Cloning of the Alcaligenes eutrophus alcohol dehydrogenase gene.
Mutants of Alcaligenes 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 Tn5 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 Tn5-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 lambda L47 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) SalI 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 Tn5-mob in class 3 mutants mapped near the structural gene for alcohol dehydrogenase on the same 2.5-kb SalI fragment.
Kuhn
M
M
Institut für Mikrobiologie, Georg-August-Universität Göttingen, Federal Republic of Germany.
Jendrossek
D
D
Fründ
C
C
Steinbüchel
A
A
Schlegel
H G
HG
D000093:Acetoin / Q000378:metabolism; D000332:Aerobiosis; D000421:Alcaligenes / Q000201:enzymology / Q000235:genetics*; D000426:Alcohol Dehydrogenase / Q000235:genetics*; D002072:Butylene Glycols / Q000378:metabolism; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D005285:Fermentation; D005796:Genes; D005798:Genes, Bacterial; D009154:Mutation; D009693:Nucleic Acid Hybridization
D002072:Butylene Glycols; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; C026978:2,3-butylene glycol; D000093:Acetoin; D000426:Alcohol Dehydrogenase