Fluoro-recognition: New in vivo fluorescent assay for toluene dioxygenase probing induction by and metabolism of polyfluorinated compounds

Kelly G. Aukema, Madison D. Bygd, Lambros J. Tassoulas, Jack E. Richman, Lawrence P. Wackett

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Abstract

The present study examined the regulatory and metabolic response of the aromatic degrader Pseudomonas putida F1 and its tod operon, controlling toluene degradation, to fluorinated aromatic and aliphatic compounds. The tod operon is upregulated by inducer binding to the TodS sensing domain of a two-component regulator. The induced enzymes include toluene dioxygenase that initiates catabolic assimilation of benzenoid hydrocarbons. Toluene dioxygenase was shown to oxidize 6-fluoroindole to a meta-stable fluorescent product, 6-fluoroindoxyl. The fluorescent output allowed monitoring relative levels of tod operon induction in whole cells using microtiter well plates. Mono- and polyfluorinated aromatic compounds were shown to induce toluene dioxygenase, in some cases to a greater extent than compounds serving as growth substrates. Compounds that are oxidized by toluene dioxygenase and undergoing defluorination were shown to induce their own metabolism. 1,2,4-Trifluorobenzene caused significant induction and computational modelling indicated productive binding to the TodS sensor domain of the TodST regulator. Toluene dioxygenase also showed preferential binding of 1,2,4-trifluorobenzene such that defluorination was favoured. Fluorinated aliphatic compounds were shown to induce toluene dioxygenase. An aliphatic ether with seven fluorine atoms, 1,1,1,2-tetrafluoro-2-trifluoromethoxy-4-iodobutane (TTIB), was an excellent inducer of toluene dioxygenase activity and shown to undergo transformation in cultures of P. putida F1.

Original languageEnglish (US)
Pages (from-to)5202-5216
Number of pages15
JournalEnvironmental microbiology
Volume24
Issue number11
DOIs
StatePublished - Nov 2022

Bibliographical note

Funding Information:
The authors thank Kristin Boardman and Wen Cai for assistance with initial experiments, and Tom Niehaus for the use of the Cary spectrophotometer. The authors thank Rebecca Parales for providing the recombinant E. coli pDTG602a strain. The authors acknowledge the support of MnDRIVE Industry and the Environment (to Madison D. Bygd and Jack E. Richman). The authors acknowledge support of National Institutes of Health Biotechnology training grant (5T32GM008347–27) and the Informatics Institute of the University of Minnesota for support (to Lambros J. Tassoulas).

Funding Information:
The authors thank Kristin Boardman and Wen Cai for assistance with initial experiments, and Tom Niehaus for the use of the Cary spectrophotometer. The authors thank Rebecca Parales for providing the recombinant pDTG602a strain. The authors acknowledge the support of MnDRIVE Industry and the Environment (to Madison D. Bygd and Jack E. Richman). The authors acknowledge support of National Institutes of Health Biotechnology training grant (5T32GM008347–27) and the Informatics Institute of the University of Minnesota for support (to Lambros J. Tassoulas). E. coli

Publisher Copyright:
© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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