1. Halophilic Archaea at Zodletone Spring
Kristen Savage

2. Zodletone Geochemistry
Source
High concentrations of dissolved S (8-10mM)
0.2 M NaCl
Barium and sulfate
Dissolved Sulfur
Barite and calcite

3. Archaeal Diversity

4. 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 )

5. 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

6. 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 %
Mat Salinity 2-5%
Soil (top layer)
5 cm- >30%
30 cm- >25%
Salinity (--)
moisture content (--)
(Elshahed et al. 2004)

7. 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

8. 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

9. 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

10. 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

11. Novel Group II

12. 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

13. 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

14. Viability and Recovery DX2530%
0.5%
1.0%
2.0%
3.0%
4.0%
5.0%
Hours + 4 12 24 48
312 -
Halophilic Medium
Sterile H2O

15. 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

16. Acknowledgements Dr. Krumholz Dr. Elshahed Dr. Oren Dr. Ventosa
Tracy Sisk