Nutritional inter-dependencies and a carbazole-dioxygenase are key elements of a bacterial consortium relying on aSphingomonasfor the degradation of the fungicide thiabendazole

Abstract

22Background: Thiabendazole (TBZ), is a benzimidazole fungicide and anthelminthic whose high 23 persistence and toxicity pose a serious environmental threat. In our quest for environmental mitigation 24 we previously isolated the first TBZ-degrading bacterial consortium and provided preliminary evidence 25 for its composition and the degrading role of a Sphingomonas. Here, we employed a multi-omic 26 approach combined with DNA-stable isotope probing (SIP) to determine the genetic make-up of the 27 key consortium members, to disentangle nutritional and metabolic interdependencies, to identify the 28 transformation pathway of TBZ and to understand the genetic network driving its transformation. 29Results: Time-series SIP in combination with amplicon sequencing analysis verified the key role of 30 Sphingomonas in TBZ degradation by assimilating over 80% of the 13 C-labelled phenyl moiety of TBZ. 31Non-target mass spectroscopy (MS) analysis showed the accumulation of thiazole-4-carboxamidine as 32 a single dead-end transformation product and no phenyl-containing derivative, in line with the phenyl 33 moiety assimilation in the SIP analysis. Time series metagenomic analysis of the consortium 34 supplemented with TBZ or succinate led to the assembly of 18 metagenome-assembled genomes 35 (MAGs) with >80% completeness, six (Sphingomonas 3X21F, γ-Proteobacterium 34A, 36Bradyrhizobiaceae 9B and Hydrogenophaga 19A, 13A, and 23F) being dominant. Meta-transcriptomic 37 and -proteomic analysis suggested that Sphingomonas mobilize a carbazole dioxygenase (car) operon 38 during the initial cleavage of TBZ to thiazole-4-carboxamidine and catechol, the latter is further 39 transformed by enzymes encoded in a catechol ortho-cleavage (cat) operon; both operons being up-40 regulated during TBZ degradation. Computational docking analysis of the terminal oxygenase 41 component of car, CarAa, showed high affinity to TBZ, comparable to carbazole, reinforcing its high 42 potency for TBZ transformation. These results suggest no interactions between consortium members in 43 TBZ transformation, performed solely by Sphingomonas. In contrast, gene expression network analysis 44 revealed strong interactions between Sphingomonas MAG 3X12F and Hydrogenophaga MAG 23F, 45with Hydrogenophaga activating its cobalamin biosynthetic pathway and Sphingomonas its cobalamin 46 salvage pathway along TBZ degradation. 47 Conclusions:Our findings suggest interactions between consortium members which align with the 48 "black queen hypothesis": Sphingomonas detoxifies TBZ, releasing consortium members by a toxicant; 49 in return for this, Hydrogenophaga 23F provides cobalamin to the auxotrophic Sphingomonas. 50 Background 54 Thiabendazole (TBZ) is a benzimidazole compound which is used as a post-harvest fungicide to control 55 fungal infestations on fruits during storage [1] and as a broad spectrum anthelminthic to control 56 endoparasites in livestock farming [2]. It acts by binding to tubulin monomers inhibiting the 57 polymerization of microtubules and, thus,...

Study of the depuration efficiency and microbiology of biobed systems which receive pesticide contaminated effluents from various agro-food processing units

“…Bacteria belonging to these orders are known for their capacity to degrade pesticides in general, and fungicides used in FPPs in particular. For example, a Sphingomonas haloaromaticamans strain and a Sphingomonas-based bacterial consortium (Sotirios et al, 2020) were able to degrade OPP and TBZ respectively, while Pseudomonas and Burkholderia spp. were previously reported as diphenylamine degraders Shin and Spain, 2009).…”