Shotgun proteomics suggests involvement of additional enzymes in dioxin degradation by Sphingomonas wittichii RW1

Abstract

Chlorinated congeners of dibenzo-p-dioxin and dibenzofuran are widely dispersed pollutants that can be treated using microorganisms, such as the Sphingomonas wittichii RW1 bacterium, able to transform some of them into non-toxic substances. The enzymes of the upper pathway for dibenzo-p-dioxin degradation in S. wittichii RW1 have been biochemically and genetically characterized, but its genome sequence indicated the existence of a tremendous potential for aromatic compound transformation, with 56 ring-hydroxylating dioxygenase subunits, 34 extradiol dioxygenases and 40 hydrolases. To further characterize this enzymatic arsenal, new methodological approaches should be employed. Here, a large shotgun proteomic survey was performed on cells grown on dibenzofuran, dibenzo-p-dioxin and 2-chlorodibenzo-p-dioxin, and compared with growth on acetate. Changes in the proteome were monitored over time. In total, 502 proteins were observed and quantified using a label-free mass spectrometry-based approach; all data were deposited to the ProteomeXchange (PXD000403). Our results confirmed the roles of the dioxin dioxygenase DxnA1A2, trihydroxybiphenyl dioxygenase DbfB, meta-cleavage product hydrolase DxnB and reductase RedA2, and corroborated the proposed involvement of the Swit_3046 dioxygenase and DxnB2 hydrolase. Trends across substrates and over the course of growth do not support concerted pathway regulation and suggest the involvement of an additional hydrolase and several TonB-dependent receptors.

Emerin self‐assembly mechanism: role of the LEM domain

“…For mapping fragments generated by limited proteolysis, excised polyacrylamide bands were first rinsed with MilliQ water, destained, and then subjected to in-gel protein reduction with DTT followed by protein alkylation with iodoacetamide, as described previously [40]. They were digested with either sequencing-grade Trypsin Gold and ProteaseMax surfactant (Promega, Madison, WI, USA) for 1 h at 323 K or sequencing-grade chymotrypsin for 1 h at 298 K as previously reported [41].…”

Proteogenomic insights into the core-proteome of female reproductive tissues from crustacean amphipods

“…For protein content standardization across species, and based on gel densitometry analysis and pre-testing in nanoLC-MS/MS with a total ion counting procedure, samples were further diluted in 0.1% trifluoroacetic acid for Gammarus organisms: 1:20 for G. fossarum and 1:15 for G. pulex and G. roeseli. NanoLC-MS/MS experiments were performed with a LTQ-Orbitrap XL hybrid mass spectrometer (ThermoFisher) coupled to an UltiMate 3000 LC system (Dionex-LC Packings) [34]. On a reverse-phase precolumn C18 PepMap 100 column (LC Packings), 10 μL peptide samples were loaded and desalted online.…”

Sphingomonas wittichii RW1 gene reporters interrogating the dibenzofuran metabolic network highlight conditions for early successful development in contaminated microcosms

“…Sphingomonas wittichii RW1 metabolizes dibenzofuran (DBF) and dibenzodioxins, which are frequently found in areas contaminated with polycyclic aromatic hydrocarbons (PAH) and after incineration processes (Safe, ). Strain RW1 has been studied in detail for its DBF and dioxin metabolic pathways (Wittich et al ., ; Wilkes et al ., ; Armengaud et al ., ; Hartmann and Armengaud, ), and has also been applied to some success in bioaugmentation studies (Megharaj et al ., ; Halden et al ., ; Nam et al ., ).…”