Synergistic dual ring-cleavage pathways enable efficient degradation of chlorobenzenes by Pseudomonas putida BS-1 in groundwater.

The accumulation of toxic chlorocatechols, which pose greater ecotoxicity than their parent compounds, represents a critical bottleneck in the biodegradation of chlorobenzenes. Addressing this challenge, the isolation of indigenous Pseudomonas putida BS‑1 revealed a novel synergistic dual‑cleavage pathway that ensures rapid intermediate detoxification during p‑dichlorobenzene (p‑DCB), chlorobenzene, and benzene degradation. The genetic basis for this mechanism was identified as a novel plasmid-borne tod-clc gene cluster harboring two clc copies (designated as clc_1 and clc_2), which uniquely integrates the ortho- and meta-cleavage pathway modules. Under pollutant stress, this cluster was synergistically upregulated, with tod genes upregulated 204-7292‑fold and clc genes 8-67‑fold. Gene knockout and heterologous expression confirmed that the encoded TodE (meta) and ClcA (ortho) enzymes function cooperatively to cleave (chloro)catechol intermediates, with corresponding chloride release detected. Owing to this efficient mechanism, strain BS‑1 demonstrated high remediation efficacy in authentic contaminated groundwater, achieving complete removal of p-DCB (6.83 mg·L-1) within 24 h and 98.7% removal of o-dichlorobenzene (13.31 mg·L-1) within 72 h. This work elucidates a novel synergistic dual-pathway strategy, providing both an effective microbial resource for in situ groundwater remediation and new insights for synthetic biology to construct robust degraders.