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Halophilic Archaea at Zodletone Spring Kristen Savage Zodletone Geochemistry Source •High concentrations of dissolved S2(8-10mM) •0.2 M NaCl •Barium and sulfate •Dissolved Sulfur •Barite and calcite Archaeal Diversity Source Archaea Crenarcharcheota 6% UAG 36% Methanogens 54% Halophiles 4% Mat Archaea Crenarchaeota 12% Uncultured 6% Halophiles 36% Methanogens 46% Phylogeny of Halophilic Archaea • 5 different groups within the order Halobacteriales • Groups I & II represent novel genera • Groups III & V related to previously described genera • Group IV represented all source clones (Elshahed et al. 2004) Halophilic Archaea http://141.150.157.117:8080/prokPUB/index.htm • Dominate hypersaline environments • Require at least 8% NaCl for growth (20-26% NaCl) • Aerobic • Heterotrophic • Characterized by red, pink or orange coloration • Order Halobacteriales – 15 genera, 44 species • Variety of shapes and sizes Spring Salinity 30 25 9 20 Moisture (%) 15 6 10 3 5 0 30 0 35 (B) 30 25 25 20 20 15 15 10 (Elshahed et al. 2004) Salinity (--) 5 moisture content (-•-) 0 Salinity (%) • Stream Salinity 0.7-1.0% • Mat Salinity 2-5% • Soil (top layer) • 5 cm- >30% • 30 cm- >25% 12 (A) 10 5 0 1 2 3 4 5 0 Depth from surface (in cm intervals) A Continued Search for Halophiles • Clone libraries indicated the presence of a diverse and novel halophilic community • Originally18 strains were isolated from the mats present at the stream • Studies indicated that these isolates were of the same species • In order to stimulate the growth of different isolates the medium was prepared with 3 different salt concentrations (18, 25 and 30%) and 11 different carbon sources. Ampicillin and Kanamycin were used to select against halotolerant bacteria Preliminary Screening • Sequenced approximately 30 isolates • Isolates clustered into four different groups • Two groups (III and IV) clustered with previously defined genera (Halogeometricum and Haloferax) • Groups I and II represented novel genera, but clustered closely with some uncultured clones Diversity of Halophilic Isolates • BZ256 is 98% similar to ZAR25 (Clone Group III) • Novel Group II isolates cluster with ZAR31(Clone Group I) • Clone groups II, IV, and V had no cultured representatives Novel Group II NaCl Range (optimum) MgCl2 pH Range (optimum) Temperature Range (opt.) Colony Morphology Cell Morphology Doubli ng Time % Relatedness DX253 5-30% (18%) 5-200 mM 4.5-7.5 (6.5) 25-45C (30C) GY252 5-30% (15%) 5-200 mM 4.5-9 (6.0) 25-45C (25C) Large round co lonies, raised, transparent, dark pink Medium cocci, tetrads and clumps 13.25 hr/gen 92% Natronorub rum tibetense Small round colon ies, raised, transparent, light pink Smaller cocci, singles and pairs 8.61 hr/gen 89% Natronorub rum tibetense Sugars 0.8 O.D. = 600nm Sugar Alcohol 0.7 0.6 Organic Acids 0.5 0.4 Amino Acids 0.3 0.2 PY MAN SO GYR MAN SO GYR SNT SNT PY FA CT FA CT LT MA MA AC LT GAL FS LAC ST XY DX SUC AA GN AL GLT AG Positive 0 Negative 0.1 Substrate GY252 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 Substrate AC GAL LAC FS ST DX XY SUC AA GN GLT AL AG Positive 0 Negative • Poor growth on amino acids • Preferred substrates: Glucose, sucrose and starch • GY252 had a larger range of substrate utilization 0.9 O.D. = 600nm Metabolism Group II DX253 Viability and Recovery • Determine the ability of cells to recover from low salt conditions • Prepared HM (0-5%) NaCl and sterile H2O solutions (0-5%) NaCl • Inoculated washed cells and attempted to recover the cells in standard HM liquid at various time points Viability and Recovery DX253 Sterile H2O Halophilic Medium 0% 0.5% 1.0% 2.0% 3.0% 4.0% 5.0% Hours + + + + + + + 0 + + + + + + + 4 + + + + + + + 12 + + + + + + + 24 + + + + + + + 48 + + + + + + + 312 + + + + + + + 0 + + + + + + + 4 + + + + + + + 12 + + + + + + + 24 + + + + + + + 48 - - + + + + + 312 Conclusions • Haloarchaea at Zodletone Spring are numerous, diverse and culturable • Extreme halophiles may be more ubiquitous in nature than previously believed • May play an important role is sulfur cycling at Zodletone and may be essential to other cycling processes in other environments Acknowledgements • • • • • Dr. Krumholz Dr. Elshahed Dr. Oren Dr. Ventosa Tracy Sisk