Introduction

1. Oklahoma State University - Dept. Microbiol. & Molec. Genetics 1110 Innovation Way Dr. Stillwater, OK 74078 (405) 744-3193 james.p.davis@okstate.edu and mostafa@okstate.edu 2008 BMBGSA Graduate Research Symposium FISHing for Uncultured Bacteria: Diversity, Enumeration, and Visualization of Candidate Division SR1 in Various Anaerobic Environments James P. Davis, and Mostafa S. Elshahed Oklahoma State University, Stillwater, OK Funding Provided by: NSF Microbial Observatories Program OSU Start-up funds to Dr. Mostafa Elshahed Fluorescent In Situ Hybridization 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. Develop probes for FISH, and determine morphology of SR1. Abstract Maximum Sequence Divergence & Diversity Candidate division SR1 has primarily been detected in sulfide/sulfur-rich, anaerobic environments, such as, Zodletone Spring (S.W.Oklahoma), hydrothermal vents, and Sulfur River sediment (Parker Cave, KY). Other than 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 (Zodletone Spring, fresh water pond sediments, bovine rumen and feces, and secondary treatment of a waste water treatment plant), and aerobic habitats (tall grass prairie soils). A universal bacterial forward primer with an 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 by phylogenetic analysis. Interestingly, the environment with the highest diversity (Zodletone Springs) had the lowest abundance, and the environment with the lowest diversity (bovine rumen) had the highest abundance. The diversity of candidate division SR1 was shown to be, 26 OTUs in 157 clones, in Zodletone Spring, and the diversity in bovine rumen was 2 OTUs in 33 clones. We developed an approach to quantify members of SR1 in the environment, using quantitative PCR (qPCR). The bovine rumen had the highest abundance (1.24 x 106 copies/g of rumen fluid), and Zodletone Spring had the lowest abundance (7.96 x 104 copies/g of sediment). 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. Using Fluorescent In Situ Hybridization (FISH), SR1 cells were visualized in three environments (Zodletone Spring, Theta Pond, and Sperm Pool (Yellow Stone National Park) microbial mat), and were shown to have a long, rod morphology, in all sites tested. 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. A B Table 2: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. C D 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. E F G H Table 3: 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. The Shannon Index for each site shows that Zodletone Spring has the greatest number of unique sequences, and bovine rumen has the least amount. J Figure 2: Whole cell hybridization of paraformaldehyde-fixed cells with Alexafluor 488-labeled SR1-specific probe (445f). Pictures A, C, E, G, I are DAPI-stained, and pictures B, D, F, H, J are Alexafluor stained. A, B are clone positive controls. C, D are negative controls (using Desulfotomaculum gothermicum). E, F are from Sperm Pool microbial mat (Yellow Stone National Park). G, H are from Theta Pond Sediment, and I, J are from Zodletone Spring sediment. The SR1 specific label showed a long, rod morphology in all sites. The negative control has one nucleotide mismatch with the SR1 probe, which shows the specificity of the SR1 probe, and the stringency of the conditions. 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. 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. Summary & Conclusion 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. Candidate Division SR1 was found in anaerobic environments, regardless the sulfide/sulfur concentrations but not aerobic environments. The sequences obtained from this study significantly increases the number of SR1 sequences in databases. SR1 is present in numbers ranging between 7.96x104 and 1.24x106 per gram sediment. Zodletone Spring had the highest diversity of the sites surveyed. Bovine rumen sequences had the lowest diversity. SR1 diversity was highest in high sulfide-sulfur environments, and SR1 diversity was lowest in low sulfide-sulfur environments. SR1 abundance was highest in low sulfide/sulfur, high temperature environments, and lowest in high sulfide/sulfur, low temperature environments. FISH showed cells belonging to SR1 in all three environments tested. The numbers appear to be in agreement with abundance numbers. SR1 morphology is long, rods. Table 4: 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. Geochemistry & Physical Properties of the sites 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 Farmsoil 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. Table 5: The geochemistry of each site shows that Zodletone Spring has the highest concentration of sulfide and zero valence sulfur. Rumen had the least concentration for all the variables tested. All units are in mM. References Elshahed, M. S., J. M. Senko, F. Z. Najar, S. M. Kenton, B. A. Roe, T. A. Dewers, J. R. Spear, and L. R. Krumholz. 2003. Bacterial Diversity and Sulfur Cycling in a Mesophilic Sulfide-Rich Spring. Appl. Environ. Microbiol. 69:5609-5621. Harris, J. K., Kelley, S.T., and N.R. Pace. 2004. New Perspective on Uncultured Bacterial Phylogenetic Division OP11 Appl. Environ. Microbiol. 70:845-849. Hsieh, Y. P., Shieh, Y.N. 1997. Analysis of reduced inorganic sulfur by diffusion methods: improved apparatus and evaluation for sulfur isotopic studies. Chem. Geol. 137:255-261. Li, L., C. Kato, and K. Horikoshi. 1999. Bacterial Diversity in deep-sea sediments from different depths. Biodiversity and Conservation 8:659-677. Perner, M., Seifert, R., Weber, S., Koschinsky, A., Schmidt, K., Strauss, H., Peters, M., Haase, K., and J. F. Imhoff. 2007. Microbial CO(2) fixation and sulfur cycling associated with low-temperature emissions at the Lilliput hydrothermal field, southern Mid-Atlantic Ridge (9 degrees S). Environ. Microbiol. 9:1186-1201 Zhu, X., Castleberry, S.R., Nanny, M.A., Butler, E.C. 2005. Effects of pH and Catalyst Concentration on Photocatalytic Oxidation of Aqueous Ammonia and Nitrite in Titanium Dioxide Suspensions. Environ. Sci. Tech. 39:3784-3791. Table 6: The physical properties show little difference between Zodletone Spring and Duck Pond. Bovine rumen has a significantly higher temperature, and has slight differences in salinity and pH.