Comparative proteogenomics reveals ecological and evolutionary insights into the organohalide-respiring <i>Dehalobacter restrictus</i> strain T

1,1,1-Trichloroethane (1,1,1-TCA) and chloroform (CF) are persistent groundwater contaminants because of their widespread industrial use as organic solvents and improper disposal in the past. Obligate organohalide-respiring bacteria (OHRB), such as <i>Dehalobacter</i> (<i>Dhb</i>), play crucial roles in biotransforming and detoxifying natural or anthropogenic halogenated organics including 1,1,1-TCA and CF through reductive dechlorination. Despite their significance, only five <i>Dhb</i> strains have been associated with the reductive dechlorination of 1,1,1-TCA or CF. Therefore, cultivating and characterizing novel <i>Dhb</i> strains from various environments of different origins worldwide is crucial for understanding the ecology and evolution of <i>Dhb</i> and the associated reductive dehalogenase (RDase) genes. This study reports the enrichment and investigation of a novel <i>Dhb</i> population capable of reducing 1,1,1-TCA to 1,1-dichloroethane, CF to dichloromethane, 1,1,2-TCA to vinyl chloride/1,2-dichloroethane, and 1,2,4-trichlorobenzene to 1,2-dichlorobenzene. The capability for dechlorinating both aliphatic and aromatic compounds was observed for the first time in the sediment sourced from the Xi River situated in the North China Plain. Comparative genomic analysis of <i>Dhb</i> strains revealed genome contraction might have resulted in the loss of various gene family members, contributing to the syntrophy interactions (e.g., cobalamin, hydrogen, and acetate) of <i>Dhb</i> with other anaerobes (e.g., fermenters and acetogens). Proteogenomic and phylogenetic analysis confirmed the highly expressed 1,1,1-TCA/CF-dechlorinating RDase, designated as TcaA, shared 94.7-96.7% amino acid sequence similarities with RDases, such as ThmA, CfrA, and TmrA. This study expands knowledge on <i>Dhb</i> biogeography and evolution while providing insights into potential syntrophy interactions supporting organohalide respiration by <i>Dhb</i>. The findings have implications for developing the novel biotechnologies for the remediation of halogenated alkane-contaminated sites.IMPORTANCEOrganohalide-respiring bacteria (OHRB) are essential for breaking down harmful pollutants in the environment. This study investigates a newly discovered OHRB capable of degrading multiple contaminants, including persistent 1,1,1-trichloroethane and chloroform. By understanding its unique abilities and interactions with other microbes, we gain valuable insights into how these bacteria evolve and function, enabling the development of improved bioremediation strategies to clean up polluted sites.