Isolation and characterization of sporulation lacZ fusion mutants of Bacillus megaterium

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Abstract
Summary: A derivative of Bacillus megaterium QM B1551 cured of all seven resident plasmids was mutated to Lac−. Transposon Tn917 carrying lacZ and cat was then used to isolate four spo::lacZ-cat fusion mutants by screening for colonies expressing the fusion in stationary phase. The sporulation frequencies of the mutants ranged from 10−7 to 10−2. Macrolide, lincosamide and streptogramin B resistance (MLSr) 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 β-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 t1-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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