Plated onto halophilic medium plates (1.5% agar). Phylogeny 16S rRNA sequence analysis of strain M6 showed a 96.7-98.0% similarity to other validly described species of this genus M6 was 89% similar to Halogeometricum borinquense the most closely related species outside the genus Strain M-6 Gram negative Motile Extremely pleomorphic Scanning Electron Microscopy revealed flat rods and cocci Colonies were small (2-3 mm) and salmon pink in color at 37°C A scanning electron micrograph of a exponential phase (Left) and A stationary phase (right) culture of strain M6. Salinity Measurements Depth from surface (in cm intervals) 5 10 15 20 25 30 35 1 2 3 4 Moisture (%) 12 6 9 Salinity (%) (A) (B) Salinity (--) moisture content (--) Core A: 5 cm from the stream bank Core B: 30 cm from the stream bank Salt profiles revealed that there were areas found throughout the mat and on the banks where moisture decreased and salinity increased presumably due to evaporation. It is probable that these areas are providing microenvironments that facilitate the growth of these Halophilic Archaea Abstract Zodletone spring is located in Southwestern Oklahoma and is characterized by a high concentration of dissolved sulfur and sulfide and a stream salinity of 1%. We recently encountered an unexpected abundance of members of the extreme halophilic Archaea of the order Halobacteraceae in the spring microbial mats. Measurement of the salinity at various locations of the spring and along vertical gradients revealed that shallow and superficial areas, where microbial mats are usually encountered, have a much higher salinity (up to 32% NaCl concentration) and lower moisture content compared to the values detected in the spring water, presumably as a result of evaporation. In this study we characterize one of the isolates, strain M6, as a new species. Strain M6 cells were pleomorphic and were capable of growth at salt concentrations ranging from 6% to saturation, and at least 1 mM Mg2+ was required for growth. Strain M6 was characterized by its ability to anaerobically reduce elemental sulfur to sulfide, and was capable of growth in defined media with a range of carbon substrates including benzoate. 16S rRNA sequence analysis indicated that it belongs to the family Halobacteriaceae, genus Haloferax, with similarity values between 96.7-98% to other described members of the genus, and 89% to Halogeometricum borinquense, its closest relative outside the genus Haloferax. Polar lipid analysis further supported placement of strain M6 in the genus Haloferax. Members of this genus are most commonly found in areas of high salt including salterns and the Dead Sea. Biochemical and physiological characterization allowed differentiation of strain M6 from the other members of the genus Haloferax, and we propose a new species, Haloferax sulfurifontis, to accommodate this strain. This work demonstrates that members of the Halobacteriaceae are not restricted to their typical hypersaline habitats, but can also inhabit lower salinity environments where localized NaCl concentrations are high enough to allow their survival. Strain M6 can reduce S° anaerobically Interestingly, Strain M6 was capable of reducing sulfur under anaerobic conditions The abundance of this organism in this environment coupled with the ability of it to reduce elemental sulfur may suggest that it has its’ own role in the sulfur cycle at Zodletone t0 4 days Medium: Yeast extract, sulfur, ferrous ammonium Sulfate 100 200 300 Sulfide produced (µM) 10 20 30 Time (Days) (--) Elemental sulfur (-∆-) elemental sulfur, but no substrate (-o-) Medium with no elemental sulfur (--) Uninoculated medium Characterization Strain M-6 has a lower temperature optimum than other species of the genus Haloferax M-6 has a wide range of NaCl concentration in which it can grow M-6 can recover from low NaCl concentrations which could explain why it is able to survive in this low salt environment M-6 was capable of growth on a wide range of carbon substrates including benzoate Introduction Zodletone Spring An artesian sulfur containing, mesophilic spring located in the Anadarko Basin of Southwestern Oklahoma Surface water contains high sulfide concentrations (8-10mM) and S°. Short chain gaseaous alkanes (methane, ethane and propane) impact the source water. The spring salinity ranges from 0.7-1.0% Sunlight is an important energy source driving the formation of purple and green phototrophic mats throughout the spring. The spring flows for approximately 20 meters before discharging into a creek. Extreme Halophilic Archaea in Zodletone spring (Family Halobacteriaceae) Molecular analysis of 16s rRNA of the spring community indicated that 36% of Archaeal mat clones belonged to 5 different groups within the family Halobacteriaceae (1). Members of this family are known to be aerobic heterotrophs. Halobacteriaceae require high salt concentration ranging from 3.5 M NaCl to saturation (5.2 M) Isolated from hypersaline environments (I.e. Salterns, the Dead Sea, hypersaline lakes) Zodletone: Microbial Mats Methods Serially diluted (up to 10-7) mat material into a yeast extract based halophilic medium(2) treated with kanamycin and ampicillin (75µg/ml). Three different NaCl concentrations were used (7,12 and 18%). Plated onto halophilic medium plates (1.5% agar). The plates were incubated at 37°C under light until pigmented colonies began to grow (about 2 weeks) Individual colonies were picked from the plates, restreaked twice and microscopically checked for purity. The strain of interest was further characterized following the guidelines provided by Oren et al. (2). All biochemical testing followed procedures outlined in Reference (3). DNA was extracted from the colonies and PCR amplified using Archaeal specific primers Conclusions A novel species of the genus Haloferax was isolated from Zodletone Spring for which we propose the name Haloferax sulfurifontis Characterization of strain M6 supported its’ affiliation with the genus Haloferax Closer analysis of the salt profiles of Zodletone indicated tha,t although overall spring salinity did not exceed 1%, there are areas in the mat and on the banks where moisture decreases and the concentration of NaCl increases. These hypersaline pockets may provide a suitable environment for the growth of extreme halophilic Archaea. Clone libraries indicate that there are diverse members of the halophilic Archaea present in the mat, however, only one strain was successfully isolated This research proves that extreme halophilic Archaea may be more abundant in low salt environments than previously believed if conditions allow for areas of high salt concentration to develop. Growth and Survival of strain M6 under Various NaCl Concentrations 0.0E+00 2.5E+07 5.0E+07 7.5E+07 1.0E+08 Cells number (celss /ml) 5 10 15 20 25 Time (days) (--) distilled water (-o-) 1% (--) 2% (-∆-) 3% (--) 4% (--) 5% (--) 15% 0.4 0.8 1.2 1.6 2 Growth rate (day-1) 30 35 40 Salt concentration (%) Optimal NaCl concentration for growth of M6 is 12.5 - 15% When NaCl concentrations are as low as 4% M6 cells can maintain their viablity DNA-DNA Hybridization DNA-DNA hybridization studies showed that strain M6 had a hybridization value between 0-24% to all other species of the genus Haloferax Current and Future Work Isolating novel halophilic Archaea using eleven different substrates and three different salt concentrations (18, 25 and 30%) Preliminary investigation suggest that two of the obtained isolates belong to novel genera within the family Halobacteriales (only 92% and 93% similar to their closest cultured relatives). Few selected strains will be further characterized References Elshahed, M.S., Najar, F.Z., Roe, B.A., Oren, A., Dewers, T.A. and Krumholz, L.R. (2004). Survey of archaeal diversity reveals an abundance of halophilic Archaea in a low-salt, sulfide- and sulfur-rich sprin. Appl. Environ. Microbiol. 70: 2230-2239. Oren, A., Ventosa, A. & Grant, W. D. (1997). Proposed minimal standards for description of new taxa in the order Halobacteriales. Int J Syst Bacteriology 47, 233-238. Gerhardt, P., Murray, R. G. E., Wood, W. A. & Krieg, N. R. (1994). Methods for General and Molecular Bacteriology, pp 227-248. Washington, D.C.: American Society for Microbiology