Of the two V. cholerae chromosomes, the larger one (Chr1) carries most of the housekeeping genes and is considered the primary chromosome. The smaller chromosome (Chr2) seems to have evolved from a plasmid. Plasmids, although prevalent in bacteria, rarely serve as the source of replicons that drive chromosomal replication. One reason could be that the firing of plasmid origins is generally not restricted to a specific time in the cell cycle, whereas timely firing is the norm for chromosomal origins in all domains of life. Comparison of plasmid and Chr2 systems could thus be valuable to understand how the timing of a biological process has evolved from being random to be specific in the cell cycle. Chr2 replication seems also to depend on Chr1 replication. Our discovery of this finding (in 2014) demonstrated that chromosomes do communicate and encouraged studies to understand the mechanism of coordination in many labs. In eukaryotes, in addition to incomplete and overreplication, uncoordinated replication causes developmental abnormalities and cancer. Our progress in understanding these processes is reported below. 1. Transition from random to cell cycle regulated replication initiation: The initial characterization of Chr2 replication suggested that its regulation is more complex than that of its presumed progenitor plasmid. Many, if not all, of these regulations are mediated through the Chr2 specific initiator protein, RctB. We have embarked on a systematic structure-function analysis of the protein not only to understand Chr2 replication control in greater depth but also for drug design against V. cholerae (Section 2 below). In the past year, we succeeded in identifying a new domain of RctB that interacts with the molecular chaperone DnaK (Hsp70). DnaK increases RctB binding to the replication origin of Chr2 and this observation suffices to explain why DnaK is required for Chr2 replication. DnaK also helps RctB binding to regions outside of the replication origin for regulatory purposes. Identification of the DnaK interacting domain of RctB (K-domain) provided valuable insights on how DnaK helps in controlling the two different kinds of DNA binding activity of RctB. Mutations in the K-domain inactivates the replication initiator activity of RctB and intragenic suppressors that allow initiator activity seem to have done so by reducing replication inhibitory activities of the protein. These results have been rewarding in identifying the major negative regulatory mechanisms of Chr2 replication. 2. Towards generation of Vibrio-specific antimicrobial agents: The Chr2 initiator, RctB, is conserved only in the Vibrio family and appears ideally suited for developing potential drugs specifically against Vibrios. In the case of cholera, although oral rehydration treatment is the mainstay, antimicrobial therapy becomes mandatory at times, and V. cholerae is no exception in developing resistance to multiple antibiotics. Design of new drugs is greatly facilitated when the 3-D structural information of target proteins are available. Replication initiators in general have proven refractory to structural studies most likely because they have unstructured regions and require remodeling by chaperone proteins for activity, which seems to be the case for RctB. In collaboration with Alex Wlodawer (CCR), Lisa Jenkins (CCR) and Rodolfo Ghirlando (NIDDK), we succeeded in recent months solving the structure of one half of RctB. Many of the regulatory mutations are mapped in this region. Determination of the structure of the remaining regions that serve more essential functions has proven refractory to date, possibly because such regions could be intrinsically unfolded (from NMR evidence obtained in collaboration with Yawen Bai, CCR). The folding of these regions may require the presence of their interacting partners. 3. Identification of a novel check point control in bacteria. We hypothesized that the timely replication and segregation of the two V. cholerae chromosomes would require communication between them, so that both the processes can complete prior to cell division. An evidence for inter chromosomal communication was obtained when we identified a locus on Chr1 (crtS for Chr2 replication triggering site) that can bind RctB and significantly stimulate Chr2 replication. The location of crtS on Chr1 is such that it would replicate just before the time of Chr2 replication initiation. This affords a straight forward mechanism for communication. Chr1 replication initiates first. When the fork passes through crtS, it activates the Chr2 initiator that triggers Chr2 replication. Replication of crtS thus relieves the check point that prevents Chr2 replication. We have now shown that blocking of Chr1 replication also blocks Chr2 replication, demonstrating that Chr2 replication indeed depends on Chr1 replication. Strikingly, two unreplicated copies of crtS could also allow Chr2 replication, suggesting that crtS has significant activity without itself being replicated and a role of replication is to increase this activity by doubling the crtS gene dosage. The activity appears to be remodeling of RctB, since crtS could relieve the dependence of the initiator on molecular chaperones. The study provides a novel example of how doubling of gene dosage by replication is utilized for other regulatory purposes.

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Ramachandran, Revathy; Ciaccia, Peter N; Filsuf, Tara A et al. (2018) Chromosome 1 licenses chromosome 2 replication in Vibrio cholerae by doubling the crtS gene dosage. PLoS Genet 14:e1007426
Jha, Jyoti K; Chattoraj, Dhruba K (2016) Inactivation of Individual SeqA Binding Sites of the E. coli Origin Reveals Robustness of Replication Initiation Synchrony. PLoS One 11:e0166722
Jha, Jyoti K; Ramachandran, Revathy; Chattoraj, Dhruba K (2016) Opening the Strands of Replication Origins-Still an Open Question. Front Mol Biosci 3:62
Baek, Jong Hwan; Chattoraj, Dhruba K (2014) Chromosome I controls chromosome II replication in Vibrio cholerae. PLoS Genet 10:e1004184
Baek, Jong Hwan; Rajagopala, Seesandra V; Chattoraj, Dhruba K (2014) Chromosome segregation proteins of Vibrio cholerae as transcription regulators. MBio 5:e01061-14
Ramachandran, Revathy; Jha, Jyoti; Chattoraj, Dhruba K (2014) Chromosome segregation in Vibrio cholerae. J Mol Microbiol Biotechnol 24:360-70
Venkova-Canova, Tatiana; Baek, Jong Hwan; Fitzgerald, Peter C et al. (2013) Evidence for two different regulatory mechanisms linking replication and segregation of vibrio cholerae chromosome II. PLoS Genet 9:e1003579
Kadoya, Ryosuke; Chattoraj, Dhruba K (2012) Insensitivity of chromosome I and the cell cycle to blockage of replication and segregation of Vibrio cholerae chromosome II. MBio 3:
Jha, Jyoti K; Baek, Jong Hwan; Venkova-Canova, Tatiana et al. (2012) Chromosome dynamics in multichromosome bacteria. Biochim Biophys Acta 1819:826-9
Venkova-Canova, Tatiana; Saha, Anik; Chattoraj, Dhruba K (2012) A 29-mer site regulates transcription of the initiator gene as well as function of the replication origin of Vibrio cholerae chromosome II. Plasmid 67:102-10

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