Roles of horizontal gene transfer and gene integration in evolution of 1,3-dichloropropene- and 1,2-dibromoethane-degradative pathways

The haloalkane-degrading bacteria Rhodococcus rhodochrous NCIMB13064, Pseud omonas pavonaceae 170, and Mycobacterium sp. strain GP1 share a highly cons erved haloalkane dehalogenase gene (dhaA). Here, we describe the extent of the conserved dhaA segments in these three phylogenetically distinct bacter ia and an analysis of their flanking sequences. The dhaA gene of the 1-chlo robutane-degrading strain NCIMB13064 was found to reside within a 1-chlorob utane catabolic gene cluster, which also encodes a putative invertase (invA ), a regulatory protein (dhaR), an alcohol dehydrogenase (adhA), and an ald ehyde dehydrogenase (aldA). The latter two enzymes may catalyze the oxidati ve conversion of n-butanol, the hydrolytic product of 1-chlorobutane, to n- butyric acid, a growth substrate for many bacteria. The activity of the dha R gene product was analyzed in Pseudomonas sp. strain GJ1, in which it appe ared to function as a repressor of dhaA expression. The 1,2-dibromoethane-d egrading strain GP1 contained a conserved DNA segment of 2.7 kb, which incl uded dhaR, dha4, and part of invA. A 12-nucleotide deletion in dhaR led to constitutive expression of dhaA in strain GP1, in contrast to the inducible expression of dhaA in strain NCIMB13064. The 1,3-dichloropropene-degrading strain 170 possessed a conserved DNA segment of 1.3 kb harboring little mo re than the coding region of the dhaA gene. In strains 170 and GP1, a putat ive integrase gene was found next to the conserved dhaA segment, which sugg ests that integration events were responsible for the acquisition of these DNA segments. The data indicate that horizontal gene transfer and integrase -dependent gene acquisition were the key mechanisms for the evolution of ca tabolic pathways for the man-made chemicals 1,3-dichloropropene and 1,2-dib romoethane.