Halophiles are the archaeal or bacterial species that can tolerate high salt concentrations. They are categorized as slight, moderate, or extreme, by the extent of their halo-tolerance. Slight halophiles prefer 0.3 to 0.8 M (1.7 to 4.8%), moderate halophiles 0.8 to 3.4 M (4.7 to 20%), and extreme halophiles 3.4 to 5.1 M (20 to 30%) salt content (Ollivier et al. 1994). The possible uses of halophilic microorganisms such as treatment of saline and hypersaline wastewaters and the production of beta-carotene, ectoine, exopolysaccharides, bioplastics and biofuel are being investigated.
Factors responsible for Halostability
A large repertoire of halophilic organisms has been identified. Search for genomes with the key words "halophilic" in NCBI genome search results in a total of 70 hits for halophiles. Like the genome of hyperthermophiles and thermophiles, the halophiles also have high GC content of around 60 to 70% which can avoid ultraviolet-induced thymidine dimer formation and mutations (Siddiqui and Thomas 2008). At the DNA level, compared to non-halophilic genomes, the halophiles exhibit distinct dinucleotides (CG, GA/TC, and AC/GT) at the first and second codon positions, reflecting an abundance of aspartate, glutamine, threonine, and valine residues in halophile proteins, which leads to their stability (Paul et al. 2014). The presence of high levels of CG dinucleotides leads to an increase in stacking energy and, thus, genome stability. By the above reports it can be concluded that the basis of extreme-stability selection purely based on GC content is fuzzy.
To investigate the molecular features responsible for protein halostability is of great significance for understanding the structure basis of protein halostability. Halophilic proteins are often also thermotolerant but possess less hydrophobicity (Setati 2010). In contrast to decrease in hydrophobic amino acid frequency, increase in smaller residues in halophilic proteins is also reported (Paul et al. 2008). The halophilic proteins also have very low bulky hydrophobic residues (Siglioccolo et al. 2011; Nayek et al. 2014). It has also been reported that weakening of the hydrophobic interactions in the protein core and conserved hydrophobic contacts result in protein stability in the presence of salts in preventing the proteins from aggregation at high salt concentrations (Siglioccolo et al. 2011). In halophilic proteins, lower content of bulky hydrophobic residues might indicate mandatory more polar protein interior than their mesophilic counterparts (Nayek et al. 2014). Similar to psychrophilic proteins, halophilic proteins are reported to prefer less aromatic residues for their stabilization under high salt concentrations (Longo and Blaber 2015). The aromatic substitutions in PV2 protein lead to the ability to move the folding properties from halophilic to mesophilic conditions (Longo and Blaber 2015).
The protein halostability is mainly due to the increased trend of negative surface charge due to increased acidic amino acid content (Asp and Glu) which compensates for the extreme ionic conditions (Reed et al. 2013). It has been noted that compared to non-halophilic homologs, the halostable proteins contain a greater proportion of negatively charged amino acids relative to positively charged amino acids (Rao and Argos 1981; Tokunaga et al. 2008). Correspondingly, there is an increased usage of negatively charged acidic amino acids Glu and Asp (Lanyi 1974). Acidic amino acid residues dominate their surface because they can hold the essential hydration shell layer for stability and catalysis intact at the surface of the protein. This is reflected in the low isoelectric values of halophilic proteins (Siddiqui and Thomas 2008).
One interesting report by Pavlov et al. (2002) reveals that the presence of helix-hairpin-helix motif in DNA polymerases was reported to increase its stability at high salt concentration (Pavlov et al. 2002). Recently, Alzbutas et al. (2015) reported that the DNA binding domains are potential activators of enzymatic activity at high ionic strength of extremely halotolerant bacterium Thioalkalivibrio sp. K90mix Dnase (Alzbutas et al. 2015). The activation is mainly due to the increase in the number of negatively charged residues on the surface of proteins adapted to high ionic strength (de Vega et al. 2010; Pavlov et al. 2012; Alzbutas et al. 2015). Such adaptation would hinder the binding to negatively charged DNA, a step critical for catalysis. The DNA binding domain has duplicate binding helix-hairpin-helix motifs that were examined to be responsible for the resistance to high ionic strength (Alzbutas et al. 2015).
For halophilic adaptation of protein, electrostatic stabilization was suggested as the key determinant of their stability. However, contribution of specific electrostatic interactions (i.e. salt-bridges) to overall stability of halophilic proteins is yet to be understood (Nayek et al. 2014). Therefore, the major focus of Physical Chemistry was in understanding the stability conferred by electrostatic interactions to render proteins stable in high salt concentration (Pieper et al. 1998). For example, Malate dehydrogenase from Haloarcula marismortui shown greater number of salt-bridges than its mesophilic counterpart which enhanced enzyme stability at high salt concentrations (Dym et al. 1995). Again, the destabilization of halophilic proteins at low-salt concentration was reported to be due to strong electrostatic repulsion (Elcock and McCammon 1998).
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