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

Abstract

Calcofluor white is a fluorescent dye that binds to glycans and can be used to detect extracellular polysaccharide in Myxococcus xanthus and many other bacteria. We observed that an esg mutant showed less binding to calcofluor white than wild-type cells. Unlike S-motility mutants that share this phenotypic characteristic, the esg mutant exhibited S motility. This led us to identify a collection of nine new transposon insertion mutants, designated Cds (for calcofluor white binding deficient and S motile), which exhibited a phenotype similar to that of the esg strain. The Cds phenotype was found in 0.6% of the random insertion mutants that were screened. The Cds mutants were also found to be defective in cell-cell agglutination and developmental aggregation. 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. Analysis of total M. xanthus carbohydrate demonstrated that polysaccharide content increased by about 50% when wild-type cells entered stationary phase. This induction was reduced or eliminated in all of the Cds mutants. 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. Preliminary genetic characterization demonstrated that the transposon insertion mutations in three of the Cds mutants (SR53, SR171, and SR200) were loosely linked. The results of this study suggest that many genes are involved in the production of calcofluor white binding polysaccharide material found in the extracellular matrix and that the polysaccharide is fibrillar. These results are also consistent with the findings of earlier studies which indicated that fibrils function to join agglutinating cells and to form multicellular fruiting aggregates.The myxobacteria display a wide range of social adaptations during their life cycle (13). These social behaviors, which include coordinated movements and multicellular development, are being intensively studied in the gram-negative soil bacterium Myxococcus xanthus. Multicellular behaviors in M. xanthus involve thousands of cells and are dependent on gliding motility and cell-cell contact-mediated interactions (31). The most notable multicellular behavior and the defining characteristic of the myxobacteria is fruiting body formation (development). Under conditions of high cell density and nutrient depletion on a solid surface, vegetative cells move toward discrete foci and form multicellular mounds that develop into fruiting bodies (31). The individual rod-shaped cells are converted to spherical, environmentally resistant myxospores within the fruiting body. It is believed that M. xanthus requires at least five extracellular signals (A, B, C, D, and E signals) to coordinate this multicellular developmental process (13).Movement on a solid surface by M....

Inorganic polyphosphate in the social life of Myxococcus xanthus : Motility, development, and predation

“…Biogenesis of EPS involves genes eps and eas (35), a DnaK homolog (encoded by sglK) (36), chemotaxis homologues (dif ) genes (37), and some uncharacterized dsp genes. EPS-deficient mutants fail to bind calcofluor white dye (38) and are defective in cellular agglutination, S-motility, and fruiting body formation (25,26), phenotypes similar to those in the PPK1 mutant. Especially notable is that the S-motility of M. xanthus resembles the twitching motility of P. aeruginosa, which also depends on PPK1, mutants of which produce less alginate, a major component of P. aeruginosa EPS (K. Ishige and A.K., unpublished data).…”

Transposon Insertions ofmagellan-4That Impair Social Gliding Motility inMyxococcus xanthus

“…Four of these 68 mutants are unable to form fruiting bodies, a phenotype of many S motility mutants. Also, they do not bind calcofluor white, which is an indicator dye for EPS production (Ramaswamy et al 1997). The integrated plasmids in three of these four integrative disruption mutants were found to flank a 37-kb region of the M. xanthus genome, which they designated the ''eps'' region.…”

Myxococcus xanthus dif Genes Are Required for Biogenesis of Cell Surface Fibrils Essential for Social Gliding Motility

“…FACS analysis was performed with a Coulter Epics Elite ESP flow cytometer. The carbohydrate moiety of fibrils was examined by the binding of calcofluor white as previously described (26). Physical examination of fibrils was performed by scanning electron microscopy (SEM) of M. xanthus cells deposited on glass chips as previously described (5).…”

“…M. xanthus fibrils consist of approximately equal amounts of proteins and carbohydrates (4). The presence of fibrils correlates well with the specific carbohydrate content and phosphoenolpyruvate carboxykinase activity (22,26). Recent observations with new techniques indicate that the presence of fibrils on M. xanthus is probably more extensive than previously thought (22).…”

“…M. xanthus fibrils are thought to be peritrichous, filamentous structures 10 to 30 nm in diameter and can be many times the length of the cells (13,22). They have been observed to link neighboring cells or to link cells to the substratum they glide over (2,5,26). The fibril-defective dsp mutants are deficient in S motility (2,3,29).…”