Transcript Diversity of uncultured candidate division SR1, in

Diversity of uncultured
candidate division SR1 in
anaerobic habitats
James P. Davis
Microbial & Molecular Genetics
Oklahoma State University
Overview
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Background
Materials and Methods
Results
Summary & Conclusion
Future Work
Introduction
Bacterial Diversity
– Culture-independent
surveys, based on 16S
gene analysis, indicates
that there are many
novel, yet-uncultured,
bacteria in the
environment
An “Unculturable” Majority
– Apart from 16S gene
based analysis, little is
known regarding:
metabolic pathways,
physiological activities,
and community
interactions
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Candidate Division SR1
• Few 16S sequences are present in
databases
• Found mainly in anaerobic habitats
– Deep-sea hydrothermal vents and
sediments
– Sulfur-rich sediments
– Termite gut
– Human oral cavity
SR1 Relevance
• Why SR1?
– Interest was prompted because SR1 was
detected in Zodletone Springs using
general bacterial primers
• Purpose
– Survey different environments for SR1 and
to determine the diversity in each
environment
Hypothesis
• Candidate division SR1 will be detected
in a variety of anaerobic environments,
including those with high and low sulfur
contents.
Materials & Methods
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Primer Design
Environmental Sampling
DNA Extraction
Polymerase Chain Reaction (PCR)
Cloned PCR Products
Sequencing
Preliminary Phylogenetic Analysis
Survey of SR1 in a variety of
anaerobic environments
• Primer Design
– 4 different primers
targeting 16S rRNA
genes of SR1 sequences
were designed
– These division specific
primers were used
together and in
conjunction with general
bacterial primers
• Sampling
– Bovine rumen and feces
– Zodletone Springs
Sediment
– Hydrocarboncontaminated Soil
– Kessler Farm Field Soil
– Anaerobic Fresh-Water
Pond Sediments
– Waste Water Treatment
Plant
Results – Division Specific
Primers
SR1 Primers
DNA
Zodletone Sed.
Bovine Rumen
Bovine Feces
WWTP
Theta Pond Sed.
Duck Pond Sed.
Kessler Soil
HC Contam soil
SR1-445F /
SR1-1075R
[+]
+
[+]
–
[+]
[+]
–
[+]
Uni-8F /
SR1-1075R
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+
[+]
–
–
–
–
–
Table 1: [+] Indentified only by Nested PCR
Uni-8F /
SR1-914R
+
–
[+]
–
–
–
–
–
Uni-27F /
SR1-914R
–
+
–
–
–
[+]
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Preliminary evidence
that distinct SR1
members are present
in different
environments
•High level of diversity
among the Zodletone
clones – 11 sequences
in 5 operational
taxonomic units
• The 5 Rumen clones
are all the same OTU
•The data shows that the
primer design was
effective because all the
sequences belonged to
SR1
Summary & Conclusion
• SR1 specific primers were designed to target the 16S
rRNA gene and were validated
• These primers identified SR1 in most environments
tested
• Preliminary phylogenetic analysis indicates a high
level of diversity among the SR1 community in
Zodletone, while there is less diversity in the Bovine
Rumen
• SR1 was detected in 2 non-sulfur rich environments
(Duck & Theta Pond). This suggests the SR1 is not
restricted to sulfur-rich sites like Zodletone Springs
and Sulfur River
Future Work
• Continue sequencing more clones of
SR1 in all environments
• Investigate other uncultured divisions
that are prevalent in anaerobic
environments such as, TM7, WS5, SC3,
OP11, OD1, and OD2
Acknowledgements
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Miller, Fathepure, Shaw Labs
DeSilva Lab
Dr. Duncan
Cody Shiek