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Novel approach for improving the productivity of antibiotic-producing strains by inducing combined resistant mutations.

ABSTRACT: We developed a novel approach for improving the production of antibiotic from Streptomyces coelicolor A3(2) by inducing combined drug-resistant mutations. Mutants with enhanced (1.6- to 3-fold-higher) actinorhodin production were detected at a high frequency (5 to 10%) among isolates resistant to streptomycin (Str(r)), gentamicin (Gen(r)), or rifampin (Rif(r)), which developed spontaneously on agar plates which contained one of the three drugs. Construction of double mutants (str gen and str rif) by introducing gentamicin or rifampin resistance into an str mutant resulted in further increased (1.7- to 2.5-fold-higher) actinorhodin productivity. Likewise, triple mutants (str gen rif) thus constructed were found to have an even greater ability for producing the antibiotic, eventually generating a mutant able to produce 48 times more actinorhodin than the wild-type strain. Analysis of str mutants revealed that a point mutation occurred within the rpsL gene, which encodes the ribosomal protein S12. rif mutants were found to have a point mutation in the rpoB gene, which encodes the beta-subunit of RNA polymerase. Mutation points in gen mutants still remain unknown. These single, double, and triple mutants displayed in hierarchical order a remarkable increase in the production of ActII-ORF4, a pathway-specific regulatory protein, as determined by Western blotting analysis. This reflects the same hierarchical order observed for the increase in actinorhodin production. The superior ability of the triple mutants was demonstrated by physiological analyses under various cultural conditions. We conclude that by inducing combined drug-resistant mutations we can continuously increase the production of antibiotic in a stepwise manner. This new breeding approach could be especially effective for initially improving the production of antibiotics from wild-type strains.

SUBMITTER: Hu H

PROVIDER: S-EPMC92810 | biostudies-literature | 2001 Apr

REPOSITORIES: biostudies-literature

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Novel approach for improving the productivity of antibiotic-producing strains by inducing combined resistant mutations.

Hu H H Ochi K K

Applied and environmental microbiology 20010401 4

We developed a novel approach for improving the production of antibiotic from Streptomyces coelicolor A3(2) by inducing combined drug-resistant mutations. Mutants with enhanced (1.6- to 3-fold-higher) actinorhodin production were detected at a high frequency (5 to 10%) among isolates resistant to streptomycin (Str(r)), gentamicin (Gen(r)), or rifampin (Rif(r)), which developed spontaneously on agar plates which contained one of the three drugs. Construction of double mutants (str gen and str rif ...[more]

PMID: 11282646

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Project description:AimTo investigate the resistance of Helicobacter pylori (H. pylori) to ciprofloxacin (CIP), levofloxacin (LVX) and moxifloxacin (MOX) in the Beijing area and to elucidate the resistance mechanisms.MethodsSeventy-nine H. pylori clinical strains, isolated from patients who had undergone upper gastrointestinal endoscopy in Peking University First Hospital from 2007 to 2009, were tested for their susceptibility to CIP, LVX and MOX using the E-test method. H. pylori strain 26695 was included in the susceptibility testing as a control strain. According to the minimal inhibitory concentration (MIC) values, a strain was classified as resistant to CIP, LVX or MOX when the MIC was > 1 microg/mL. We amplified by polymerase chain reaction (PCR) and sequenced the quinolone resistance-determining regions of the gyrA and gyrB genes from 29 quinolone-resistant and 16 quinolone-susceptible H. pylori strains selected at random.ResultsIn this study, the resistance rates of H. pylori to CIP, LVX or MOX were 55.7% (44/79), and the primary resistance rates were 26.6% (21/79). Patients with secondary resistance had received LVX in previous eradication treatments, but not MOX or CIP. Forty-five strains, including 29 CIP, LVX or MOX-resistant strains (MIC: 1.5-32 microg/mL) and 16 susceptible strains, were selected randomly from the 79 strains and used in PCR analysis. Among these 45 strains, 27 resistant strains had mutations in the gyrA gene, including 11 strains with mutations corresponding to Asp-91 (MIC: 2-32 microg/mL), one of which also had a mutation corresponding to Val-150, and 16 strains had mutations at Asn-87 (MIC: 4-32 microg/mL), three of which also had mutations corresponding to Arg-140 or Val-150. In addition, Arg-140, Val-150 or Ala-97 mutations were separately detected in three susceptible strains. Analysis of the gyrB gene showed that one strain of low resistance had a mutation corresponding to Ser-457 that coexisted with an Asp-91 mutation. There was a significant difference in the occurrence of mutations in the gyrA gene between CIP, LVX and MOX-resistant and -susceptible strains (P < 0.05), but 2 resistant strains were found to possess no quinolone resistance-determining region mutations.ConclusionResistance is primarily mediated through point mutations in gyrA. Whether other mechanisms are responsible for resistance in strains without mutations in the QRDR should be detected.

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Novel approach for improving the productivity of antibiotic-producing strains by inducing combined resistant mutations.

Publications

Novel approach for improving the productivity of antibiotic-producing strains by inducing combined resistant mutations.

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