Browsing by Author "Wathugala, Nandun Dulmini"

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    Conjugative Complexities: Defining the requirements for the transfer of Rhizobium leguminosarum plasmid pRleVF39b
    (2019-03-20) Wathugala, Nandun Dulmini; Hynes, Michael F.; Turner, Raymond Joseph; Harrison, Joe J.
    The genome of Rhizobium leguminosarum is compartmentalized into a chromosome and multiple large (> 100 kb) plasmids. Four conjugation systems have been identified that can mediate horizontal transfer of these plasmids, and type IVA-1 was identified on R. leguminosarum VF39SM plasmid pRleVF39b. This category of system is found among multiple Rhizobiaceae members from different geographical locations, on both non-symbiotic and symbiotic plasmids. The type IVA-1 conjugation system on pRleVF39b is distinctive due to the presence of a shorter relaxase gene (traA) compared to canonical relaxases, a repressor, TrbR, belonging to the xenobiotic response element (Xre)-family, and the lack of some common genes found in the other conjugation systems. There are 15 annotated Orfs adjacent to known transfer genes in the type IV conjugation locus that might have important functions in plasmid transfer. A series of deletions in each of the hypothetical genes was created to identify essential and facilitating genes for the transfer of pRleVF39b. The effect of these mutations on transfer of pRleVF39b to plasmid-free Agrobacterium recipients was analyzed. orf23, orf24, orf26, orf27, orf28 and orf29 mutants reduced the plasmid transfer frequency by 10-fold or more compared to wild type, suggesting a key role in conjugation. To define the TrbR regulon of pRleVF39b, putative promoter (P) regions of orf17, trbR, orf24, orf25, orf26 and orf29 were fused to a reporter gene gusA. The expression of promoters in WT VF39, VF39a-, VF39b-, trbR mutant and an orf29 mutant was analyzed using β-glucuronidase assays. PtrbR expression was not reduced due to trbR mutation. Thus, TrbR does not autoregulate itself. P24 showed ~30-fold increase and P25 showed ~10-fold increase in expression in VF39b- and trbR backgrounds, indicating that the operons of orf24 and orf25 are part of the TrbR regulon. These results were validated using qRT-PCR experiments. It was also identified that orf16 and orf17 are not part of the TrbR regulon. The absence of conjugative plasmid pRleVF39a in donors negatively affected plasmid transfer but this result was contradicted by qRT-PCR experiments that showed higher levels of mRNA transcripts for orf23, orf24, orf26, orf27 and orf28 in a pRleVF39a cured background.