Ramírez-Saad H C, Sessitsch A, de Vos W M, Akkermans A D
Laboratory of Microbiology, Wageningen University, The Netherlands.
Syst Appl Microbiol. 2000 Dec;23(4):591-8. doi: 10.1016/S0723-2020(00)80035-1.
Bacterial community shifts in a peat-forest soil spiked with 3-chlorobenzoate (3CBA) or 2,5-dichlorobenzoate (2,5DCB) were monitored by PCR-amplification of the V6 to V8 regions of the 16S rRNA and rDNA, followed by separation of the amplicons by temperature gradient gel electrophoresis. 3CBA disappeared to non-detectable levels after 15 days by a biologically mediated process, while 2,5DCB remained at the initial concentration values. The experiments were conducted under microcosms systems. Addition of the chlorinated benzoates to the soil resulted in a rapid decrease of the microbial diversity, as judged by a time-dependent reduction in the number of amplicons detected by temperature gradient gel electrophoresis. Few amplicons specifically enriched in the spiked soils were cloned and characterised by sequence analysis. The identity of the cloned DNA and the corresponding soil amplicons was confirmed by hybridisation with a radioactively labelled V6-probe. Analysis of the 16S rDNA sequences indicated that Burkholderia-related bacteria dominated the enriched soil populations under 3CBA stress. In addition, enrichment cultures growing on 3CBA as sole C-source were obtained from the respective spiked soil, which were found to contain bacteria with identical 16S rDNA sequences as those induced by 3CBA stress in soil.
通过对16S rRNA和rDNA的V6至V8区域进行PCR扩增，随后通过温度梯度凝胶电泳分离扩增子，监测添加了3 - 氯苯甲酸（3CBA）或2,5 - 二氯苯甲酸（2,5DCB）的泥炭林土壤中的细菌群落变化。3CBA在15天后通过生物介导的过程消失至检测不到的水平，而2,5DCB则保持在初始浓度值。实验在微观系统下进行。向土壤中添加氯化苯甲酸导致微生物多样性迅速下降，这通过温度梯度凝胶电泳检测到的扩增子数量随时间的减少来判断。对在添加了氯化苯甲酸的土壤中特异性富集的少数扩增子进行克隆，并通过序列分析进行表征。通过与放射性标记的V6探针杂交，确认了克隆DNA与相应土壤扩增子的一致性。对16S rDNA序列的分析表明，在3CBA胁迫下，伯克霍尔德氏菌相关细菌在富集的土壤种群中占主导地位。此外，从各自添加了3CBA的土壤中获得了以3CBA作为唯一碳源生长的富集培养物，发现其中所含细菌的16S rDNA序列与土壤中3CBA胁迫诱导的细菌序列相同。
