Sam Houston State University, USA
Madhusudan Choudhary is a Associate Professor in the Department of Biological Sciences at Sam Houston State University, Texas, USA. He did his Ph.D. from McMaster University (Canada) and postdoctoral study at Duke University (USA). He has previously worked as Research Assistant Professor in the Department of Microbiology and Molecular Genetics at University of Texas Medical Center-Housrton, Texas. He has published over 50 research publications in national and international journals and has been serving as an editorial board member of JMBE and Microbial Life.
Rhodobacter sphaeroides is a facultative anaerobic bacterium which belongs to the α-3 subdivision of Proteobacteria. It has a complex genome, consisting of two chromosomes, chromosome I (CI) and chromosome II (CII), which are approximately 3Mb and 0.9Mb, respectively. The objective of this study was to identify and characterize the origins of replication of the two chromosomes, and analyze them with respect to chromosomal or plasmid origin type. Using bioinformatics approaches, such as Z-curve and GC-skew analyses, three and five putative chromosomal origin regions were identified on CI and CII, respectively. The flanking regions of these putative regions were analyzed for the conservation of genes known to be located near confirmed replicative origins of other bacterial species. Each of the putative regions were amplified and cloned into a pLO1 vector, which contains a Kanamycin resistance gene and acts as a suicide vector in R. sphaeroides. These recombinant pLO1 plasmids were mobilized into R. sphaeroides using biparental mating of E. coli S17-1 and R. sphaeroides. Resulting transconjugants were characterized for the autonomous replication of the plasmid in R. sphaeroides. Conservation of genes proximal to the replication origins as well as biological characterization of these putative origin sequences confirmed the repective origins of the two chromosmes. Results also revealed that the replicative origin of the primary chromosome is a typical bactyerial chromosomal type, while secondary chromosome is located near parA and parB genes, an arrangement shared by a number of megaplasmids in other bacteria.