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Mutants that had a genetic lesion increasing the production of a-amylase and protease simultaneously were isolated from a transformable strain of Bacillus subtilis Marburg by N-methyl-N'-nitro-N-nitrosoguanidine treatment. These mutants produced two to three times more a-amylase and five to 16 times more protease than their parent and were tentatively referred to as AP mutants. As this mutation seems to have occurred at a single gene of the bacterial chromosome and was not located near the a-amylase structural gene, the gene was designated as "pap." When pap-and amyR2 (an a-amylase regulator gene) or pap-and ProH coexisted in the same cell, synergistic effects of the two genetic characters were observed on the a-amylase and protease production, respectively. Upon introduction of the pap mutation, the following phenotypic changes were observed in addition to changes in a-amylase and protease productivity. (i) Mutants lost the character of competence for the transformation. (ii) When cells were cultured at 30 C for 30 h, mutant cells became filament owing to the formation of chains of cells. (iii) Autolysis of cells was decreased in the mutants. When pap was transferred to the wild strain by deoxyribonucleic acid-mediated transformation, the transformants showed all these phenotypic alterations simultaneously.

A mutant of Bacillus subtilis 6160 that had been isolated by its hyperproduction of a-amylase and protease lacked flagella and motility, and its content of autolytic enzyme(s) was reduced to one-third to one-fourth that of the parent. These phenotypic differences were completely co-transferred by the deoxyribonucleic acid (DNA) of the mutant when five DNA recipient strains of B. subtilis were transformed. The revertants, isolated by motility with a frequency of approximately 10-f, recovered a normal level of autolytic activity and showed reduced productivity of a-amylase and protease. This point mutation allowed normal flagellin synthesis, spore formation, and rate of growth. The comparison of cell envelope of the mutant with that of the parent indicated that there was no significant difference except loss of flagella. Therefore the association at the cell surface of a group of extracellular proteins consisting at least of a-amylase, proteases, flagellin, and autolytic enzyme(s) seem to be coordinately regulated by the gene or seem to be affected coordinately by certain undetected alterations of the cell envelope.Bacillus subtilis produces a variety of extracellular enzymes. For control of the production of extracellular a-amylase in B. subtilis, at least two kinds of genetic characters (amyR and pap) were revealed by Yamaguchi et al. (27,28), Yuki (33), Yoneda et al. (29,30), and Sekiguchi et al. (21). amyR regulated specifically the production of a-amylase, whereas pap affected the production of a-amylase and proteases, but not ribonuclease, simultaneously. B. subtilis YN9, one of the mutants isolated from a transformable strain of B. subtilis by N-methyl-N'nitro-N-nitrosoguanidine treatment, carries a pap mutation and simultaneously produced elevated amounts of a-amylase, alkaline protease, and neutral protease. However, the extent of the elevation was different for each enzyme. Furthermore, the mutant lost its transformability and was different in its cell shape, and the protein component of the membrane preparation was different from that of the parental strain (29). Young (32) and Tomasz and Spizizen (22) reported that the transformability and cell shape of B. subtilis and Pneumococcus were closely related to their autolytic activities.The production of extracellular enzymes seems to vary with alterations not only in environmental conditions but also in the structure and function of the cell envelope (8). Since the autolytic enzyme(s) is responsible for many functions such as the morphogenesis of the cell (5) and the turnover of the cell wall (16), it is quite plausible that the enzyme(s) affects the production of the extracellular enzymes.In this paper we show that the content of the autolytic enzyme(s) in the mutant YN9 is one-third to one-fourth that in the parent 6160.Furthermore, the mutant YN9 lacks flagella and motility, whereas the chemical nature of its cell envelope was similar to that of the parent 6160. The correlation among the clear and significant differences, i.e., the hyperproduction of ...

MATERIALS AND METHODS Bacterial strains. Strains of B. subtilis used in this study were derivatives of strain 168 of Burkholder and xiles (2). Strains 6160 (metB5 purB6 trp-160 amyEm+ amyRl) and 1-131 (met his aro-116 amyEm+ amyRl) were donated from Y. Ikeda and S. Yuki, respectively. Strain NA64 (metB5 purB6 410

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