Isolation of recombination-defective and UV-sensitive mutants of Bacillus megaterium

English, Joey; Vary, Patricia S.

doi:10.1128/jb.165.1.155-160.1986

Cited by 29 publications

(17 citation statements)

References 24 publications

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“…Thus, we looked for recA and its function in DNA repair in Bacillus megaterium. Interestingly, this biotechnologically relevant species (Vary, 1994) has been reported to resist DNA damage much more efficiently than B. subtilis (English & Vary, 1986). As a result of our work, B. megaterium -unlike several other Bacillus species -was found to harbour two recA genes, both being damage-inducible and functional in DNA repair.…”

Section: Introductionmentioning

confidence: 53%

Evidence for two recA genes mediating DNA repair in Bacillus megaterium

2005

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Isolation and subsequent knockout of a recA-homologous gene in Bacillus megaterium DSM 319 resulted in a mutant displaying increased sensitivity to mitomycin C. However, this mutant did not exhibit UV hypersensitivity, a finding which eventually led to identification of a second functional recA gene. Evidence for recA duplicates was also obtained for two other B. megaterium strains. In agreement with potential DinR boxes located within their promoter regions, expression of both genes (recA1 and recA2) was found to be damage-inducible. Transcription from the recA2 promoter was significantly higher than that of recA1. Since a recA2 knockout could not be achieved, functional complementation studies were performed in Escherichia coli. Heterologous expression in a RecA null mutant resulted in increased survival after UV irradiation and mitomycin C treatment, proving both recA gene products to be functional in DNA repair. Thus, there is evidence for an SOS-like pathway in B. megaterium that differs from that of Bacillus subtilis.

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Section: Introductionmentioning

confidence: 53%

Evidence for two recA genes mediating DNA repair in Bacillus megaterium

2005

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“…After 24-48 h incubation, colonies were replica plated to minimal medium and LB-MLS medium to determine cotransduction frequency. Mutagenesis by nitrosoguanidine and by UV irradiation have been described previously (English & Vary, 1986).…”

Section: Methodsmentioning

confidence: 99%

“…After 12-24 h further incubation, transformants could be observed. Media, procedures for preparing lysates, and transductions with phage MP 13 have been described previously (English & Vary, 1986) except that 6 g KH2P0, 1-I was also added to the minimal glucose medium. Transductants appearing on minimal plates were picked to minimal medium with inducing Em.…”

Section: Methodsmentioning

confidence: 99%

“…Luria-Bertani (LB) medium (Levine. 1957) was used in transposition experiments, and S N B medium (English & Vary, 1986) was used for routine growth, sporulation, and preparation of phage lysates. Antibiotic concentrations were 10 pg tetracycline (Tc) ml-I, 0.15 pg erythromycin (Em) ml-I for induction, 5 pg erythromycin ml-l and 250 pg lincomycin (MLS) ml-' for transposon selection, and 3.5 pg chloramphenicol (Cm) ml-l for selection of fusions.…”

Section: Methodsmentioning

confidence: 99%

“…In the last few years, our laboratory has developed genetic systems for B. megaterium and has mapped over 45 genes into linkage groups covering approximately 70% of the chromosome (English & Vary, 1986;Sussman et al, 1988;Vary, 1979;Vary & Tao, 1988; S. L. Palm & P. S. Vary, unpublished). Although the sporulation process has been studied extensively in this species (Chatelain, 1975;Ellar et al, 1967Ellar et al, , 1975Greene et al, 1971), only a few spo mutants have been reported.…”

Section: Introductionmentioning

confidence: 99%

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Isolation and characterization of sporulation lacZ fusion mutants of Bacillus megaterium

Tao

Vary

1991

Journal of General Microbiology

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A derivative of Bacillus megaterium QM B1551 cured of all seven resident plasmids was mutated to Lac-. Transposon Tn917 carrying lac2 and cat was then used to isolate four spo: : fucZ-cat fusion mutants by screening for colonies expressing the fusion in stationary phase. The sporulation frequencies of the mutants ranged from lo-' to Macrolide, lincosamide and streptogramin B resistance (MLS') of the mutants cotransduced 100% with the sporulation defect and all four mutations mapped near trp by transduction in the order: trp-hisH-spo. All four mutants isolated were defective early in sporulation since P-galactosidase could be detected between zero and 2 h after the end of exponential growth. Electron microscopy of two of the mutants expressing the enzyme at t,-t2 revealed a defect prior to or just at the beginning of septum formation in one, and after completion of septum formation in the other. Little or no synthesis of dipicolinic acid, glucose dehydrogenase or alkaline phosphatase was detected in the mutants, but each was neutral protease positive. These results show that the mutations were in at least two early genes expressed before glucose dehydrogenase production. This study represents the first genetic characterization of sporulation mutants in B. megaterium and also demonstrates that gene fusion technology can be used in this species.

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Bacillus Megateriumand Other Bacilli: Industrial Applications

Bunk

Biedendieck

Jahn

et al. 2010

Encyclopedia of Industrial Biotechnology

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Strains of the genus Bacillus are Gram‐positive microorganisms. Many of them, such as Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus amyloliquefaciens, Brevibacillus brevis , and Bacillus clausii offer several advantages in industrial applications. They all have a very efficient protein secretion system, grow on several different and cheap carbon sources, lack endotoxins, and are nonpathogenic. Proteins produced from and with the bacilli are of great industrial importance. Next to B. subtilis , B. megaterium has attracted the most attention in recent years. A toolbox for recombinant protein production, secretion, and purification has been developed that is based on various vectors with different origins of replication, selection markers, promoters, affinity tag sequences, and sequences for a signal peptide. The number of proteins recombinantly produced by B. megaterium has increased rapidly. The genomes of two B. megaterium strains namely QM B1551, which naturally contains seven different plasmids but also has a plasmidless derivative, and DSM319, a naturally plasmidless strain, have now been completely sequenced. Systems biotechnology investigations using transcriptome, proteome, and metabolome analyses in combination with bioinformatics‐based modeling approaches have already started.

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Isolation of recombination-defective and UV-sensitive mutants of Bacillus megaterium

Cited by 29 publications

References 24 publications

Evidence for two recA genes mediating DNA repair in Bacillus megaterium

Evidence for two recA genes mediating DNA repair in Bacillus megaterium

Isolation and characterization of sporulation lacZ fusion mutants of Bacillus megaterium

Bacillus Megateriumand Other Bacilli: Industrial Applications

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