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 K-m values for acetaldehyde and other non-halogenated aldehydes (0.8-4 mu M), but much higher K-m values for chloroacetaldehyde (1.7 mM) and bromoacetaldehyde (10.5 mM), while V-max values were similar for halogenated and non-halogenated aldehydes. Cultures chat 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 K-m for bromoacetaldehyde and for inactivation of the enzyme by bromoacetaldehyde.