sn-Glycerol-3-phosphate dehydrogenase and its interaction with nitrate reductase in wild-type and hem mutant strains of Staphylococcus aureus. J. Bacteriol. 133 : 621-625.
Lowy, F. D. 1998. Staphylococcus aureus infections. N. Engl. J. Med. 339 : 520-532.
Lu, C. D., H. Winteler, A. Abdelal, and D. Haas. 1999. The ArgR regulatory protein, a helper to the anaerobic regulator ANR during transcriptional activation of the arcD promoter in Pseudomonas aeruginosa. J. Bacteriol. 181 : 2459-2464.
Maghnouj, A., A. Abu-Bakr, S. Baumberg, V. Stalon, and W. C. Vander. 2000. Regulation of anaerobic arginine catabolism in Bacillus licheniformis by a protein of the Crp/Fnr family. FEMS Microbiol. Lett. 191 : 227-234.
Maghnouj, A., T. F. de Sousa Cabral, V. Stalon, and W. C. Vander. 1998. The arcABDC gene cluster, encoding the arginine deiminase pathway of Bacillus licheniformis, and its activation by the arginine repressor argR. J. Bacteriol. 180 : 6468-6475.
Moisan, H., E. Brouillette, C. L. Jacob, P. Langlois-Begin, S. Michaud, and F. Malouin. 2006. Transcription of virulence factors in Staphylococcus aureus small-colony variants isolated from cystic fibrosis patients is influenced by SigB. J. Bacteriol. 188 : 64-76.
Patil, K. R., and J. Nielsen. 2005. Uncovering transcriptional regulation of metabolism by using metabolic network topology. Proc. Natl. Acad. Sci. USA 102 : 2685-2689.
Peacock, S. J., T. J. Foster, B. J. Cameron, and A. R. Berendt. 1999. Bacterial fibronectin-binding proteins and endothelial cell surface fibronectin mediate adherence of Staphylococcus aureus to resting human endothelial cells. Microbiology 145 : 3477-3486.
Persson, M., E. Glatz, and B. Rutberg. 2000. Different processing of an mRNA species in Bacillus subtilis and Escherichia coli. J. Bacteriol. 182 : 689-695.
Pöhlmann-Dietze, P., M. Ulrich, K. B. Kiser, G. Doring, J. C. Lee, J. M. Fournier, K. Botzenhart, and C. Wolz. 2000. Adherence of Staphylococcus aureus to endothelial cells: influence of capsular polysaccharide, global regulator agr, and bacterial growth phase. Infect. Immun. 68 : 4865-4871.
Proctor, R. A., B. Kahl, C. von Eiff, P. E. Vaudaux, D. P. Lew, and G. Peters. 1998. Staphylococcal small colony variants have novel mechanisms for antibiotic resistance. Clin. Infect. Dis. 27(Suppl. 1): S68-S74.
Proctor, R. A., and G. Peters. 1998. Small colony variants in staphylococcal infections: diagnostic and therapeutic implications. Clin. Infect. Dis. 27 : 419-422.
Proctor, R. A., C. von Eiff, B. C. Kahl, K. Becker, P. McNamara, M. Herrmann, and G. Peters. 2006. Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat. Rev. Microbiol. 4 : 295-305.
Roggenkamp, A., A. Sing, M. Hornef, U. Brunner, I. B. Autenrieth, and J. Heesemann. 1998. Chronic prosthetic hip infection caused by a small-colony variant of Escherichia coli. J. Clin. Microbiol. 36 : 2530-2534.
Schaaff, F., G. Bierbaum, N. Baumert, P. Bartmann, and H. G. Sahl. 2003. Mutations are involved in emergence of aminoglycoside-induced small colony variants of Staphylococcus aureus. Int. J. Med. Microbiol. 293 : 427-435.
Senn, M. M., M. Bischoff, C. von Eiff, and B. Berger-Bächi. 2005. σB activity in a Staphylococcus aureus hemB mutant. J. Bacteriol. 187 : 7397-7406.
Sifri, C. D., A. Baresch-Bernal, S. B. Calderwood, and C. von Eiff. 2006. Virulence of Staphylococcus aureus small colony variants in the Caenorhabditis elegans infection model. Infect. Immun. 74 : 1091-1096.
Spiro, S. 1994. The FNR family of transcriptional regulators. Antonie Leeuwenhoek 66 : 23-36.
Thakker, M., J. S. Park, V. Carey, and J. C. Lee. 1998. Staphylococcus aureus serotype 5 capsular polysaccharide is antiphagocytic and enhances bacterial virulence in a murine bacteremia model. Infect. Immun. 66 : 5183-5189.
Tuchscherr, L. P., F. R. Buzzola, L. P. Alvarez, R. L. Caccuri, J. C. Lee, and D. O. Sordelli. 2005. Capsule-negative Staphylococcus aureus induces chronic experimental mastitis in mice. Infect. Immun. 73 : 7932-7937.
Vaudaux, P., P. Francois, C. Bisognano, W. L. Kelley, D. P. Lew, J. Schrenzel, R. A. Proctor, P. J. McNamara, G. Peters, and C. von Eiff. 2002. Increased expression of clumping factor and fibronectin-binding proteins by hemB mutants of Staphylococcus aureus expressing small colony variant phenotypes. Infect. Immun. 70 : 5428-5437.
von Eiff, C., K. Becker, D. Metze, G. Lubritz, J. Hockmann, T. Schwarz, and G. Peters. 2001. Intracellular persistence of Staphylococcus aureus small-colony variants within keratinocytes: a cause for antibiotic treatment failure in a patient with Darier's disease. Clin. Infect. Dis. 32 : 1643-1647.
von Eiff, C., C. Heilmann, R. A. Proctor, C. Woltz, G. Peters, and F. Götz. 1997. A site-directed Staphylococcus aureus hemB mutant is a small-colony variant which persists intracellularly. J. Bacteriol. 179 : 4706-4712.
von Eiff, C., P. McNamara, K. Becker, D. Bates, X.-H. Lei, M. Ziman, B. R. Bochner, G. Peters, and R. A. Proctor. 2006. Phenotype microarray profiling of Staphylococcus aureus menD and hemB mutants with the small-colony-variant phenotype. J. Bacteriol. 188 : 687-693.
Yin, S., R. S. Daum, and S. Boyle-Vavra. 2006. VraSR two-component regulatory system and its role in induction of pbp2 and vraSR expression by cell wall antimicrobials in Staphylococcus aureus. Antimicrob. Agents Chemother. 50 : 336-343.

Information & Contributors


Published In

cover image Journal of Bacteriology
Journal of Bacteriology
Volume 188Number 2215 November 2006
Pages: 7765 - 7777
PubMed: 16980462


Received: 30 May 2006
Accepted: 24 August 2006
Published online: 15 November 2006


Request permissions for this article.



Jochen Seggewiß
Institute of Medical Microbiology, University of Münster, Münster, Germany
Karsten Becker
Institute of Medical Microbiology, University of Münster, Münster, Germany
Oliver Kotte
Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland
Martin Eisenacher
Integrated Functional Genomics (IFG), Interdisciplinary Center for Clinical Research (IZKF), University of Münster, Münster, Germany
Mohammad Reza Khoschkhoi Yazdi
Institute of Medical Microbiology, University of Münster, Münster, Germany
Andreas Fischer
Institute of Medical Microbiology, University of Münster, Münster, Germany
Peter McNamara
Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, Wisconsin
Nahed Al Laham
Institute of Medical Microbiology, University of Münster, Münster, Germany
Richard Proctor
Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, Wisconsin
Georg Peters
Institute of Medical Microbiology, University of Münster, Münster, Germany
Matthias Heinemann
Institute of Molecular Systems Biology, ETH Zürich, Zürich, Switzerland
Christof von Eiff [email protected]
Institute of Medical Microbiology, University of Münster, Münster, Germany


Supplemental material for this article may be found at .

Metrics & Citations



  • For recently published articles, the TOTAL download count will appear as zero until a new month starts.
  • There is a 3- to 4-day delay in article usage, so article usage will not appear immediately after publication.
  • Citation counts come from the Crossref Cited by service.


If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. For an editable text file, please select Medlars format which will download as a .txt file. Simply select your manager software from the list below and click Download.

View Options

Figures and Media






Share the article link

Share with email

Email a colleague

Share on social media

American Society for Microbiology ("ASM") is committed to maintaining your confidence and trust with respect to the information we collect from you on websites owned and operated by ASM ("ASM Web Sites") and other sources. This Privacy Policy sets forth the information we collect about you, how we use this information and the choices you have about how we use such information.
FIND OUT MORE about the privacy policy