Browsing by Author "Hiniduma Gama Achchi, Kasuni Maheshika Hemananda"

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    Genomic and Proteomic Approaches to Understanding Conjugation in Rhizobium leguminosarum VF39
    (2023-03-02) Hiniduma Gama Achchi, Kasuni Maheshika Hemananda; Hynes, Michael; Chua, Gordon; Fraser, Marie; Schryvers, Anthony; Torres Tejerizo, Gonzalo
    The root nodule bacterium Rhizobium leguminosarum strain VF39 contains six large single copy plasmids and a single circular chromosome. These six plasmids are named pRleVF39a to pRleVF39f in order of increasing size. pRleVF39a and pRleVF39b are self-transmissible plasmids. pRleVF39b has a type IVa rhizobial conjugation system and is characterized by presence of a shorter relaxase gene (traA) than other systems. It also has a gene encoding a negative transcriptional regulator (trbR) belonging to the Xre family and shows a lack of some common genes found in the other conjugation systems. The transfer region of pRleVF39b contains 15 hypothetical protein encoding genes with no known function. It was hypothesized that some of these proteins interact with TraA to form a relaxase complex. Thus, TraA protein-protein interactions were analysed by performing a pull-down assay followed by LC-MS/MS identification. The analysis has shown TraA interaction with the proteins encoded in trb region of pRleVF39b, and some other chromosomally encoded proteins with known TraA interaction. These interactions appear to be largely dependent on the presence of the coupling protein, TraG. Transposon sequencing experiments were performed to identify any additional genes potentially involved in pRleVF39a and pRleVF39b plasmid transfer. The results revealed 67 genes potentially involved in pRleVF39b plasmid transfer and 53 genes putatively involved in pRleVF39a plasmid transfer. The previously known but not well-studied repressor (traM) and activator (traR) in the pRleVF39a conjugation system and hyp1 and hyp2 genes located immediately downstream of traR were identified as potential regulators of pRleVF39a plasmid transfer. The pRleVF39b gene list contains 23 genes identified from previous studies and 44 genes newly identified from this work. The effect of the mutation of traG, encoding the coupling protein, on transfer of pRleVF39b to plasmid-free Agrobacterium recipients was analyzed. A traG marked mutant reduced the plasmid transfer frequency by ~200 fold compared to the wild type. Transfer frequency of the pRleVF39b plasmid was zero when the traG marked mutant was made in a pRleVF39a cured background, suggesting that residual transfer by traG mutants made in the wild type background might be a result of cointegrates between pRleVF39a and pRleVF39b.