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 P-glucanase activity. A number of N-methyl-N'-nitro-N-nitrosoguanidine-induced mutants defective in P-glucanase activity at an elevated temperature (44 "C) were isolated and characterized. All mutants analysed by PAGE had no active 22 kDa P-glucanase in their supernatants and showed no cross-reactivity with antibodies raised against purified 22 kDa P-glucanase. However, formation of other exoenzymes, including the other minor P-glucanases, was not affected in these mutant strains. The mutations (bgl-2, bgl-22, bgl-35, bgl-202) were mapped by phage PBSlmediated transduction and found to be located betweenpurA 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 P-glucanase synthesis in Escherichia coli as well as complementing a P-glucanase deficient mutant, bgl-35, of B. subtilis to synthesize active 22 kDa P-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-2, bgl-12, bgl-35 and bgl-202, suggesting that the gene affected encodes for the 22 kDa /I-glucanase and lies within the order ctrA-sacA-bgl-purA . Abbreviarion: NTG, N-methyl-N-nitro-N-nitrosoguanidine. 0001-2803 0 1986 SGM enzyme. Agricultural and Biological Chemistry 40. Electrophoretic transfer of proteins from polyacryl-577-586.amide gels to nitrocellulose sheets: procedure and TAKAHASHI, J . (1963). Transducing phages for Bacillus some applications.