Identification of novel toluene monooxygenase genes in a hydrocarbon-polluted sediment using sequence- and function-based screening of metagenomic libraries

Abstract

The microbial potential for toluene degradation within sediments from a tar oil-contaminated site in Flingern, Germany, was assessed using a metagenomic approach. High molecular weight environmental DNA from contaminated sediments was extracted, purified, and cloned into fosmid and BAC vectors and transformed into Escherichia coli. The fosmid library was screened by hybridization with a PCR amplicon of the α-subunit of the toluene 4-monooxygenase gene to identify genes and pathways encoding toluene degradation. Fourteen clones were recovered from the fosmid library, among which 13 were highly divergent from known tmoA genes and several had the closest relatives among Acinetobacter species. The BAC library was transferred to the heterologous hosts Cupriavidus metallidurans (phylum Proteobacteria) and Edaphobacter aggregans (phylum Acidobacteria). The resulting libraries were screened for expression of toluene degradation in the non-degradative hosts. From expression in C. metallidurans, three novel toluene monooxygenase-encoding operons were identified that were located on IncP1 plasmids. The E. aggregans-hosted BAC library led to the isolation of a cloned genetic locus putatively derived from an Acidobacteria taxon that contained genes involved in aerobic and anaerobic toluene degradation. These data suggest the important role of plasmids in the spread of toluene degradative capacity and indicate putative novel tmoA genes present in this hydrocarbon-polluted environment.

Microbial Degradation Pathways of Acrylic Paint Thinner Gas in Biotrickling Filter and Comparison of Kinetic Models Under Different Gas Concentrations

“…The Bacillus cereus can produce 2,3-catechol dioxygenase and toluene monooxygenase [33]; Bacillus subtilis can produce carbonyl reductase [34]; Acinetobacter calcoaceticus can produce toluene monooxygenase [35], toluene dioxygenase [36] and 2,3-catechol dioxygenase [37].…”

Improvements in Extraction Methods of High-molecular-weight DNA from Soils by Modifying Cell Lysis Conditions and Reducing Adsorption of DNA onto Soil Particles

“… Williamson et al (2005) successfully cloned DNA fragments, measuring up to 190‍ ‍kb, isolated from soil samples into bacterial artificial chromosome (BAC) vectors. Several libraries of HMW DNA isolated from soil have been constructed, and various functional genes have been analyzed ( Brady and Clardy, 2000 ; Rondon et al , 2000 ; MacNeil et al , 2001 ; Quaiser et al , 2002 ; O’Mahony et al , 2015 ; Bouhajja et al , 2017 ; Duarte et al , 2017 ). Clone libraries enable the expression and analysis of target genes and the use of some products from gene clusters.…”

“…This method targets the microbial cells strongly attached to soil particles, and the DNA yield is markedly higher than that from the indirect method ( Steffan et al , 1988 ). Among the procedures reported for the direct extraction method, that described by Zhou et al (1996) using a high-salt buffer has been applied in several experiments with or without modifications ( Brady and Clardy, 2000 ; Rondon et al , 2000 ; Williamson et al , 2005 ; Tao et al , 2011 ; Bouhajja et al , 2017 ; Verma et al , 2017 ). We recognized the efficiency of the method described by Zhou et al (1996) and attempted to identify the most suitable conditions in each step for the extraction of HMW DNA from our soil samples in order to increase yield and molecular weight.…”

“…The freeze-thawing treatment step was omitted based on the findings of Moré et al (1994) . Lysozyme is frequently used as a cell lysis enzyme to extract DNA from soil samples ( Tsai and Olson, 1991 ; Tebbe and Vahjen, 1993 ; Frostegård et al , 1999 ; Brady and Clardy, 2005 ; Bouhajja et al , 2017 ; Verma et al , 2017 ). Although lysozyme generally reacts at 37°C, a temperature of 45°C was selected in our modified procedure based on the results of a pretest ( Fig.…”

“…The electrophoresis of crude HMW DNA followed by a gel extraction procedure is effective in most cases. Several purification methods of crude HMW DNA from soil samples have been reported to date ( Zhou et al , 1996 ; Brady and Clardy, 2000 ; Rondon et al , 2000 ; MacNeil et al , 2001 ; Williamson et al , 2005 ; Kielak et al , 2009 ; de Castro et al , 2011 ; O’Mahony et al , 2015 ; Bouhajja et al , 2017 ; Verma et al , 2017 ), and need to be applied depending on the purpose of the experiment. The improved procedures for HMW DNA extraction from soil samples described in the present study and examples of purification steps are shown in Fig.…”