Halothermothrix orenii is of interest because it survives hot, salty, anaerobic environments and produces thermohalophilic enzymes for biotechnology. It is also a high hydrogen producer and is used in bioremediation of hot salty oil fields.

Survival strategies of Halothermothrix orenii in hot salty environments -Thermohalophiles, grow optimally at temperature > 60 ° C with NaCl concentrations >5%, and are unique amongst extremophiles as they have adapted to more than one environmental stresses.   If one were to consider the habitats in which thermohalophiles could thrive, than halite-saturated lakes of Australia, Africa and the Middle East, geothermally heated brine below the Red Sea and petroleum reservoirs, would immediately come to mind (Patel et al., 1995). However, thermohalophiles appear to be rare in nature and despite the intensive searches made by many research groups only two strains have so far been isolated: Halothermothrix orenii of the order Haloanaerobiales (Cayol et al., 1994) grows optimally at 60 ° C (maximum of 70 ° C) with 10% NaCl (NaCl growth range between 4 - 20%), and, Thermohalobacter berrensis of the order Clostridiales (Cayol et al., 2000) grows optimally at 65 ° C with 5% NaCl, domain Bacteria with no reports yet on hyperthermohalophilic Bacteria . Additionally, both are strict anaerobes and therefore have to contend with a further stress of presence of oxygen. It appears that the simultaneous exposure to hot salty anaerobic conditions could severely limit life and hence the rarity of thermohalophile diversity. To determine the bottlenecks to life under hot, salty and anaerobic conditions, we have focused our attention on H. orenii and are extending our studies the salty environment of the non-volcanically geothermally heated Great Artesian Basin (GAB) of Australia aquifer.

(i) H. orenii proteins lack excess of positively charged amino acids: Genome tag analysis (Mijts and Patel, 2001) indicated that H. orenii did not contain high proportions of positively charged acidic amino acids. Analysis of crystal structures of two extracellular thermoactive and halophilic recombinant amylases (AmyA and AmyB) from H. orenii also showed a lack of positively charged surface amino acids (Mijts, 2001; Mijts and Patel, 2001) suggesting that H. orenii possessed a "salt out" survival strategy. This finding was unexpected as the immediate phylogenetic relatives of H. orenii Patel et al., 1995), are the mesophilic halophiles and hyperhalophiles, all of which contain an excess of positively charged surface amino acids in their proteins and therefore posses a "salt in" strategy for survival.

(ii) Protein structure and stability under thermohalophilic conditions : The biochemical properties and the structure of the recombinant amylases, AmyA and AmyB have been studied (Mijts and Patel, 2002; Li et al., 2002; Tan et al., 2003; Sivakumar et al., 2006). Both enzymes are not close phylogenetic relatives: AmyB is closely related to amylases from the mesophilic Bacillus species whereas AmyA is related to the amylases from the thermophiles Dictyoglomus thermophilium and Thermotoga maritimea .   Though both AmyA and AmyB are optimally active at 65 ° C, AmyA retains high levels of activity (90% activity in 25% NaCl) than AmyB (15% activity in 25% NaCl. AmyA but not AmyB forms a unique reversible oligomeric, thermostable protein resistant to boiling in the absence of NaCl but in the presence of as little as 5mM NaCl or CaCl 2 thermostability decreased dramatically. This change cannot be attributed to any structural change as protein crystallography shows that the structure is maintained over the entire salinity range indicating that there may be other unknown subtle interplay between temperature and salts that maintain structural integrity. Comparative analysis of AmyA with other amylases from thermophiles and halophiles has shown that there is marked conservation in the buried residues but the surface accessible residues are unique. It can be hypothesised that the oligomeric form maintains the structural integrity of AmyB when the protein is exposed to higher than normal salinities and temperatures and the reversion to a monomeric form when the adverse conditions have passed. Mutational studies, initially of the surface amino acids, will be performed to determine the mechanism underlying oligomerization and adaptation of proteins to high. Fortunately, the recombinant AmyB can be purified (>99%) in a single step by heating E. coli lysate in the presence of NaCl and can be easily used for crystal production.

(iii) Compatable solutes are involved in osmoregulation : Sucrose phosphate synthase (SPS) has been cloned and characterized from H. orenii (Huynh et al., 2005; Huynh, 2005). The SPS is found in an operon involved in the synthesis and regulation of sucrose. Sucrose is a source of nutrient in plants and is also an osmoregulator which regulates the rate of water evaporation by opening and closing stomata cells. Sucrose may be an important osmoregulator in H. orenii which allows it to maintain an osmotic balance of the cell cytoplasm with the outside environment.

(iv) Proteins of H. orenii are easier to crystallize : We have to date cloned, expressed, purified and crystallized 4 out of the 4 proteins. This is an unusually high hit rate as only up to 40% of the proteins from a given microbe have been shown to crystallize in high throughput studies. Interestingly, the SPS produced crystals within 36 hours.   It is most likely that proteins from thermohalophiles may be easy to crystallize.

(v) Sequencing ratioale: The sequencing of the genome of H. orenii will provide an insight into survival and adaptation strategies of thermohalophiles. It will also assist in determining the molecular structural bottlenecks that could limit life under hot salty anaerobic conditions. The availability of the genome sequence will assist in determining whether proteins from thermohalophiles are inherently easier to purify and crystallize than those from thermophiles.  

References:

Cayol, J.-L., Ollivier, B., Patel, B.K.C., Prensier, G., Guezennec, J., Garcia, J.-L. (1994). Isolation and Characterization of Halothermothrix orenii gen. nov., sp. nov., a Halophilic, Thermophilic, Fermentative, Strictly Anaerobic Bacterium. International Journal of Systemic Bacteriology 44, 534-540.

Cayol, J. L., Ducerf, S., Patel, B.K.C., Garcia, J.-L., Thomas, P. and Ollivier, B. (2000). Thermohalobacter berrensis gen. nov., sp. nov., a thermophilic, strictly halophilic bacterium from a solar saltern.   International Journal of Systematic and Evolutionary Microbiology. 50, 559-564

Huynh, F. (2004). Structure-Function relationships of proteins from the halothermophile, Halothermothrix orenii . Griffith University, BSc (Hons) thesis.

Huynh, F., Tien-Chye Tan, T.-C., Swaminathan, K. and Patel, B. K. C. (2005). Expression, purification and preliminary crystallographic analysis of sucrose phosphate synthase (SPS) from Halothermothrix orenii . Acta Crystallographica. Section F

Li., N., Patel, B.K.C., Mijts, B. N. and Swaminanthan, K. (2002). Crystallization of an alpha-amylase, AmyA, from the thermophilic halophile Halothermothrix orenii . Acta Crystallography D58, 2125-2126.

Mijts, B. N. (2001). Genes and Enzymes of Halothermothrix orenii . Griffith University, PhD thesis

Mijts, B. N. and Patel, B.K.C. (2001). Random sequence analysis of genomic DNA of an anaerobic, thermophilic, halophilic bacterium, Halothermothrix orenii . Extremophiles 5, 61-69

Mijts, B. N. and Patel, B.K.C. (2002). Cloning, sequencing and expression of an alpha-amylase gene, amyA, from the thermophilic halophile Halothermothrix orenii and purification and biochemical characterization of the recombinant enzyme. Microbiology 2002 148: 2343-2349.  

Patel, B. K. C., Andrews, K. T., Ollivier, B., Mah, R. A. & Garcia, J. L. (1995). Reevaluating the classification of Halobacteroides and Haloanaerobacter species based on sequence comparisons of the 16S ribosomal RNA gene. FEMS Microbiology Letters 134, 115-119.

Sivakumar, N., Li, N., Tang, J.W.,Patel, B.K.C. and Swaminathan, K. (2006). Crystal structure of AmyA lacks acidic surface and provide insights into protein stability at poly-extreme conditions. FEBS Letters 580, 2646-2652.

Tan, T.-C., Yien, Y.Y., Patel, B.K.C., Mijts, B.N. and Swaminathan, K (2003). Crystallization of a novel -amylase, AmyB, from the thermophilic halophile, Halothermothrix orenii. Acta Crystallography. D59, 2257-2258.