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.

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.

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) 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 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.

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.