(160ax) Investigating the Role of pBBR1’s Mobilization Protein in Plasmid Maintenance in Nonmodel Bacteria

Stable plasmid maintenance is critical to testing and employing programmable biological devices.
While the mechanisms are well-characterized for commonly used vectors in model
microorganisms, the same is not true for replicons when utilized in non-model bacteria, many of
which harbor important biochemical capabilities. The discovery that the plasmid pBBR1’s
mobilization protein, Mob, is required for maintenance in Rhodopseudomonas palustris CGA009,
which is an extremely metabolically versatile soil bacterium known for its utilization of all four
forms of metabolism. Its ability to consume recalcitrant feedstocks such as lignin breakdown
products, and produce hydrogen, prompted the investigation into the protein’s function in this and
other non-model bacteria. Mobilization proteins are relaxases that facilitate horizontal gene
transfer between bacteria through the type IV secretion system by nicking the DNA before
conjugation and rejoining the DNA afterwards. While some mobilization proteins have also been
shown to be crucial for its plasmid’s replication, pBBR1 has been successfully employed without
Mob in the pentose and hexose-consuming bacterium, Paraburkholderia sacchari LMG 19450
LFM101. The Mob gene from the plasmid pBBR1 shares a very similar amino acid sequence with
the mobilization protein from promiscuous streptococcal plasmid pMV158, whose nicking
behavior was severely impaired by replacing the catalytic histidine and other active site residues.
Using an analogous approach, the effect of the active site mutations on plasmid relaxation and
subsequent maintenance in R. palustris, P. sacchari, and the symbiotic Bradyrhizobium sp.
ORS278 will be presented. This study provides important groundwork for harnessing the
extraordinary biochemical capabilities of non-model bacteria.