Unraveling acridine degradation mechanisms in PAH-contaminated soils using DNA-SIP combined with metagenomics and soil transcriptomics

Polycyclic aromatic nitrogen heterocycles (PANHs), also known as azaarenes, are common co-contaminants at sites contaminated with polycyclic aromatic hydrocarbons (PAHs). Recent non-target analysis of PAH-contaminated soil samples has revealed an unexpected abundance and diversity of PANHs, with acridine standing out as a predominant compound within this group. Despite its known toxicity and prevalence in contaminated soils, the microbial communities and biochemical mechanisms responsible for acridine degradation remain poorly understood. We conducted DNA-stable isotope probing (DNA-SIP) using newly synthesized uniformly labeled 13 C-acridine to comprehensively assess the bacterial taxa and functional genes involved in acridine biodegradation in a creosote-contaminated soil. Metagenomic analysis of 13 C-enriched DNA from soil incubations identified a member of the genus Sphingobium as the primary acridine degrader. Transcriptomic analysis based on its 16S rRNA gene expression demonstrated a strong correlation with acridine removal from the soil. Shotgun metagenomic sequencing enabled the reconstruction of one metagenome-assembled genome (MAG). Functional annotation of this MAG revealed five gene clusters potentially involved in acridine biodegradation, and their actual contribution was assessed by gene expression analysis in soil incubations. Based on these findings, we reconstructed the metabolic pathway for putative acridine degradation in PAH-contaminated soil. • Methodological refinements in DNA-SIP protocol increased DNA recovery. • DNA-SIP identified an uncultured Sphingobium as the main acridine degrader in soil. • Acridine metabolic pathway reconstructed by metagenomics and soil transcriptomics. • Acridine assimilation mediated by nag -like naphthalene RHD and anthranilate pathway. • Gene mining revealed widespread distribution of acridine-linked nagAc RHD in soils.