Acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) and the fast-migrating protein (FMP) were purified to homogeneity from crude ex tracts of acetoin-grown cells of Alcaligenes eutrophus. Ao:DCPIP OR consisted of a and , subunits (Mrs, 35,5 00 and 36,000, respectively), and a tetrameric a2,12 structure was most likely for the native protein. The mole cular weight of FMP subunits was 39,000. The N-terminal amino acid sequences of the three proteins were det ermined, and oligonucleotides were synthesized on the basis of the codon usage of A. eutrophus. With these, the structural genes for the a and ,1 subunits of Ao:DCPIP OR and FMP, which were referred to as acoA, aco)B, and acoC, respectively, were localized on one single EcoRI restriction fragment which has been clonei recently (C. Frund, H. Priefert, A. Steinbuchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). The nucleotide sequences of a 5.3-kbp region of this fragment and one adjacent fragment were determined, an d the structural genes for acoA (1,002 bp), acoB (1,017 bp), and acoC (1,125 bp) were identified. Together woith the gene acoX, whose function is still unknown and which is represented by a 1,080-bp open reading fr ame, these genes are probably organized in one single operon (acoXABC). The transcription start site was identified 27 bp upstream of acoX; this site was preceded by a region which exhibited complete homology to the enterobacterial a54-dependent promoter consensus sequence.The amino acid sequences deduced from aco. 4 and acoB for the a subunit (Mr,35,243) and the 0 subunit (Mr, 35,788) exhibited significant homologies to th e primary structures of the dehydrogenase components of various 2-oxo acid dehydrogenase complexes, whei 'eas those deduced from acoC for FMP (Mr, 38,941) revealed homology to the dihydrolipoamide acetyltrarw,ferase of Escherichia coli. The occurrence of a new enzyme type for the degradation of acetoin is discussed.Alcaligenes eutrophus has been studied with respe ct to the degradation of acetoin. Acetoin-grown cells of A. eb rtrophus are devoid of 2,3-butanediol dehydrogenase and acetoin dehydrogenase (diacetyl forming; EC 1.1.1.5), and mutants which lack 2,3-butanediol dehydrogenase (73) are stilil able to utilize acetoin as the sole carbon source for grovvth (72). Therefore, this bacterium lacks the 2,3-butanedi oil cycle which was described by Juni and Heym (36) (23,59). As insertional inactivation of this gene caused a pleiotropic effect and as these mutants were unable to synthesize proteins which are essential for degradation of acetoin, it was concluded that expression of the corresponding genes is rpoN dependent (23).Physiological studies localized the genes for Ao:DCPIP OR and FMP on restriction fragments A and C, which are closely linked in the genome (23), whereas the gene for acetaldehyde dehydrogenase II was localized on restriction fragment D (52a). The present study was aimed at isolation of FMP and Ao:DCPIP OR and identification and characterization of their structural genes. ...