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In this study we addressed the question how a mevalonate (MVA)-auxotrophic Staphylococcus aureusΔmvaS mutant can revert to prototrophy. This mutant couldn’t grow in the absence of MVA. However, after a long lag-phase of 4–6 days the mutant adapted from auxotrophic to prototrophic phenotype. During that time, it acquired two point mutations: One mutation in the coding region of the regulator gene spx, which resulted in an amino acid exchange that decreased Spx function. The other mutation in the upstream-element within the core-promoter of the mevalonolactone lactonase gene drp35. This mutation led to an increased expression of drp35. In repeated experiments the mutations always occurred in spx and drp35 and in the same order. The first detectable mutation appeared in spx and allowed slight growth; the second mutation, in drp35, increased growth further. Phenotypical characterizations of the mutant showed that small amounts of the lipid-carrier undecaprenol are synthesized, despite the lack of mvaS. The growth of the adapted clone, ΔmvaSad, indicates that the mutations reawake a rescue bypass. We think that this bypass enters the MVA pathway at the stage of MVA, because blocking the pathway downstream of MVA led to growth arrest of the mutant. In addition, the lactonase Drp35 is able to convert mevalonolactone to MVA. Summarized, we describe here a mutation-based two-step adaptation process that allows resuscitation of growth of the ΔmvaS mutant. [ABSTRACT FROM AUTHOR]

Excretion of cytoplasmic proteins ( ECP) is a common physiological feature in bacteria and eukaryotes. However, how these proteins without a typical signal peptide are excreted in bacteria is poorly understood. We studied the excretion pattern of cytoplasmic proteins using two glycolytic model enzymes, aldolase and enolase, and show that their excretion takes place mainly during the exponential growth phase in S taphylococcus aureus very similar to that of Sbi, an IgG-binding protein, which is secreted via the Sec-pathway. The amount of excreted enolase is substantial and is comparable with that of Sbi. For localization of the exit site, we fused aldolase and enolase with the peptidoglycan-binding motif, LysM, to trap the enzymes at the cell wall. With both immune fluorescence labeling and immunogold localization on electron microscopic thin sections aldolase and enolase were found apart from the cytoplasmic area particularly in the cross wall and at the septal cleft of dividing cells, whereas the non-excreted Ndh2, a soluble NADH:quinone oxidoreductase, is only seen attached to the inner side of the cytoplasmic membrane. The selectivity, the timing and the localization suggest that ECP is not a result of unspecific cell lysis but is mediated by an as yet unknown mechanism. [ABSTRACT FROM AUTHOR]

The mechanisms behind carbon dioxide (CO2) dependency in non-autotrophic bacterial isolates are unclear. Here we show that the Staphylococcus aureus mpsAB operon, known to play a role in membrane potential generation, is crucial for growth at atmospheric CO2 levels. The genes mpsAB can complement an Escherichia coli carbonic anhydrase (CA) mutant, and CA from E. coli can complement the S. aureus delta-mpsABC mutant. In comparison with the wild type, S. aureus mps mutants produce less hemolytic toxin and are less virulent in animal models of infection. Homologs of mpsA and mpsB are widespread among bacteria and are often found adjacent to each other on the genome. We propose that MpsAB represents a dissolved inorganic carbon transporter, or bicarbonate concentrating system, possibly acting as a sodium bicarbonate cotransporter. The mechanisms behind CO2 dependency in non-autotrophic bacterial isolates are unclear. Here the authors show that the Staphylococcus aureus mpsAB operon is crucial for growth at atmospheric CO2 levels, and provide evidence indicating that MpsAB represents a bicarbonate concentrating system. [ABSTRACT FROM AUTHOR]

Rhodomyrtone (Rom) is an acylphloroglucinol antibiotic originally isolated from leaves of Rhodomyrtus tomentosa . Rom targets the bacterial membrane and is active against a wide range of Gram-positive bacteria but the exact mode of action remains obscure. Here we isolated and characterized a spontaneous Rom-resistant mutant from the model strain Staphylococcus aureus HG001 (Rom R ) to learn more about the resistance mechanism. We showed that Rom-resistance is based on a single point mutation in the coding region of farR [regulator of fatty acid (FA) resistance] that causes an amino acid change from Cys to Arg at position 116 in FarR, that affects FarR activity. Comparative transcriptome analysis revealed that mutated farR affects transcription of many genes in distinct pathways. FarR represses for example the expression of its own gene ( farR ), its flanking gene farE (effector of FA resistance), and other global regulators such as agr and sarA . All these genes were consequently upregulated in the Rom R clone. Particularly the upregulation of agr and sarA leads to increased expression of virulence genes rendering the Rom R clone more cytotoxic and more pathogenic in a mouse infection model. The Rom-resistance is largely due to the de-repression of farE . FarE is described as an efflux pump for linoleic and arachidonic acids. We observed an increased release of lipids in the Rom R clone compared to its parental strain HG001. If farE is deleted in the Rom R clone, or, if native farR is expressed in the Rom R strain, the corresponding strains become hypersensitive to Rom. Overall, we show here that the high Rom-resistance is mediated by overexpression of farE in the Rom R clone, that FarR is an important regulator, and that the point mutation in farR (Rom R clone) makes the clone hyper-virulent.