Cloning, sequence and expression of a linear plasmid-based and a chromosomal homolog of chloroacetaldehyde dehydrogenase-encoding genes in Xanthobacter autotrophicus GJ10.
The degradation of 1,2-dichloroethane (DCE) by Xanthobacter autotrophicus GJ10 proceeds via chloroacetaldehyde (CAA), a toxic intermediate in the cells if it is not metabolized further by the NAD(+)-dependent CAA dehydrogenases. Here, we describe the cloning, sequence and expression in Escherichia coli of aldA, a plasmid-located CAA dehydrogenase-encoding gene of GJ10 as well as a chromosomal homolog, designated aldB. The DNA-predicted amino acid (aa) sequences of the two proteins (505 aa in AldA and 506 aa in AldB) are 84% identical. The cloned aldA and aldB genes were verified by their expression in the E. coli T7 polymerase/promoter and the pUC lac promoter systems. The expression level of AldA and its enzymatic activity towards CAA were both higher than those of AldB. In a hybrid construct, the 3'end of aldB was able to complement, although not completely, the corresponding portion of aldA to produce a functional gene. Both AldA and AldB proteins of GJ10 share the highest degree of sequence identity with an acetaldehyde dehydrogenase (ALDH) encoded by acoD of Alcaligenes eutrophus (77.3-78% identity). Together with at least three other ALDHs of prokaryotic origin, these proteins apparently form a special class of ALDHs whose expressions are dependent on RpoN factors. By pulsed-field gel electrophoresis the 225-kb pXAU1 plasmid encoding aldA was shown to be linear.
Bergeron
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Biotechnology Research Institute, National Research Council Canada, Montréal, Québec, Canada.
Labbé
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Turmel
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Lau
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PC
D000079:Acetaldehyde / Q000031:analogs & derivatives / Q000378:metabolism; D000444:Aldehyde Dehydrogenase / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001673:Biodegradation, Environmental; D002262:Carboxy-Lyases / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D016946:Gram-Negative Aerobic Rods and Cocci / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic
C004656:chloroacetaldehyde; D000444:Aldehyde Dehydrogenase; D002262:Carboxy-Lyases; C023746:acetolactate decarboxylase; D000079:Acetaldehyde