Investigations into σ ^B -Modulated Regulatory Pathways Governing Extracellular Virulence Determinant Production in Staphylococcus aureus

Abstract

The commonly used Staphylococcus aureus laboratory strain 8325-4 bears a naturally occurring 11-bp deletion in the B -regulating phosphatase rsbU. We have previously published a report (M. J. Horsburgh, J. L. Aish, I. J. White, L. Shaw, J. K. Lithgow, and S. J. Foster, J. Bacteriol. 184:5457-5467, 2002) on restoring the rsbU deletion, producing a B -functional 8325-4 derivative, SH1000. SH1000 is pleiotropically altered in phenotype from 8325-4, displaying enhanced pigmentation, increased growth yields, and a marked decrease in secreted exoproteins. This reduction in exoprotein secretion appears to result from a sixfold reduction in agr expression. In this study we have undertaken transposon mutagenesis of SH1000 to identify components involved in the modulation of extracellular proteases and ␣-hemolysin compared to 8325-4. In total, 13 genes were identified displaying increased ␣-hemolysin transcription and extracellular proteolysis. Phenotypic analysis revealed that each mutant also had decreased pigmentation and a general increase in protein secretion. Interestingly this phenotype was not identical in each case but was variable from mutant to mutant. None of the genes identified encoded classic regulatory proteins but were predominantly metabolic enzymes involved in amino acid biosynthesis and transport. Further analysis revealed that all of these mutations were clustered in a 35-kb region of the chromosome. By complementation and genetic manipulation we were able to demonstrate the validity of these mutations. Interestingly transcriptional analysis revealed that rather than being regulated by B , these genes appeared to have a role in the regulation of B activity. Thus, we propose that the loss of individual genes in this chromosomal hot spot region results in a destabilization of cellular harmony and disruption of the B regulatory cascade.

Analysis of the Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrum of Staphylococcus aureus Identifies Mutations That Allow Differentiation of the Main Clonal Lineages

“…Furthermore, three of the markers that distinguish the clonal complexes CC22, CC8, and CC30 are under SigB control and are not expressed in SigB-negative strains (22). On Columbia blood agar, rsbU-or sigB-negative colonies are detected by their white color, since staphyloxanthin biosynthesis is dependent on SigB (22), and strong hemolysis (33). Upon analysis of the MALDI spectra, the loss of activity of SigB is easily inferred from the absence of the large SigB-dependent signals at m/z 6,889 and m/z 5,525 and, therefore, should be included in the interpretation of the data.…”

NsaRS is a cell-envelope-stress-sensing two-component system of Staphylococcus aureus

“…Levels of b-galactosidase activity were measured as described previously (Shaw et al, 2006(Shaw et al, , 2007. Fluorescence was measured using a BioTek Synergy 2 plate reader and calibrated with standard concentrations of 4-methyl umbelliferone (MU).…”

Extra Cytoplasmic Sigma Factors in Staphylococcus aureus; their Role and Significance in the Survival of Cocci

“…The σB has been shown to be a pleiotropic regulator of virulence determinant production and to be required for pathogenesis [16]. In vitro studies of transcription of S. aureus SigB have been shown to participate in the transcription of the sar locus, a key regulator of virulence gene expression.…”

“…Co-culture studies reveal that the σS mutant is significantly outcompeted by its parental strain, which is only exacerbated during prolonged growth or in the presence of stressor compounds. Interestingly, transcriptional analysis determined that under standard conditions, S. aureus SH1000 does not initiate expression of sigS [16]. Sigma H (σH): A member of double stranded DNA viruses, a template phage takes on a bacterial RNA polymerase with an essential σ factor to initiate the sequence of reactions.…”

“…S. aureus [20] σS Starvation, stresses that DNA damage and cell wall damage, oxidative stress S. aureus [16,20] σH Phage 11 infection S. aureus [10] …”