Wawer C, Muyzer G
Molecular Ecology Group, Max-Planck-Institute for Marine Microbiology, Bremen, Germany.
Appl Environ Microbiol. 1995 Jun;61(6):2203-10. doi: 10.1128/aem.61.6.2203-2210.1995.
The genetic diversity of Desulfovibrio species in environmental samples was determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified [NiFe] hydrogenase gene fragments. Five different PCR primers were designed after comparative analysis of [NiFe] hydrogenase gene sequences from three Desulfovibrio species. These primers were tested in different combinations on the genomic DNAs of a variety of hydrogenase-containing and hydrogenase-lacking bacteria. One primer pair was found to be specific for Desulfovibrio species only, while the others gave positive results with other bacteria also. By using this specific primer pair, we were able to amplify the [NiFe] hydrogenase genes of DNAs isolated from environmental samples and to detect the presence of Desulfovibrio species in these samples. However, only after DGGE analysis of these PCR products could the number of different Desulfovibrio species within the samples be determined. DGGE analysis of PCR products from different bioreactors demonstrated up to two bands, while at least five distinguishable bands were detected in a microbial mat sample. Because these bands most likely represent as many Desulfovibrio species present in these samples, we conclude that the genetic diversity of Desulfovibrio species in the natural microbial mat is far greater than that in the experimental bioreactors.
通过对PCR扩增的[NiFe]氢化酶基因片段进行变性梯度凝胶电泳（DGGE），测定环境样品中脱硫弧菌属物种的遗传多样性。在对三种脱硫弧菌属物种的[NiFe]氢化酶基因序列进行比较分析后，设计了五种不同的PCR引物。这些引物以不同组合在多种含氢化酶和不含氢化酶的细菌的基因组DNA上进行了测试。发现一对引物仅对脱硫弧菌属物种具有特异性，而其他引物对其他细菌也给出了阳性结果。通过使用这对特异性引物，我们能够扩增从环境样品中分离的DNA的[NiFe]氢化酶基因，并检测这些样品中脱硫弧菌属物种的存在。然而，只有在对这些PCR产物进行DGGE分析后，才能确定样品中不同脱硫弧菌属物种的数量。对来自不同生物反应器的PCR产物进行DGGE分析显示最多有两条带，而在一个微生物垫样品中检测到至少五条可区分的带。由于这些条带很可能代表这些样品中存在的同样多的脱硫弧菌属物种，我们得出结论，天然微生物垫中脱硫弧菌属物种的遗传多样性远大于实验生物反应器中的。
