CONSTRUCTION AND CHARACTERIZATION OF A MUTANT OF ALKALIPHILIC BACILLUS-FIRMUS OF4 WITH A DISRUPTED CTA OPERON AND PURIFICATION OF A NOVEL CYTOCHROME BD

The caa(3)-type terminal oxidase of Bacillus firmus OF4 has been propo sed to play an important role in the growth and bioenergetics of this alkaliphile (A. A. Guffanti and T. A. Krulwich, J. Biol. Chem. 267:958 0-9588, 1992), A mutant strain was generated in which the cta operon e ncoding the oxidase was disrupted by insertion of a spectinomycin resi stance cassette, The mutant was unable to oxidize ascorbate in the pre sence of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), Absorption s pectra of membranes confirmed the loss of the enzyme and indicated the presence of a cytochrome bd-type terminal oxidase, The mutant could g row on glucose but was unable to grow on malate or other nonfermentati ve carbon sources, despite the presence of the cytochrome bd, The cyto chrome bd was purified from the mutant, The enzyme consisted of two su bunits and, with menadiol as substrate, consumed oxygen with a specifi c activity of 12 mu mol of O-2 . min(-1). mg(-1). In contrast to both cytochromes bd of Escherihia 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.