Identification and characterization of Myxococcus xanthus mutants deficient in calcofluor white binding

The esg locus of Myxococcus xanthus was analyzed and shown to consist of two genetic complementation groups. In an attempt to characterize the defects in esg, it was found that the mutant bound the dye calcofluor white more poorly than wild-type cells. Unlike S-motility mutants that share this phenotype, esg exhibited S-motility. This led to the identification of nine new transposon insertion mutants, designated Cds (calcofluor white binding deficient, S-motile), which exhibited a phenotype similar to the esg strain. The Cds mutants were also found to be defective in cell-cell agglutination, polysaccharide production and developmental aggregation. The degree of polysaccharide deficiency in the Cds mutants correlated with the degree of loss of agglutination and dye binding as well as with the severity of the developmental aggregation defect. Extracellular matrix fibrils composed of roughly equal amounts of polysaccharide and protein have been shown to be involved in agglutination, and electron microscopic examination showed that esg and the other Cds mutants lack the wild-type level of fibrils. Preliminary genetic characterization demonstrated that the transposon insertion mutations in three of the Cds mutants (SR53, SR171, and SR200) were loosely linked.; In an attempt to further characterize the Cds phenotypes, it was found that SR171 (a Cds mutant) drastically reduced developmentally regulated tps gene expression similar to esg. Total fatty acid analysis revealed that SR171 is deficient in branched-chain fatty acids (BCFA). Addition of IVA to growing cells rescued fruiting body formation and increased sporulation and tps gene expression by several folds. SR171 differed from esg in pigmentation and sporulation. Extracellular complementation was observed when SR171 was mixed with wild-type and esg mutant. A 6kbp DNA fragment cloned from the wild-type cosmid library was able to partially complement genetically, the aggregation defect in SR171. Restriction analysis revealed that the SR171 locus was different from esg locus. The esg locus encodes the E1{dollar}alpha{dollar} and E1{dollar}beta{dollar} subunits of a branched-chain keto acid dehydrogenase (BCKAD) involved in the production of BCFA. The results suggest that SR171 locus appears to either encode for additional components of the BCKAD or components of a second enzyme involved in BCFA synthesis.