Summary R-prime plasmids were constructed from a derivative of
Rhizobium strain NGR234 (ANU280) and were shown to contain overlapping genomic DNA segments involved in biosynthesis of exopolysaccharides (EPS). The R-primes originally constructed carried the mutant allele from Tn
5-induced EPS-deficient (Exo
–) mutant ANU2811. This plasmid-located mutant allele was dominant to the corresponding wild-type allele as merodiploid strains were Exo
–. Exo
+ revertants occurred at a low rate (1×10
-7) and these were shown to result from double reciprocal recombination events, which led to the isolation of R-prime plasmids carrying functional wild-type
exo alleles. R-prime plasmids that carry overlapping segments of DNA from parental strain ANU280 complemented 28 of the 30 group 2 Exo
– mutants of strain ANU280. Complementation of these Exo
– mutants also restored their symbiotic abilities of effective nodulation. Subsequent in vivo recombination between the wild-type alleles located on the R-prime and the corresponding mutated allele on the genome, was used to generate a new family of R-primes, which carried mutations in the
exo genes. The 30 group 2 Exo
– mutants were classified into 7 distinct genetic groups based upon complementation and physical mapping data. Five of the seven
exo loci were gentically linked and located on a 15-kb region of DNA. Mutations at two loci were dominant only when the mutations were R-prime plasmid-located while a mutation at a second locus was
cis-dominant to two other
exo loci. At least five genes involved in the synthesis of acidic exopolysaccharide synthesis have been identified.
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