Metabolic and Evolutionary Insights in the Transformation of Diphenylamine by a Pseudomonas putida Strain Unravelled by Genomic, Proteomic, and Transcription Analysis.

Evangelia S. Papadopoulou,Chiara Perruchon,Constantina Rousidou,Sotirios Vasileiadis,Athanassios Molassiotis,Georgia Tanou,Dimitrios G. Karpouzas,Martina Samiotaki

doi:10.3389/fmicb.2018.00676

Abstract

Diphenylamine (DPA) is a common soil and water contaminant. A Pseudomonas putida strain, recently isolated from a wastewater disposal site, was efficient in degrading DPA. Thorough knowledge of the metabolic capacity, genetic stability and physiology of bacteria during biodegradation of pollutants is essential for their future industrial exploitation. We employed genomic, proteomic, transcription analyses and plasmid curing to (i) identify the genetic network of P. putida driving the microbial transformation of DPA and explore its evolution and origin and (ii) investigate the physiological response of bacterial cells during degradation of DPA. Genomic analysis identified (i) two operons encoding a biphenyl (bph) and an aniline (tdn) dioxygenase, both flanked by transposases and (ii) two operons and several scattered genes encoding the ortho-cleavage of catechol. Proteomics identified 11 putative catabolic proteins, all but BphA1 up-regulated in DPA- and aniline-growing cells, and showed that the bacterium mobilized cellular mechanisms to cope with oxidative stress, probably induced by DPA and its derivatives. Transcription analysis verified the role of the selected genes/operons in the metabolic pathway: DPA was initially transformed to aniline and catechol by a biphenyl dioxygenase (DPA-dioxygenase); aniline was then transformed to catechol which was further metabolized via the ortho-cleavage pathway. Plasmid curing of P. putida resulted in loss of the DPA and aniline dioxygenase genes and the corresponding degradation capacities. Overall our findings provide novel insights into the evolution of the DPA degradation pathway and suggests that the degradation capacity of P. putida was acquired through recruitment of the bph and tdn operons via horizontal gene transfer.

Highlights

Diphenylamine (DPA) is an industrial chemical used as a precursor in the production of azo-dyes (Lye and Freeman, 1999) or non-steroidal anti-inflammatory drugs (Masubuchi et al, 1999) and due to its strong antioxidant properties, as stabilizer for propellants (Drzyzga, 2003) and as preservative in fruit-packaging plants (Rudell et al, 2005)
We propose that the P. putida DPA1 was originally an aniline-degrading bacterium which recently acquired the bph operon under the selection pressure exerted in soil by DPA contamination
Overall our findings suggest that the metabolic pathway of DPA in P. putida DPA1 is composed of three sections: (a) DPA is initially oxidized to catechol and aniline (b) the latter is further oxidized to catechol which (c) is transformed to acetylCoA and succinyl-CoA through the ortho-cleavage pathway

Summary

Introduction

Diphenylamine (DPA) is an industrial chemical used as a precursor in the production of azo-dyes (Lye and Freeman, 1999) or non-steroidal anti-inflammatory drugs (Masubuchi et al, 1999) and due to its strong antioxidant properties, as stabilizer for propellants (Drzyzga, 2003) and as preservative in fruit-packaging plants (Rudell et al, 2005). The latter two applications have been deemed responsible for the long-term contamination of soil, surface water and groundwater systems with DPA (Entenmann and Schacke, 1994; Tsaboula et al, 2016). Good knowledge of the (i) intermediate and final transformation products derived from DPA biodegradation, (ii) phenotypic stability and (iii) homeostasis of P. putida cells during the biodegradation process are essential for its future industrial exploitation

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