A New Catabolic Plasmid in Xanthobacter and Starkeya spp. from a 1,2-Dichloroethane-Contaminated Site

Abstract

ABSTRACT1,2-Dichloroethane (DCA) is a problematic xenobiotic groundwater pollutant. Bacteria are capable of biodegrading DCA, but the evolution of such bacteria is not well understood. In particular, the mechanisms by which bacteria acquire the key dehalogenase genes dhlA and dhlB have not been well defined. In this study, the genomic context of dhlA and dhlB was determined in three aerobic DCA-degrading bacteria (Starkeya novella strain EL1, Xanthobacter autotrophicus strain EL4, and Xanthobacter flavus strain EL8) isolated from a groundwater treatment plant (GTP). A haloalkane dehalogenase gene (dhlA) identical to the canonical dhlA gene from Xanthobacter sp. strain GJ10 was present in all three isolates, and, in each case, the dhlA gene was carried on a variant of a 37-kb circular plasmid, which was named pDCA. Sequence analysis of the repA replication initiator gene indicated that pDCA was a member of the pTAR plasmid family, related to catabolic plasmids from the Alphaproteobacteria, which enable growth on aromatics, dimethylformamide, and tartrate. Genes for plasmid replication, mobilization, and stabilization were identified, along with two insertion sequences (ISXa1 and ISPme1) which were likely to have mobilized dhlA and dhlB and played a role in the evolution of aerobic DCA-degrading bacteria. Two haloacid dehalogenase genes (dhlB1 and dhlB2) were detected in the GTP isolates; dhlB1 was most likely chromosomal and was similar to the canonical dhlB gene from strain GJ10, while dhlB2 was carried on pDCA and was not closely related to dhlB1. Heterologous expression of the DhlB2 protein confirmed that this plasmid-borne dehalogenase was capable of chloroacetate dechlorination. IMPORTANCEEarlier studies on the DCA-degrading Xanthobacter sp. strain GJ10 indicated that the key dehalogenases dhlA and dhlB were carried on a 225-kb linear plasmid and on the chromosome, respectively. The present study has found a dramatically different gene organization in more recently isolated DCA-degrading Xanthobacter strains from Australia, in which a relatively small circular plasmid (pDCA) carries both dhlA and dhlB homologs. pDCA represents a true organochlorine-catabolic plasmid, first because its only obvious metabolic phenotype is dehalogenation of organochlorines, and second because acquisition of this plasmid provides both key enzymes required for carbon-chlorine bond cleavage. The discovery of the alternative haloacid dehalogenase dhlB2 in pDCA increases the known genetic diversity of bacterial chloroacetate-hydrolyzing enzymes. O rganochlorine chemicals have been widely used as pesticides, solvents, and for plastic manufacture. Many of these compounds are xenobiotic, and, as a result, their environmental breakdown is often slow (1). The environmental persistence of organochlorines is a problem, since many of these chemicals are damaging to human and environmental health (2). One organochlorine of concern is 1,2-dichloroethane (DCA), which is often present as a groundwater contaminant (3, 4). DCA is not known to exi...

Perspectives of genetically engineered microbes for groundwater bioremediation

“…The initial dehalogenase was isolated and crystallized, revealing for the rst time the enzymatic mechanism of a fundamental reaction in organic chemistry: the S N 2 nucleophilic displacement reaction of an alkylhalide with water. 21 The structures also showed that the haloalkane dehalogenases are members of a large group of related enzymes, the a/b-hydrolase fold family, which also includes lipases and [22][23][24][25][26] The dehalogenase gene is plasmid localized, and a plasmid was also found in other organisms that grow on DCA. [23][24][25][26] These dehalogenases catalyze cofactor-independent hydrolytic reactions.…”

“…30 A chloroacetate dehalogenase gene has also been found on a plasmid. 26 Genes encoding catabolic enzymes for synthetic compounds are oen located on plasmids, frequently associated with transposons or insertion elements which enable gene transfer or gene activation. Insertion elements also ank regions encoding haloalkane dehalogenase.…”

“…Insertion elements also ank regions encoding haloalkane dehalogenase. 24,26,30 Second, the haloalkane dehalogenase is constitutively expressed, which is unusual for enzymes active in a growth-supporting catabolic pathway and indicates there has been insufficient time to evolve a regulatory system. Control of haloalkane dehalogenase gene expression by its substrate would require a second protein that recognizes DCA, in addition to the haloalkane dehalogenase itself.…”

Microbial Synthesis and Transformation of Inorganic and Organic Chlorine Compounds

“…On the other hand, haloalkane dehalogenases are also widely present in organisms and ecosystems with no track record of degrading synthetic organohalogens (Hesseler et al, 2011; Chaloupkova et al, 2014; Guan et al, 2014; Carlucci et al, 2016; Weigold et al, 2016), suggesting a role in the metabolism of natural halogenated compounds. Another intriguing observation is that identical dehalogenase genes are repeatedly obtained by classical enrichment using synthetic compounds whereas these genes do not appear in general genomic databases (Poelarends et al, 1998; Poelarends et al, 2000; Govender and Pillay, 2011; Munro et al, 2016), indicating that enrichment identifies a widely distributed subpopulation of dehalogenase-producing organisms that does not represent the majority of the dehalogenase activity present in natural environments. These specific dehalogenase genes may be widespread: identical 1,2-dichloroethane dehalogenase (DhlA) genes were discovered in the Netherlands, South Africa, Korea, and Australia.…”

“…These specific dehalogenase genes may be widespread: identical 1,2-dichloroethane dehalogenase (DhlA) genes were discovered in the Netherlands, South Africa, Korea, and Australia. However, they are located on different plasmids and associated with different insertion sequences, illustrating the high mobility of the genes (Song et al, 2004; Munro et al, 2016). The mechanism of distribution and the ancestry of 1,2-dichloroethane isolates remain unknown.…”