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Biochemical and genetic analyses of a catalase from the anaerobic bacterium Bacteroides fragilis.

ABSTRACT: A single catalase enzyme was produced by the anaerobic bacterium Bacteroides fragilis when cultures at late log phase were shifted to aerobic conditions. In anaerobic conditions, catalase activity was detected in stationary-phase cultures, indicating that not only oxygen exposure but also starvation may affect the production of this antioxidant enzyme. The purified enzyme showed a peroxidatic activity when pyrogallol was used as an electron donor. It is a hemoprotein containing one heme molecule per holomer and has an estimated molecular weight of 124,000 to 130,000. The catalase gene was cloned by screening a B. fragilis library for complementation of catalase activity in an Escherichia coli catalase mutant (katE katG) strain. The cloned gene, designated katB, encoded a catalase enzyme with electrophoretic mobility identical to that of the purified protein from the B. fragilis parental strain. The nucleotide sequence of katB revealed a 1,461-bp open reading frame for a protein with 486 amino acids and a predicted molecular weight of 55,905. This result was very close to the 60,000 Da determined by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified catalase and indicates that the native enzyme is composed of two identical subunits. The N-terminal amino acid sequence of the purified catalase obtained by Edman degradation confirmed that it is a product of katB. The amino acid sequence of KatB showed high similarity to Haemophilus influenzae HktE (71.6% identity, 66% nucleotide identity), as well as to gram-positive bacterial and mammalian catalases. No similarities to bacterial catalase-peroxidase-type enzymes were found. The active-site residues, proximal and distal hemebinding ligands, and NADPH-binding residues of the bovine liver catalase-type enzyme were highly conserved in B. fragilis KatB.

SUBMITTER: Rocha ER

PROVIDER: S-EPMC177000 | biostudies-other | 1995 Jun

REPOSITORIES: biostudies-other

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Biochemical and genetic analyses of a catalase from the anaerobic bacterium Bacteroides fragilis.

Rocha E R ER Smith C J CJ

Journal of bacteriology 19950601 11

A single catalase enzyme was produced by the anaerobic bacterium Bacteroides fragilis when cultures at late log phase were shifted to aerobic conditions. In anaerobic conditions, catalase activity was detected in stationary-phase cultures, indicating that not only oxygen exposure but also starvation may affect the production of this antioxidant enzyme. The purified enzyme showed a peroxidatic activity when pyrogallol was used as an electron donor. It is a hemoprotein containing one heme molecule ...[more]

PMID: 7768808

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Project description:In this study, we show that Bacteroides species utilize Fe(III)-xenosiderophores as the only source of exogenous iron to support growth under iron-limiting conditions in vitro anaerobically. Bacteroides fragilis was the only species able to utilize Fe(III)-ferrichrome while Bacteroides vulgatus ATCC 8482 and Bacteroides thetaiotaomicron VPI 5482 were able to utilize both Fe(III)-enterobactin and Fe(III)-salmochelin S4 as the only source of iron in a dose-dependent manner. We have investigated the way B. fragilis assimilates Fe(III)-ferrichrome as initial model to understand the utilization of xenosiderophores in anaerobes. B. fragilis contains two outer membrane TonB-dependent transporters (TBDTs), FchA1 and FchA2, which are homologues to Escherichia coli ferrichrome transporter FhuA. The disruption of fchA1 gene had only partial growth defect on Fe(III)-ferrichrome while the fchA2 mutant had no growth defect compared to the parent strain. The genetic complementation of fchA1 gene restored growth to parent strain levels indicating that it plays a role in Fe(III)-ferrichrome assimilation though we cannot rule out some functional overlap in transport systems as B. fragilis contains abundant TBDTs whose functions are yet not understood. However, the growth of B. fragilis on Fe(III)-ferrichrome was abolished in a feoAB mutant indicating that Fe(III)-ferrichrome transported into the periplasmic space was reduced in the periplasm releasing ferrous iron prior to transport through the FeoAB transport system. Moreover, the release of iron from the ferrichrome may be linked to the thiol redox system as the trxB deletion mutant was also unable to grow in the presence of Fe(III)-ferrichrome. The genetic complementation of feoAB and trxB mutants completely restored growth on Fe(III)-ferrichrome. Taken together, these findings show that Bacteroides species have developed mechanisms to utilize ferric iron bound to xenosiderophores under anaerobic growth conditions though the regulation and role in the biology of Bacteroides in the anaerobic intestinal environment remain to be understood.

Dataset Information

Biochemical and genetic analyses of a catalase from the anaerobic bacterium Bacteroides fragilis.

Publications

Biochemical and genetic analyses of a catalase from the anaerobic bacterium Bacteroides fragilis.

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