Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids and chromosomes from prokaryotic organisms. All known plasmid‐encoded par loci specify three components: a cis‐acting centromere‐like site and two trans‐acting proteins that form a nucleoprotein complex at the centromere (i.e. the partition complex). The proteins are encoded by two genes in an operon that is autoregulated by the par‐encoded proteins. In all cases, the upstream gene encodes an ATPase that is essential for partitioning. Recent cytological analyses indicate that the ATPases function as adaptors between a host‐encoded component and the partition complex and thereby tether plasmids and chromosomal origin regions to specific subcellular sites (i.e. the poles or quarter‐cell positions). Two types of partitioning ATPases are known: the Walker‐type ATPases encoded by the par/sop gene family (type I partitioning loci) and the actin‐like ATPase encoded by the par locus of plasmid R1 (type II partitioning locus). A phylogenetic analysis of the large family of Walker type of partitioning ATPases yielded a surprising pattern: most of the plasmid‐encoded ATPases clustered into distinct subgroups. Surprisingly, however, the par loci encoding these distinct subgroups have different genetic organizations and thus divide the type I loci into types Ia and Ib. A second surprise was that almost all chromosome‐encoded ATPases, including members from both Gram‐negative and Gram‐positive Bacteria and Archaea, clustered into one distinct subgroup. The phylogenetic tree is consistent with lateral gene transfer between Bacteria and Archaea. Using database mining with the ParM ATPase of plasmid R1, we identified a new par gene family from enteric bacteria. These type II loci, which encode ATPases of the actin type, have a genetic organization similar to that of type Ib loci.
Bacterial plasmids are stabilized by a number of different mechanisms. Here we describe the molecular aspects of a group of plasmid-encoded gene systems called the proteic killer gene systems. These systems mediate plasmid maintenance by selectively killing plasmid-free cells (post-segregational killing or plasmid addiction). The group includes ccd of F, parD/pem of R1/R100, parDE of RP4/RK2, and phd/doc of P1. All of these systems encode a stable toxin and an unstable antidote. The antidotes prevent the lethal action of their cognate toxins by forming tight complexes with them. The antidotes are degraded by cellular proteases. Thus, the different decay rates of the toxins and antidotes seem to be the molecular basis of toxin activation in plasmid-free cells. The operons encoding the toxins and antidotes are autoregulated at the level of transcription either by a complex formed by the toxins and the cognate antidotes or by the antidote alone. The cellular targets of the killer proteins have been determined to be DNA gyrase in the case of ccd of F and DnaB in the case of parD of R1. Surprisingly, the Escherichia coli chromosome encodes at least two of these peculiar gene systems.
The highly conserved SMC (Structural Maintenance of Chromosomes) proteins function in chromosome condensation, segregation, and other aspects of chromosome dynamics in both eukaryotes and prokaryotes. A null mutation in the Caulobacter crescentus smc gene is conditionally lethal and causes a cell cycle arrest at the predivisional cell stage. Chromosome segregation in wild-type and smc null mutant cells was examined by monitoring the intracellular localization of the replication origin and terminus by using f luorescence in situ hybridization. In wild-type cells, the origin is located at the f lagellated pole of swarmer cells and, immediately after the initiation of DNA replication in stalked cells, one of the origins moves to the opposite pole, giving a bipolar localization of the origins. The terminus moves from the end of the swarmer cell opposite the origin to midcell. A subpopulation of the smc null mutant cells had mislocalized origins or termini, showing that the smc null mutation gives DNA segregation defects. Nucleoid morphology was also abnormal. Thus, we propose that the Caulobacter chromosomal origins have specific cellular addresses and that the SMC protein plays important roles in maintaining chromosome structure and in partitioning. The specific cell cycle arrest in the smc null mutant indicates the presence of a cell cycle checkpoint that senses perturbations in chromosome organization or segregation.
The mechanisms responsible for prokaryotic DNA segregation are largely unknown. The partitioning locus (par) encoded by the Escherichia coli plasmid R1 actively segregates its replicon to daughter cells. We show here that the ParM ATPase encoded by par forms dynamic actin-like ®laments with properties expected for a force-generating protein. Filament formation depended on the other components encoded by par, ParR and the centromere-like parC region to which ParR binds. Mutants defective in ParM ATPase exhibited hyper®lamentation and did not support plasmid partitioning. ParM polymerization was ATP dependent, and depolymerization of ParM ®laments required nucleotide hydrolysis. Our in vivo and in vitro results indicate that ParM polymerization generates the force required for directional movement of plasmids to opposite cell poles and that the ParR±parC complex functions as a nucleation point for ParM polymerization. Hence, we provide evidence for a simple prokaryotic analogue of the eukaryotic mitotic spindle apparatus.
The in vivo intracellular location of components of the Caulobacter replication apparatus was visualized during the cell cycle. Replisome assembly occurs at the chromosomal origin located at the stalked cell pole, coincident with the initiation of DNA replication. The replisome gradually moves to midcell as DNA replication proceeds and disassembles upon completion of DNA replication. Although the newly replicated origin regions of the chromosome are rapidly moved to opposite cell poles by an active process, the replisome appears to be an untethered replication factory that is passively displaced towards the center of the cell by the newly replicated DNA. These results are consistent with a model in which unreplicated DNA is pulled into the replication factory and newly replicated DNA is bidirectionally extruded from the complex, perhaps contributing to chromosome segregation.
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