SummaryStaphylococcus aureus peptidoglycan is cross-linked via a characteristic pentaglycine interpeptide bridge. Genetic analysis had identified three peptidyltransferases, FemA, FemB and FemX, to catalyse the formation of the interpeptide bridge, using glycyl t-RNA as Gly donor. To analyse the pentaglycine bridge formation in vitro , we purified the potential substrates for FemA, FemB and FemX, UDP-MurNAc-pentapeptide, lipid I and lipid II and the staphylococcal t-RNA pool, as well as His-tagged Gly-tRNA-synthetase and His-tagged FemA, FemB and FemX. We found that FemX used lipid II exclusively as acceptor for the first Gly residue. Addition of Gly 2,3 and of Gly 4,5 was catalysed by FemA and FemB, respectively, and both enzymes were specific for lipid II-Gly 1 and lipid II-Gly 3 as acceptors. None of the FemABX enzymes required the presence of one or two of the other Fem proteins for activity; rather, bridge formation was delayed in the in vitro system when all three enzymes were present. The in vitro assembly system described here will enable detailed analysis of late, membrane-associated steps of S. aureus peptidoglycan biosynthesis.

The alternative sigma factor B of Staphylococcus aureus controls the expression of a variety of genes, including virulence determinants and global regulators. Genetic manipulations and transcriptional start point (TSP) analyses showed that the sigB operon is transcribed from at least two differentially controlled promoters: a putative A -dependent promoter, termed sigB p1 , giving rise to a 3.6-kb transcript covering sa2059-sa2058-rsbU-rsbV-rsbW-sigB, and a B -dependent promoter, sigB p3 , initiating a 1.6-kb transcript covering rsbV-rsbWsigB. TSP and promoter-reporter gene fusion experiments indicated that a third promoter, tentatively termed sigB p2 and proposed to lead to a 2.5-kb transcript, including rsbU-rsbV-rsbW-sigB, might govern the expression of the sigB operon. Environmental stresses, such as heat shock and salt stress, induced a rapid response within minutes from promoters sigB p1 and sigB p3 . In vitro, the sigB p1 promoter was active in the early growth stages, while the sigB p2 and sigB p3 promoters produced transcripts throughout the growth cycle, with sigB p3 peaking around the transition state between exponential growth and stationary phase. The amount of sigB transcripts, however, did not reflect the concentration of B measured in cell extracts, which remained constant over the entire growth cycle. In a guinea pig cage model of infection, sigB transcripts were as abundant 2 and 8 days postinoculation as values found in vitro, demonstrating that sigB is indeed transcribed during the course of infection. Physical interactions between staphylococcal RsbU-RsbV, RsbV-RsbW, and RsbW-B were inferred from a yeast (Saccharomyces cerevisiae) two-hybrid approach, indicating the presence of a partner-switching mechanism in the B activation cascade similar to that of Bacillus subtilis. The finding that overexpression of RsbU was sufficient to trigger an immediate and strong activation of B , however, signals a relevant difference in the regulation of B activation between B. subtilis and S. aureus in the cascade upstream of RsbU.Staphylococcus aureus is one of the leading causes for nosocomial-and community-acquired infections (11,46). Its capacity to cause a wide spectrum of diseases and to survive in unfavorable conditions is due to a network of global regulatory elements enabling it to rapidly sense changes and to respond appropriately. These elements comprise two-component regulatory systems, including the agr locus, the SarA protein family, and alternative factors (reviewed in reference 16 and references within).Computational analysis of the published staphylococcal genomes suggests that S. aureus harbors only two alternative sigma factors, B and H (45). B of S. aureus was demonstrated to influence the expression of a variety of genes (6,26,33,43,78,79), including virulence factors (23,27,34,38,43,50,51,52,62,78,79) and regulatory elements (5,6,21,26,34,47,60,66). Moreover, it affects methicillin and glycopeptide resistance (4,56,65,74), biofilm production (58), and internalization into endothelial ce...

Faster growing and more virulent strains of methicillin resistant Staphylococcus aureus (MRSA) are increasingly displacing highly resistant MRSA. Elevated fitness in these MRSA is often accompanied by decreased and heterogeneous levels of methicillin resistance; however, the mechanisms for this phenomenon are not yet fully understood. Whole genome sequencing was used to investigate the genetic basis of this apparent correlation, in an isogenic MRSA strain pair that differed in methicillin resistance levels and fitness, with respect to growth rate. Sequencing revealed only one single nucleotide polymorphism (SNP) in the diadenylate cyclase gene dacA in the faster growing but less resistant strain. Diadenylate cyclases were recently discovered to synthesize the new second messenger cyclic diadenosine monophosphate (c-di-AMP). Introduction of this mutation into the highly resistant but slower growing strain reduced resistance and increased its growth rate, suggesting a direct connection between the dacA mutation and the phenotypic differences of these strains. Quantification of cellular c-di-AMP revealed that the dacA mutation decreased c-di-AMP levels resulting in reduced autolysis, increased salt tolerance and a reduction in the basal expression of the cell wall stress stimulon. These results indicate that c-di-AMP affects cell envelope-related signalling in S. aureus. The influence of c-di-AMP on growth rate and methicillin resistance in MRSA indicate that altering c-di-AMP levels could be a mechanism by which MRSA strains can increase their fitness levels by reducing their methicillin resistance levels.

Isogenic Staphylococcus aureus strains with different capacities to produce B activity were analyzed for their ability to attach to fibrinogen-or fibronectin-coated surfaces or platelet-fibrin clots and to cause endocarditis in rats. In comparison to the B -deficient strain, BB255, which harbors an rsbU mutation, both rsbUcomplemented and B -overproducing derivatives exhibited at least five times greater attachment to fibrinogenand fibronectin-coated surfaces and showed increased adherence to platelet-fibrin clots. No differences in adherence were seen between BB255 and a ⌬rsbUVWsigB isogen. Northern blotting analyses revealed that transcription of clfA, encoding fibrinogen-binding protein clumping factor A, and fnbA, encoding fibronectinbinding protein A, were positively influenced by B . B overproduction resulted in a statistically significant increase in positive spleen cultures and enhanced bacterial densities in both the aortic vegetations and spleens at 16 h postinoculation. In contrast, at 72 h postinoculation, tissues infected with the B overproducer had lower bacterial densities than did those infected with BB255. These results suggest that although B appears to increase the adhesion of S. aureus to various host cell-matrix proteins in vitro, it has limited effect on pathogenesis in the rat endocarditis model. B appears to have a transient enhancing effect on bacterial density in the early stages of infection that is lost during progression.

BackgroundSecDF is an accessory factor of the conserved Sec protein translocation machinery and belongs to the resistance-nodulation-cell division (RND) family of multidrug exporters. SecDF has been shown in Escherichia coli and Bacillus subtilis to be involved in the export of proteins. RND proteins can mediate resistance against various substances and might be of relevance in antimicrobial therapy. The role of RND proteins in Staphylococcus aureus has not yet been determined.ResultsMarkerless deletion mutants were constructed to analyze the impact of the so far uncharacterized RND proteins in S. aureus. While the lack of Sa2056 and Sa2339 caused no phenotype regarding growth and resistance, the secDF mutant resulted in a pleiotropic phenotype. The secDF mutant was cold sensitive, but grew normally in rich medium at 37°C. Resistance to beta-lactams, glycopeptides and the RND substrates acriflavine, ethidium bromide and sodium dodecyl sulfate was reduced. The secDF mutant showed an aberrant cell separation and increased spontaneous and Triton X-100 induced autolysis, although the amounts of penicillin-binding proteins in the membrane were unchanged. The impact of secDF deletion on transcription and expression of specific virulence determinants varied: While coagulase transcription and activity were reduced, the opposite was observed for the autolysin Atl. A reduction of the transcription of the cell wall anchored protein A (spa) was also found. The accumulation of SpA in the membrane and lowered amounts in the cell wall pointed to an impaired translocation.ConclusionsThe combination of different effects of secDF deletion on transcription, regulation and translocation lead to impaired cell division, reduced resistance and altered expression of virulence determinants suggesting SecDF to be of major relevance in S. aureus. Thus SecDF could be a potential target for the control and eradication of S. aureus in the future.