Elucidating the metabolic pathway and initial degradation gene for -chloro--xylenol biodegradation in DMU114.

Unlabelled: The antimicrobial agent -chloro--xylenol (PCMX), an emerging environmental pollutant, poses ecological risks; however, its biodegradation mechanisms remain unresolved. Here, we elucidate the metabolic pathway and functional genes involved in the initial catabolic step of PCMX in a newly isolated bacterium, DMU114. Pure-culture and synthetic consortium assays confirmed the pivotal role of in PCMX degradation, despite its relatively low abundance in the PCMX-enriched consortium. Genomic analysis and heterologous expression identified a constitutively expressed flavin-dependent monooxygenase CxyAB as the key enzyme initiating PCMX degradation. High-resolution liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses demonstrated that strain DMU114 degraded PCMX via a potential three-step pathway: -hydroxylation to 4-chloro-3,5-dimethylcatechol, dechlorination to 2-hydroxy-3,5-dimethyl-[1,4]benzoquinone, and dual - and -cleavage of the aromatic ring. Homologs of CxyA are phylogenetically widespread in environmentally relevant genera, including , , , and , indicating their potential role in natural PCMX attenuation. This work provides the first genetic dissection of PCMX mineralization, offering critical insights into its environmental fates and bioremediation strategies targeting antimicrobial contaminants.

Importance: The widespread use of the antimicrobial agent -chloro--xylenol (PCMX) in consumer products has raised environmental concerns due to its aquatic toxicity. However, the microbial mechanisms driving its natural breakdown remain poorly understood. This study reveals how a newly isolated bacterium, DMU114, mineralizes PCMX, a process critical for mitigating its ecological risks. This study, for the first time, elucidates the PCMX's complete degradation pathway and identifies the functional genes for its initial conversion step. The degradation gene identified is widespread in environmentally relevant bacteria, suggesting that natural ecosystems may already harbor the potential to neutralize PCMX contamination. These findings advance our ability to predict PCMX's environmental fate and provide a foundation for engineering microbial solutions to combat antimicrobial pollution.