Identification and molecular characterization of the Alcaligenes eutrophus H16 aco operon genes involved in acetoin catabolism

Abstract

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. ...

Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus

“…Proteins were separated under denaturating and nondenaturating conditions in polyacrylamide gels and stained with Coomassie brilliant blue as described previously (36). To stain gels for acetaldehyde dehydrogenase activity, the gels were incubated in the presence of NAD, p-nitroblue tetrazolium chloride, phenazine methosulfate, and acetaldehyde as described previously (21).…”

“…For the determination of the transcription start site, a nuclease protection assay was used. Hybridization and the S1 nuclease treatment were performed as described previously (36 RESULTS N-terminal sequence analysis of purified AcDH-II. The AcDH-II of A. eutrophus was purified from ethanol-grown cells of the type strain TF93 and from the ethanol-utilizing mutant MK1000 as described previously (21).…”

“…The dideoxychain termination method of Sanger et al (41) was applied with modifications described previously (36). Synthetic oligonucleotides were used as primers, and the primer-hopping strategy (46) was employed to determine the DNA sequence of both strands.…”

“…Plasmid DNA and DNA restriction fragments were isolated and characterized by standard methods according to references compiled in the previous study (36).…”

“…Recently, we have identified and localized the genes, which are essentially involved in the catabolism of acetoin in A. eutrophus, on five different genomic EcoRI restriction fragments (A, B, C, D, and E) (13). The structural genes for the a-and P-subunits of acetoin:2,6-dichlorophenolindophenol oxidoreductase (acoA and acoB, respectively); for the protein FMP (acoC), which seems to be a dihydrolipoamide acetyltransferase; and for a protein of yet unknown function (acoX) are probably organized in one single operon (acoXABC) which is localized on fragments A and C (36). Fragment B harbors an rpoN-like gene, and mutants carrying a TnS insertion in this gene exhibited an extremely pleiotropic phenotype (13,38).…”