(500f) Investigating the Role of pBBR1’s Mobilization Protein in Plasmid Maintenance in Non-Model Bacteria

Stable plasmid maintenance is crucial to accurate characterization and testing of biological devices. Model bacteria are often transformed with plasmids to utilize biochemical capabilities they do not naturally possess, and the mechanisms for replication of plasmids in these model bacteria are well understood. However, the plasmid maintenance mechanisms for non-model bacteria are not as well known. The mobilization gene found in the plasmid BBR1, Mob, has shown to be crucial for plasmid stability in Rhodopseudomonas palustris CGA009 (hereafter R. palustris). R. palustris is an extremely metabolically versatile bacterium that is capable of all four modes of metabolism, catabolizes recalcitrant feedstocks such as lignin monomers, produces hydrogen and the industrially relevant bioplastic, polyhydroxybutyrate (PHB), and possesses both carbon dioxide and nitrogen fixation abilities. Strangely, BBR1 has been successfully employed in Paraburkholderia sacchari LMG 19450 LFM101 (hereafter P. sacchari), a pentose and hexose consuming bacterium, without the presence of Mob. Mobilization proteins facilitate horizontal gene transfer between two different bacteria through the Type IV secretion system by nicking DNA before conjugation and rejoining the DNA afterwards. A previously annotated mobilization protein from the streptococcal plasmid pMV158 has shown to have a very similar amino acid sequence to Mob. The nicking behavior of the pMV158 mobilization protein was severely impaired by replacing catalytic histidines and other active site residues. Using analogous methods, the effects of mutation on the active site residues of Mob will be explored in R. palustris and P. sacchari. This study will provide groundwork to engineering non-model bacteria and harnessing their unique biochemical capabilities.