Construction and characterization of a mutant of alkaliphilic Bacillus firmus OF4 with a disrupted cta operon and purification of a novel cytochrome bd.
The caa3-type terminal oxidase of Bacillus firmus OF4 has been proposed to play an important role in the growth and bioenergetics of this alkaliphile (A. A. Guffanti and T. A. Krulwich, J. Biol. Chem. 267:9580-9588, 1992). A mutant strain was generated in which the cta operon encoding the oxidase was disrupted by insertion of a spectinomycin resistance cassette. The mutant was unable to oxidize ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). Absorption spectra of membranes confirmed the loss of the enzyme and indicated the presence of a cytochrome bd-type terminal oxidase. The mutant could grow on glucose but was unable to grow on malate or other nonfermentative carbon sources, despite the presence of the cytochrome bd. The cytochrome bd was purified from the mutant. The enzyme consisted of two subunits and, with menadiol as substrate, consumed oxygen with a specific activity of 12 micromol of O2 x min(-1) x mg(-1). In contrast to both cytochromes bd of Escherichia coli, the enzyme did not utilize TMPD as an electron source. A number of additional features, including subunit size and spectral properties, distinguish this cytochrome bd from its counterparts in E. coli and Azotobacter vinelandii.
Gilmour
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Department of Biochemistry, Mount Sinai School of Medicine of CUNY, New York, New York 10029, USA.
Krulwich
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TA
D000468:Alkalies; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D003573:Cytochrome b Group; D003580:Cytochromes / Q000302:isolation & purification*; D004579:Electron Transport; D045222:Electron Transport Chain Complex Proteins; D003576:Electron Transport Complex IV / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008566:Membranes / Q000737:chemistry; D016254:Mutagenesis, Insertional; D009876:Operon; D010088:Oxidoreductases / Q000302:isolation & purification*; D010101:Oxygen Consumption; D018892:Proton-Motive Force; D013053:Spectrophotometry
D000468:Alkalies; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli; D003576:Electron Transport Complex IV