Caulobacter crescentus has a single dnaK gene that is highly homologous to the hsp70 family of heat shock genes. Analysis of the cloned and sequenced dnaK gene has shown that the deduced amino acid sequence could encode a protein of 67.6 kilodaltons that is 68% identical to the DnaK protein of Escherichia coli and 49% identical to the Drosophila and human hsp70 protein family. A partial open reading frame 165 base pairs 3' to the end of dnaK encodes a peptide of 190 amino acids that is 59% identical to DnaJ of E. coli. Northern blot analysis revealed a single 4.0-kilobase mRNA homologous to the cloned fragment. Since the dnaK coding region is 1.89 kilobases, dnaK and dnaJ may be transcribed as a polycistronic message. S1 mapping and primer extension experiments showed that transcription initiated at two sites 5' to the dnaK coding sequence. A single start site of transcription was identified during heat shock at 42 degrees C, and the predicted promoter sequence conformed to the consensus heat shock promoters of E. coli. At normal growth temperature (30 degrees C), a different start site was identified 3' to the heat shock start site that conformed to the E. coli sigma 70 promoter consensus sequence. S1 protection assays and analysis of expression of the dnaK gene fused to the lux transcription reporter gene showed that expression of dnaK is temporally controlled under normal physiological conditions and that transcription occurs just before the initiation of DNA replication. Thus, in both human cells (I. K. L. Milarski and R. I. Morimoto, Proc. Natl. Acad. Sci. USA 83:9517-9521, 1986) and in a simple bacterium, the transcription of a hsp70 gene is temporally controlled as a function of the cell cycle under normal growth conditions.
Caulobacter crescentus cells respond to a sudden increase in temperature by transiently inducing the synthesis of several polypeptides. Two of the proteins induced, Hsp62 and Hsp70, were shown to be analogous to the heat shock proteins of Escherichia coli, GroEL and DnaK, respectively, by immunological cross-reactivity with antibodies raised against the E. coli proteins. Two-dimensional gel electrophoretic resolution of extracts of cells labeled with [35S]methionine during heat shock led to the identification of 20 distinct Hsps in C. crescentus which are coordinately expressed, in response to heat, at the various stages of the cell division cycle. Thus, a developmental control does not seem to be superimposed on the transient activation of the heat shock genes. Nonetheless, under normal temperature conditions, four Hsps (Hsp70, Hsp62, Hsp24b, and Hsp23a) were shown to be synthesized, and their synthesis was cell cycle regulated.
The groESL operon is under complex regulation inCaulobacter crescentus. In addition to strong induction after exposure to heat shock, under physiological growth conditions, its expression is subject to cell cycle control. Transcription and translation of the groE genes occur primarily in predivisional cells, with very low levels of expression in stalked cells. The regulatory region of groESL contains both a ς32-like promoter and a CIRCE element. Overexpression ofC. crescentus ς32 gives rise to higher levels of GroEL and increased levels of the groESL transcript coming from the ς32-like promoter. Site-directed mutagenesis in CIRCE has indicated a negative role for thiscis-acting element in the expression of groESLonly at normal growth temperatures, with a minor effect on heat shock induction. Furthermore, groESL-lacZ transcription fusions carrying mutations in CIRCE are no longer cell cycle regulated. Analysis of an hrcA null strain, carrying a disruption in the gene encoding the putative repressor that binds to the CIRCE element, shows constitutive synthesis of GroEL throughout theCaulobacter cell cycle. These results indicate a negative role for the hrcA gene product and the CIRCE element in the temporal control of the groESL operon.
Caulobacter crescentus carries a flagellum and is motile only during a limited time in its cell cycle. We have asked if the biochemical machinery that mediates chemotaxis exists coincident with the cell's structural ability to respond to a chemotactic signal. We first demonstrated that one function of the chemotaxis machinery, the ability to methylate the carboxyl side chains of a specific set of membrane proteins (methyl-accepting chemotaxis proteins, MCPs), is present in C. crescentus. This conclusion is based on the observations that (i) methionine auxotrophs starved of methionine can swim only in the forward direction (comparable to smooth swimming in the enteric bacteria), (ii) a specific set of membrane proteins was found to be methylated in vivo and the incorporated [3H]methyl groups were alkali sensitive, (iii) this same set of membrane proteins incorporated methyl groups from Sadenosylmethionine in vitro, and (iv) out of a total of eight generally nonchemotactic mutants, two were found to swim only in a forward direction and one of these lacked methyltransferase activity. Analysis of in vivo and in vitro methylation in synchronized cultures showed that the methylation reaction is lost when the flagellated swarmer cell differentiates into a stalked cell. In vivo methylation reappeared coincident with the biogenesis of the flagellum just prior to cell division. In vitro reconstitution experiments with heterologous cell fractions from different cell types showed that swarmer cells contain methyltransferase and their membranes can be methylated. However, newly differentiated stalked cells lack methyltransferase activity and membranes from these cells cannot accept methyl groups. These results demonstrate that MCP methylation is confined to that portion of the cell cycle when flagella are present.
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