Most of our projects are centered on understanding how the replication of chrII is controlled in the cell cycle of V. cholerae. The chromosome is believed to have originated from a plasmid, yet it initiates replication at a particular time of the cell cycle, similar to other bona fide chromosomes but unlike plasmids whose timing of replication is generally not fixed in the cell cycle. We are trying to understand how the random timing of initiation has become fixed in the cell cycle. ChrII replication seems to also depend on chrI replication. How the two chromosomes communicate to coordinate replication is largely unknown and this knowledge is basic to our understanding of how multiple chromosomes are maintained in bacteria, such as V. cholerae. Finally, chrII replication is controlled by a segregation protein. The discovery of the influence of segregation on replication is a recent development in the field of chromosome dynamics in bacteria. Our progress in understanding these processes is reported below.Transition from plasmid to chromosomal mode of replication:We discovered that the chrII initiator binds specifically to two kinds of site: One kind of site consists of iterons, equivalent to repeats that characterize replication origin of a family of plasmids. The other kind of site, called 39-mers, consists of unrelated sequence. The chrII iterons, however, have to be methylated at their adenine residue before they can bind the initiator, unlike the situation in plasmid iterons. The 39-mers are the primary negative regulators of replication. They enhance interactions that inactivate the origins. The iterons in turn can also dampen the inhibitory activity of the 39-mers. How the interplay between the two kinds of site might coordinate chrII replication with the cell cycle has been modeled. The model also takes into account the fact that the origin iterons, which becomes hemimethylated after origin firing, are kept in an inactivated state due to the binding of a hemimethylation-specific protein, SeqA, for two-thirds of the cell cycle. This does not happen to plasmid iterons because they are not methylated. The chrII control system thus has features additional to those present in plasmid systems, which most likely allow cell cycle specific replication. Control of chrII replication by chrI:We hypothesize that the timely replication and segregation of the two chromosomes of V. cholerae requires communication between them, so that both of the chromosomes can complete the processes prior to cell division. Preliminary evidence for inter-chromosomal communication 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 chrI replication can prevent/delay chromosome II replication but the reverse is not true. The mechanism of how chrI dictates chrII replication is currently being investigated. ChrII replication is also controlled by a segregation protein:That segregation can influence replication was not known until recently. Our knowledge of chromosome segregation in bacteria comes primarily from studies of plasmids, where genes dedicated to plasmid segregation (par genes) were first found. Homologues of plasmid par genes have now been identified near the origin of replication in most bacteria, including V. cholerae. Both of the Vibrio chromosomes have their own par genes. Upon deletion of one of the two par genes (parB) of chrI replication of that chromosome was specifically promoted. A similar finding has also been made in B. subtilis. In both B. subtilis and V. cholerae, the universal bacterial initiator, DnaA, was found to be the direct target of Par proteins. How the Par proteins stimulate the activity of DnaA has been studied in B. subtilis, and the mechanism is likely to be similar in V. cholerae. For this reason, we are not continuing with this project, but rather asking whether segregation proteins could also influence chrII replication.We have found that the centromere-binding protein ParB2 of chrII can spread beyond the centromere and cover a strong replication inhibitory site (a 39-mer) of the origin. This likely interferes with the 39-mer interaction with the initiator RctB, an interaction required for replication inhibition. Unexpectedly, ParB2 can also promote replication without requiring the centromere by directly binding to a different 39-mer. ParB2 and RctB compete for binding to this site, which could compromise its inhibitory activity. ParB2 thus appears to promote replication by competing with the initiator for binding to two strong replication inhibitory sites, by spreading into one and binding specifically to the other. Spreading represents a novel replication control mechanism acting from a distance;non-centromeric binding is a novel mechanism for control by a segregation protein. These studies establish several ways by which segregation proteins could influence replication. We suggest that involving a segregation protein directly in replication control is a feature of the putative adaptation of an acquired plasmid to permanent residency as a second chromosome in V. cholerae.Generation of Vibrio specific antimicrobial agents:Given the increasing prevalence of multi-drug resistance in pathogenic vibrios, there is a need for new targets and drugs to combat these pathogens. The chrII-specific initiator RctB is conserved only in the family Vibrionaceae. The protein appears ideally suited for developing potential anti-vibrio specific drugs. Drug design is greatly facilitated if the 3-D structural information of the target protein is known. Towards this goal we have started a systematic domain analysis of RctB. Replication initiators in general have proven refractory to structural studies because they require remodeling by chaperone proteins and/or binding to specific DNA for activity. Initial attempts to form crystals of RctB have failed. We are attempting deletion analysis to isolate functional domains. These smaller derivatives might be more amenable to structural studies. Cell size and the initiation of DNA replication in bacteria: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 have determined the timing of replication initiation (initiation age) in the cell cycle of these small size mutants. In B. subtilis, the age remains unchanged in the mutants, indicating that the achievement of a particular cell size is not obligatory for initiation. 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 for by an increase in the replication elongation rate, allowing the replication cycle to complete on time. The initiation delay could be avoided by overproducing the initiator protein, DnaA. These results are consistent with DnaA being the rate-limiting component of replication initiation in E. coli, and that the accumulation of the initiator to a level critical for initiation depends upon growth. DnaA is also rate-limiting in B. subtilis but appears not to be controlled in a growth dependent manner. Thus, although DnaA is likely to be required for initiation in all bacteria, the mechanisms governing its supply appear not to be conserved. Understanding the mechanistic basis of the difference will require detailed understanding of the replication control systems of the two bacteria, which would be too ambitious for our resources. We do not plan to proceed with this project.
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