PURIFICATION OF A CYTOCHROME BD TERMINAL OXIDASE ENCODED BY THE ESCHERICHIA-COLI APP LOCUS FROM A DELTA-CYO DELTA-CYD STRAIN COMPLEMENTED BY GENES FROM BACILLUS-FIRMUS OF4

Escherichia coli GK100, with deletions in the operons encoding its two terminal oxidases, cytochrome bo and cytochrome bd, was complemented for growth on succinate by a recombinant plasmid (pMS100) containing a 3,4-kb region of DNA from alkaliphilic Bacillus firmus OF4. The compl ementing DNA was predicted to encode five proteins, but neither sequen ce analysis nor complementation experiments with subclones provided in sight into the basis for the complementation. Cytochrome difference sp ectra of everted membrane vesicles from the transformed strain had cha racteristics of a cytochrome bd spectrum but with features different f rom those observed for alkaliphile membranes. To determine the bacteri al source and identity of the structural genes for the cytochrome bd i n the transformed mutant, the complex was extracted and partially puri fied. On sodium dodecyl sulfate-polyacrylamide gels, two polypeptides were resolved from the preparation, 43 (subunit I) and 27 (subunit II) kDa. An internal peptide from subunit I was sequenced, and it yielded the same primary sequence as is found in positions 496 to 510 of E. c oli appC. Consistent with the microsequencing results, pMS100 failed t o complement a triple mutant of E. coli carrying a deletion in appB as well as in the cyo and cyd loci. The deduced sequence of AppBC had be en predicted to be very similar to the sequence of CydAB (J. Dassa et al., Mol. Gen. Genet. 229:341-352, 1991), but this is the first demons tration that the former is indeed a cytochrome bd terminal oxidase. Th e enzyme catalyzed oxygen uptake coupled to quinol or N,N,N',N'-tetram ethyl-p-phenylenediamine oxidation, and the activity was sensitive to cyanide. No cross-reactivity to subunit specific polyclonal antibodies directed against the two individual subunits of cyd-encoded cytochrom e bd was detected. Since this is the second cytochrome bd discovered i n E. coli, it is proposed that the two complexes be designated cytochr ome bd-I (cydAB-encoded enzyme) and cytochrome bd-II (appBC-encoded en zyme). In addition, cbdAB is suggested as a more appropriate gene desi gnation for cytochrome bd than either appBC or cyxAB.