Molecular Dissection of the Essential Features of the Origin of Replication of the Second Vibrio cholerae Chromosome

Abstract

Vibrionaceae family members are interesting models for studying DNA replication initiation, as they contain two circular chromosomes. Chromosome II (chrII) replication is governed by two evolutionarily unique yet highly conserved elements, the origin DNA sequence oriCII and the initiator protein RctB. The minimum functional region of oriCII, oriCII-min, contains multiple elements that are bound by RctB in vitro, but little is known about the specific requirements for individual elements during oriCII initiation. We utilized undirected and site-specific mutagenesis to investigate the functionality of mutant forms of oriCII-min and assessed binding to various mutant forms by RctB. Our analyses showed that deletions, point mutations, and changes in RctB target site spacing or methylation all impaired oriCII-min-based replication. RctB displayed a reduced affinity for most of the low-efficacy origins tested, although its characteristic cooperative binding was generally maintained. Mutations that removed or altered the relative positions of origin components other than RctB binding sites (e.g., AT-rich sequence, DnaA target site) also abolished replicative capacity. Comprehensive mutagenesis and deep-sequencing-based screening (OriSeq) allowed the identification of a previously uncharacterized methylated domain in oriCII that is required for origin function. Together, our results reveal the remarkable evolutionary honing of oriCII and provide new insight into the complex interplay between RctB and oriCII.

A Cytosine Methytransferase Modulates the Cell Envelope Stress Response in the Cholera Pathogen

“…The V . cholerae Dam homologue has been shown to be critical for replication of one of the organism’s two chromosomes, and is consequently essential for survival [ 20 , 21 ]. In contrast, vc1769 ( hsdM ), vca0198 (vchM) and vca0447 have been found to be not essential, either through targeted mutagenesis [ 22 ] or in transposon insertion sequencing screens [ 23 – 25 ].…”

Vibrio cholerae chromosome 2 copy number is controlled by the methylation-independent binding of its monomeric initiator to the chromosome 1 crtS site

“…On the other hand, we show here the first evidence of DnaA binding to ori2 ( Supplementary Figure S8 ). The DnaA box found in ori2 was shown to be essential to initiate Chr2 replication ( 14 ). Nevertheless, the exact implication of DnaA in ori2 replication control is still unknown.…”

“…The minimum origin of replication contains the rctB encoding gene and six repeated 12-mers (iterons) onto which RctB binds to promote replication initiation with the unwinding of a contiguous AT-rich sequence ( 13 ). It also contains putative IHF and DnaA binding sites, the latter being required for replication at ori2 ( 14 , 15 ). The ori2 regulatory region contains a transcribed but non-translated ORF ( rctA) and two types of RctB binding sites: five regulatory iterons and two 39-mer motifs, one of which is found in rctA ( 16 ) .…”

The Pelagic Bacterium Paraphotobacterium marinum Has the Smallest Complete Genome Within the Family Vibrionaceae

“…The cholera pathogen Vibrio cholerae El Tor N16961, for example, contains two chromosomes, with the large one of 3.0 Mbp and the small one of 1.1 Mbp ( Heidelberg et al, 2000 ). Because V. cholerae is the most clinically important vibrio causing cholera, a severe diarrheal disease, it becomes a model organism to study the chromosome replication initiation ( Baek and Chattoraj, 2014 ; Gerding et al, 2015 ), cell division mechanisms ( Galli et al, 2016 ), and other fundamental biological investigations. Vibrio parahaemolyticus KX-V237, another pathogenic vibrio causing gastroenteritis, also contains two circular chromosomes, with the large one of 3.3 Mbp and the small one of 1.9 Mbp ( Tagomori et al, 2002 ).…”