Mutations conferring acid sensitivity in the acid-tolerant strains Rhizobium meliloti WSM419 and Rhizobium leguminosarum biovar viciae WSM710

The genetic basis of acid-tolerance in rhizobia is being investigated by the isolation and molecular characterisation of Tn 5-induced acid-sensitive mutants. Strains of Rhizobium meliloti (the most acid-sensitive species of rhizobia) respond to increasing concentrations of calcium in the growth medium both by growing faster and by being able to grow at progressively lower pH. Two classes of acid-sensitive mutant of R. meliloti WSM419 (a relatively acid-tolerant inoculant strain) have been isolated: those that are calcium repairable and those that are not. Tn5 and flanking rhizobial DNA containing sequences involved in pH tolerance have been cloned from five acid-sensitive mutants into pUC18 or pBR322. Restriction mapping of the fragments has shown that the genes involved in acid tolerance are located on four unique EcoRI fragments. A 0.7-kb EcoRI- SalI fragment located very close to the site of insertion of Tn5 was used as a probe to help map the act206 gene region. Attempts to clone the wild-type act206 gene from WSM419 have proved unsuccessful. Preliminary hybridization studies using the 0.7-kb EcoRI- SalI probe suggest that there are sequences homologous to the act206 region present in several species of bacteria. We are also investigating the genetics of acid-tolerance in R. leguminosarum WSM710 which grows at significantly lower pH than WSM419. Two Tn 5-induced acid-sensitive mutants have also been isolated from WSM710. The Tn 5 and flanking sequences from these mutants have been cloned into pBR322; the two genes are on separate EcoRI fragments.