Isolation and characterization of a hydrogen peroxide resistant mutant of Bacillus subtilis

Abstract

A mutant of Bacillus subtilis has been isolated by continuous selection in increasing concentrations of H202. It grew with a doubling time of 85 min in minimal medium containing I 5 0 mM H202, whereas the wild-type parent lysed in 100 mM H20,. The mutant was also more resistant to organic peroxides than the wild-type. Further resistance to H202 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 H202 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.

Mutation of the Bacillus subtilis alkyl hydroperoxide reductase (ahpCF) operon reveals compensatory interactions among hydrogen peroxide stress genes

“…The recovered HindIII fragment overlaps the end of the gluconate operon (gntRKPZ [17]) and contains the complete ahpC gene and the 5Ј end (the first 136 codons) of ahpF. The predicted AhpC protein contains 187 amino acids and matches exactly the N-terminal sequence of a 23-kDa H 2 O 2 -inducible protein identified previously and postulated to be AhpC (18). The first 21 residues of the predicted AhpF protein are identical to the experimentally determined N-terminal sequence of a 54-kDa H 2 O 2 -inducible protein proposed to be AhpF (18).…”

“…The predicted AhpC protein contains 187 amino acids and matches exactly the N-terminal sequence of a 23-kDa H 2 O 2 -inducible protein identified previously and postulated to be AhpC (18). The first 21 residues of the predicted AhpF protein are identical to the experimentally determined N-terminal sequence of a 54-kDa H 2 O 2 -inducible protein proposed to be AhpF (18). The complete sequence of ahpF was inferred by combining the sequence of this HindIII fragment with that of a HindIII fragment sequenced by Zhang and Aronson (51) and was later verified when the complete sequence of this region of the chromosome was determined as part of the B. subtilis genome project (22) and by Antelmann et al (4).…”

“…MrgA, AhpC, AhpF, and KatA are overproduced in a spontaneous H 2 O 2 -resistant strain, MA991 (10,18). In an effort to isolate regulatory factors controlling the expression of mrgA in B. subtilis, we screened for mini-Tn10 insertions which led to elevated expression of mrgA under repressing conditions (rich medium).…”

Derepression of the Bacillus subtilis PerR Peroxide Stress Response Leads to Iron Deficiency

“…B. subtilis MA991 was previously isolated by serial passage into higher and higher levels of H 2 O 2 (35). This strain expresses elevated levels of several PerR regulon members, including KatA, AhpCF, and MrgA.…”

Regulation of inducible peroxide stress responses

“…The link between mrgA and the peroxide stimulon emerged from two findings: mrgA encodes a homologue of the E. coli peroxide-inducible DNA-binding protein, Dps (Almirón et al, 1992), and MrgA is a major peroxideinducible protein identified by amino-terminal sequencing (Hartford and Dowds, 1994). The PerR regulon, defined using both genome-scale searches for PerR binding sites (Per boxes) and transcriptional profiling, includes mrgA, the major vegetative catalase (katA), alkyl hydroperoxide reductase (ahpCF), the haem biosynthesis operon (hemAXCDBL), fur, perR and a zinc uptake system (zosA) Herbig and Helmann, 2002;Gaballa and Helmann, 2002).…”