Plasmid-Encoded Catabolic Genes in Rhizobium leguminosarum bv. trifolii: Evidence for a Plant-Inducible Rhamnose Locus Involved in Competition for Nodulation

Abstract

Cosmids carrying genes involved in utilization of rhamnose, sorbitol, and adonitol were isolated from a genomic library of Rhizobium leguminosarum by complementation of plasmid-cured derivatives of strain Rlt100 that were unable to grow on these carbon sources. Transposon mutagenesis was used to identify regions of each cosmid necessary for catabolism of the respective carbon source; partial DNA sequencing, as well as analysis of gene fusions created with transposon Tn5-B20, helped to determine the orientation and possible function of genes required for growth on the three substrates. Representative Tn5 insertions in the cosmids were recombined into the wild-type strain Rlt100 by gene replacement to generate isogenic strains unable to use either rhamnose, sorbitol, or adonitol. These strains were tested for their nodulation competitiveness compared with Rlt100 in co-inoculation experiments on clover plants. While sorbitol and adonitol catabolic mutants were unaltered in their competitive behavior, the nodulation competitiveness of three different rhamnose utilization mutants was significantly impaired. This result, coupled with the fact that the rhamnose catabolic genes were inducible by clover root extracts, suggests an important role for rhamnose catabolism in the early stages of the interaction of R. leguminosarum with clover plants. Hybridization studies with probes derived from the rhamnose, sorbitol, and adonitol catabolic loci demonstrated that these genes are plasmid encoded in virtually all R. leguminosarum strains, including representatives from all three biovars from a variety of different geographic locations.