Cloning, sequence and expression of a linear plasmid-based and a chromosomal homolog of chloroacetaldehyde dehydrogenase-encoding genes in Xanthobacter autotrophicus GJ10

• Published in: Gene 1998-01, Vol.207 (1), p.9-18
• Main Authors: Bergeron, Hélène, Labbé, Diane, Turmel, Chantal, Lau, Peter C.K
• Format: Article
• Language: English
• Subjects: Acetaldehyde - analogs & derivatives; Acetaldehyde - metabolism; Aldehyde Dehydrogenase - genetics; Aldehyde dehydrogenase evolution; Amino Acid Sequence; Base Sequence; Biodegradation; Biodegradation, Environmental; Carboxy-Lyases - genetics; Chlorinated aliphatic hydrocarbon; Cloning, Molecular; Dehalogenase; Escherichia coli - genetics; Gene Expression; Genes, Bacterial; Gram-Negative Aerobic Rods and Cocci - genetics; Molecular Sequence Data; Plasmids; Promoter Regions, Genetic; Pulsed-field gel electrophoresis; σ 54-dependent promoter
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/S0378-1119(97)00598-2

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.