Performance of the pBHR1 mobilization protein MobV and its role in stable plasmid expression in <i>Rhodopseudomonas palustris</i> CGA009

ABSTRACT Mobilizable plasmids are typically used in metabolic engineering studies, especially for their small size, to express heterologous proteins in new host organisms to manipulate their metabolism. Rhodopseudomonas palustris is a non-model soil bacterium of interest that is well-known for its extensive metabolic versatility, being able to accumulate a wide range of industrially relevant bioproducts, such as polyhydroxybutyrate, n -butanol, hydrogen, and other lignin-derived compounds. However, many of these non-model organisms are more genetically recalcitrant, and the rules of genetic stability, or even plasmid stability, can change drastically from organism to organism. This study investigates the effects of pBHR1’s native mobilization protein, MobV, on the retention of pBBR1 origin plasmids in R. palustris , and the effects of supercoil regulation on both plasmid stability, as well as plasmid-based gene expression. Mobilization proteins participate in horizontal gene transfer between bacterial species. Through two functional assays, a relaxation and a conjugation assay, we determine that the relaxation mechanism is similar to a previously annotated mobilization protein, MobM, and confirm that R. palustris is able to participate in conjugation using its two native type IV secretion systems, respectively. Using flow cytometry, we determine that mutations to various homologous active sites deleteriously impact plasmid expression. Finally, through RT-qPCR, we also determine that the presence of the mobilization protein confers a large positive effect on copy number, where its absence reduces the copy number from 44.27 ± 2.00 copies per cell to 22.86 ± 0.63. IMPORTANCE Plasmid instability remains a major barrier to genetic engineering in non-model gram-negative bacteria, such as Rhodopseudomonas palustris . During previous efforts to optimize plasmid vectors for this species, we observed rapid post-transformation unstable plasmid-based expression when using a minimized pBHR1 backbone lacking the native mobilization protein MobV. Restoring MobV eliminated this instability, suggesting an uncharacterized role in plasmid maintenance. In this study, we systematically dissect the contribution of MobV to plasmid expression in R. palustris by performing site-directed mutagenesis for several histidine residues. By comparing MobV to the well-characterized relaxase MobM, and generating active-site mutants, we link specific catalytic residues to plasmid persistence, stable genetic expression, and MobV activity. These findings clarify a previously overlooked mechanism of plasmid maintenance in R. palustris and provide design principles for constructing stable, high-performing vectors in non-model gram-negative hosts. This work therefore supports more reliable metabolic engineering strategies in organisms of growing biotechnological interest.