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Thioredoxin reductase (encoded by trxB) protects Staphylococcus aureus against oxygen or disulfide stress and is indispensable for growth. Among the different sara family mutants analyzed, transcription of trxB was markedly elevated in the sarA mutant under conditions of aerobic as well as microaerophilic growth, indicating that SarA acts as a negative regulator of trxB expression. Gel shift analysis showed that purified SarA protein binds directly to the trxB promoter region DNA in vitro. DNA binding of SarA was essential for repression of trxB transcription in vivo in S. aureus. Northern blot analysis and DNA binding studies of the purified wild-type SarA and the mutant SarAC9G with oxidizing agents indicated that oxidation of Cys-9 reduced the binding of SarA to the trxB promoter DNA. Oxidizing agents, in particular diamide, could further enhance transcription of the trxB gene in the sarA mutant, suggesting the presence of a SarA-independent mode of trxB induction. Analysis of two oxidative stress-responsive sara regulatory target genes, trxB and sodM, with various mutant sarA constructs showed a differential ability of the SarA to regulate expression of the two above-mentioned genes in vivo. The overall data demonstrate the important role played by SarA in modulating expression of genes involved in oxidative stress resistance in S. aureus. doi: 10.1128/JB.01202-09

Expression of genes involved in the pathogenesis of Staphylococcus aureus is controlled by global regulatory loci, including two-component regulatory systems and transcriptional regulators. The staphylococcal-specific SarA family of transcription regulators control large numbers of target genes involved in virulence, autolysis, biofilm formation, stress responses and metabolic processes, and are recognized as potential therapeutic targets. Expression of some of these important regulators has been examined, mostly in laboratory strains, while the pattern of expression of these genes in other strains, especially clinical isolates, is largely unknown. In this report, a comparative analysis of 10 sarA-family genes was conducted in six different S. aureus strains, including two laboratory (RN6390, SH1000) and four clinical (MW2, Newman, COL and UAMS-1) strains, by Northern and Western blot analyses. Transcription of most of the sarA-family genes showed a strong growth phase-dependence in all strains tested. Among these genes, no difference was observed in expression of the sarA, sarV, sarT and sarU genes, while a major difference was observed in expression of the sarX gene only in strain RN6390. Expression of mgrA, rot, sarZ, sarR and sarS was observed in all strains, but the level of expression varied from strain to strain.

The scavenging of reactive oxygen species (ROS) within cells is regulated by several interacting factors, including transcriptional regulators. Involvement of sarA family genes in the regulation of proteins involved in the scavenging of ROS is largely unknown. In this report, we show that under aerobic conditions, the levels of sodM and sodA transcription, in particular the sodM transcript, are markedly enhanced in the sarA mutant among the tested sarA family mutants. Increased levels of sod expression returned to near the parental level in a single-copy sarA complemented strain. Under microaerophilc conditions, transcription of both sodM and sodA was considerably enhanced in the sarA mutant compared to the wild-type strain. Various genotypic, phenotypic, and DNA binding studies confirmed the involvement of SarA in the regulation of sod transcripts in different strains of Staphylococcus aureus. The sodA mutant was sensitive to an oxidative stress-inducing agent, methyl viologen, but the sarA sodA double mutant was more resistant to the same stressor than the single sodA mutant. These results suggest that overexpression of SodM, which occurs in the sarA background, can rescue the methyl viologen-sensitive phenotype observed in the absence of the sodA gene. Analysis with various oxidative stress-inducing agents indicates that SarA may play a greater role in modulating oxidative stress resistance in S. aureus. This is the first report that demonstrates the direct involvement of a regulatory protein (SarA) in control of sod expression in S. aureus.

The expression of genes involved in the pathogenesis of Staphylococcus aureus is controlled by global regulatory loci, including two-component regulatory systems and transcriptional regulators (e.g., sar family genes). Most members of the SarA family have been partially characterized and shown to regulate a large numbers of target genes. Here, we describe the characterization of sarZ, a sarA paralog from S. aureus, and its regulatory relationship with other members of its family. Expression of sarZ was growth phase dependent with maximal expression in the early exponential phase of growth. Transcription of sarZ was reduced in an mgrA mutant and returned to a normal level in a complemented mgrA mutant strain, which suggests that mgrA acts as an activator of sarZ transcription. Purified MgrA protein bound to the sarZ promoter region, as determined by gel shift assays. Among the sarA family of genes analyzed, inactivation of sarZ increased sarS transcription, while it decreased agr transcription. The expression of potential target genes involved in virulence was evaluated in single and double mutants of sarZ with mgrA, sarX, and agr. Northern and zymogram analyses indicated that the sarZ gene product played a role in regulating several virulence genes, particularly those encoding exoproteins. Gel shift assays demonstrated nonspecific binding of purified SarZ protein to the promoter regions of the sarZ-regulated target genes. These results demonstrate the important role played by SarZ in controlling regulatory and virulence gene expression in S. aureus.

The 375-bp sarA open reading frame is driven by three promoters, P1, P3, and P2. Using gel shift and DNase I footprinting assays, we found that SarA binds to two 26-bp sequences and one 31-bp sequence within the P1 and P3 promoters, respectively. Together with the results of transcription analyses, our data indicate that SarA binds to its own promoter to down-regulate sarA expression.

The pathogenesis of Staphylococcus aureus infections is dependent upon expression of various virulence factors, which are under the control of multiple regulatory systems, including two-component regulatory systems and transcriptional regulators such as the SarA family of proteins. As a part of a continuing effort to understand the regulatory mechanisms that involve the SarA protein family, the regulation and physical characterization of rot transcription is described here. The rotgene, a member of the sara family of genes, was previously characterized and has been shown to regulate a large number of genes. The rot locus is composed of multiple overlapping transcripts as determined by primer extension and was proposed to encode an open reading frame of 133 residues. Transcription of rot was significantly increased in the sara mutant. Gel shift and transcriptional studies revealed that SarA could bind to the rot promoter region, probably acting as a repressor for rot transcription. The data indicate that the expression of rot transcription is significantly repressed only by SarA among the sara family of mutants tested at the post-exponential phase of growth.

The expression of genes involved in the pathogenesis of Staphylococcus aureus is known to be controlled by global regulatory, loci, including agr, sarA, saeRS, arlRS, and sarA-like genes. As part of our continuing efforts to understand the regulatory mechanisms that involve sarA-like genes, we describe here the characterization of a novel transcriptional regulator called SarX, a member of the SarA protein family. The transcription of sarX was growth phase dependent and was expressed maximally during the stationary phase of growth, which was significantly decreased in the mgrA mutant. MgrA acted as an activator of sarX expression as confirmed by transcriptional fusion and Northern blot analyses. Purified MgrA protein bound to the upstream region of the sarX promoter as demonstrated by gel shift assay. The expression levels of various potential target genes involved in virulence and regulation, specifically those affected by sarA and mgrA, were analyzed with isogenic sarX mutant strains. Our data indicated that SarX acted as a repressor of the agr locus and consequently target genes regulated by the agr system. We propose that SarX is an important regulator in the SarA protein family and may be part of the common pathway by which agr and members of the sara gene family control virulence in S. aureus.

The biofilm-associated protein Bap is a staphylococcal surface protein involved in biofilm formation. We investigated the influence of the global regulatory locus sarA on bap expression and Bap-dependent biofilm formation in three unrelated Staphylococcus aureus strains. The results showed that Bap-dependent biofilm formation was diminished in the sarA mutants by an agr-independent mechanism. Complementation studies using a sarA clone confirmed that the defect in biofilm formation was due to the sarA mutation. As expected, the diminished capacity to form biofilms in the sarA mutants correlated with the decreased presence of Bap in the bacterial surface. Using transcriptional fusion and Northern analysis data, we demonstrated that the sarA gene product acts as an activator of bap expression. Finally, the bap promoter was characterized and the transcriptional start point was mapped by the rapid amplification of cDNA ends technique. As expected, we showed that purified SarA protein binds specifically to the bap promoter, as determined by gel shift and DNase I footprinting assays. Based on the previous studies of others as well as our work demonstrating the role for SarA in icaADBC and bap expression (J. Valle, A. Toledo-Arana, C. Berasain, J. M. Ghigo, B. Amorena, J. R. Penades, and I. Lasa, Mol. Microbiol. 48:1075-1087), we propose that SarA is an essential regulator controlling biofilm formation in S. aureus.

Staphylococcus epidermidis biofilm formation is associated with the production of the polysaccharide intercellular adhesin (PIA)--poly-N-acetylglucosamine polysaccharide (PNAG) by the products of the icaADBC operon. Recent evidence indicates that SarA, a central regulatory element that controls the production of Staphylococcus aureus virulence factors, is essential for the synthesis of PIA/PNAG and the ensuing biofilm development in this species. Based on the presence of a sarA homolog, we hypothesized that SarA could also be involved in the regulation of the biofilm formation process in S. epidermidis. To investigate this, we constructed nonpolar sarA deletions in two genetically unrelated S. epidermidis clinical strains, O-47 and CH845. The SarA mutants were completely defective in biofilm formation, both in the steady-state conditions of a microtiter dish assay and in the flow conditions of microfermentors. Reverse transcription-PCR experiments showed that the mutation in the sarA gene resulted in downregulation of the icaADBC operon transcription in an IcaR-independent manner. Purified SarA protein showed high-affinity binding to the icaA promoter region by electrophoretic mobility shift assays. Consequently, mutation in sarA provoked a significant decrease in the amount of PIA/PNAG on the cell surface. Furthermore, heterologous complementation of S. aureas sara mutants with the sara gene of S. epidermidis completely restored biofilm formation. In summary, SarA appeared to be a positive regulator of transcription of the ica locus, and in its absence, PIA/PNAG production and biofilm formation were diminished. Additionally, we present experimental evidence showing that SarA may be an important regulatory element that controls S. epidermidis virulence factors other than biofilm formation.

The expression of genes involved in the pathogenesis of Staphylococcus aureus is known to be controlled by global regulatory loci, including agr, sarA, sae, arlRS, lytSR, and sarA-like genes. Here we described a novel transcriptional regulator called sarV of the SarA protein family. The transcription of sarV is low or undetectable under in vitro conditions but is significantly augmented in sara and mgrA (norR or rat) (SA0641) mutants. The sarA and mgrA genes act as repressors of sarV expression, as confirmed by transcriptional fusion and Northern analysis data. Purified SarA and MgrA proteins bound specifically to separate regions of the sarV promoter as determined by gel shift and DNase I footprinting assays. The expression of 19 potential target genes involved in autolysis and virulence, phenotypes affected by sara and mgrA, was evaluated in an isogenic sarV mutant pair. Our data indicated that the sarV gene product played a role regulating some virulence genes and more genes involved in autolysis. The sarV mutant was more resistant to Triton X-100 and penicillin-induced lysis compared to the wild type and the sara mutant, whereas hyperexpression of sarV in the parental strain or the sarV mutant rendered the resultant strain highly susceptible to lysis. Zymographic analysis of murein hydrolase activity revealed that inactivation of the sarV gene results in decreased extracellular murein hydrolase activity compared to that of wild-type S. aureus. We propose that sarV may he part of the common pathway by which mgrA and sarA gene products control autolysis in S. aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) regulates resistance to β-lactams via preferential production of an alternative penicillin-binding protein (PBP), PBP2a. PBP2a binds many β-lactam antibiotics with less affinity than PBPs which are predominant in methicillin-susceptible (MSSA) strains. A novel, rather frequent in vitro phenotype was recently identified among clinical MRSA bloodstream isolates, termed "NaHCO3-responsiveness". This phenotype features β-lactam susceptibility of certain MRSA strains only in the presence of NaHCO3. Two distinct PBP2a variants, 246G and 246E, have been linked to the NaHCO3-responsive and NaHCO3-non-responsive MRSA phenotypes, respectively. To determine the mechanistic impact of PBP2a variants on β-lactam susceptibility, binding profiles of a fluorescent penicillin probe (Bocillin-FL) to each purified PBP2a variant were assessed and compared to whole-cell binding profiles characterized by flow cytometry in the presence vs. absence of NaHCO3. These investigations revealed that NaHCO3 differentially influenced the binding of the fluorescent penicillin, Bocillin-FL, to the PBP2a variants, with binding intensity and rate of binding significantly enhanced in the 246G compared to the 246E variant. Of note, the NaHCO3-β-lactam (oxacillin)-responsive JE2 strain, which natively harbors the 246G variant, had enhanced Bocillin-FL whole-cell binding following exposure to NaHCO3. This NaHCO3-mediated increase in whole-cell Bocillin-FL binding was not observed in the NaHCO3-non-responsive parental strain, COL, which contains the 246E PBP2a variant. Surprisingly, genetic swaps of the mecA coding sites between JE2 and COL did not alter the NaHCO3-enhanced binding seen in JE2 vs. COL. These data suggest that the non-coding regions of mecA may be involved in NaHCO3-responsiveness. This investigation also provides strong evidence that the NaHCO3-responsive phenotype in MRSA may involve NaHCO3-mediated increases in both initial cell surface β-lactam binding, as well as ultimate PBP2a binding of β-lactams. [ABSTRACT FROM AUTHOR]

The human pathogen staphylococcus aureus causes a variety of infections both minor and life-threatening and generates a host of exotoxins including those which can cause food poisoning and toxic shock syndrome. Extracellular virulence determinants expressed by S. aureus are controlled by pleiotropic elements such as sar and agr. Studies were conducted to probe the regulation of sar expression in a pair of isogenic sar strains of S. aureus using transcriptional fusion with the xylE reporter gene. Results show that sar locus regulation is complex.

Two alternative models for elucidating the mechanism of action of the replication terminator proteins are proposed. The steric hindrance model suggests that the complex between the terminator proteins and the terminator DNA sets up a polar, steric barrier to fork movement. The other model triggers a specific interaction between the terminator protein and the replicative helicase. Evidence strongly supports the second model.

The size of the Azotobacter chroococcum Mu chromosome is evaluated to be 5300 kb. The size is derived by resolving all the DNA restriction fragments of the bacteria, that are digested by restriction endonucleases AseI and DraI and separated by pulsed-field gel electrophoresis. 1690 kb fragment from DraI and 930 kb and 780 kb fragments from AseI hybridize during digestion.

Summary: Staphyloxanthin, a carotenoid in S. aureus, is a powerful antioxidant against oxidative stresses. The crtOPQMN operon driving pigment synthesis is under the control of σB. CspA, a cold shock protein, is known to control σB activity. To ascertain genes that regulate cspA, we screened a transposon library that exhibited reduced cspA expression and pigmentation. We found that the adaptor protein YjbH activates cspA expression. Spx, the redox‐sensitive transcriptional regulator and a proteolytic target for YjbH and ClpXP, complexes with αCTD of RNAP prior to binding the cspA promoter to repress cspA activity. Increased cspA expression in trans in the inactive spx C10A mutant of JE2 did not enhance pigment production while it did in JE2, suggesting that cspA is downstream to Spx in pigmentation control. As the staphyloxanthin pigment is critical to S. aureus survival in human hosts, we demonstrated that the cspA and yjbH mutants survived less well than the parent in whole blood killing assay. Collectively, our studies suggest a pathway wherein YjbH and ClpXP proteolytically cleave Spx, a repressor of cspA transcription, to affect σB‐dependent carotenoid expression, thus providing a critical link between intracellular redox sensing by Spx and carotenoid production to improve S. aureus survival during infections. [ABSTRACT FROM AUTHOR]