Haloferax sulfurifontis sp. nov., a Halophilic Archaeon Isolated from a Low Salt Sulfide-rich Spring K. N. Savage 1, M. S. Elshahed 1, A. Oren 2, L. R. Krumholz 1 1 University of Oklahoma, Norman, OK, 2 The Hebrew University of Jerusalem, Jerusalem, ISRAEL

Zodletone Spring 8 lmin m from source to creek High sulfide concentrations (8- 10mM) 0.2 M NaCl F, Br, Li, B, Sr, Ba, SO 4 2- BaSO 4 and CaCO 3 Dissolved sulfur

Sulfur Cycling S 2- S° SO 4 2- H2SH2S H2SH2S H2SH2S H2SH2S H2SH2S H2SH2S H2SH2S H2SH2S H2SH2S

Sulfur Cycling H2SH2S H2SH2S H2SH2S H2SH2S H2SH2S H2SH2S H2SH2S H2SH2S H2SH2S SO 4 2- S° HS - SO Sulfur Disproportionation Sº HS - SO 4 2-

Archaeal Diversity

Halophilic Archaea 14 validly described genera Isolated from hypersaline environments (i.e. The Dead Sea) Rods, cocci and pleomorphic forms Require high salt concentrations from 3.5 M NaCl to saturation (5.2 M) Contain pigment material (bacteriorhodopsin and halorhodopsin) which gives them a distinct pink or red color

Isolation of Haloarchaea 36% of the mat and 4% of the source library Isolated halophiles from the mat using a high salt plus antibiotic medium with different salt concentrations (7, 12 and 18%) Serially diluted mat material and plated onto HM plates (immediately and after incubation) Cultures were incubated at 37ºC under light

A Novel Species of Haloferax 16S rRNA sequence analysis of strain M6 showed a % similarity to other validly described species of this genus M6 was 89% similar to Halogeometricum borinquense the most closely related species outside the genus DNA-DNA hybridization confirmed its novel speciation having a hybridization value of only 24% to Haloferax gibbonsii

Phylogeny

Haloferax sulfurifontis Isolated from Zodletone spring Extreme pleomorphism Colonies were small (2-3 mm) and salmon pink in color at 37°C Growth occurred in a wide range of salt concentrations (6% to saturation) Required at least 1 mM Mg 2+ for growth Anaerobically reduces Sº

Haloferax sulfurifontis

A Continued Search for Halophiles Clone libraries indicated the presence of a diverse 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 encourage the growth of different isolates the media was prepared at 3 different salt concentrations (18, 25 and 30%) and 11 different carbon sources were used instead of yeast extract

Future Work Microscopic inspection of colonies to determine cell morphologies present and trends Further purification and isolation Colony PCR and sequencing Anaerobic reduction of S° Strain characterizations

Acknowledgements Dr. Krumholz Dr. Elshahed Aharon Oren and Antonio Ventosa Roe Laboratory Funding for this project is by the National Science Foundation