Nutritional inter-dependencies and a carbazole-dioxygenase are key elements of a bacterial consortium relying on a Sphingomonas for the degradation of the fungicide thiabendazol

Background: Thiabendazole (TBZ), is a benzimidazole fungicide and anthelminthic whose high persistence and toxicity pose a serious environmental threat. In our quest for environmental mitigation we previously isolated the first TBZ-degrading bacterial consortium and provided preliminary evidence for its composition and the degrading role of a Sphingomonas. Here, we employed a multi-omic approach combined with DNA-stable isotope probing (SIP) to determine the genetic make-up of the key consortium members, to disentangle nutritional and metabolic interdependencies, to identify the transformation pathway of TBZ and to understand the genetic network driving its transformation. Results: Time-series SIP in combination with amplicon sequencing analysis verified the key role of Sphingomonas in TBZ degradation by assimilating over 80% of the 13C-labelled phenyl moiety of TBZ. Non-target mass spectroscopy (MS) analysis showed the accumulation of thiazole-4-carboxamidine as a single dead-end transformation product and no phenyl-containing derivative, in line with the phenyl moiety assimilation in the SIP analysis. Time series metagenomic analysis of the consortium supplemented with TBZ or succinate led to the assembly of 18 metagenome-assembled genomes (MAGs) with >80% completeness, six (Sphingomonas 3X21F, γ-Proteobacterium 34A, Bradyrhizobiaceae 9B and Hydrogenophaga 19A, 13A, and 23F) being dominant. Meta-transcriptomic and -proteomic analysis suggested that Sphingomonas mobilize a carbazole dioxygenase (car) operon during the initial cleavage of TBZ to thiazole-4-carboxamidine and catechol, the latter is further transformed by enzymes encoded in a catechol ortho-cleavage (cat) operon; both operons being up-regulated during TBZ degradation. Computational docking analysis of the terminal oxygenase component of car, CarAa, showed high affinity to TBZ, comparable to carbazole, reinforcing its high potency for TBZ transformation. These results suggest no interactions between consortium members in TBZ transformation, performed solely by Sphingomonas. In contrast, gene expression network analysis revealed strong interactions between Sphingomonas MAG 3X12F and Hydrogenophaga MAG 23F, with Hydrogenophaga activating its cobalamin biosynthetic pathway and Sphingomonas its cobalamin salvage pathway along TBZ degradation. Conclusions: Our findings suggest interactions between consortium members which align with the "black queen hypothesis": Sphingomonas detoxifies TBZ, releasing consortium members by a toxicant; in return for this, Hydrogenophaga 23F provides cobalamin to the auxotrophic Sphingomonas.