164. Identification, Isolation, and Genome Amplification of Abundant Non-Cultured Bacteria from Novel Phylogenetic Kingdoms in Two Extreme Surface Environments 

Cheryl R. Kuske, Susan M. Barns, John D. Dunbar, Jody A. Davis, and Greg Fisher 
Environmental Molecular Biology Group, LS-7, M888, Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545 
Kuske@lanl.gov 

Microbial genome sequencing projects have produced a wealth of information on microbial genetics, biochemistry, and evolution with important medical, environmental, agricultural and industrial applications, but have focused primarily on species we can easily culture. Cultured bacteria are only a small fraction of the total bacterial diversity present in the environment. Non-cultured organisms of considerable genetic and biochemical diversity are present in arid and extreme surface environments. Microbial processes in these environments are of critical importance to the biosphere and the non-cultured bacteria residing there are a valuable resource for novel genomic information. 

We have initiated a project to (1) identify novel bacterial kingdoms in 16S ribosomal RNA gene (rDNA) libraries from two extreme surface environments to expand our current understanding of the scope of environmental bacterial diversity, (2) determine the abundance and activity of these novel organisms in the environment by using rRNA-targeted fluorescent probes, and (3) collect cells of novel phylogenetic groups that are abundant and active in the environment by flow cytometry and isolate or amplify DNA from the pools of bacterial cells. 

Our preliminary results demonstrate the extensive diversity of bacteria present in two extreme terrestrial surface environments. Sequence analysis of 60 clones from two 16S rDNA libraries indicated that bacterial diversity in these two environments is extensive. RFLP fingerprint analysis of 800 clones in the two libraries demonstrated that diversity in the remaining clones was also great and that so far only 5% of them have fingerprints similar to those clones already sequenced. In fact, almost every clone produced a unique pattern, and there was very little overlap in patterns between the two environments. Preliminary RFLP and 16S rDNA sequence analysis indicate that most of the bacterial species represented in the clone libraries fall outside the known, previously-described bacterial taxa. Thus these libraries are a rich resource for identifying and isolating novel bacterial species with novel genes. We are developing fluorescently tagged oligonucleotide probes for detection and collection of some of these bacterial groups from environmental samples. 

The pooled DNA of isolated, non-cultured bacteria will be a valuable resource of genetic material for comparative analyses of conserved and novel gene families, and for targeted genome sequencing. Identification and sequence analysis of genes from abundant bacterial species will greatly enhance our understanding of their functional roles in the environment and will significantly expand the set of unique genes and proteins available for DOE missions, as well as for medical, industrial and agricultural applications.