Effect of a ctrA promoter mutation, causing a reduction in CtrA abundance, on the cell cycle and development of Caulobacter crescentus

摘要

Background Polar development during the alphaproteobacterium Caulobacter crescentus cell cycle is integrated to the point that individual mutations can have pleiotropic effects on the synthesis of polar organelles. Disruption of the genes encoding the histidine kinase PleC, or its localization factor PodJ, disrupts synthesis or functionality of pili, flagella and adhesive holdfast. However, the mechanism by which these mutations affect polar development is not well understood. The aim of this study was to identify new regulators that control multiple aspects of polar organelle development. Results To identify mutants with pleiotropic polar organelle synthesis defects, transposon mutagenesis was performed and mutants were selected based resistance to the pili-tropic bacteriophage ΦCbK. Mutants were then screened for defects in motility and holdfast production. Only a single podJ/pleC-independent mutant was isolated which had defects in all three phenotypes. Directed phage assays confirmed the phage resistance phenotype, while the strain demonstrated a similar dispersal radius as a podJ mutant in swarm agar, and treatment with a fluorescent lectin that labels the holdfast showed no staining for this mutant. The transposon had inserted into the promoter region of ctrA, a gene encoding a master transcriptional regulator of the cell cycle, disrupting native transcription but still allowing reduced transcriptional activity and protein production of this essential protein. Transcriptional fusions showed that essential genes controlled by CtrA exhibited minor to moderate changes in expression in the ctrA promoter mutant, while the pilA gene, encoding the subunit of the pilus filament, had a drastic decrease in gene expression. Introduction of a plasmid-born copy of ctrA under its native promoter complemented the phage resistance and holdfast defects, as well as a moderate cell morphology defect, but not the swarming defect. Conclusions A mutation was identified that caused pleiotropic defects in polar organelle synthesis, and revealed the surprising result that some CtrA-dependent promoters are more sensitive to changes in CtrA concentration than others. However, the fact that no pleiotropic mutations were found in new regulators suggests that downstream signaling of PleC/PodJ is either essential, redundant, or branching such that all three phenotypes were not simultaneously affected.