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The article presents a study on the expression pattern of sarA-family of transcription regulators on Staphylococcus aureus strains. The study used S. aureus strains including RN6390 and SH1000 laboratory and MW2, COL, Newman and UAMS-1 clinical strains by western blot analyses. The study showed no difference in sarV, sarT, sarV and sarU gene expression while major difference was found in sarX gene on the RN6390 strain and the level of expression of mgrA, sarZ, rot and sarS on each strain varied.

The sarA locus in Staphylococcus aureus controls the expression of many virulence genes. The sarA regulatory molecule. SarA, is a 14.7-kDa protein (124 residues) that binds to the promoter region of target genes. Here we report the 2.6 A-resolution x-ray crystal structure of the dimeric winged helix SarA protein, which differs from the published SarA structure dramatically. In the crystal packing, multiple dimers of SarA form a scaffold, possibly via divalent cations. Mutations of individual residues within the DNA- binding helix-turn-helix and the winged region as well as within the metal-binding pocket implicate basic residues R84 and R90 within the winged region to be critical in DNA binding, whereas acidic residues D88 and E89 (wing), D8 and E11 (metal-binding pocket), and cysteine 9 are essential for SarA function. These data suggest that the winged region of the winged helix protein participates in DNA binding and activation, whereas the putative divalent cation binding pocket is only involved in gene function. [ABSTRACT FROM AUTHOR]

The expression of virulence determinants in Staphylococcus aureus is controlled by global regulatory loci (e.g., sara and agr). One of these determinants, protein A (spa), is activated by sarS, which encodes a 250-residue DNA-binding protein. Genetic analysis indicated that the agr locus likely mediates spa repression by suppressing the transcription of sarS. Contrary to Sara and SarR, which require homodimer formation for proper function, SarS is unusual within the Sara protein family in that it contains two homologous halves, with each half sharing sequence similarity to Sara and SarR. Here we report the 2.2 Å resolution X-ray crystal structure of the SarS protein. SarS has folds similar to those of SarR and, quite plausibly, the native Sara structure. Two typical winged-helix DNA-binding domains are connected by a well-ordered loop. The interactions between the two domains are extensive and conserved. The putative DNA-binding surface is highly positively charged. In contrast, negatively charged patches are located opposite to the DNA-binding surface. Furthermore, sequence alignment and structural comparison revealed that MarR has folds similar to those of SarR and SarS. Members of the MarR protein family have previously been implicated in the negative regulation of an efflux pump involved in multiple antibiotic resistance in many gram-negative species. We propose that MarR also belongs to the winged-helix protein family and has a similar mode of DNA binding as SarR and SarS and possibly the entire Sara protein family member. Based on the structural differences of SarR, SarS, and MarR, we further classified these winged-helix proteins to three subfamilies, SarA, SarS, and MarR. Finally, a possible transcription regulation mechanism is proposed. [ABSTRACT FROM AUTHOR]

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a leading pathogen causing severe endovascular infections. The prophage-encoded protein Gp05 has been identified as a critical virulence factor that contributes to MRSA persistence during vancomycin (VAN) treatment in an experimental endocarditis model. However, the underlining mechanisms driving this persistence phenotype remain poorly understood., Methods: The current study aimed to elucidate the genetic factors contributing to Gp05-associated MRSA persistence by utilizing RNA sequencing (RNA-seq) on an isogenic MRSA strain set, including a clinical persistent bacteremia isolate (PB 300-169), its isogenic chromosomal gp05 deletion mutant, and gp05-complemented strains., Results: RNA-seq analysis revealed significantly downregulation of the graSR-vraFG regulatory system and its downstream genes, mprF and dltABCD, in the gp05 deletion mutant compared to the wild-type and gp05-complemented strains. Notably, this downregulation led to a substantial shift in cell membrane composition, with a marked increase in negatively charged phosphatidylglycerol (PG) and a concomitant decrease in positively charged lysyl-PG (LPG). These changes in membrane lipid composition resulted in increased susceptibility of the gp05 deletion mutant to human cationic antimicrobial peptide (CAMP) LL-37, polymorphonuclear neutrophil (PMN) and VAN. Similar findings were observed in an isogenic gp05 overexpression strain set with different genetic background (MRSA USA300 JE2)., Conclusions: These findings suggest that Gp05 plays a pivotal role in MRSA persistence by modulating cell surface components and surface charge. This study provides new insights into the molecular mechanisms underlying Gp05-mediated persistence in MRSA endovascular infections and highlights potential therapeutic targets to combat persistent MRSA infections., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Methicillin-resistant Staphylococcus aureus (MRSA) infections are an increasing concern due to their intrinsic resistance to most standard-of-care β-lactam antibiotics. Recent studies of clinical isolates have documented a novel phenotype, termed NaHCO 3 responsiveness, in which a substantial proportion of MRSA strains exhibit enhanced susceptibility to β-lactams such as cefazolin and oxacillin in the presence of NaHCO 3 . A bicarbonate transporter, MpsAB ( m embrane p otential-generating s ystem), was recently found in S. aureus, where it plays a role in concentrating NaHCO 3 for anaplerotic pathways. Here, we investigated the role of MpsAB in mediating the NaHCO 3 responsiveness phenotype. Radiolabeled NaH 14 CO 3 uptake profiling revealed significantly higher accumulation in NaHCO 3 -responsive vs nonresponsive MRSA strains when grown in ambient air. In contrast, under 5% CO 2 conditions, NaHCO 3 -responsive (but not nonresponsive) strains exhibited repressed uptake. Oxacillin MICs were measured in four prototype strains and their mpsABC deletion mutants in the presence of NaHCO 3 supplementation under 5% CO 2 conditions. NaHCO 3 -mediated reductions in oxacillin MICs were observed in the responsive parental strains but not in mpsABC deletion mutants. No significant impact on oxacillin MICs was observed in the nonresponsive strains under the same conditions. Transcriptional and translational studies were carried out using both quantitative reverse transcription-PCR (qRT-PCR) and mpsA -green fluorescent protein (GFP) fusion constructs; these investigations showed that mpsA expression and translation were significantly upregulated during mid-exponential-phase growth in oxacillin-NaHCO 3 -supplemented medium in responsive versus nonresponsive strains. Taken together, these data show that the NaHCO 3 transporter MpsABC is a key contributor to the NaHCO 3 -β-lactam responsiveness phenotype in MRSA. IMPORTANCE MRSA infections are increasingly difficult to treat, due in part to their resistance to most β-lactam antibiotics. A novel and relatively common phenotype, termed NaHCO 3 responsiveness, has been identified in which MRSA strains show increased susceptibility in vitro and in vivo to β-lactams in the presence of NaHCO 3 . A recently described S. aureus NaHCO 3 transporter, MpsAB, is involved in intracellular NaHCO 3 concentration for anaplerotic pathways. We investigated the role of MpsAB in mediating the NaHCO 3 responsiveness phenotype in four prototype MRSA strains (two responsive and two nonresponsive). We demonstrated that MpsABC is an important contributor to the NaHCO 3 -β-lactam responsiveness phenotype. Our study adds to the growing body of well-defined characteristics of this novel phenotype, which could potentially translate to alternative targets for MRSA treatment using β-lactams., Competing Interests: The authors declare no conflict of interest.

Recently, more emphasis has been given to understand molecular genetics and the contribution of a gene in the disease process. In fact, increased understanding of bacterial pathogenesis and intracellular communication has revealed many potential strategies for development of novel agents to treat bacterial infection. Therefore, to study the function and the involvement of a particular gene in pathogenesis, the inactivation or interruption is very important. In this section, various methods leading to inactivation of the gene in Staphylococcus aureus will be discussed.