2006 Fiscal Year Final Research Report Summary
Assessing the activity of sulphate-reducing bacteria in polluted waters by means of molecular tool
| Project/Area Number |
17580169
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General fisheries
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| Research Institution | Fukui Prefectural University |
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Principal Investigator |
KONDO Ryuji Fukui Prefectural University, Faculty of Biotechnology, Associate Professor, 生物資源学部, 准教授 (30244528)
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| Project Period (FY) |
2005 – 2006
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| Keywords | sulphate-reducing bacteria / dissimilatory sulphite reductase / sulphate reduction rate / quantitative competitive RT-PCR |
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| Research Abstract |
We developed a new PCR primer set and a new quantitative method using PCR are useful tool for the detection and the enumeration of sulpahte-reducing bacteria (SRB) in natural environments. A PCR primer set targeting the gene coding for portions of α-subunit of dissimilatory sulphite reductase (dsrA) of SRB was designed. PCR amplification using the single set of dsr-specific primers resulted in PCR products of the expected size from 27 SRB strains tested, including Gram-negative and positive species. Clones derived from environmental DNA using the primers were sequenced and all were closely related with the predicted dsrA of SRB. These results indicate that PCR using the newly designed primer set are useful for the selective detection of SRB from a natural sample. This primer set was used to estimate cell numbers by dsrA selective competitive PCR using a competitor, which was about 20% shorter than the targeted region of dsrA. This procedure was applied to sediment and water samples from anaerobic aquatic environments. High densities of SRB were detected by the competitive PCR assuming that all SRB have a single copy of dsrA. Our results show that the newly developed competitive PCR technique targeted to dsrA is a powerful tool for rapid and reproducible estimation of SRB numbers in situ and is superior to the use of culture-dependent techniques.
We also developed a competitive RT-PCR method for the quantification of dsrA mRNA in Desulfovibrio desulfuricans. The amount of dsrA mRNA was determined at different growth conditions to find the relationship between the dsrA mRNA content per cell and the rate of metabolism (cell specific sulphate reduction rate, csSRR). The maximum dsrA mRNA content in SRB cell correlated with csSRR, indicating that dsrA mRNA may be of value as molecular proxy assessing cellular sulphate reduction rates in nature.
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[Book] 海の環境微生物学2005
Author(s)
近藤竜二 ほか
Total Pages
239
Publisher
恒星社厚生閣
Description
「研究成果報告書概要(和文)」より
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