In genetic studies on the catabolism of acetoin in Alcaligenes eutrophus, we used TnS::mob-induced mutants which were impaired in the utilization of acetoin as the sole carbon source for growth. The transposonharboring EcoRI restriction fragments from 17 acetoin-negative and slow-growing mutants (class 2a) and from six pleiotropic mutants of A. eutrophus, which were acetoin-negative and did not grow chemolithoautotrophically (class 2b), were cloned from pHC79 gene banks. The insertions of TnS were mapped on four different chromosomal EcoRI restriction fragments (A, C, D, and E) in class 2a mutants. The native DNA fragments were cloned from a XL47 or from a cosmid gene bank. Evidence is provided that fragments A (21 kilobase pairs [kb]) and C (7.7 kb) are closely linked in the genome; the insertions of TnS covered a region of approximately 5 kb. Physiological experiments revealed that this region encodes for acetoin:dichlorophenol-indophenol oxidoreductase, a fast-migrating protein, and probably for one additional protein that is as yet unknown. In mutants which were not completely impaired in growth on acetoin but which grew much slower and after a prolonged lag phase, fragments D (7.2 kb) and E (8.1 kb) were inactivated by insertion of TnS::mob. No structural gene could be assigned to the D or E fragments. In class 2b mutants, insertions of Tn5 were mapped on fragment B (11.3 kb). This fragment complemented pleiotropic hno mutants in trans; these mutants were impaired in the formation of a rpoN-like protein. The expression of the gene cluster on fragments A and C seemed to be rpoN dependent.
Mutants of Akcaligenes eutrophus which are altered with respect to the utilization of 2,3-butanediol and acetoin were isolated after transposon mutagenesis. The suicide vehicle pSUP5011 was used to introduce the drug resistance transposable element TnS into A. eutrophus. Kanamycin-resistant transconjugants of the 2,3-butanediol-utilizing parent strains CF10141 and AS141 were screened for mutants impaired in the utilization of 2,3-butanediol or acetoin. Eleven mutants were negative for 2,3-butanediol but positive for acetoin; they were unable to synthesize active fermentative alcohol dehydrogenase protein (class 1). Forty mutants were negative for 2,3-butanediol and for acetoin (class 2). Tn5-mob was also introduced into a Smr derivative of the 2,3-butanediol-nonutilizing parent strain H16. Of about 35,000 transconjugants, 2 were able to grow on 2,3-butanediol. Both mutants synthesized the fermentative alcohol dehydrogenase constitutively (class 3). The TnS-labeled EcoRI fragments of genomic DNA of four class 1 and two class 3 mutants were cloned from a cosmid library. They were biotinylated and used as probes for the detection of the corresponding wild-type fragments in a AL47 and a cosmid gene bank. The gene which encodes the fermentative alcohol dehydrogenase in A. eutrophus was cloned and localized to a 2.5-kilobase (kb) Sall fragment which is located within a 11.5-kb EcoRI-fragment. The gene was heterologously expressed in A. eutrophus JMP222 and in Pseudomonas oxalaticus. The insertion of TnS-mob in class 3 mutants mapped near the structural gene for alcohol dehydrogenase on the same 2.5-kb Sall fragment.Recently, the ability of strictly respiratory bacteria to form fermentation enzymes under conditions of restricted oxygen supply was demonstrated (2, 28, 37). A whole physiological group of bacteria, the aerobic chemolithoautotrophic bacteria, relies only on respiratory energy generation, and not a single member has been reported to be able to grow on organic compounds anaerobically on the basis of fermentative energy generation. Since fermentation enzymes were not expected to be present in these bacteria, the question of the metabolic role of these enzymes and the significance of these cryptic genes in strict aerobes was raised. The hydrogen-oxidizing bacterium Alcaligenes eutrophus serves as a model for our study on enzyme derepression in strictly respiratory bacteria under conditions of oxygen deficiency.In A. eutrophus an NAD-dependent lactate and an NAD(P)-dependent alcohol dehydrogenase appear to function as a safety valve for the release of excess reducing power in the absence of exogenous hydrogen acceptors such as oxygen or nitrate (38-41). A. eutrophus is even able to evolve molecular hydrogen (43) and to synthesize poly-,-hydroxybutyric acid (29) to dispose of the reductant if a suitable electron acceptor is lacking. The cytoplasmic, NAD-reducing hydrogenase is responsible for the evolution of hydrogen (19).The fermentative alcohol dehydrogenase is a tetramer of relative molecular mass 156,000 an...
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