Rhizobium etli TAL182, a competitive strain for the nodulation of Phaseolus beans, occupied more than 99% of the nodules when co-inoculated in various proportions with Rhizobium TAL1145 or Rhizobium tropici CIAT899. Two overlapping cosmid clones, pUHR68 and pUHR69, containing genes for nodulation competitiveness from TAL182, were isolated by functional complementation of strain TAL1145. Using one of these cosmid clones, we constructed two Tn5-insertion mutants of TAL182 defective in nodulation competitiveness. The Tn5 insertions in both mutants were localized in identical positions within a 4.6-kb HindIII fragment. One mutant, RUH120, was complemented for nodulation competitiveness by this HindIII fragment. The cloned DNA in pUHR68 is a part of a plasmid, 150 MDa in size, in TAL182 and does not show homology with TAL1145 genomic DNA. The 4.6-kb HindIII fragment contains a gene(s) required for nodulation competitiveness on beans, which is present only in some R. etli strains and absent in other Rhizobium spp.
Rhizobium etli strain TAL182 is a competitive strain for effective nodulation of beans. From this strain, a novel gene was isolated, slp, which is 669 bp in size and required for nodulation competition on the common bean. The slp knockout mutant of TAL182 is defective in nodulation competition, shows reduced growth in the presence of 200 mM NaCl, KCl or LiCl and is complemented by the cloned slp gene. The deduced amino acid sequence of slp shows 66-72% similarity to stomatin proteins of Homo sapiens, Mus musculus and Caenorhabditis elegans. Expression of slp in Escherichia coli from a T7 promoter shows a 26 kDa protein which cross-reacts with human-stomatin-specific polyclonal antibody. Like the human stomatin protein, the slp-deduced protein, Slp, is very hydrophilic except for a single hydrophobic membrane-spanning domain. Among various bean-nodulating rhizobia, slp is present in R. etli, Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici type A strains but is absent in R. tropici type B strains. It is also absent in Bradyrhizobium and several other Rhizobium spp.
Rhizobium etli strain TAL182 and R. leguminosarum bv phaseoli strain 8002, both of which produce melanin pigment, were tested for their nodulation competitiveness on beans by paired inoculation with two strains which do not produce melanin: R. tropici strain CIAT899 and Rhizobium sp. strain TAL1145. An assay was developed to distinguish nodules formed by the melanin-producing and non-producing strains. Strain TAL182 had discrete competitive superiority over CIAT899 and TAL1145 for nodulation of beans. Nodulation competitiveness was not correlated with the ability to produce melanin pigment or the host range of the Rhizobium strains tested.
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