In the past year, we have brought to closure projects on replication control of a low copy number E. coli plasmid, P1. Nilangshu Das with help from a mathematician, Johan Paulsson (Cambridge U., UK), has characterized control-defective initiator mutants that confer relatively unhindered replication profiency to the plasmid. The properties of the mutants are best explained assuming multiple modes of control involving transcriptional autorepression of the initiator gene, initiator inactivation by dimerization, and origin inactivation by pairing. The control is best when the three mechanisms cooperate. A new development in the field of E. coli DNA replication is the finding of weak (incognito) binding sites for the initiator DnaA in the origin of replication, called I-sites. These sites recognize only by ATP-bound DnaA. The generality of I-sites in DNA replication remained to be established. Richard Fekete has identified a possible I-site in the replication origin in plasmid P1. Since ATP-DnaA level decreases after the initiation of chromosomal replication, this may be a way for the plasmid to correlate its replication with that of the chromosome and add a new paradigm in plasmid replication control.Knowing the importance for controlling initiator proteins in DNA replication control, Debasish Pal has studied the regulation of the RctB initiator for chromosome II replication of V. cholerae. A strong promoter for the gene was identified and found to be autorepressed as well as regulated by unstream sequences and global regulators, IHF and Dam methylase. Increasing RctB in trans increased the copy number of a miniplasmid carrying the chrII origin, implying that RctB can be rate-limiting for chrII replication. The multiple modes of control on RctB are expected to reduce fluctuations in the initiator concentration and thereby help maintain chromosome copy number homeostasis. To study the regulation of replication of chrII, Tatiana Venkova-Canova has defined the origin and its negative control elements. The origin resembles those of P1 type plasmids but the negative control locus is more extended and complex. It is already clear that the regulation of replication has diverged significantly from the ones operating in plasmids or in E. coli.Preeti Srivastava is studying chromosome dynamics in V. cholera using fluorescence microscopy and flow cytometry. So far she has tracked the movement of the origins and terminii of both the chromosomes. The migration patterns seem to differ between the two chromosomes as well as from what has been reported in other systems. One of the novel findings is the cohesion of the terminus region of the two chromosomes, which suggests the possibility that there could be segregational information in those regions.

National Institute of Health (NIH)
Division of Basic Sciences - NCI (NCI)
Intramural Research (Z01)
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Basic Sciences
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Chattoraj, Dhruba K (2007) Tryptophanase in sRNA control of the Escherichia coli cell cycle. Mol Microbiol 63:1-3
Srivastava, Preeti; Demarre, Gaelle; Karpova, Tatiana S et al. (2007) Changes in nucleoid morphology and origin localization upon inhibition or alteration of the actin homolog, MreB, of Vibrio cholerae. J Bacteriol 189:7450-63
Srivastava, Preeti; Chattoraj, Dhruba K (2007) Selective chromosome amplification in Vibrio cholerae. Mol Microbiol 66:1016-28
Srivastava, Preeti; Fekete, Richard A; Chattoraj, Dhruba K (2006) Segregation of the replication terminus of the two Vibrio cholerae chromosomes. J Bacteriol 188:1060-70
Das, Nilangshu; Valjavec-Gratian, Majda; Basuray, Ashish N et al. (2005) Multiple homeostatic mechanisms in the control of P1 plasmid replication. Proc Natl Acad Sci U S A 102:2856-61
Pal, Debasish; Venkova-Canova, Tatiana; Srivastava, Preeti et al. (2005) Multipartite regulation of rctB, the replication initiator gene of Vibrio cholerae chromosome II. J Bacteriol 187:7167-75
Fekete, Richard A; Chattoraj, Dhruba K (2005) A cis-acting sequence involved in chromosome segregation in Escherichia coli. Mol Microbiol 55:175-83