1. Oklahoma State University - Dept. Microbiol. & Molec. Genetics
1110 Innovation Way Dr.
Stillwater, OK 74078
and
Diversity and Quantification of Candidate Division SR1 in Various Anaerobic Environments
James P. Davis and Mostafa S. Elshahed
Oklahoma State University, Stillwater, OK
N-228
Purpose
Develop primers for phylogenetic analysis and quantification of SR1 in the environment.
Determine the diversity of SR1 in various anaerobic environments.
Ascertain a link between diversity and abundance with concentration of various sulfur species at environmental sites.
Abstract
Candidate division SR1 has mainly been detected in several anaerobic, predominantly sulfide/sulfur rich environments, such as, Zodletone Spring (S.W. Oklahoma), hydrothermal vents, and Sulfur River sediment (Parker Cave, KY). With the exception of 16S rRNA gene sequences, little is known regarding abundance, and global ecological distribution of members of this division. In this study, we surveyed and quantified members of SR1 in a variety of anaerobic, sulfur-rich (Zodletone Spring), anaerobic (fresh water pond sediments, bovine rumen and feces, and secondary treatment of a waste water treatment plant), and aerobic habitats (tall grass prairie soil). A universal bacterial forward primer with a SR1 group specific reverse primer, was used to selectively amplify SR1 sequences. Candidate division SR1 was detected in all anaerobic environments regardless of sulfur content, with the exception of the waste-water treatment plant. Multiple novel, sub-phyla level, lineages were detected through phylogenetic analysis. SR1 was shown to have the highest diversity in Zodletone Spring (27 OTUs in 158 clones) and the lowest diversity in bovine rumen (2 OTUs in 33 clones). We developed a quantitative PCR approach for quantifying members of SR1 in various environments. Surprisingly, the environments with the lowest phylogenetic diversity had the highest abundance, with 7.96 x 104 copies/g of sediment in Zodletone Spring sediment compared with 1.24 x 106 copies/g of sediment in bovine rumen. The geochemistry of three sites (Zodletone Spring, Duck Pond, and bovine rumen) was measured to determine a link between diversity/abundance and chemical composition of the environment. SR1 diversity was higher in sites with higher concentrations of sulfide and zero valence sulfur. This work confirms the presence of SR1 in most of the anaerobic sites tested, and provides a quantitative assessment of the division in multiple ecosystems. This study significantly increases the number of SR1 16S rRNA gene sequences available in public databases, and links SR1 diversity with sulfide/sulfur concentration.
Maximum Sequence Divergence & Species Richness
SR1 Diversity & Shannon Index
Environment
Clones
OTUs
Zodletone Spring
157 26
Duck Pond 30 4
Theta Pond 3
Bovine Feces 14 2
Bovine Rumen 19
Environment
MSD
Zodletone Spring
30.58%
Bovine rumen/feces
4.21%
Duck Pond
24.90%
Theta Pond
9.34%
Table 2a:The maximum sequence divergence (MSD) shows that Zodletone Spring sequences have the highest order of divergence, while bovine rumen/feces sequences have the lowest divergence.
Table 4: Zodletone Spring has the highest diversity with 26 OTUs in 157 clones. Bovine Rumen/Feces has the lowest diversity with 2 OTUs in 33 clones.
Table 1: Primers were developed based on known 16S rRNA gene sequences. A known universal bacteria primer was used in conjunction with SR1 specific primers.
Existing SR1 Sequence
36.22%
All Sequences
40.79%
Table 2b: SR1 sequences from this study increased the MSD of all known SR1 sequences (from public databases) by 4.6%.
Primer Name
Primer Sequence
Function
27F
5’-AGAGTTTGATCMTGGCTCAG-3’
Phylogeny
445F
5’-GAAGAMGMATGACGGTAC-3’
Phylogeny/qPCR
668R
5’-CCACCKGAAATTCCACTA-3’
qPCR
914R
5’-GYTCCCCCGCCTATCCYT-3’
1075R
5’-TTAACYRGACACCTTGCG-3’
Environment
Avg
95%lci
95%hci
Zodletone Spring
2.56
2.36
2.75
Bovine Rumen
0.34
0.02
0.65
Duck Pond
0.94
0.69
1.19
Table 5: The Shannon Index for each site shows that Zodletone Spring has the greatest number of unique sequences, and bovine rumen has the least amount.
Environment
Coverage
Zodletone Spring
94.94%
Bovine rumen/feces
100%
Duck Pond
93.33%
Table 3: Species richness shows near saturation of novel sequences in all the sites. This indicates that there are theoretically few to no new SR1 sequences to be detected in the sites.
Detection of SR1
SR1 was detected in all the anaerobic sites except the waste water treatment plant. SR1 was not
detected in either of the aerobic sites.
Geochemistry & Physical Properties of the sites
SR1 Quantification in Various Environments
Introduction
In the last two decades, culture-independent surveys, based on 16S rRNA gene analysis, indicate that there are many novel, yet-uncultured divisions, as well as, classes and orders.
Other than the 16S rRNA gene analysis, little is known about the metabolic pathways, physiological activities, and community interactions of these uncultured groups.
One member of the uncultured majority is candidate division SR1. There are few SR1 rRNA sequences in sequence databases. Members of this division are found mainly in sulfur/sulfide-rich, anaerobic habitats, such as, deep sea hydrothermal vents and ocean sediments, sulfur-rich sediments, and in the termite gut, and human oral cavity.
A detailed study of ecological diversity and abundance required development of sequence-specific SR1 primers for phylogenetic analysis and quantitative PCR (qPCR) analysis.
Environment
Gene copy number per gram of sediment
Standard Deviation
Zodletone Spring
7.96x104
± 1.81x104
Bovine Rumen
1.24x106
± 1.44x105
Duck Pond
9.41x104
± 1.8x104
Phylogenetic Tree
Figure 1: A phylogenetic tree was constructed using the representative operational taxonomic unit (OTU) sequences along with existing SR1 sequences. The tree shows five, novel, SR1 sub-groups, as well as, sequences from this study fitting in existing SR1 groups.
Table 6: A standard curve was generated using a vector with and SR1 sequence insert. Using SR1 sequence specific primers, the standard curve and the qPCR output, the 16S rRNA gene copy number per gram of sediment was calculated. Zodletone Spring has the lowest abundance amongst the sites, and bovine rumen has the highest abundance.
Environmental Sites Surveyed
Anaerobic Sites:
Zodletone Spring is an anaerobic, fissure spring located in Southwestern Oklahoma. The spring bubbles natural gas constantly. The spring flows about 20m into Stinking Creek (named due to the prevalent sulfide odor of the site). The sediment depth is ~20cm.
Bovine rumen fluid and feces of a fistulated steer at the livestock research facility at Oklahoma State University was used for sampling. The steer was fed a barley-grain feed.
Duck Pond (Norman, OK) and Theta Pond (Stillwater, OK) are fresh water ponds.
Waste water treatment plant (Stillwater, OK) secondary treatment.
Aerobic Sites:
5. Kessler Farm soil from a tall grass prairie reserve (McClain County, OK) that has been undisturbed for over 25 years.
Hydrocarbon contaminated soil from a tall grass prairie reserve in Osage County, OK.
Summary & Conclusion
References
1, Candidate Division SR1 was found in anaerobic environments, regardless the sulfide/sulfur concentrations but not aerobic environments.
2. The sequences obtained from this study significantly increases the number of SR1 sequences in databases.
3. SR1 is present in numbers ranging between 10-5 xxxx /gm sediment
3. Zodletone Spring had the highest diversity of the sites surveyed. Bovine rumen sequences had the lowest diversity.
4. SR1 diversity was highest in high sulfide-sulfur environments and lowest diversity in low sulfide-sulfur environments.
5. SR1 abundance was highest in low sulfide-sulfur/high temperature environments, lowest in high sulfide/sulfur, low temperature environments.
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