TY - JOUR
T1 - Development of a PCR method for the detection and quantification of benzoyl-CoA reductase genes and its application to monitored natural attenuation
AU - Hosoda, Akifumi
AU - Kasai, Yuki
AU - Hamamura, Natsuko
AU - Takahata, Yoh
AU - Watanabe, Kazuya
N1 - Funding Information:
We would like to thank Mike Manefield for valuable discussions relating to this manuscript. We are also grateful to Fusako Numazaki for technical assistance. This study was supported by the New Energy and Industrial Technology Development Organization (NEDO).
PY - 2005/12
Y1 - 2005/12
N2 - Benzoyl coenzyme A reductase (BCR) catalyzes dearomatization of benzoyl coenzyme A (benzoyl-CoA), which is the central step in the anaerobic degradative pathways for a variety of aromatic compounds. This study developed a PCR method for the detection and quantification of BCR genes in bacterial strains and environmental samples. PCR primers were designed by aligning known BCR genes in Thauera, Azoarcus and Rhodopseudomonas species, and their utility was assessed by amplifying BCR fragments from aromatic-hydrocarbon degrading anaerobes and other bacteria. BCR fragments with the expected sizes were obtained from denitrifying and phototrophic aromatics degraders. The positive signals were also obtained from Geobacter metallireducens and xylene-degrading sulfate-reducing bacterium (strain mXyS1) but not from other aromatics-degrading sulfate-reducing bacteria and aerobic bacteria. When the PCR was used for analyzing a natural attenuation (NA) site, the positive signal was obtained only from gasoline-contaminated groundwater; sequence analysis of these amplicons revealed that most of them exhibited substantial similarities to the known BCRs. Quantitative competitive PCR analysis estimated BCR-gene copies to account for 10-40% of bacterial 16S rRNA gene copies in the contaminated groundwater, indicating that bacteria possessing BCR genes were highly enriched in the contaminated groundwater. In microcosm bioremediation tests using the contaminated groundwater, the copy number of BCR gene was approximately 10-fold increased in the course of aromatics degradation under denitrifying conditions but not under sulfidogenic conditions. These results suggest the utility of the PCR method for assessing the potential of denitrifying bacteria for aromatic-compound degradation in groundwater.
AB - Benzoyl coenzyme A reductase (BCR) catalyzes dearomatization of benzoyl coenzyme A (benzoyl-CoA), which is the central step in the anaerobic degradative pathways for a variety of aromatic compounds. This study developed a PCR method for the detection and quantification of BCR genes in bacterial strains and environmental samples. PCR primers were designed by aligning known BCR genes in Thauera, Azoarcus and Rhodopseudomonas species, and their utility was assessed by amplifying BCR fragments from aromatic-hydrocarbon degrading anaerobes and other bacteria. BCR fragments with the expected sizes were obtained from denitrifying and phototrophic aromatics degraders. The positive signals were also obtained from Geobacter metallireducens and xylene-degrading sulfate-reducing bacterium (strain mXyS1) but not from other aromatics-degrading sulfate-reducing bacteria and aerobic bacteria. When the PCR was used for analyzing a natural attenuation (NA) site, the positive signal was obtained only from gasoline-contaminated groundwater; sequence analysis of these amplicons revealed that most of them exhibited substantial similarities to the known BCRs. Quantitative competitive PCR analysis estimated BCR-gene copies to account for 10-40% of bacterial 16S rRNA gene copies in the contaminated groundwater, indicating that bacteria possessing BCR genes were highly enriched in the contaminated groundwater. In microcosm bioremediation tests using the contaminated groundwater, the copy number of BCR gene was approximately 10-fold increased in the course of aromatics degradation under denitrifying conditions but not under sulfidogenic conditions. These results suggest the utility of the PCR method for assessing the potential of denitrifying bacteria for aromatic-compound degradation in groundwater.
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U2 - 10.1007/s10532-005-0826-5
DO - 10.1007/s10532-005-0826-5
M3 - Article
C2 - 15865350
AN - SCOPUS:16844386076
SN - 0923-9820
VL - 16
SP - 591
EP - 601
JO - Biodegradation
JF - Biodegradation
IS - 6
ER -