Targeted disruption of the Myxococcus xanthus orotidine 5'-monophosphate decarboxylase gene: effects on growth and fruiting-body development

Abstract

The Myxococcus xanthus gene coding for orotidine 5'-monophosphate (OMP) decarboxylase (EC 4.1.1.23) was cloned. The M. xanthus uraA gene efficiently complemented an Escherichia coli OMP decarboxylase mutant, permitting it to grow in the absence of uracil. Electroporation of M. xanthus with a circular plasmid carrying a selectable uraA::kan gene disruption resulted in homologous recombination at the chromosomal uraA locus. Chromosomal integration of the gene disruption plasmid created heterozygous (uraA+/uraA::kan) tandem duplications. These tandem duplications were unstable and segregated auxotrophic uraA::kan daughters at frequencies of 2 x 10(-4) to 8 x 10(-4) per viable cell. Rare uraA::kan segregants were easily obtained by selecting for resistance to the toxic analog 5-fluoroorotic acid. Our experiments suggest that the cloned uraA gene could facilitate the use of gene duplications in the genetic analysis of M. xanthus development. The uraA mutants could utilize uracil, uridine, or uridine 5'-phosphate for growth, indicating that M. xanthus has pyrimidine salvage pathways. During multicellular development, uraA::kan gene disruption mutants sporulated to wild-type levels but formed smaller and more numerous aggregates than did their uraA+ parent, regardless of whether uracil was added to the medium. Pyrimidine deprivation of uraA mutants, under conditions that otherwise supported vegetative growth, failed to induce fruiting-body development or sporulation.