DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

122. Detection of Non-Cultured Bacterial Divisions in Environmental Samples using 16S rRNA-Based Fluorescent in situ Hybridization

Cheryl R. Kuske, Susan M. Barns, and Stephan Burde

Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545

Kuske@lanl.gov

Microbial genome sequencing projects have focused primarily on species that can be easily cultured. However, readily cultured bacteria are only a small fraction of the total bacterial diversity present in the environment. Diverse bacteria representing novel divisions have been identified in many natural environments using 16S rDNA sequence analysis. 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 identified novel bacterial divisions from 16S ribosomal RNA gene libraries generated from DNA of a volcanic cinder field and an arid sandstone soil. Using RFLP and sequence analysis, we have analyzed 800 bacterial rDNA sequences obtained from the two arid environments. The majority of sequences were members of recently identified bacterial divisions that have no, or very few, cultivated members (Kuske et al. 1997. AEM 63:3614-3621, Hugenholtz et al. 1998 J.Bact. 180:366-376). Using PCR primers specific for two of these divisions, Acidobacterium and OP11, and their subgroups, we have detected both divisions in local hot/warm spring microbial mats and sediments. Analysis of cell abundance of members of these groups is under investigation using fluorescently labeled rRNA probes and fluorescence microscopy. We plan to collect bacterial cells directly from the environmental samples using flow cytometry and cell sorting. The pooled DNA of 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.