β‐1.3.‐1.4‐Glucanase in spore‐forming microorganisms. VI. Genetic instability of β‐glucanase production in a high‐producer strain of Bacillus amyloliquefaciens grown in a chemostat

Abstract

A Bacillus amyloliquefaciens strain high-producing for 8-1.3-1.4-glucanase has gradually lost the ability to produce this enzyme during long-time continuous rultivation, independent of the culture conditions. Mutant strains isolated after long-term cultivation exhibited changed behaviour concerning extracellular enzyme formation and sporulation. By agarose gel electrophoresls of alkaline DNA extracts isolated from original and mutant strains we demonstrate that the observed pleiotropic phenomena are not caused by the loss of a complete plasmid present in the original strain. From extracts of both the original and mutant strains plasmid DNAs with approximately the same molecular weight of about 35 Mdal were isolated.Recently NOACK et al. (1980) and ROTH and NOACK (1981) showed that degeneration of Strepto.myces hygroscopicus with respect to its capacity to produce antibiotics during continuous culture is dependent on the chosen cultivation conditions. By comparison of the results obtained with the plasmid-containing strain E. coli GY 2364 pBR325 the authors proposed that an extrachromosoinal element may be involved in the control of product formation. On the basis of studies concerning a-amylase production by Bacillus subtilis in continuous culture it has been suggested that the factor responsible for production of this enzyme is also a plasmid (FENCL and PAZ-LAROVA 1980). This extrachromosoinal genetic determinant was present in highproductive clones only and could be further characterized as a 3.45 Mdal circular plasmid DNA with various target sites for restriction endonucleases (PAZLAROVA d nl. 1980). Another extracellular enzyme, endo-/3-1.3-1.4-glucanase, formed by several Bacilliis species (BORRISS et al. 1980) is important for use in brewing industry (BEUBLER et al. 1968). I n the present coininunication first results are reported concerning the stability of @-glucanase production by clones isolated after growth of a high-producing B. amyloliquefaciens strain in continuous culture. N a t e~i a l s and methodsA ,Q-ghicanase high-producing strain from B. aniyloliquefuciens ATCC 15 841 was used. The strain was obtained after treatment with nitrosoguanidine according to BALASSA (1969) and growth on rifaniycin (10 pg/ml)-containing agar. The strain was further characterized by resistance to streptomycin (0.5 mg/ml) and novobiocin (5 pglml).Liquid medium used for chemostat cultivation contained (amounts in g per 1) : Na,HPO, . The maximal growth rate in this medium was estimated as 0.9 h-1.