Detection of Noncultured Bacterial Divisions
in Environmental Samples using 16S rRNA-Based Fluorescent in Situ Hybridization

Environmental Molecular Biology Group; M888 Life Sciences Division; Los Alamos National Laboratory; Los Alamos, NM 87545
505/665-4800, Fax: -6894, kuske@lanl.gov

Microbial genome sequencing projects have focused primarily on species that can be easily cultured. Readily cultured bacteria, however, 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 noncultured 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. Using PCR primers specific for two of these divisions (Acidobacterium and OP11) and their subgroups, we have detected both divisions in local hot or 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 noncultured bacteria will be a valuable resource of genetic material for comparative analyses of conserved and novel gene families and for targeted genome sequencing.

Phylogenetic Analysis of Hyperthermophilic Natural Populations Using Ribosomal RNA Sequences

Plant and Microbial Biology; University of California; Berkeley, CA 947203102 

Current Address: Department of Molecular, Cellular, and Developmental Biology; University of Colorado; Boulder, CO 803030347
303/7351864, Fax: /492-7744, nrpace@colorado.edu

It has become clear over the past few decades that substantial microbial diversity occurs at very high temperatures. Hyperthermophilic organisms (temperature optima >800°C) promise a wealth of unknown biochemistry and biotechnological potential and challenge our comprehension of biomolecular structure. Nonetheless, relatively little is known about the diversity of life at high temperatures because of a traditional problem in microbial ecology: the inability to cultivate naturally occurring organisms. Molecular techniques recently have been developed, however, that allow the detection and some characterization of organisms without cultivation. Limited surveys of hyperthermophilic communities using such techniques have revealed the existence of an unexpected plethora of organisms, some profoundly different from known ones. This program's main objective was to continue the phylogenetic and quantitative characterization, without cultivation, of ecosystem constituents that are known to be associated with particular hightemperature sites. Main focus was on the Yellowstone geothermal system.

These methods for characterizing organisms in the environment revolve about the use of rRNA sequences for phylogenetic analysis of population constituents. We obtained rRNA genes by directly cloning environmental DNA or by cloning products of polymerase chain reaction (PCR) amplification using primers complementary to universally conserved or phylogenetic groupspecific sequences in rDNAs. Comparison of sequences to known rRNA sequences revealed phylogenetic relationships of organisms in the community to known organisms. In a second approach, fluorescently labeled oligonucleotide hybridization probes that bind selectively to rRNA were used for microscopic phylogenetic analysis of single cells. Results were highlighted by ongoing results from Yellowstone hotsprings.

Program results have contributed significantly to the emerging view of microbial diversity. Previous and ongoing studies have revealed a great wealth of archaeal diversity in sediments and scinters of hotsprings 70 to 95°C. These results have revised our understanding of archaea's phylogenetic depth and have allowed the recognition of archaea as a new kingdom. Surveys of bacterial "phylotypes" have expanded substantially our understanding of bacterial diversity; 12 of the current 36 to 38 bacterial divisions were first articulated in this program. Some of the newly discovered evolutionary lineages are sufficiently abundant that they must be significant in this ecosystem. Selected sequencetypes were explored further using fluorescently labeled oligonucleotide hybridization probes to visualize the organisms in their natural setting. Scanning electron microscope investigations showed that a succession of morphotypes forms biofilms on surfaces in hotsprings. Hybridization probes, in concert with available confocal microscopy, eventually will allow the reconstruction of threedimensional aspects of this geothermal ecosystem.

We and others have found that types of organisms formerly thought to be restricted to high temperatures are in fact abundant at low temperatures and common in our environment. We used PCR primers characteristic of Crenarchaeota (thermophilic in all cultivated instances) to show that such organisms are common in sediments and soils at low temperatures, so they are likely to occur globally; such organisms also have been detected as abundant in the marine environment. Although not yet cultivated, these organisms are sufficiently abundant in the environment that they are likely to have impact on the biosphere's chemistry. Similarly with representatives of bacteria, we encountered many kinds of organisms previously thought restricted to low temperature ecosystems. Indeed bacteria, not the commonly thought archaea, were found to dominate high-temperature ecosystems.

Overall the program has been contributory and conspicuous in the field of life in extreme environments. The period of performance for this program was July 15, 1995, to September 14, 1997.

This is a completed project.

The Ribosomal Database Project II: 
Providing an Evolutionary Framework

Center for Microbial Ecology; Michigan State University; East Lansing, MI 48824
Tiedje: 517/3539021, Fax: -2917, tiedej@pilot.msu.edu
http://rdp.cme.msu.edu/index.jsp

The Ribosomal Database Project II (RDPII) provides rRNA-related data and tools important for researchers from a number of fields. These RDPII products are used widely in molecular phylogeny and evolutionary biology, microbial ecology, bacterial identification, microbial population characterization, and in understanding the diversity of life. As a valueadded database, RDPII offers the research community aligned and annotated rRNA sequence data, analysis services, and phylogenetic inferences derived from these data. These services are available through the RDPII Web site.

Release 7.1 (September 1999) contained more than 10,000 aligned and annotated small subunit (SSU) rRNA sequences. A special focus of this release was the identification and annotation of sequences from type material. Over 3000 type sequences representing 636 distinct prokaryotic genera were included in release 7.1. These type sequences provide a mechanism for users to place new sequences in taxonomic and phylogenetic frameworks. This release also included the introduction of an interactive assistant to help with the planning and analysis of TRFLP experiments (TAP TRFLP).

We are now preparing release 8, scheduled for March 2000. We are enhancing the alignment to match a new set of guidelines for more consistent treatment of secondary structure regions. This release will contain over 20,000 aligned prokaryotic SSU rRNA sequences, including the vast majority of those available through GenBank release 114 (October 15, 1999). Initially, release 8 will be made available without manual curation of annotation information. We are establishing an RDP advisory panel to help us set new annotation standards to better serve our users with available curation resources. Release 8 also will mark a turning point for RDP. It will be the first release since 1994 in which the time has decreased between sequences becoming available through GenBank and being released in aligned format by RDP. We expect both the time and frequency of releases to continue to improve through 2000.