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

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 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 http://141.150.157.117:8080/prokPUB/index.htm

Spring Salinity Stream Salinity 0.7-1.0% Mat Salinity 2-5% 12 30 Depth from surface (in cm intervals) 5 10 15 20 25 35 1 2 3 4 Moisture (%) 6 9 Salinity (%) (A) (B) Stream Salinity 0.7-1.0% Mat Salinity 2-5% Soil (top layer) 5 cm- >30% 30 cm- >25% Salinity (--) moisture content (--) (Elshahed et al. 2004)

Isolation of Haloarchaea Isolated halophiles from the mat using a high salt (7, 12 and 18%) plus antibiotic medium Serially diluted mat material and plated onto HM plates Cultures were incubated at 37ºC under light

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

Metabolism Group II Poor growth on amino acids Sugars Sugar Alcohol O.D. = 600nm Organic Acids Amino Acids Poor growth on amino acids Preferred substrates: Glucose, sucrose and starch GY252 had a larger range of substrate utilization O.D. = 600nm

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 0% 0.5% 1.0% 2.0% 3.0% 4.0% 5.0% Hours + 4 12 24 48 312 - Halophilic Medium Sterile H2O

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