Mutation of Bacillus subtilis causing hyperproduction of alpha-amylase and protease, and its synergistic effect

Abstract

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.

Coproduction of several exoenzymes in Bacillus subtilis

“…The strain YY88 secretes high levels of a-amylase, alkaline and neutral protease because of two regulatory mutations pap and amy R2 [4]. From about 180 000 colonies tested, 114 were selected as a-amylase or protease-negative mutants by the absence of halos on nutrient agar plates containing 1% starch or 1% casein.…”

“…Yoneda and Maruo [4] have reported a mutation in B. subtilis to cause hyperproduction of a-amylase, neutral and alkaline protease. The pleiotropic effect is mediated through the single pap mutation.…”

Serine protease subtilisin stimulates levansucrase synthesis in Bacillus subtilis Marburg

“…it cannot be excluded that non-growing cells are less sensitive to subtilisin addition than growing cells. It must be pointed out that sacU h mutations enhance a-amylase synthesis only two-or three-fold while increasing levansucrase synthesis ten-fold (constitutive strains or 30-fold (inducible strains) [1,8,10] and as shown in this paper maximal stimulation of levansucrase synthesis of sacU + strains by subtilisin addition never reached the level of synthesis of sacU h strains. One may consider that subtilisin mimics the true effector poorly and that only concentrations of subtilisin higher than 1 mg/ml could virtually transform sacU + strain to perfect sacU h phenocopies.…”

“…Several laboratories have described pleiotropic mutants that overproduce exocellular proteases, a-amylase and levansucrase and show altered cell envelope properties such as low transformability, lack of flagella and low autolytic activities [1,[6][7][8][9]. The relevant mutations, pap, sacU h and amyB were mapped at the same locus and must affect the same gene [10].…”

“…One must also explain why subtilisin does not modify other phenotypic traits, such as c~-amylase production, which are affected by sacU h mutations. It is possible that sacU h mutations affect levansucrase and a-amylase production via two different mechanisms but other explanations can be proposed: levansucrase is synthesized during exponential growth (1) and a-amylase when growth has stopped [8,10], so. it cannot be excluded that non-growing cells are less sensitive to subtilisin addition than growing cells.…”