A Fosmid Cloning Strategy for Detecting the Widest Possible Spectrum of Microbes from the International Space Station Drinking Water System

Genomics & Informatics

Sangdun Choi¹  Mi Sook Chang²  Christine Chung²  Kasthuri Venkateswaran³  David A. Newcombe³  Tara Stuecker³ 

[1] Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Korea.;Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.;Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA 91109, USA.;

关键词: fosmid;    international space station;    multiple displacement amplification;    rolling-circle amplification;   

DOI  :  10.5808/GI.2012.10.4.249

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown