HALOPHILES

INTRODUCTION

Aquatic environments:
Fresh waters: less than 0.05% w/v dissolved salts Sea waters: total salinity 3.2-3.8% w/v Saturated salt solutions: up to 30% and above

In freshwater- extracellular solute conc. is low dilution stress swelling osmotic lysis. Lysis is prevented by c.w. develop positive hydrostatic pressure restricting water entry and cell enlargement. Wall less bac. counteract such water uptake by using contractile vacuoles to concentrate and remove water. In sea water, microbes faces osmotic and salt combined stress. Microbes counterbalance by the intracellular accumulation of solutes to an equivalent osmotic level. Extreme environments includes non ionic environments of high osmotic strength.

MICROBIAL DIVERSITY IN HYPERSALINE ENVIRONMENT

Bacteria
Halobacterium (Gram negative elongated rods, > 2M NaCl) Halococcus (thick walled Gram positive coccus, > 2M NaCl) Ectothiorhodospira (Anaerobic, phototroph, > 1.5 M NaCl, optimum 3M NaCl)

Cyanobacteria
Synechococcus Synechocystis Dactylococcopsis

Eukaryotic photosynthetic flagellates

Chlamydomonas Dunaliella Asteromonas

Halobacterium and Halococcus: aerobic (heterotrophic) mode of growth - absolute requirement of salt about 2 M or higher - pink or red pigmentation lack of peptidoglycan preference of aminoacids as source of fixed carbon for energy metabolism.

Halobacterium ruburum: capacity of photophosphorylation via pigment bacteriorhodopsin under anaerobic of microaerophilic conditions.

Ectothiorhodospira halochloris: spiral shaped rods flagellated, bipolar contain stacked intracytoplasmic membranes (sites of pigment localization) grow best with H2O as ele donor and CO2 as C-source.

Dactylococcopsis salina cyanobact slender, fusiform rods (~80mm long) granular cytoplasm no growth at salt conc below sea water.

Synechococcus cyanobact - no sheath surrounding cell single plane halo philic/tolerant.

Synechocystis cyanobact lack of sheath cell division in two or three planes.

Dunaliella biflagellate, unicellular green algae lack rigid polysaccharide c.w. grow over wide range of salt concentrations halo phile/tolerant.

PRINCIPLES OF HALOTOLERANCE AND HALOPHILISM

Adaptations are classified in to three broad catagories. Insulation:
Adaptations reducing the impact of the external environment by preventing entry of salt. Feature of c.w and/or p.m.

Protection:

Protecting intracellular function and metabolic activity from inhibiting effect of Na+.

Modification:

Aspect of cellular metabolism showing optimal function in high salt conditions.

INSULATION

High conc of Na+ - toxic to most biological systems. Most bac, fungi and alga possess
memb. with low permeability to Na+ than to K+. Active Na+ extrusion mechanism.

Halobacterium cells
contains 0.36-3.6 M NaCl on growing media with 3.3 to 5.1 M. more K + than Na+. Na + -K + ratio is 0.1-0.2(0.7 in stationary phase). Show activ Na + extrusion (efflux linked to proton entry) P.m. has low passive Na + permeability.

Dunaliella has
low internal Na + conc growing in high salt media may be two compartment system with inner well insulated & outer leaky compartment. So, low internal Na + levels and low passive + permeability.

Intracellular accumulation of K + also occurs in halotolerant cyanobac and in Dunaliella (not in same extent as Halobacterium). All halophilic and halotolerant mos show some degree of insulation of the cell interior from external Na + varying between orgs.

Alternative compounds also used to generate intracellular solutes providing turgor pressure (esp in cells lacking rigid cell wall).
In Halobacterium and Halococcus this additional solute is K + . Photoautotrophs accumulates organic solutes rather than inorganic ions to achieve osmotic bal.

Dunaliella synthesizes glycerol when grown on saline medium. In some cyanobacteria, quaternary ammonium compound glycine betaine is principle intracellular solute while in some glutamate betaine is major (in Calothrix genus).
Polyols and other organic molecules are not inhibitory for enzyme activity at various concentrations.

Term Compatible solute has been used to describe all compounds showing this feature of compatibility. First demonstrated for polyols in sugar tolerant yeasts and for glycerol in Dunaliella. In Cynechocystis glycine betaine acts as compatible solute did not inhibit enzymes at 2 M conc. Halophile/tolerant mos requires enzyme to function in environment of high ionic strength with KCl (less effective inhibitor than NaCl) acting as compatible solute.

Except halobacterium and halococcus, organic solutes generally found as intracellular solutes. Diverse range of carbohydrates, amino acids, quaternary ammonium and tertiary sulphonium compounds and related molecules are employed by salt stressed organisms to effect osmotic salt stress.

Osmotic adjustment occurs in response to changes in external water states rather than to specific ionic effects. Level of compatible solute is sensitive to changes in external osmolarity. It increases in response to upshock (increase in external salt conc.) and decrease in response to downshock (decrease in external salt conc.). Osmoregulation: change in intracellular solute concentration in response to outer environment and the processes involved in this adjustment.

PROTECTION

Halotolerant and halophilic mos maintains low intracellular Na+ concentrations by combination of low plasma membrane Na+ permeability and active Na+ extrusion. As compared to freshwater and marine bac (<0.05M), halotolerant cyanobac contain 0.1 0.3 M internal Na+ levels. Mos growing at high external NaCl conc may be faced with combination of
Lowered intracellular water potential and Increased internal Na+ content ( insulation is less than 100% effective)

Compatible solute theory had been extended to include aspects of salt and osmotic stress. Water like hydroxyl groups of polyols (glycerol and mannitol) can replace water molecules and thus maintain hydrophobicity enforced water structure within cytoplasm under lowered cellular water potential. Similar functions can be played from quaternary ammonium compounds and imino acid proline. Glycine betaine can partially protect enzymes (e.g. melate dehydrogenase in Hordeum vulgare) and in some cases it can increase enzyme activity (e.g. in Synechocystic DUN52, 1 to 1.2 M betaine increased activity of glutamine synthatase by 24% and 28% respectively) Above discussion is applied to salt adapted cells.

Salt inactivation can occur on initial transfer to saline medium(rapid sodium entry within 2 min).
Most of the cells responds to this upshock and new low steady state is achieved within 30 min. this time course may be extended till extrusion of Na+. Na+ extrusion is energy dependent and sensitive to external K+ concentration. It shows that intracellular osmotica may be involved in protection against NaCl inactivation in short term following upshock. So, orgs containing high compatible solutes may well show less Na+ inhibition upon transfer to high salt medium than orgs containing lower levels of organic osmotica. It can be considered as possible training of mos to grow in hypersaline media.

For protection against Na+ toxicity orgs will be dependent on the extent of insulation against NaCl entry at O.M. Compatible (organic) osmotica may be also involved in

Maintainance of cytoplasmic water structure Protection against transient increase in intracellular salt levels.

MODIFICATION