Investigations into σB-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 σB-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 σB-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 α-hemolysin compared to 8325-4. In total, 13 genes were identified displaying increased α-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 σB, these genes appeared to have a role in the regulation of σB 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 σB regulatory cascade.

Membrane Association and Multimerization of Secreton Component PulC

ABSTRACT The PulC component of the Klebsiella oxytocapullulanase secretion machinery (the secreton) was found by subcellular fractionation to be associated with both the cytoplasmic (inner) and outer membranes. Association with the outer membrane was independent of other secreton components, including the outer membrane protein PulD (secretin). The association of PulC with the inner membrane is mediated by the signal anchor sequence located close to its N terminus. These results suggest that PulC forms a bridge between the two membranes that is disrupted when bacteria are broken open for fractionation. Neither the signal anchor sequence nor the cytoplasmic N-terminal region that precedes it was found to be required for PulC function, indicating that PulC does not undergo sequence-specific interactions with other cytoplasmic membrane proteins. Cross-linking of whole cells resulted in the formation of a ca. 110-kDa band that reacted with PulC-specific serum and whose detection depended on the presence of PulD. However, antibodies against PulD failed to react with this band, suggesting that it could be a homo-PulC trimer whose formation requires PulD. The data are discussed in terms of the possible role of PulC in energy transduction for exoprotein secretion.

Modulation of mannosylphosphodolichol synthase and dolichol kinase activity in Trichoderma, related to protein secretion.

It has been postulated that exoprotein secretion in Trichoderma is related to their O-glycosylation. In the present paper the involvement of phosphodolichol in this process is described and the key role of mannosylphosphodolichol (MPD) synthase in protein O-mannosylation is discussed. The effect of water soluble phospholipid precursors such as choline and Tween 80, known also to increase secretion of cellulases when added to the medium, on MPD-synthase activity is presented. This effect is positive in the Trichoderma reesei QM 9414 (a low producing strain) but has no influence on the enzyme activity from the RUT C-30 strain selected to overproduce secretion of exoproteins and known to contain an increased cellular amount of endoplasmic reticulum. The positive effect of addition of choline and Tween to the medium on the level of dolichol kinase activity is also demonstrated. The influence of cultivation temperature on the activity of the various enzymes involved in dolichol-dependent protein glycosylation i.e. MPD-synthase, dolichyl kinase and MPD/Protein mannosyl transferase was tested. For all enzymes cultivation at 35 degrees C led to the elevated activity, which was most striking for dolichol kinase, whereas for MPD-synthase and MPD/Protein mannosyl transferase the difference was only apparent in the assay when endogenous phosphodolichol was used as a substrate. Furthermore, lipid extract from the membranes cultivated at elevated temperature, when added to the enzyme obtained from Trichoderma grown at 25 degrees C, enhanced the dolichol kinase activity measured in the absence of exogenous dolichol.(ABSTRACT TRUNCATED AT 250 WORDS)