Metabolism and host specificity in the Rhizobium leguminosarum species complex

Forming symbiosis with legumes and fixing nitrogen, rhizobia can be ranked among the most ecologically significant bacteria in the world. Two features of rhizobia viz. metabolism and host specificity are important determinants of their growth and their ability to form symbiosis with plants. Investigations of these two features are common in studies conducted using laboratory based experimental and computer based bioinformatics methods.

Rhizobium is the largest genus of root-nodulating bacteria. Hence, abundant data can be collected by investigating members of this group. 72 isolates of root-nodule bacteria were collected from a field adjacent to Wentworth College, University of York from plants of genus Vicia and Trifolium; 36 from each. Their metabolic assays showed diversity. Analysis of genome data revealed the presence of five contiguous genes in isolates from Vicia, as well as in the reference strain Rlv. 3841; but absent in the isolates from Trifolium. Limited information was available on the five genes.

Work in this thesis promotes our understanding of R. leguminosarum genotype and phenotype. Cross nodulation assays showed that all biovar viciae strains retained their ability to form symbiosis plants of genera Vicia, Pisum and Lathyrus. Metabolic study and celC phylogeny showed that clusters of different species - type strains of R. pisi and R. fabae, and type strains of R. phaseoli and R. etli were closely related to each other. Studies on distribution of the five biovar-specific genes showed that they were widely distributed in biovar viciae strains with one exception – the type strain of R. fabae did not have any of five bvs genes, which suggested these bvs genes were not essential for nodulating Vicia plants. Single gene mutation of the five genes in Rlv. 3841 and subsequent competition nodulation tests suggested that inactivation of the bvs4 and bvs5 genes could lead to decreased competitiveness in mutants as compared to wild type on Vicia sativa, Vicia faba and Pisum sativum plants. Complementation of bvs5 gene restored competitiveness. Enzyme assays for the two genes confirmed the activities described in their annotation viz. aliphatic nitrilase for bvs4 and sulfite oxidase for bvs5. Mutation of bvs4 results in a loss of bvs5¬-encoded enzyme activity, indicating that these two genes are probably in an operon.

In conclusion, the study shows that relations among rhizobial strains and species can be observed by studying bacterial metabolism. The work hints at a novel competitive mechanism that requires further investigation.