Isolation, identification, and transcriptional specificity of the heat shock sigma factor sigma32 from Caulobacter crescentus

Abstract

We report the identification of the Caulobacter crescentus heat shock factor 32 as a 34-kDa protein that copurifies with the RNA polymerase holoenzyme. The N-terminal amino acid sequence of this protein was determined and used to design a degenerate oligonucleotide as a probe to identify the corresponding gene, rpoH, which encodes a predicted protein with a molecular mass of 33,659 Da. The amino acid sequence of this protein is similar to those of known bacterial heat shock sigma factors of Escherichia coli (41% identity), Pseudomonas aeruginosa (40% identity), and Citrobacter freundii (38% identity). The isolated C. crescentus gene complements the growth defect of an E. coli rpoH deletion strain at 37؇C, and Western blot (immunoblot) analysis confirmed that the gene product is related to the E. coli 32 protein. The purified RpoH protein in the presence of RNA polymerase core enzyme specifically recognizes the heat shock-regulated promoter P1 of the C. crescentus dnaK gene, and base pair substitutions in either the ؊10 or ؊35 region of this promoter abolish transcription. S1 nuclease mapping indicates that rpoH transcripts originate from two promoters, P1 and P2, under the normal growth conditions. The P2 promoter is similar to the 32 promoter consensus, and the P2-specific transcript increases dramatically during heat shock, while the P1-specific transcript remains relatively constant. These results suggest that although the structure and function of C. crescentus 32 appear to be very similar to those of its E. coli counterpart, the C. crescentus rpoH gene contains a novel promoter structure and may be positively autoregulated in response to environmental stress.The Caulobacter crescentus cell cycle generates two different daughter cells: a motile swarmer cell which possesses a single polar flagellum, and a nonmotile stalked cell. The progeny swarmer and stalked cell types differ not only in morphology and motility but also in their programs of gene expression and capacities to reinitiate DNA replication. Formation of the new swarmer cell results from a series of discrete morphogenic events during the cell cycle that occur at one pole of the dividing cell (reviewed in references 8 and 38). This developmental sequence depends on completion of successive cell cycle checkpoints, and there is now evidence that the regulation of cell division and some developmental events is mediated by two-component signal transduction pathways (reviewed in reference 27).Many developmental events in C. crescentus are regulated at the level of transcription initiation. The specificity of promoter recognition is conferred by the sigma subunit of RNA polymerase (RNAP), which binds to the core RNAP (E) containing ␤, ␤Ј, and ␣ 2 . In eubacterial systems examined to date, multiple sigma factors have been identified, each of which programs RNAP to recognize a distinct promoter sequence. Although the predominant sigma subunit ( 70 in gram-negative bacteria) directs expression of the bulk of the genome, highly specialized genes involved in ...

Three disparately regulated genes for σ32‐like transcription factors in Bradyrhizobium japonicum

“…Because of the presence of a 32 -type promoter preceding the ragAB rpoH 3 operon, one may envisage either autoregulation by RpoH 3 , or regulation by one of the other RpoH proteins of B. japonicum (see above). Autoregulation of an rpoH gene was found in C. crescentus (Reisenauer et al, 1996;Wu and Newton, 1996). Ideally, in vitro transcription studies are now required to elucidate the exact function(s) of all three RpoH proteins in the complex network of regulatory stress factors in B. japonicum.…”

“…Other genes are duplicated, as was reported for nodD (Gö ttfert et al, 1992), fixK (Anthamatten et al, 1992;Fischer, 1994), and for another sigma factor, namely 54 (Kullik et al, 1991 (Landick et al, 1984); Citrobacter freundii, Serratia marcescens, Agrobacterium tumefaciens, and Zymomonas mobilis (Nakahigashi et al, 1995); Haemophilus influenzae (Fleischmann et al, 1995); Vibrio cholerae (J. Das, unpublished; Accession No. U44432); Pseudomonas aeruginosa (Benvenisti et al, 1995;Naczynski et al, 1995); Caulobacter crescentus (Reisenauer et al, 1996;Wu and Newton, 1996); Myxococcus xanthus RpoC (Apelian and Inouye, 1993). The proteobacterial subdivisions (␥, ␣, and ␦) are indicated on the right.…”

An essential, multicomponent signal transduction pathway required for cell cycle regulation in Caulobacter

“…6A, lane 2), the size predicted from the in vivo transcription initiation start site (28). Holoenzymes containing purified 32 (26) or 54 (30) did not recognize the fliL promoter, however (data not shown). Importantly, transcription from the fliL promoter was stimulated by the addition of CtrA in the presence of DivJ and ATP (Fig.…”

The Caulobacter heat shock sigma factor gene rpoH is positively autoregulated from a sigma32-dependent promoter

“…The heat shock response has also been studied in Caulobacter crescentus, a gram-negative aquatic bacterium that divides asymmetrically to produce a new differentiated motile swarmer cell and the mother stalked cell at the end of each cell cycle (reviewed in references 4, 13, and 25). The expression of several heat shock genes, including dnaK (14), groESL (1), and hcrA/grpE (29), has been examined in this bacterium, and isolation of the heat shock sigma factor gene rpoH has recently been described (28,34). Transcription from the 32 -like promoter of groESL is temporally controlled during the cell cycle at the physiological temperature of 30ЊC (2).…”