Mapping and Properties of bgl ( -Glucanase) Mutants of Bacillus subtilis

Summary: Among secreted proteins of Bacillus subtilis 168 four polypeptides, separated by electrophoresis in polyacrylamide gradient gels under denaturing conditions, were shown to have lichenanhydrolysing enzyme activity; one of them, a 22 kDa protein, represents the main β-glucanase activity. A number of N-methyl-N-nitro-N-nitrosoguanidine-induced mutants defective in β-glucanase activity at an elevated temperature (44°C) were isolated and characterized. All mutants analysed by PAGE had no active 22 kDa β-glucanase in their supernatants and showed no cross-reactivity with antibodies raised against purified 22 kDa β-glucanase. However, formation of other exoenzymes, including the other minor β-glucanases, was not affected in these mutant strains. The mutations (bgl-1, bgl-12, bgl-35, bgl-101) were mapped by phage PBS1-mediated transduction and found to be located between purA and sacA, close to the hutH marker of the B. subtilis chromosome. A 3.8 kb EcoRI fragment of the B. subtilis chromosome which directs β-glucanase synthesis in Escherichia coli as well as complementing a β-glucanase deficient mutant, bgl-35, of B. subtilis to synthesize active 22 kDa β-glucanase was mapped after homologous recombination by means of an integratable plasmid. The cointegrated chloramphenicol-resistance marker of the plasmid was found at the same map position as the mutations bgl-1, bgl-12, bgl-35 and bgl-101, suggesting that the gene affected encodes for the 22 kDa β-glucanase and lies within the order sacA–sacA–bgl–purA.