A strategy for determiing the physiological potential of uncultivated soil bacteria

Huizinga, K., J.L. Rodriguez, B.P. Keough, J.A. Breznak, and T.M. Schmidt

Presented at the ASM in Seattle (2003-09-18 to 2017-12-05 )

Soil harbors approximately 10^9-10^10 bacterial cells per gram of soil, however only about 1% of these cells have been cultured at this time. Without pure cultures it is difficult to assess the physiological potential and impact these microorganisms have in the soil environment. The creation of a bacterial artificial chromosome (BAC) library is a molecular method used to capture genomic DNA in microbially diverse environments. The genomic content of organisms in environmental samples can be analyzed which yields new insight into their in situ metabolic capabilities. BAC vectors can accommodate large pieces (100-500 kb) of DNA, but until now, there has not been a way to preferentially capture phylogenetically useful information (i.e., rRNA gene sequences) linked to genes indicating an organisms physiological potential. A unique BAC vector, pSuperPhyloBAC, has been constructed that contains an I-CeuI restriction site. I-CeuI is an intron-encoded endonuclease that cuts within the 23S rRNA gene of most bacteria. A cloning strategy has been developed that would allow genomic DNA that has been cut with I-CeuI to be inserted into pSuperPhyloBAC. Due to the high degree of conservation in bacterial rRNA operons, this will allow for the creation of a unique BAC library in which each clone would contain a partial 23S rRNA gene and a complete 16S rRNA gene. This library will allow for rapid screening of clones to isolate those containing DNA from microbial groups of interest along with genes indicating their physiological potential. This library will allow us to begin to bridge the gap between phylogeny and function in an environment with few members in pure culture.

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