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Research Abstracts
DOE Microbial Genome Program
Report
Section 3: Resources for Genomic
Comparison
Detection of Noncultured Bacterial Divisions
in Environmental Samples using 16S rRNA-Based Fluorescent in Situ Hybridization
Cheryl R. Kuske, Susan M. Barns, and Stephan Burde
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
Norman R. Pace
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
James R. Cole, Bonnie L. Maidak, Timothy G. Lilburn, Charles T. Parker, Paul Saxman,
Bing Li, George M. Garrity, Sakti Pramanik, Thomas M. Schmidt, and James
M. Tiedje
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/html/
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. |