85N. Tuteja and S. Singh Gill (eds.), Plant Acclimation to Environmental Stress,DOI 10.1007/978-1-4614-5001-6_4, Springer Science+Business Media New York 2013
Life is based on chemistry which must be allowed to function for life to continue. Extremophiles adopt two distinct approaches within extreme environments; they might adapt to function in the physical and chemical limits of their environment or maintain mesophilic conditions intracellular, withstanding the external pressures. Among the extremophiles, halophiles are an interesting class of organisms adapted to moderate and hyper saline environments.
Halophiles are represented by all three domains of life. Among the bacteria; the phyla Cyanobacteria, Proteobacteria, Firmicutes, Actinobacteria, Spirochaetes, and Bacteroidetes are commonly reported. In Archaea, the most salt-requiring microorganisms belong to Halobacteria. Halobacterium and most of its relatives require over 100150 g/L salt for growth and structural stability. Halophilic microorganisms use two strategies to balance their cytoplasm osmotically with their medium. The rst involves accumulation of molar concentrations of KCl. This strat-egy requires adaptation of the intracellular enzymatic machinery, as proteins should maintain their proper conformation and activity at near-saturating salt concentrations (Oren 2008 ) .
The minimum salt concentration required for growth, the salinity optimum, and the upper salt limit toleratedwithin the microbial world highlighted towards a continuum of properties, which makes it nearly impossible to de ne by sharp boundaries. Moreover, the minimum, optimum, and maximum salt concentrations often depend on the medium composition and growth temperatures. The most widely used de nitions were formulated 30 years ago (Kushner 1978 ) which distin-guished halophiles into following categories: extreme halophiles (growing best in
S. P. Singh (*) V. Raval M. K. Purohit Department of Biosciences , Saurashtra University , Rajkot 360 005 , India e-mail: email@example.com
Chapter 4 Strategies for the Salt Tolerance in Bacteria and Archeae and its Implications in Developing Crops for Adverse Conditions
Satya P. Singh , Vikram Raval, and Megha K. Purohit
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media containing 2.55.2 M salt), borderline extreme halophiles (growing best is media containing 1.54.0 M salt), moderate halophiles (growing best in media containing 0.52.5 M salt), and halotolerant microorganisms that do not require salt for growth but grow well up to reasonably high salt concentrations. The extreme halotolerant groups have growth range above 2.5 M salt. These de nitions, though with loose boundaries, are valuable in the classi cation of microorganisms according to their relationship with salt (Oren 2002, 2006 ; Ventosa et al. 1998 ) . Majority of halophilic organisms accumulate organic compounds, e.g. betaine, ectoines, and glycerol. These solutes could be quite useful for commercial applications.
Only few reports on the occurrence of plasmids and their physiological and ecological signi cance from haloalkaliphiles are available. Attempts are being made towards developing vectors and expression systems of halophilic origin to express their genes and investigate the regulation of gene expression.
Towards this end, exploration of diversity, phylogeny and biochemical and genetic characteristics of extracellular enzymes from haloalkaliphilic bacteria and actinomycetes dwelling in relatively moderate saline habitats have generated interesting clues on the functioning of enzymes under multitude of extremities (Dodia et al. 2008a, b ; Gupta et al. 2005 ; Joshi et al. 2008 ; Nowlan et al. 2006 ; Patel et al. 2005, 2006 ; Purohit and Singh 2009 ; Ram et al. 2010 ; Siddhapura et al. 2010 ; Thumar and Singh 2007, 2009 ) .
2 Strategies for Salt Adaptation
The halophilic proteins are highly acidic in nature and majority would denature in low salt concentration. The other strategies include exclusion of salt from the cytoplasm and to synthesize and accumulate organic compatible solutes that do not interfere with enzymatic activity. The organisms using the organic-solutes-in strategy often adapt to a surprisingly broad salt concentration range (Cho 2005 ) . Most halophilic bacteria and the halophilic methanogenic archaea a number of such organic solutes like glycine betaine, ectoine and other amino acid derivatives, sugars and sugar alcohols. The high-salt-in strategy is not limited to the Halobacteriaceae. The Halanaerobiales (Firmicutes) also accumulate salt rather than organic solutes. A third, phylogenetically unrelated group of organisms accumulates KCl: the extremely halophilic Salinibacter (Bacteroidetes), recently isolated from saltern crystallizer brines. Analysis of its genome revealed its resemblance with Halobacteriaceae, which probably is a result of horizontal gene transfer. The exam-ple of Salinibacter indicates towards further discovery of unusual halophiles.
Bacteria and Archaea have developed two basic mechanisms to cope with osmotic stress. The salt-in-cytoplasm mechanism involves adjusting the salt concentration in the cytoplasm. The organic-osmolyte mechanism involves a ccumulation of uncharged and highly water-soluble organic compounds to maintain an osmotic equilibrium with the surrounding medium. The osmo-adaptation of prokaryotes through the organic-osmolyte strategy introduces a model of the ne-tuning of osmo regulatory osmolyte synthesis (Kunte 2006 ) .
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Mechanisms of adaptation of halophilic microorganisms at high salt points out towards Trpers four postulates, as presented in Alicante symposium (Truper et al. 1991 ) . It deals with the presence, distribution and biosynthesis of organic osmotic solutes. A basic property of all halophilic microorganisms relate to the fact that their cytoplasm has to be atleast iso-osmotic with their surrounding medium. Biological membranes are permeable to water, and active energy-dependent inward transport of water to compensate for water loss by osmotic processes is energeti-cally not feasible. Moreover, cells that keep a turgor need even to maintain their intracellular osmotic pressure higher than that of their environment (Brown 1976, 1990 ) . There are two fundamentally different strategies used by halophilic microor-ganisms to balance their cytoplasm osmotically within their medium. The rst involves accumulation of molar concentrations of potassium and chloride. This strategy high-salt-in strategy requires extensive adaptation of the intracellular enzy-matic machinery to the presence of salt, as the proteins should maintain their proper conformation and activity at near-saturating salt concentrations (Lanyi 1974 ) . The second strategy is to exclude salt from the cytoplasm and to synthesize and/or a ccumulate organic compatible solutes that do not interfere with enzymatic a ctivity. Few adaptations of the cells proteome are needed, and organisms using the organic-solutes-in strategy often adapt to a surprisingly broad salt concentration range. Most halophilic bacteria and some halophilic methanogenic archaea use such organic solutes. A variety of such solutes are known, including glycine betaine, ectoine and other amino acid derivatives, sugars and sugar alcohols. Far more widespread in nature is the second strategy of halo-adaptation based on the biosynthesis and/or accumulation of organic osmotic solutes. Cells that use this strat-egy exclude salt from their cytoplasm as much as possible. The high concentrations of organic compatible solutes do not greatly interfere with normal enzymatic activ-ity. Such organisms can often adapt to a surprisingly broad salt concentration range (Ventosa et al. 1998 ) . The list of organic compounds known to act as osmotic solutes in halophilic microorganismsprokaryotic as well as _eukaryoticis extensive. Most compatible solutes are based on amino acids and amino acid derivatives, sugars, or sugar alcohols. Many are either uncharged or zwitter ionic (Galinski 1986 ; Roberts 2005, 2006 ) .
Although the high-salt-in strategy is energetically less costly to the cell than the biosynthesis of large amounts of organic osmotic solutes (Oren 1999 ) , this strategy is not widely used among the different phylogenetic and physiological groups of halo-philes. It is best known from the extremely halophilic Archaea of the family Halobacteriaceae, and species such as Halobacterium salinarum and Haloarcula marismortui . These organisms have emerged as popular model organisms to examine the implications of the accumulation of high intracellular KCl concentrations.
Our understanding of the biology of the Halobacteriaceae has greatly increased in recent years due to the elucidation and analysis of the genome sequences of Halobacterium NRC-1 (Kennedy et al. 2001 ; Ng et al. 2000 ) , Haloarcula marismortui (Baliga et al. 2004 ) , Natronomonas pharaonis (Falb et al. 2005 ) , and Halquadratum walsbyi (Bolhuis et al. 2006 ) . The strategy of salt adaptation is not limited to the aerobic halophilic archaea. The anaerobic fermentative Halanaerobiales
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(Bacteria, Firmicutes) also use potassium chloride (KCl) rather than organic solutes to osmotically balance their cytoplasm and are also have adapted their intracellular machinery to tolerate the salt (Oren 1986, 2006 ) .
3 Mechanisms for Stress Tolerance
The organisms living in extreme conditions possess special adaptation strategies that make them interesting not only for fundamental research but also towards exploration of their applications (Horikoshi 2008 ) . These organisms may hold secret, for the origin of life and unfold many basic questions about the stability of the macromolecules, under extreme conditions. Therefore, their studies would provide important clues for adaptation under salinity. To cope with high and often changing salinity of their environment, the aerobic halophilic bacteria, similar to all other microorganisms, need to balance their cytoplasm with the osmotic pressure exerted by the external medium (Oren 2008, 2010 ) . Osmotic balance can be achieved by the accumulation of salts, organic molecules, or similar mechanism. Alternatively, the cell is able to control water movement in and out and maintain a hypo osmotic state of their intracellular space.
The extremely halophilic archaea and bacteria, adopt various strategies; molar con-centrations of chloride is pumped into the cells by co-transport with sodium ions and/or using the light-driven primary chloride pump halorhodopsin (Shazia 2004 ) . Distribution of charged amino acids could also serve as one of the major approaches. Certain organisms show a speci c requirement of chloride for growth, endospore germination, motility and agellar synthesis, and glycine betaine transport (Muller and Oren 2003 ) .
3.1 Chloride Pumps
A high requirement for chloride was demonstrated in two groups of bacteria; anaerobic Halanaerobiales and the aerobic extremely halophilic Salinibacter rubber , that accumulate inorganic salts intracellularly rather than using organic osmotic solutes. Thus, it is clear that chloride has speci c functions in halo- adaptation in different groups of halophilic microorganisms (Muller and Oren 2003 ) .
Osmoregulation is a fundamental phenomenon developed by bacteria, fungi, plants, and animals to overcome osmotic stress. The most widely distributed strategy of response to hyperosmotic stress is the accumulation of compatible solutes, which protect the cells and allow growth. Adaptation of bacteria to high solute concentra-tions involves intracellular accumulation of organic compounds called osmolytes .
894 Strategies for the Salt Tolerance in Bacteria
Osmolytes are referred as compatible solutes because they can be accumulated to high intracellular concentrations without adversely affecting cellular processes. The solutes can be either taken up from the environment or synthesized de novo , and they act by counterbalancing external osmotic strength and thus preventing water loss from the cell and plasmolysis. Since cytoplasmic membrane is highly permeable to water, the imbalances imposed between turgor pressure and the osmolality gradient across the bacterial cell wall are of shorter duration. Bacteria respond to osmotic upshifts in three overlapping phases: dehydration (loss of some cell water); adjustment of cytoplasmic solvent composition and rehydration and cellular remodeling. Responses to osmotic downshifts also proceed in three phases: water uptake (phase I), extrusion of water and co-solvents (phase II), and cytoplasmic co-solvent re-accumulation and cellular remodeling (phase III) (Munns 2005 ) .
3.3 Compatible Solutes
The accumulation of organic solutes is a prerequisite for osmotic adjustment of the organism. Archaea synthesize unusual solutes, such as b -amino acids, N e -acetyl- b -lysine, mannosylglycerate, and di- myo -inositol phosphate. Among them, uptake of solutes such as glycine betaine is preferred over de novo synthesis. Most interestingly, some solutes are not only produced in response to salt but also to temperature stress (Muller et al. 2005 ) .
3.4 Glycine Betaine
The ability of the organism to survive in both high salt concentrations and low temperatures is attributed mainly to the accumulation of the compatible solute glycine betaine, one of the most effective compatible solutes widely used by bacteria. This solute is N-trimethyl derivative of glycine and can be accumulated intracellularly at high concentration through synthesis, uptake, or both. Bacillus subtilis has been shown to possess three transport systems for glycine betaine: the secondary uptake system opuD and two binding-protein-dependent transport systems, opuA and opuC (proU). The secondary transport system betP is involved in glycine betaine accumulation in Corynebacterium glutamicum (Sleator et al. 1999 ) . Further, characterization and disruption of betL, a gene which plays an important role in glycine betaine uptake in L. monocytogenes has been studied. Studies on some of the candidate genes from microbes for salinity tolerance highlight their functions. L. monocytogenes can survive a variety of environmental stresses. Growth at 10 % NaCl concentrations and temperature, as low as 20 C has been reported. The ability of the organism to survive both high salt and low temperatures is attributed mainly to the accumulation of the compatible solute glycine betaine.
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The genetic basis of glycine betaine uptake in other gram-positive ba...