The Physiology and Symbiotic Characterisation of Paraburkholderia sprentiae WSM5005

Talitha H Rogers

Paraburkholderia sprentiae WSM5005 is a β-rhizobia isolated from nodules of the perennial legume, Lebeckia ambiguia, with both bacterial and legume partners native to the fynbos region of South Africa. Preliminary studies of this strain have shown its genome to consist of a chromosome, a second large replicon (presumed to be a second chromosome), three plasmids, and a diverse arrangement of nitrogen fixation (nif/fix) genes, which is a structure that differs markedly to genomes of well-studied α-rhizobia. Furthermore WSM5005, like other analysed β-rhizobia, lacks fixNOQP and fixGHIS, encoding the high-affinity terminal oxidase cytochrome cbb3, which is essential to N2 fixation in α-rhizobia symbioses. These findings present a clear knowledge gap in the physiology and genomic architecture of β-rhizobia, and proposed a paradigm shift in rhizobial research. This study aimed to characterise the physiology of WSM5005 in symbiosis with L. ambigua, to explore whether the genome architecture confers a functional role in symbiotic N2-fixation, and what respiratory mechanisms allow for N2-fixation in the absence of cytochrome cbb3. Bioinformatic analysis showed WSM5005 harbours a multipartite genome consisting of a primary chromosome, a chromid, and three accessory replicons (pPS1, pPS2, pPS3). Genome interrogation confirmed the absence of a cytochrome cbb3 complex, while alternative terminal oxidases cytochrome bo3, -bd (type I), and -bd (type II), were found distributed across the chromosome, chromid, pPS1, and pPS2. A large proportion of genes were found to be duplicated, including nif/fix genes. Essential genes were found to be enriched on the chromosome and chromid, while the distribution of nif/fix genes were predominantly confined to a 99% homologous ~40Kb repeat region, duplicated across the chromid and pPS2. RNAseq was used to probe functionality and expression of duplications in WSM5005 free-living cells and bacteroids inside legume root nodules, followed by comparative analyses of genes pertaining to N2-fixation, central metabolism, and microaerobic respiration. All duplicated copies of nif and fix genes were expressed in bacteroid and the induction of genes relating to central metabolism and nitrogen metabolism were suggestive of an alternate carbon flux to what has been previously observed in α-rhizobia. Mutational studies showed WSM5005 lacking a functional cytochrome bd continued to fix N2 at wildtype rates, however, free-living experiments demonstrated significant changes in cell survival. Together, the results presented demonstrate WSM5005 to be an effective, N2-fixing microsymbiont. While the functional role of the chromid remains unclear, expression of the symbiotic genome shows the strain to possess respiratory flexibility and redundancy, while suggesting additional pathways for carbon storage to compliment and enable this. Fully characterising the intrinsic link between carbon metabolism and the functional respiratory chains in WSM5005 bacteroids will, in turn, provide significant insight into N2-fixation in β-rhizobia.

The Physiology and Symbiotic Characterisation of Paraburkholderia sprentiae WSM5005

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