Influence of a functional sigB operon on the global regulators sar and agr in Staphylococcus aureus.

The growth phase-dependent activity profile of the alternate transcription factor sigma(B) and its effects on the expression of sar and agr were examined in three different Staphylococcus aureus strains by Northern blot analyses and by the use of reporter gene fusion experiments. Significant sigma(B) activity was detectable only in the clinical isolates MSSA1112 and Newman, carrying the wild-type rsbU allele, but not in the NCTC8325 derivative BB255, which is defective in rsbU. sigma(B) activity peaked in the late exponential phase and diminished towards the stationary phase when bacteria were grown in Luria-Bertani medium. Transcriptional analysis and a sarP1-sarP2-sarP3 (sarP1-P2-P3)-driven firefly luciferase (luc+) reporter gene fusion demonstrated a strong sigma(B) activity- and growth phase-dependent increase in sar expression that was totally absent in either rsbU or Delta rsbUVWsigB mutants. In contrast, expression of the agr locus, as measured by RNAIII levels and by an hldp::luc+ fusion, was found to be higher in the absence of sigma(B) activity, such as in rsbU or Delta rsbUVWsigB mutants, than in wild-type strains. Overexpression of sigma(B) in BB255 derivatives resulted in a clear increase in sarP1-P2-P3::luc+ expression as well as a strong decrease in hldp::luc+ expression. The data presented here suggest that sigma(B) increases sar expression while simultaneously reducing the RNAIII level in a growth phase-dependent manner.

Sigma(B) activity depends on RsbU in Staphylococcus aureus.

Derivatives of the widely used laboratory strain Staphylococcus aureus NCTC8325, which are natural rsbU mutants, were shown to be unable to produce RsbU, a positive regulator of the alternative sigma factor sigma(B). The lack of RsbU prevented the heat-dependent production of sigma(B)-controlled transcripts and resulted in reduced H2O2 and UV tolerance, enhanced alpha-hemolysin activity, and the inability to produce the alkaline shock protein Asp23. After 48 h of growth, rsbU mutant strains failed to accumulate staphyloxanthin, the major stationary-phase carotenoid. Transcription of Asp23 was found to be exclusively controlled by sigma(B), making it an excellent target for the study of sigma(B) activity in S. aureus. Reporter gene experiments, using the firefly luciferase gene (luc+) fused to the sigma(B)-dependent promoter(s) of asp23, revealed that sigma(B) is almost inactive in 8325 derivatives. cis complementation of the 8325 derivative BB255 with the wild-type rsbU gene from strain COL produced the rsbU(+) derivative GP268, a strain possessing a sigma(B) activity profile comparable to that of the rsbU(+) wild-type strain Newman. In GP268, the heat inducibility of sigma(B)-dependent genes, Asp23 production, alpha-hemolysin activity, pigmentation, and susceptibility to H2O2 were restored to the levels observed in strain Newman, clearly demonstrating that RsbU is needed for activation of sigma(B) in S. aureus.

Involvement of multiple genetic loci in Staphylococcus aureus teicoplanin resistance.

Teicoplanin resistance was transformed from a teicoplanin-resistant Staphylococcus aureus into the susceptible strain BB255 to give strain BB938. The cell wall composition, amidation of the iD-glutamate, and peptide crosslinking were identical in BB938 as in BB255 except for a 60% increased length of the glycan chain. Transductional crosses revealed that at least two distinct loci contributed in a cumulative fashion to teicoplanin resistance. One of these loci correlated with a mutation inactivating the anti-sigma factor RsbW. This mutation must have occurred during transformation and selection for teicoplanin resistance in BB938. Genetic manipulations involving the sigB operon showed that transcription factor SigB contributed to decreased teicoplanin susceptibility.

Inactivation of a novel three-cistronic operon tcaR-tcaA-tcaB increases teicoplanin resistance in Staphylococcus aureus.

A novel teicoplanin-associated operon termed tcaR-tcaA-tcaB was identified by Tn917-mediated insertional mutagenesis. Resistance to teicoplanin rose 4-fold by insertional inactivation of tcaA or by deletion of the entire operon. tcaA encodes a hypothetical transmembrane protein with a metal-binding motif, possibly a sensor-transducer. tcaB codes for a membrane-associated protein, which has sequence homologies to a bicyclomycin resistance protein. The two genes are preceded by tcaR encoding a putative regulator with sequence homologies to the transcriptional regulator MarR. The fact that tcaA inactivation as well as deletion of tcaRAB produced the same increase in teicoplanin resistance confirmed the association of tcaRAB with teicoplanin susceptibility. Cotransductional crosses showed that the level of teicoplanin resistance produced by these insertions was strain-dependent and that in the methicillin-resistant strain COL, it was paired with a remarkable decrease in methicillin resistance. This allowed to postulate that tcaRAB may be involved in some way in cell wall biosynthesis, and that teicoplanin may interact with TcaA and/or TcaB either directly or indirectly.

Deletion of the alternative sigma factor sigmaB in Staphylococcus aureus reveals its function as a global regulator of virulence genes.

A deletion of the sigB operon was constructed in three genetically distinct Staphylococcus aureus strains, and the phenotypes of the resulting mutants were analyzed. Compared to the corresponding wild-type strains, the DeltasigB mutants showed reduced pigmentation, accelerated sedimentation, and increased sensitivity to hydrogen peroxide during the stationary growth phase. A cytoplasmic protein missing in the DeltasigB mutants was identified as alkaline shock protein 23, and an extracellular protein excreted at higher levels in one of the DeltasigB mutants was identified as staphylococcal thermonuclease. Interestingly, most sigB deletion phenotypes were only seen in S. aureus COL and Newman and not in 8325, which was found to contain an 11-bp deletion in the regulator gene rsbU. Taken together, our results show that sigmaB is a global regulator which modulates the expression of several virulence factors in S. aureus and that laboratory strain 8325 is a sigmaB-defective mutant.

The alternative sigma factor sigmaB in Staphylococcus aureus: regulation of the sigB operon in response to growth phase and heat shock

In the human pathogen Staphylococcus aureus, many proteins involved in the infection process are preferentially produced during the stationary growth phase. Using a DNA probe corresponding to the Bacillus subtilis gene encoding the stationary-phase sigma factor SigB (sigmaB), we identified a gene in S. aureus with similarity to B. subtilis sigB. The sigB region was mapped on the SmaI I fragment of the S. aureus chromosome and contains a total of six open reading frames (orf1-6). The deduced amino acid sequences of orf2, orf3, orf4, and orf5 show 64, 67, 71, and 77% similarity to the B. subtilis proteins RsbU, RsbV, RsbW, and SigB, respectively, with SigB representing the sigma factor and the Rsb proteins representing regulators of sigma B. Furthermore, the relative position of the corresponding genes is conserved in B. subtilis, which strongly suggests that we identified the sigB operon of S. aureus, encoding an alternative sigma factor in this organism. The proposed gene products of the two remaining open reading frames show 48-62% similarity to the PemK, ChpAK, and ChpBK growth inhibitors of Escherichia coli (ORF1) and 61% similarity to the ribosomal protein S1 of Haemophilus influenzae (ORF6). Northern blot analysis of the sigB region in S. aureus revealed that four different transcripts are expressed under different conditions of growth phase and stress. These results indicate a complex transcriptional regulation that differs between S. aureus and B. subtilis.