(27bk) Investigating the Relaxase Behavior and Replication Functionality of the Mobilization Protein Mobv in the Plasmid pBBR1

Plasmids are used in synthetic biology as a convenient method of employing heterologous genes in bacteria. Transforming non-model bacteria and measuring the expression of these heterologous genes, however, can be difficult or unreliable as plasmids can be lost across generations. Interestingly, the incorporation of the mobilization protein, MobV, onto BBR1 plasmids employed in Rhodopseudomonas palustris CGA009, has shown to be crucial to the bacterium’s ability to retain plasmids, possibly owing to its relaxase behavior. Mobilization proteins facilitate horizontal gene transfer between bacteria by nicking the DNA before conjugation and rejoining the DNA afterwards. The objective of this study is to probe the role of the mobilization protein MobV from the broad host range plasmid pBBR1 in R. palustris, an extremely metabolically versatile organism known for its capability of all four forms of metabolism as well as its ability to consume recalcitrant feedstocks such as lignin breakdown products. Specifically, this study explores if a tyrosine participates in MobV’s nicking mechanism, which is common among relaxases, or if pBBR1’s MobV belongs to a unique group that utilizes a histidine to cut the DNA. In-vitro relaxation assays of proteins with mutated potential active site residues and the plasmid will be employed to clarify this question. In addition, the effect of these mutations on plasmid copy number and the transcription of the plasmid’s replication protein will be examined through qPCR and fluorescent protein expression via flow cytometry. The significance of this study is increasing the understanding of the mechanisms by which the mobilization protein ensures stable plasmid maintenance, which is critical for harnessing this bacterium’s extraordinary biochemical capabilities.

Immethun, C. M., Kathol, M., Changa, T., & Saha, R. (2022). Synthetic Biology Tool Development Advances Predictable Gene Expression in the Metabolically Versatile Soil Bacterium Rhodopseudomonas palustris. Frontiers in Bioengineering and Biotechnology, 10. https://doi.org/10.3389/fbioe.2022.800734