Choi Sangdun, Chang Mi Sook, Stuecker Tara, Chung Christine, Newcombe David A, Venkateswaran Kasthuri
Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Korea. ; Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Genomics Inform. 2012 Dec;10(4):249-55. doi: 10.5808/GI.2012.10.4.249. Epub 2012 Dec 31.
In this study, fosmid cloning strategies were used to assess the microbial populations in water from the International Space Station (ISS) drinking water system (henceforth referred to as Prebiocide and Tank A water samples). The goals of this study were: to compare the sensitivity of the fosmid cloning strategy with that of traditional culture-based and 16S rRNA-based approaches and to detect the widest possible spectrum of microbial populations during the water purification process. Initially, microbes could not be cultivated, and conventional PCR failed to amplify 16S rDNA fragments from these low biomass samples. Therefore, randomly primed rolling-circle amplification was used to amplify any DNA that might be present in the samples, followed by size selection by using pulsed-field gel electrophoresis. The amplified high-molecular-weight DNA from both samples was cloned into fosmid vectors. Several hundred clones were randomly selected for sequencing, followed by Blastn/Blastx searches. Sequences encoding specific genes from Burkholderia, a species abundant in the soil and groundwater, were found in both samples. Bradyrhizobium and Mesorhizobium, which belong to rhizobia, a large community of nitrogen fixers often found in association with plant roots, were present in the Prebiocide samples. Ralstonia, which is prevalent in soils with a high heavy metal content, was detected in the Tank A samples. The detection of many unidentified sequences suggests the presence of potentially novel microbial fingerprints. The bacterial diversity detected in this pilot study using a fosmid vector approach was higher than that detected by conventional 16S rRNA gene sequencing.
在本研究中，使用fosmid克隆策略评估国际空间站（ISS）饮用水系统中的微生物群落（以下简称杀生剂前和水箱A水样）。本研究的目标是：将fosmid克隆策略的灵敏度与传统的基于培养和基于16S rRNA的方法进行比较，并在水净化过程中检测尽可能广泛的微生物群落谱。最初，微生物无法培养，传统PCR也未能从这些低生物量样本中扩增出16S rDNA片段。因此，使用随机引物滚环扩增来扩增样本中可能存在的任何DNA，随后通过脉冲场凝胶电泳进行大小选择。将两个样本中扩增得到的高分子量DNA克隆到fosmid载体中。随机选择数百个克隆进行测序，随后进行Blastn/Blastx搜索。在两个样本中均发现了编码伯克霍尔德氏菌（一种在土壤和地下水中丰富的物种）特定基因的序列。慢生根瘤菌属和中生根瘤菌属属于根瘤菌，根瘤菌是一个经常与植物根系相关联的大型固氮群落，在杀生剂前样本中存在。在水箱A样本中检测到了在重金属含量高的土壤中普遍存在的罗尔斯通氏菌。许多未鉴定序列的检测表明存在潜在的新型微生物指纹。使用fosmid载体方法在这项初步研究中检测到的细菌多样性高于通过传统16S rRNA基因测序检测到的细菌多样性。
