Adaptation of Pseudomonas sp. GJ1 to 2-bromoethanol caused by overexpression of an NAD-dependent aldehyde dehydrogenase with low affinity for halogenated aldehydes.
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.
Van der Ploeg
J R
JR
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
Kingma
J
J
De Vries
E J
EJ
Van der Ven
J G
JG
Janssen
D B
DB
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
D000447:Aldehydes; D004791:Enzyme Inhibitors; C038235:bromoacetaldehyde; D000431:Ethanol; D000444:Aldehyde Dehydrogenase; D000079:Acetaldehyde; C025475:ethylene bromohydrin