Genetic diversity and expression of the [NiFe] hydrogenase large-subunit gene of Desulfovibrio spp. in environmental samples
Genetic diversity and expression of the [NiFe] hydrogenase large-subunit gene of Desulfovibrio spp. in environmental samples
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DOI：
10.1128/aem.63.11.4360-4369.1997
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发表时间：
1997-11-01
影响因子：
4.4
通讯作者：
Muyzer, G
中科院分区：
文献类型：
--
作者：
Wawer, C;Jetten, MSM;Muyzer, G
脱硫弧菌[NiFe]氢化酶大亚基基因的遗传多样性和表达。测定环境样品中的细菌数量是为了同时显示脱硫弧菌种群中现有的和活跃的成员。从不同厌氧生物反应器样品中提取DNA和总RNA； RNA被转录成cDNA。随后，进行 PCR 扩增 [NiFe] 氢化酶大亚基基因的约-440 bp 片段及其 mRNA，使用变性梯度凝胶电泳分析根据 PCR 产物的序列分离 PCR 产物，从而可视化各个群落成员。与扩增的[NiFe]氢化酶转录物相对应的脱硫弧菌菌株被认为具有代谢活性，因为在纯培养物中，在指数生长的细胞中可以检测到转录物，但在稳定期的培养物中则不能检测到。 DNA 测序和比较序列分析用于根据 [NiFe] 氢化酶序列来识别检测到的生物体。特征脱硫弧菌属的基因。显示出相当大程度的分歧（约 30%），而从生物反应器的细菌群体获得的序列显示出低水平的变异，表明可能属于 Desulfovibrio sulfodismutans 种的密切相关菌株的共存。在产甲烷条件下，所有检测到的种群均处于活跃状态；在反硝化条件下，没有观察到[NiFe]氢化酶mRNA。可以使用本研究中描述的方法监测由环境条件变化引起的脱硫弧菌种群的活性和组成的变化。
The genetic diversity and expression of the [NiFe] hydrogenase large-subunit gene of Desulfovibrio spp. in environmental samples were determined in order to show in parallel the existing and active members of Desulfovibrio populations. DNA and total RNA were extracted from different anaerobic bioreactor samples; RNA was transcribed into cDNA. Subsequently, PCR was performed to amplify a ca.-440-bp fragment of the [NiFe] hydrogenase large-subunit gene and its mRNA Denaturing gradient gel electrophoresis analysis was used to separate the PCR products according to their sequence and thereby to visualize the individual community members. Desulfovibrio strains corresponding to amplified [NiFe] hydrogenase transcripts were regarded as metabolically active, because in pure cultures transcripts were detectable in exponentially growing cells but not in cultures in the stationary phase. DNA sequencing and comparative sequence analysis were used to identify the detected organisms on the basis of their [NiFe] hydrogenase sequences. The genes of characterized Desulfovibrio spp. showed a considerable extent of divergence (ca. 30%), whereas sequences obtained from bacterial populations of the bioreactors showed a low level of variation and indicated the coexistence of closely related strains probably belonging to the species Desulfovibrio sulfodismutans. Under methanogenic conditions, all detected populations were active; under denitrifying conditions, no [NiFe] hydrogenase mRNA was visible. Changes in activity and composition of Desulfovibrio populations caused by changes in the environmental conditions could be monitored by using the approach described in this study.