Improvement of α-Amylase Production by Modulation of Ribosomal Component Protein S12 in Bacillus subtilis 168

Abstract

The capacity of ribosomal modification to improve antibiotic production by Streptomyces spp. has already been demonstrated. Here we show that introduction of mutations that produce streptomycin resistance (str) also enhances ␣-amylase (and protease) production by a strain of Bacillus subtilis as estimated by measuring the enzyme activity. The str mutations are point mutations within rpsL, the gene encoding the ribosomal protein S12. In vivo as well as in vitro poly(U)-directed cell-free translation systems showed that among the various rpsL mutations K56R (which corresponds to position 42 in E. coli) was particularly effective at enhancing ␣-amylase production. Cells harboring the K56R mutant ribosome exhibited enhanced translational activity during the stationary phase of cell growth. In addition, the K56R mutant ribosome exhibited increased 70S complex stability in the presence of low Mg 2؉ concentrations. We therefore conclude that the observed increase in protein synthesis activity by the K56R mutant ribosome reflects increased stability of the 70S complex and is responsible for the increase in ␣-amylase production seen in the affected strain.

Mutations in rpsL that confer streptomycin resistance show pleiotropic effects on virulence and the production of a carbapenem antibiotic in Erwinia carotovora

“…K43N, K43T and K43R mutations resulted in streptomycin resistance, but had no impact on antibiotic production in S. coelicolor (Okamoto-Hosoya et al, 2003a). A recent study by Kurosawa et al (2006) showed that amylase and protease production in Bacillus subtilis strain 168 was enhanced in rpsL K56R mutants (equivalent to K43R of E. coli or Ecc), but production was unaffected in K56T and K56N mutants. In a study by Björkman et al (1998), Salmonella rpsL mutations were shown to have an effect on virulence in a mouse model.…”

Identification and Characterization of a Novel Multidrug Resistance Operon,mdtRP(yusOP), ofBacillus subtilis

“…These findings indicated that bacterial gene expression can be altered dramatically by modulating ribosomal proteins and/or RNA polymerase (12,16). The rpsL mutant ribosomes carrying an amino acid substitution in S12, which confers a high level of resistance to streptomycin, are more stable than those of wild-type controls at low magnesium concentrations, indicating that this increase in stability could enhance protein synthesis at the late growth phase (11,20,31,33). We later found that increased expression of the translation factor ribosome recycling factor also contributes to the enhanced protein synthesis observed during the late growth phase in the rpsL K88E mutant of S. coelicolor.…”

Dramatic Activation of Antibiotic Production in Streptomyces coelicolor by Cumulative Drug Resistance Mutations

“…Thus, this method requires no induced mutagenesis. This approach has been used to enhance the production of salinomycin in an industrial strain of Streptomyces albus (33); to activate the synthesis of dormant antibiotics (15,19); to improve chemical tolerance in Pseudomonas putida (12), a valuable bacterium for waste processing; and to enhance the synthesis of enzymes such as ␣-amylase in Bacillus subtilis (22). Interestingly, the introduction of several drug resistance mutations had a cumulative effect on antibiotic production.…”