Rifampin-resistant mutants of Myxococcus xanthus defective in development

Abstract

Rifampin, an antibiotic which is known to bind to and inhibit RNA polymerase, was used to probe the molecular regulation of development in Myxococcus xanthus. Rifampin-resistant mutants were screened for defects in fruiting-body formation. About 20% of the isolates in the initial screenings showed major defects in developmental aggregation or sporulation. Eleven independent mutants with wild-type growth rates and stable phenotypes were analyzed by transduction. In these strains, the rifampin-resistant and nonfruiting phenotypes showed cotransduction frequencies equal to or greater than 99.0 to 99.9%. The RNA polymerase activities were resistant to rifampin in vitro, indicating that the RNA polymerase is altered in these strains. Although their fruiting phenotypes are heterogeneous, these strains can be divided into two classes based on the level of aggregation. The results suggest that RNA polymerase plays a significant role in the regulation of development in M. xanthus since mutations which cause no apparent changes in vegetative growth result in striking defects in fruiting-body formation.Myxococcus xanthus is a gram-negative bacterium which grows vegetatively in soils on decaying organic material, on the bark of living trees, or by preying upon other microorganisms (7). When nutrients are depleted from a solid culture medium, cells aggregate to form mounds. Within the mounds, the rod-shaped vegetative cells convert to round or ovoid spores. Mounds of mature myxospores are referred to as fruiting bodies,We have recently investigated protein synthesis during aggregation and fruiting-body formation in M. xanthus and have found that many proteins showed significant changes in their amounts and patterns of synthesis during development (M. Inouye, S. Inouye, apd D. R. Zusman, Dev. Biol., in press). These results suggest a complex but precise program of gene expression. It is likely that the synthesis of at least some of these proteins is regulated at the transcriptional level. To study this possibility, we searched for developmental mutants with defects in RNA polymerase. RNA polymerase mutations are easily obtained in many bacteria by selecting for resistance to the antibiotic rifampin (6,10,11,15), a specific inhibitor which is known to bind to the ,B subunit of RNA polymerase (11,14). We found that a significant class of these rifampin-resistant mutants in M. xanthus were, in fact, defective in fruiting-body formation; in at least 11 of these mutants the two phenotypes are probably caused by a single mutation. The heterogeneous terminal phenotypes observed in these mutants suggests that transcriptional spec-ificities may be altered in several different ways during development. MATERIALS AND METHODSBacterial and bacteriophage strains. Three different strains that are "wild type" for fruiting-body formation were used: DZ2 (5), DZF1 (FB) obtained from D. Kaiser, who obtained it from M. Dworkin, and DZF6 (4). DZ1 (18), a nonfruiting strain, was used as a phage indicator. The phage strain MX4ts27htfhfrm (4) was used ...

Unique developmental characteristics of the swarm and short cells of Proteus vulgaris and Proteus mirabilis

“…Respiration rates for short and swarm cells of P. vulgaris responsible for the changes in the pattern of enzyme activities and outer membrane protein composition is supported by the evidence that a fraction of rifampinresistant mutants of either P. vulgaris or P. mirabilis are incapable of swarming (data not shown). Similar evidence has been found and correlated with changes in DNA-dependent RNA polymerase in M. xanthus(21) and B. subtilis…”

RNA polymerase of Myxococcus xanthus: purification and selective transcription in vitro with bacteriophage templates

“…Bacterial and bacteriophage strains. RNA polymerases were purified from M. xanthus strain DZF1(FB) (34) and E. coli BG156 (obtained from Michael Chamberlin). The bacteriophage T7 deletion mutant AD111, which lacks promoters A2 and A3 (14), was obtained from Michael Chamberlin.…”

Mutation of rpoB Shifts the Nutrient Threshold Triggering Myxococcus Multicellular Development

“…Specifically, we show that the resulting S534L substitution in RpoB greatly increases the nutrient threshold necessary to prevent development from proceeding ( Figure 1 ). Mutation of both RpoB and RpoD components of the transcription machinery has previously been found to cause fundamental defects in M. xanthus development ( Rudd and Zusman, 1979 ; Davis et al, 1995 ), whereas the S534L substitution in RpoB is found to alter what environmental conditions induce development without causing defects in fruiting body formation and viable spore production. This same mutation is found to also exert complex pleiotropy, impacting multiple other traits.…”