The replication enhancer crtS depends on transcription factor Lrp for modulating binding of initiator RctB to ori2 of Vibrio cholerae.

Replication of Vibrio cholerae chromosome 2 (Chr2) initiates when the Chr1 locus, crtS (Chr2 replication triggering site) duplicates. The site binds the Chr2 initiator, RctB, and the binding increases when crtS is complexed with the transcription factor, Lrp. How Lrp increases the RctB binding and how RctB is subsequently activated for initiation by the crtS-Lrp complex remain unclear. Here we show that Lrp bends crtS DNA and possibly contacts RctB, acts that commonly promote DNA-protein interactions. To understand how the crtS-Lrp complex enhances replication, we isolated Tn-insertion and point mutants of RctB, selecting for retention of initiator activity without crtS. Nearly all mutants (42/44) still responded to crtS for enhancing replication, exclusively in an Lrp-dependent manner. The results suggest that the Lrp-crtS controls either an essential function or more than one function of RctB. Indeed, crtS modulates two kinds of RctB binding to the origin of Chr2, ori2, both of which we find to be Lrp-dependent. Some point mutants of RctB that are optimally modulated for ori2 binding without crtS still remained responsive to crtS and Lrp for replication enhancement. We infer that crtS-Lrp functions as a unit, which has an overarching role, beyond controlling initiator binding to ori2.

The dimerization interface of initiator RctB governs chaperone and enhancer dependence of Vibrio cholerae chromosome 2 replication.

Protein function often requires remodeling of protein structure. In the well-studied iteron-containing plasmids, the initiator of replication has a dimerization interface that undergoes chaperone-mediated remodeling. This remodeling reduces dimerization and promotes DNA replication, since only monomers bind origin DNA. A structurally homologs interface exists in RctB, the replication initiator of Vibrio cholerae chromosome 2 (Chr2). Chaperones also promote Chr2 replication, although both monomers and dimers of RctB bind to origin, and chaperones increase the binding of both. Here we report how five changes in the dimerization interface of RctB affect the protein. The mutants are variously defective in dimerization, more active as initiator, and except in one case, unresponsive to chaperone (DnaJ). The results indicate that chaperones also reduce RctB dimerization and support the proposal that the paradoxical chaperone-promoted dimer binding likely represents sequential binding of monomers on DNA. RctB is also activated for replication initiation upon binding to a DNA site, crtS, and three of the mutants are also unresponsive to crtS. This suggests that crtS, like chaperones, reduces dimerization, but additional evidence suggests that the remodelling activities function independently. Involvement of two remodelers in reducing dimerization signifies the importance of dimerization in limiting Chr2 replication.

Interactions of replication initiator RctB with single- and double-stranded DNA in origin opening of Vibrio cholerae chromosome 2.

Studies of bacterial chromosomes and plasmids indicate that their replication initiator proteins bind to origins of replication at many double-stranded sites and also at AT-rich regions where single-stranded DNA is exposed during origin opening. Single-strand binding apparently promotes origin opening by stabilizing an open structure, but how the initiator participates in this process and the contributions of the several binding sites remain unclear. Here, we show that the initiator protein of Vibrio cholerae specific to chromosome 2 (Chr2) also has single-strand binding activity in the AT-rich region of its origin. Binding is strand specific, depends on repeats of the sequence 5'ATCA and is greatly stabilized in vitro by specific double-stranded sites of the origin. The stability derives from the formation of ternary complexes of the initiator with the single- and double-stranded sites. An IHF site lies between these two kinds of sites in the Chr2 origin and an IHF-induced looping out of the intervening DNA mediates their interaction. Simultaneous binding to two kinds of sites in the origin appears to be a common mechanism by which bacterial replication initiators stabilize an open origin.

Mutual interaction enables the mycobacterial plasmid pAL5000 origin binding protein RepB to recruit RepA, the plasmid replicase, to the origin.

The Mycobacterium fortuitum plasmid, pAL5000, is the most-studied member of a family of plasmids that are found in Actinobacteria. Its replication is brought about by the combined action of two plasmid-encoded replication proteins, RepA and RepB. RepB has earlier been shown to be a sigma factor homologue that possesses origin-binding activity. The mechanism by which RepA functions, and its relationship with RepB, if any, has not been explored yet. In this study, we show that RepA shares a common catalytic domain, with proteins belonging to the primase-polymerase and DNA polymerase X families. We demonstrate that RepA is functionally a DNA polymerase and that mutations that alter two conserved aspartic acid residues present within the catalytic core lead to inactivation of plasmid replication. Replication of pAL5000 was shown not to depend on the host primase, and thus it is most likely that RepA is responsible for the priming act. We further demonstrate that RepA and RepB function as a pair and that the functional cooperation between the two requires physical contact. The C-terminal domain of RepA, which is structurally a helical bundle, is responsible for unwinding the origin in a site-specific manner and also for the establishment of contacts with RepB. The results presented show that RepB functions by recruiting RepA to the origin in much the same way as sigma factors recruit RNA polymerase core enzyme to promoters.

Evolutionary link between the mycobacterial plasmid pAL5000 replication protein RepB and the extracytoplasmic function family of σ factors.

Mycobacterial plasmid pAL5000 represents a family of plasmids found mostly in the Actinobacteria. It replicates using two plasmid-encoded proteins, RepA and RepB. While BLAST searches indicate that RepA is a replicase family protein, the evolutionary connection of RepB cannot be established, as no significant homologous partner (E < 10(-3)) outside the RepB family can be identified. To obtain insight into the structure-function and evolutionary connections of RepB, an investigation was undertaken using homology modeling, phylogenetic, and mutational analysis methods. The results indicate that although they are synthesized from the same operon, the phylogenetic affinities of RepA and RepB differ. Thus, the operon may have evolved through random breaking and joining events. Homology modeling predicted the presence of a three-helical helix-turn-helix domain characteristic of region 4 of extracytoplasmic function (ECF) σ factors in the C-terminal region of RepB. At the N-terminal region, there is a helical stretch, which may be distantly related to region 3 of σ factors. Mutational analysis identified two arginines indispensable for RepB activity, one each located within the C- and N-terminal conserved regions. Apart from analyzing the domain organization of the protein, the significance of the presence of a highly conserved A/T-rich element within the RepB binding site was investigated. Mutational analysis revealed that although this motif does not bind RepB, its integrity is important for efficient DNA-protein interactions and replication to occur. The present investigation unravels the possibility that RepB-like proteins and their binding sites represent ancient DNA-protein interaction modules.

Characterization of the gluconate utilization system of Vibrio cholerae with special reference to virulence modulation.

Orthologs search identified that the Vibrio cholerae gluconate (Gnt) utilization system minimally consisted of the Entner-Doudoroff (ED) pathway (edd and eda) and three other genes, namely gntU, gntK and gntR This system appeared unique by genomic organization of component genes into two operons transcribed in opposite directions. In silico analysis indicated GntU as an inner-membrane protein functioning for transport and GntK as a kinase with cytosolic localization that generates Gnt6P, which is then metabolized through the ED pathway. Enzyme 6-phosphogluconate dehydratase encoded by edd converts Gnt6P to 2-keto-3-deoxy-6-phosphogluconate (KDPG), which is metabolized by the action of KDPG-aldolase encoded by eda Transcriptional upregulation of the Gnt utilization genes in the gntR mutant matched well to a predicted repressor role of GntR. GntR displayed DNA binding to a region in the promoters of two bi-directionally transcribed operons. Growth defect of mutants in Gnt-supplemented media confirmed obligate involvement of these genes in Gnt utilization and such defect was restored upon complementation. Defective Gnt utilization resulted in attenuation of colonization potential and reduction of cholera toxin secretion in V. cholerae The ED pathway mutants showed the highest level of virulence attenuation. Overall, this study established a minimal requirement of the V. cholerae Gnt utilization system, which played a critical role in pathogenesis.