Regulation of a heat shock sigma32 homolog in Caulobacter crescentus

Abstract

High temperature and other environmental stresses induce the expression of several heat shock proteins in Caulobacter crescentus, including the molecular chaperones DnaJ, DnaK, GrpE, and GroEL and the Lon protease. We report here the isolation of the rpoH gene encoding a homolog of the Escherichia coli RNA polymerase 32 subunit, the sigma factor responsible for the transcription of heat shock promoters. The C. crescentus 32 homolog, predicted to be a 33.7-kDa protein, is 42% identical to E. coli 32 and cross-reacts with a monoclonal antibody to E. coli 32. Functional homology was demonstrated by complementing the temperature-sensitive growth defect of an E. coli rpoH deletion mutant with the C. crescentus rpoH gene. Immunoblot analysis showed a transient rise in 32 levels after a temperature shift from 30 to 42؇C similar to that described for E. coli. In addition, increasing the cellular content of 32 by introducing a plasmid-encoded copy of rpoH induced DnaK expression in C. crescentus cultures grown at 30؇C. The C. crescentus rpoH gene was transcribed from either of two heat shock consensus promoters. rpoH transcription and 32 levels increased coordinately following heat shock, indicating that transcriptional regulation contributes to 32 expression in this organism. Both the rpoH gene and 32 protein were expressed constitutively throughout the cell cycle at 30؇C. The isolation of rpoH provides an important tool for future studies of the role of 32 in the normal physiology of C. crescentus.Heat shock proteins (HSPs) are generally either molecular chaperones that act to maintain the proper folding of cell proteins, such as DnaK or Hsp70 and GroEL or Hsp60, or proteases, such as Clp and Lon. This group of proteins, found in prokaryotes and eukaryotes, functions both at physiological temperature and during cellular responses to high temperature and other environmental stresses. Distinct modes of transcriptional induction of the HSPs have been described for Escherichia coli and Saccharomyces cerevisiae (reviewed in reference 27). In E. coli, heat shock enhances the level of the 32 subunit of RNA polymerase which then recognizes the promoters of the heat shock genes and increases their levels of transcription (10,22,24). By contrast, in S. cerevisiae a transcriptional activator, HSF, binds constitutively to a highly conserved DNA repeat, or heat shock element, in the promoter region. Elevated temperature induces a conformational change in HSF which enables it to activate the transcription of heat shock genes (27).Although the general heat shock response in bacteria is highly conserved, its regulation by 32 has previously been documented in bacteria that are members of the ␥ subdivision of proteobacteria. Only recently have 32 homologs been reported in ␣ proteobacteria (32). The role of 32 in the heat shock response is best understood for E. coli (reviewed in references 7, 19, 21, and 50). In this organism, 32 levels increase 15-to 20-fold in response to a sudden rise in temperature (25,44 promoters and a E promoter ...

Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle

“…These data suggest that the induction of genes in these clusters is driven predominantly by CtrA. For three of the genes in these two clusters (the ccrM gene encoding the CcrM DNA methyltransferase, the pilA gene encoding the major pilin subunit, and mcpA, encoding the McpA chemoreceptor), direct activation by CtrA has been verified (5,6,8,21).…”

“…1) ensures that this protein activates or represses its target genes only at specific times during the cell cycle. Seven genes have previously been identified as directly controlled by CtrA by using in vitro footprinting analyses and expression assays of lacZ transcription fusions in ctrA ts mutant strains (5)(6)(7)(8). Further, all of these genes were shown to have cell cycle-regulated mRNAs, although maximal expression occurs at widely disparate times during the cell cycle (4).…”

“…CtrA, like many response regulators, has a DNA-binding domain. The phosphorylated form of CtrA has been shown to act as a transcription factor that directly regulates genes involved in cell division, DNA methylation, flagellar biogenesis, and pili biogenesis (5)(6)(7)(8); it has also been shown to repress initiation of DNA replication by binding to five sites in the origin of replication (9). The cell cycleregulated expression of these CtrA target genes is effected by changes in active CtrA levels during the cell cycle (Fig.…”

A phospho-signaling pathway controls the localization and activity of a protease complex critical for bacterial cell cycle progression

“…In Caulobacter, the essential master regulator CtrA controls the transcription of genes involved in cell division, DNA methylation, flagellar biogenesis, and pili biogenesis (6,(19)(20)(21) and also silences the initiation of DNA replication by binding to five sites in the origin of replication (7). Because of its multiple functions, the presence and activity of CtrA is tightly regulated, temporally and spatially, by two redundant mechanisms, synthesis-proteolysis and phosphorylation-dephosphorylation (3-5, 8-12, 22-27).…”

Heat-shock sigma factor RpoH from Geobacter sulfurreducens

“…rpoH genes from a-and b-Proteobacteria do not contain characteristics found in those from c-Proteobacteria. Instead, some rpoH genes from a-Proteobacteria, such as Agrobacterium tumefaciens (Nakahigashi et al, 1999), Bradyrhizobium japonicum (Narberhaus et al, 1997) and Caulobacter crescentus (Reisenauer et al, 1996;Wu & Newton, 1996), contain an RpoH-dependent promoter that can be induced by heat shock. In G. sulfurreducens the downstream box and mRNA secondary structure found in c-Proteobacteria are absent (data not shown), while the rpoH gene has an RpoH-dependent promoter (Figs 1 and 4).…”