Comparative genomics reveal novel heat shock regulatory mechanisms in Staphylococcus aureus and other Gram‐positive bacteria

Abstract

SummaryMultiple regulatory mechanisms for coping with stress co-exist in low G + + + + C Gram-positive bacteria. Among these, the HrcA and CtsR repressors control distinct regulons in the model organism, Bacillus subtilis . We recently identified an orthologue of the CtsR regulator of stress response in the major pathogen, Staphylococcus aureus . Sequence analysis of the S . aureus genome revealed the presence of potential CtsR operator sites not only upstream from genes encoding subunits of the Clp ATP-dependent protease, as in B . subtilis , but also, unexpectedly, within the promoter regions of the dnaK and groESL operons known to be specifically controlled by HrcA. The tandem arrangement of the CtsR and HrcA operators suggests a novel mode of dual heat shock regulation by these two repressors. The S . aureus ctsR and hrcA genes were cloned under the control of the P xylA xylose-inducible promoter and used to demonstrate dual regulation of the dnaK and groESL operons by both CtsR and HrcA, using B . subtilis as a heterologous host. Direct binding by both repressors was shown in vitro by gel mobility shift and DNase I footprinting experiments using purified S .

Role for dnaK locus in tolerance of multiple stresses in Staphylococcus aureus

“…Altogether, five genes have been proposed to constitute the dnaK cluster in S. aureus (Kuroda et al, 2001;Ohta et al, 1994). The first gene of this cluster is proposed to encode a protein HrcA that negatively regulates the expression of the dnaK locus (Chastanet et al, 2003). A study by others, and nucleotide sequence analysis of the dnaK locus in S. aureus, have suggested the presence of two putative promoters: one upstream of hrcA, and another one upstream of dnaJ (Ohta et al, 1994).…”

“…subtilis (Homuth et al, 1997). It has been recently reported that the S. aureus dnaK, as well as the groEL operon, are subjected to dual negative regulation by CtsR and HrcA (Chastanet et al, 2003). However, S. aureus is able to overcome this repression during growth at higher temperatures and in other stress conditions (Chastanet et al, 2003).…”

Regulation of expression of type I signal peptidases in Listeria monocytogenes

“…The same experiment was performed with a radiolabelled probe corresponding to P sipY (a region showing no promoter activity in the previous experiment), and no displacement of radiolabelled fragment among the amount of protein extract was observed (data not shown). Cold competitor chase experiments (as described by Chastanet et al, 2003) were used to demonstrate the specificity of protein binding. The addition of increasing amounts of unlabelled DNA fragments corresponding to P sipX or P sipZ efficiently prevented binding of the radiolabelled probes (Fig.…”

Intrinsic resistance to aminoglycosides in Enterococcus faecium is conferred by the 16S rRNA m5C1404-specific methyltransferase EfmM

“…To improve protein expression, the EfmM gene was re-cloned into pET28/16 (Chastanet et al 2003) to give pAT859 (pET28/ 16VefmM). E. coli B834 cells (Novagen) containing pET28Vefm were used for the production of selenomethionylated EfmM with an N-terminal His 6 purification tag followed by a thrombin cutting site.…”