title: Adaptation of Pseudomonas sp. GJ1 to 2-bromoethanol caused by overexpression of an NAD-dependent aldehyde dehydrogenase with low affinity for halogenated aldehydes.
abstract: Pseudomonas sp. GJ1 is able to grow with 2-chloroethanol as the sole carbon and energy source, but not with 2-bromoethanol, which is toxic at low concentrations (1 mM). A mutant that could grow on 2-bromoethanol with a growth rate of 0.034 h-1 at concentrations up to 5 mM was isolated and designated strain GJ1M9. Measurement of enzyme activities showed that mutant and wild-type strains contained a PMS-linked alcohol dehydrogenase that was active with halogenated alcohols and that was threefold overexpressed in the mutant when grown on 2-chloroethanol, but only slightly overproduced when grown on 2-bromoethanol. Both strains also contained an NAD-dependent alcohol dehydrogenase that had no activity with halogenated alcohols. Haloacetate dehalogenase levels were similar in the wild-type and the mutant. Activities of NAD-dependent aldehyde dehydrogenase were only slightly higher in extracts of the mutant grown with 2-bromoethanol than in those of the wild-type grown with 2-chloroethanol. SDS-PAGE, however, showed that this enzyme amounted to more than 50% of the total cellular protein in extracts of the mutant from 2-bromoethanol-grown cells, which was fourfold higher than in extracts of the wild-type strain grown on 2-chloroethanol. The enzyme was purified and shown to be a tetrameric protein consisting of subunits of 55 kDa. The enzyme had low Km values for acetaldehyde and other non-halogenated aldehydes (0.8-4 microM), but much higher Km values for chloroacetaldehyde (1.7 mM) and bromoacetaldehyde (10.5 mM), while V(max) values were similar for halogenated and non-halogenated aldehydes. Cultures that were pregrown on 2-chloroethanol rapidly lost aldehyde dehydrogenase activity after addition of 2-bromoethanol and chloroamphenicol, which indicates that bromoacetaldehyde inactivates the enzyme. To achieve growth with 2-bromoethanol, the high expression of the enzyme thus appears to be necessary in order to compensate for the high Km for bromoacetaldehyde and for inactivation of the enzyme of bromoacetaldehyde.
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
authors.forename: J R
authors.initials: JR
authors.lastname: Van der Ploeg
authors.forename: J
authors.initials: J
authors.lastname: Kingma
authors.forename: E J
authors.initials: EJ
authors.lastname: De Vries
authors.forename: J G
authors.initials: JG
authors.lastname: Van der Ven
authors.forename: D B
authors.initials: DB
authors.lastname: Janssen
mesh_terms: D000079:Acetaldehyde / Q000031:analogs & derivatives / Q000494:pharmacology; D000222:Adaptation, Physiological; D000421:Alcaligenes / Q000201:enzymology / Q000235:genetics; D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism; D000595:Amino Acid Sequence; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000431:Ethanol / Q000031:analogs & derivatives* / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014734:Vibrio cholerae / Q000201:enzymology / Q000235:genetics
chemical_list: D000447:Aldehydes; D004791:Enzyme Inhibitors; C038235:bromoacetaldehyde; D000431:Ethanol; D000444:Aldehyde Dehydrogenase; D000079:Acetaldehyde; C025475:ethylene bromohydrin