STATIONARY-PHASE MUTANTS OF SINORHIZOBIUM-MELILOTI ARE IMPAIRED IN STATIONARY-PHASE SURVIVAL OR IN RECOVERY TO LOGARITHMIC GROWTH

A screening method was used to identify Sinorhizobium meliloti mutants which are affected in stationary-phase survival, Of 20,000 individual colonies mutagenized with transposon Tn5-B20, 10 mutant strains which showed poor or no survival in the stationary phase were identified. A nalyses of expression patterns of the promoterless lacZ genes in the m utant strains revealed individual induction patterns, Most strains wer e induced in stationary phase as well as under carbon limitation and i n pure H2O, but none of the mutants was induced under heat, alkali str ess conditions, or low oxygen tension, Plant inoculation tests reveale d that the symbiotic proficiency of the mutants was not affected. Two mutants, however, showed gene induction not only in the stationary pha se under free-living conditions but also in the bacteroid state, A lon g-term starvation test was carried out to examine the ability of the 1 0 mutants to survive prolonged stationary-phase conditions, All mutant s showed a clear decrease in the colony-forming ability under the chos en experimental conditions, Staining with green and red fluorescent nu cleic acid stain showed that the mutants fell into two different class es, Seven mutants died during stationary phase; the three other mutant s remained viable but did not resume growth after prolonged starvation , Five of the ten Tn5-B20 insertions were cloned from the genomes of t he mutant strains. Nucleotide sequence analyses established that the t ransposon had inserted in five distinctive genes. Database searches re vealed that four of the tagged loci corresponded to already characteri zed genes whose gene products are involved in important cellular proce sses such as amino acid metabolism or aerobic respiration.