Mechanisms of Chromosome Maintenance in Bacteria

点击翻译按钮获取中文摘要

Irrespective of the organism, genomic stability requires that the entire DNA be replicated once and only once per cell cycle. The intricacies of regulatory mechanisms that guarantee initiation of replication and prevent reinitiation prematurely are still being unraveled even in a well-studied system like E. coli. V. cholerae has two chromosomes. The origin of replication of chromosome I is similar to that of E. coli, while the origin of chromosome II is similar to plasmid origins that have repeated origin-specific initiator binding sites (which in plasmids are called iterons). We have determined that in spite of the distinct features of the two Vibrio origins, once-per-cell-cycle replication requires that both the origins be fully methylated at the adenine residues of their GATC sites. For chromosome I, as for oriC of E. coli, methylation was found to be inessential for replication initiation but required to control replication initiation frequency. For chromosome II, however, methylation was additionally required for initiator binding to the origin iterons, an essential function in the initiation process per se. Although methylation is widely used to control many DNA transactions, its role in mediating initiator-origin interactions is an unprecedented finding. Like plasmids, chromosome II has a dedicated locus that inhibits replication initiation. In spite of the entire origin region of chromosome II bearing many signatures of iteron-type plasmid origins, it functions like a chromosomal origin in that it restricts replication to once per cell cycle and to a particular stage of the cell cycle. Neither of these features are hallmarks of plasmid replication. We have discovered that while a typical plasmid locus consists of iterons only, the chromosome II locus has a second kind of site. The iterons require the sites to be fully methylated for initiator binding. The other type also binds the initiator but does not involve methylation. Since the methylation status of DNA changes during the replication cycle, the effectiveness of the iterons as control sites is likely to be cell-cycle dependent. In any event, the control activity of the typical iterons appears to be rather modest. They are primarily responsible for restraining the activity of the other more powerful methylation-independent sites. In this scenario, following replication, the DNA becomes hemimethylated, rendering the iterons unavailable to interact with the methylation-independent sites; the latter remain fully effective in preventing premature reinitiation. When full methylation of the iterons is restored, they become available to restrain the inhibitory action of the methylation-independent sites, thus setting the stage for replication initiation in the next cell cycle. It appears that the plasmid-type origin of chromosome II depends on methylation for initiating replication and for a chromosomal feature of its control. Multichromosomal bacteria (unlike such well-studied bacteria as E. coli and B. subtilis) may offer opportunities to investigate mechanisms for coordinating replication and segregation of the different chromosomes. An initial indication of inter-chromosomal coordination in V. cholerae has been obtained. We have been able to find conditions where replication of one of the chromosomes could be selectively prevented. It appears that preventing chromosome I replication can prevent/delay chromosome II replication but the reverse is not true. Chromosome II is smaller than chromosome I and the delay might allow the two chromosomes to complete replication at the same time, which might facilitate the coordination of their segregation with the cell division. The mechanism of delay remains to be investigated. Compared to our understanding of how chromosomes segregate in eukaryotes, much less is known about how chromosomes segregate in bacteria. Until recently, segregation studies were done primarily in plasmids, where genes dedicated to plasmid partition (par genes) could be found. Homologues of plasmid par genes have now been identified near the origin of replication in most bacteria, including V. cholerae. Both the Vibrio chromosomes have their own par genes. We have succeeded in deleting the par genes of chromosome I without causing much of a segregation defect. Rather, deletion of one the two par genes promoted replication. A similar finding has also been made in B. subtilis. The two bacteria, B. subtilis and V. cholerae, have diverged more than a billion years ago but both have retained the par genes and use them for similar purposes. The wide-spread occurrence of par genes near the replication origins suggests that the genes have a general role in connecting replication and segregation. How the par genes promote replication is under current investigation. Using both the bacterial and yeast two-hybrid systems we are trying to identify proteins that might interact with Par proteins, which might provide a clue as to their mechanism of action. Cell division must await completion of chromosome replication and movement of the two sister chromosomes to opposite cell halves. The temporal control of cell division is largely unknown in bacteria. Recently, some of the genes involved in sensing glucose concentration in the growth media have been found to regulate cell size in E. coli and in B. subtilis. Mutations in these genes make cells smaller by about 30% but do not change their growth rates. In a collaborative study, we are determining the timing of replication initiation (initiation age) in these smaller cell variants. The initiation age seems to depend on the bacterium in question. In B. subtilis, the age remains unchanged in the mutants, which means that, at the time of initiation the cell size is smaller in the case of mutants compared to the wild type. In E. coli, however, initiation is delayed until the mutants reach the size at which initiation occurs in the wild type. The delay in initiation is compensated by increase in replication elongation rate, allowing the replication cycle to complete on time. The rate-limiting component is believed to be the initiator protein, DnaA, in both bacteria. How the initiator accumulates in a cellage dependent manner in one case and cell-size dependent manner in the other remains to be investigated.