SummaryIn Caulobacter crescentus, the genes encoding the chromosome partitioning proteins, ParA and ParB, are essential. Depletion of ParB resulted in smooth filamentous cells in which DNA replication continued. Immunofluorescence microscopy revealed that the formation of FtsZ rings at the mid-cell, the earliest molecular event in the initiation of bacterial cell division, was blocked in cells lacking ParB. ParB binds sequences near the C. crescentus origin of replication. Cell cycle experiments show that the formation of bipolarly localized ParB foci, and presumably localization of the origin of replication to the cell poles, preceded the formation of FtsZ rings at the mid-cell by 20 min. These results suggest that one major function of ParA and ParB may be to regulate the initiation of cytokinesis in C. crescentus.

ParA and ParB of Caulobacter crescentus belong to a conserved family of bacterial proteins implicated in chromosome segregation. ParB binds to DNA sequences adjacent to the origin of replication and localizes to opposite cell poles shortly following the initiation of DNA replication. ParA has homology to a conserved and widespread family of ATPases. Here, we show that ParB regulates the ParA ATPase activity by promoting nucleotide exchange in a fashion reminiscent of the exchange factors of eukaryotic G proteins. Furthermore, we demonstrate that ADP-bound ParA binds single-stranded DNA, whereas the ATP-bound form dissociates ParB from its DNA binding sites. Increasing the fraction of ParA-ADP in the cell inhibits cell division, suggesting that this simple nucleotide switch may regulate cytokinesis.

SummaryIn Caulobacter crescentus the partitioning proteins ParA and ParB operate a molecular switch that couples chromosome partitioning to cytokinesis. Homologues of these proteins have been shown to be important for the stable inheritance of F-plasmids and the prophage form of bacteriophage P1. In C. crescentus , ParB binds to sequences adjacent to the origin of replication and is required for the initiation of cell division. Additionally, ParB influences the nucleotidebound state of ParA by acting as a nucleotide exchange factor. Here we have performed a genetic analysis of the chromosome partitioning protein ParB. We show that C. crescentus ParB, like its plasmid homologues, is composed of three domains: a carboxyl-terminal dimerization domain; a central DNA-binding, helix-turn-helix domain; and an aminoterminal domain required for the interaction with ParA. In vivo expression of amino-terminally deleted parB alleles has a dominant lethal effect resulting in the inhibition of cell division. Fluorescent in situ hybridization experiments indicate that this phenotype is not caused by a chromosome partitioning defect, but by the reversal of the amounts of ATPversus ADP-bound ParA inside the cell. We present evidence suggesting that amino-terminally truncated and full-length, wild-type ParB form heterodimers which fail to interact with ParA, thereby reversing the intracellular ParA-ATP to ParA-ADP ratio. We hypothesize that the amino-terminus of ParB is required to regulate the nucleotide exchange of ParA which, in turn, regulates the initiation of cell division.

SummaryThe expression of the flagellin proteins in Caulobacter crescentus is regulated by the progression of flagellar assembly both at the transcriptional and post-transcriptional levels. An early basal body structure is required for the transcription of flagellin genes, whereas the ensuing assembly of a hook structure is

The FliX/FlbD-dependent temporal transcription of late flagellar genes in Caulobacter crescentus requires the assembly of an early, class II-encoded flagellar structure. Class II flagellar-mutant strains exhibit a delay in the completion of cell division, with the accumulation of filamentous cells in culture. It is shown here that this cell-division defect is attributable to an arrest in the final stages of cell separation. Normal cell morphology could be restored in class II mutants by gain-of-function alleles of FliX or FlbD, suggesting that the timely completion of cell division requires these trans-acting factors. In synchronized cultures, inhibition of cell division by depleting FtsZ resulted in normal initial expression of the late, FlbD-dependent fliK gene; however, the cell cycle-regulated cessation of transcription was delayed, indicating that cell division may be required to negatively regulate FlbD activity. Interestingly, prolonged depletion of FtsZ resulted in an eventual loss of FlbD activity that could be bypassed by a constitutive mutant of FlbD, but not of FliX, suggesting the possible existence of a second cell cycle-dependent pathway for FlbD activation.