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Isolation and characterization of a hydrogen peroxide resistant mutant of Bacillus subtilis
1Department of Biology, St Patrick's College, Maynooth, Co. Kildare, Ireland.
Abstract:
A mutant of Bacillus subtilis has been isolated by continuous selection in increasing concentrations of H2O2. It grew with a doubling time of 85 min in minimal medium containing 150 mM H2O2, whereas the wild-type parent lysed in 100 mM H2O2. The mutant was also more resistant to organic peroxides than the wild-type. Further resistance to H2O2 could not be induced by pretreatment with low concentrations of the oxidant. The mutant synthesized a number of proteins at a much higher rate than the wild-type, including constitutive synthesis of all of the proteins which were induced by H2O2 in the wild-type. Four of these proteins were sequenced; three were identified as catalase and two subunits of alkyl hydroperoxide reductase. Two proteins whose synthesis was repressed in the mutant were sequenced, and one was identified as flagellin. The mutant grew as non-flagellated, partially septate, filaments of cells, and fragments of flagella were seen in the surrounding medium.
Insights
Researchers developed a Bacillus subtilis mutant resistant to hydrogen peroxide (H2O2) and organic peroxides. This H2O2-resistant mutant constitutively synthesizes protective enzymes like catalase and alkyl hydroperoxide reductase.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Bacillus subtilis is a model organism for studying bacterial physiology.
- Oxidative stress from hydrogen peroxide (H2O2) can damage cellular components.
- Developing resistance to oxidants is crucial for bacterial survival.
Purpose of the Study:
- To isolate and characterize a Bacillus subtilis mutant with enhanced resistance to hydrogen peroxide (H2O2).
- To investigate the molecular mechanisms underlying H2O2 resistance in the isolated mutant.
Main Methods:
- Continuous selection of Bacillus subtilis in increasing concentrations of H2O2.
- Comparative growth analysis of wild-type and mutant strains in H2O2-containing media.
- Proteomic analysis (protein sequencing) to identify differentially expressed proteins.
Main Results:
- A mutant exhibiting significant resistance to H2O2 (150 mM) and organic peroxides was isolated.
- The mutant displayed constitutive synthesis of H2O2-inducible proteins, including catalase and alkyl hydroperoxide reductase subunits.
- Repressed synthesis of flagellin was observed, correlating with non-flagellated, filamentous growth.
Conclusions:
- The isolated Bacillus subtilis mutant demonstrates robust resistance to oxidative stress.
- Constitutive expression of key oxidative stress response proteins contributes to mutant resistance.
- Altered cell morphology and reduced flagellation are associated with the H2O2-resistant phenotype.