PCR-DGGE method to assess the diversity of BTEX mono-oxygenase genes at contaminated sites

Barbara Hendrickx, Winnie Dejonghe, Folkert Faber, Wesley Boënne, Leen Bastiaens, Willy Verstraete, Eva M Top, Dirk Springael

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

tmoA and related genes encode the alpha-subunit of the hydroxylase component of the major group (subgroup 1 of subfamily 2) of bacterial multicomponent mono-oxygenase enzyme complexes involved in aerobic benzene, toluene, ethylbenzene and xylene (BTEX) degradation. A PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess the diversity of tmoA-like gene sequences in environmental samples using a newly designed moderately degenerate primer set suitable for that purpose. In 35 BTEX-degrading bacterial strains isolated from a hydrocarbon polluted aquifer, tmoA-like genes were only detected in two o-xylene degraders and were identical to the touA gene of Pseudomonas stutzeri OX1. The diversity of tmoA-like genes was examined in DNA extracts from contaminated and non-contaminated subsurface samples at a site containing a BTEX-contaminated groundwater plume. Differences in DGGE patterns were observed between strongly contaminated, less contaminated and non-contaminated samples and between different depths, suggesting that the diversity of tmoA-like genes was determined by environmental conditions including the contamination level. Phylogenetic analysis of the protein sequences deduced from the amplified amplicons showed that the diversity of TmoA-analogues in the environment is larger than suggested from described TmoA-analogues from cultured isolates, which was translated in the DGGE patterns. Although different positions on the DGGE gel can correspond to closely related TmoA-proteins, relationships could be noticed between the position of tmoA-like amplicons in the DGGE profile and the phylogenetic position of the deduced protein sequence.

Original languageEnglish
Pages (from-to)262-273
JournalFEMS microbiology ecology
Volume55
Issue number2
DOIs
Publication statusPublished - Feb 2006

Keywords

  • bacteria/classification
  • bacterial proteins/genetics
  • benzene/metabolism
  • benzene derivatives/metabolism
  • biodiversity
  • dna
  • electrophoresis, polyacrylamide gel/methods
  • molecular sequence data
  • phylogeny
  • polymerase chain reaction
  • pseudomonas stutzeri/genetics
  • sequence analysis, dna
  • sequence homology
  • soil microbiology
  • soil pollutants/metabolism
  • toluene/metabolism
  • xylenes/metabolism

Cite this

Hendrickx, B., Dejonghe, W., Faber, F., Boënne, W., Bastiaens, L., Verstraete, W., ... Springael, D. (2006). PCR-DGGE method to assess the diversity of BTEX mono-oxygenase genes at contaminated sites. FEMS microbiology ecology, 55(2), 262-273. https://doi.org/10.1111/j.1574-6941.2005.00018.x
Hendrickx, Barbara ; Dejonghe, Winnie ; Faber, Folkert ; Boënne, Wesley ; Bastiaens, Leen ; Verstraete, Willy ; Top, Eva M ; Springael, Dirk. / PCR-DGGE method to assess the diversity of BTEX mono-oxygenase genes at contaminated sites. In: FEMS microbiology ecology. 2006 ; Vol. 55, No. 2. pp. 262-273.
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title = "PCR-DGGE method to assess the diversity of BTEX mono-oxygenase genes at contaminated sites",
abstract = "tmoA and related genes encode the alpha-subunit of the hydroxylase component of the major group (subgroup 1 of subfamily 2) of bacterial multicomponent mono-oxygenase enzyme complexes involved in aerobic benzene, toluene, ethylbenzene and xylene (BTEX) degradation. A PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess the diversity of tmoA-like gene sequences in environmental samples using a newly designed moderately degenerate primer set suitable for that purpose. In 35 BTEX-degrading bacterial strains isolated from a hydrocarbon polluted aquifer, tmoA-like genes were only detected in two o-xylene degraders and were identical to the touA gene of Pseudomonas stutzeri OX1. The diversity of tmoA-like genes was examined in DNA extracts from contaminated and non-contaminated subsurface samples at a site containing a BTEX-contaminated groundwater plume. Differences in DGGE patterns were observed between strongly contaminated, less contaminated and non-contaminated samples and between different depths, suggesting that the diversity of tmoA-like genes was determined by environmental conditions including the contamination level. Phylogenetic analysis of the protein sequences deduced from the amplified amplicons showed that the diversity of TmoA-analogues in the environment is larger than suggested from described TmoA-analogues from cultured isolates, which was translated in the DGGE patterns. Although different positions on the DGGE gel can correspond to closely related TmoA-proteins, relationships could be noticed between the position of tmoA-like amplicons in the DGGE profile and the phylogenetic position of the deduced protein sequence.",
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author = "Barbara Hendrickx and Winnie Dejonghe and Folkert Faber and Wesley Bo{\"e}nne and Leen Bastiaens and Willy Verstraete and Top, {Eva M} and Dirk Springael",
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Hendrickx, B, Dejonghe, W, Faber, F, Boënne, W, Bastiaens, L, Verstraete, W, Top, EM & Springael, D 2006, 'PCR-DGGE method to assess the diversity of BTEX mono-oxygenase genes at contaminated sites' FEMS microbiology ecology, vol. 55, no. 2, pp. 262-273. https://doi.org/10.1111/j.1574-6941.2005.00018.x

PCR-DGGE method to assess the diversity of BTEX mono-oxygenase genes at contaminated sites. / Hendrickx, Barbara; Dejonghe, Winnie; Faber, Folkert; Boënne, Wesley; Bastiaens, Leen; Verstraete, Willy; Top, Eva M; Springael, Dirk.

In: FEMS microbiology ecology, Vol. 55, No. 2, 02.2006, p. 262-273.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - PCR-DGGE method to assess the diversity of BTEX mono-oxygenase genes at contaminated sites

AU - Hendrickx, Barbara

AU - Dejonghe, Winnie

AU - Faber, Folkert

AU - Boënne, Wesley

AU - Bastiaens, Leen

AU - Verstraete, Willy

AU - Top, Eva M

AU - Springael, Dirk

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AB - tmoA and related genes encode the alpha-subunit of the hydroxylase component of the major group (subgroup 1 of subfamily 2) of bacterial multicomponent mono-oxygenase enzyme complexes involved in aerobic benzene, toluene, ethylbenzene and xylene (BTEX) degradation. A PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess the diversity of tmoA-like gene sequences in environmental samples using a newly designed moderately degenerate primer set suitable for that purpose. In 35 BTEX-degrading bacterial strains isolated from a hydrocarbon polluted aquifer, tmoA-like genes were only detected in two o-xylene degraders and were identical to the touA gene of Pseudomonas stutzeri OX1. The diversity of tmoA-like genes was examined in DNA extracts from contaminated and non-contaminated subsurface samples at a site containing a BTEX-contaminated groundwater plume. Differences in DGGE patterns were observed between strongly contaminated, less contaminated and non-contaminated samples and between different depths, suggesting that the diversity of tmoA-like genes was determined by environmental conditions including the contamination level. Phylogenetic analysis of the protein sequences deduced from the amplified amplicons showed that the diversity of TmoA-analogues in the environment is larger than suggested from described TmoA-analogues from cultured isolates, which was translated in the DGGE patterns. Although different positions on the DGGE gel can correspond to closely related TmoA-proteins, relationships could be noticed between the position of tmoA-like amplicons in the DGGE profile and the phylogenetic position of the deduced protein sequence.

KW - bacteria/classification

KW - bacterial proteins/genetics

KW - benzene/metabolism

KW - benzene derivatives/metabolism

KW - biodiversity

KW - dna

KW - electrophoresis, polyacrylamide gel/methods

KW - molecular sequence data

KW - phylogeny

KW - polymerase chain reaction

KW - pseudomonas stutzeri/genetics

KW - sequence analysis, dna

KW - sequence homology

KW - soil microbiology

KW - soil pollutants/metabolism

KW - toluene/metabolism

KW - xylenes/metabolism

KW - bodemverontreiniging

UR - http://www.mendeley.com/research/pcrdgge-method-assess-diversity-btex-monooxygenase-genes-contaminated-sites-1

U2 - 10.1111/j.1574-6941.2005.00018.x

DO - 10.1111/j.1574-6941.2005.00018.x

M3 - Article

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SN - 0168-6496

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