Isolation and Characterization of NaCl-Sensitive Mutants of Caulobacter crescentus

Abstract

ABSTRACT An attempt to characterize Caulobacter crescentus genes important for the response to high concentrations of NaCl was initiated by the isolation of mutants defective in survival in the presence of 85 mM NaCl. A transposon Tn 5 library was screened, and five strains which contained different genes disrupted by the transposon were isolated. Three of the mutants had the Tn 5 in genes involved in lipopolysaccharide biosynthesis, one had the Tn 5 in the nhaA gene, which encodes a Na + /H + antiporter, and one had the Tn 5 in the ppiD gene, which encodes a peptidyl-prolyl cis-trans isomerase. All the mutant strains showed severe growth arrest in the presence of 85 mM NaCl, but only the nhaA mutant showed decreased viability under these conditions. All the mutants except the nhaA mutant showed a slightly reduced viability in the presence of 40 mM KCl, but all the strains showed a more severe reduction in viability in the presence of 150 mM sucrose, suggesting that they are defective in responding to osmotic shock. The promoter regions of each disrupted gene were cloned in lacZ reporter vectors, and the pattern of expression in response to NaCl and sucrose was determined; this showed that both agents induced ppiD and nhaA gene expression but did not induce the other genes. Furthermore, the ppiD gene was not induced by heat shock, indicating that it does not belong to the σ 32 regulon, as opposed to what was observed for its Escherichia coli homolog.