Molecular adaptations in psychrophiles:

insights from “omic’’ methods

Cold-adapted organism A) psychrophiles( <0, 15, 20 oC)

B) psychrotrophs (>0, >20, >30 oC)

Psychrophiles

A) Stenopsychrophiles

B) Eurypsychrophiles

Introduction

Habitats for cold adapted microorganisms include oceans, which cover some 70% of the Earth’s surface, are at an average temperature of -1 to +5 oC

Polar regions, including Antarctica and portions of North America constitute 20% of the world’s land surface area

Alpines, The Himalayas, The Rocky Mountains and Alps constitute a 5% of Land surface area.

Man-made habitats (such as refrigeration and freezer systems)

Cold Habitats

Low temperatures.

Low liquid water availability.

Lowered enzyme reaction and solute uptake rate.

Reduced membrane fluidity.

Stabilized inhibitory nucleic acid structures .

Intracellular crystalline ice formation.

Challenges

Physiological adaptations of psychrophiles

Modifying enzymes

Sensing environmental temperature.

Metabolism at low temperature.

Role of RNA degradosome.

Formation of viable but non culturable cells(VBNC).

Interlinked adaptive response.

Other strategies

Colwellia psychrerythraea genome analyses have revealed the presence of coding sequences(CDS) which code for polyketide-like polyunsaturated fatty acid synthases; fatty acid cis-trans isomerase which changes fatty acid composition in phospholipids.

Membrane fluidity

Genome analyses of Colwellia psychrerythraea indicate five transporters involved in the movement of quaternary ammonium compounds of the betaine, carnitine, choline.

Transporter family include ATP-binding cassette transport system for direct uptake of glycine.

Compatible Solutes

Pathway of interconversion

(Barbara ,A.M.etal.2005)

.

At low-temperature there is a stress on translation which induce the production of CspA, an RNA chaperone that binds to mRNA and destabilizes secondary structure (Jiang et al. 1997).

In Psychrobacter cryohalolentis K5, five CIPs were involved with translation and included two ribosomal proteins (S2 and the Ctc form of L25), two elongation factors (EF-Ts and the EF-Tu related TypA), and the cold shock protein CspA. (Fedorov et al. 2001).

Roles of CIPs in low temperature growth

Mechanism for coupling of transcription and translationthrough CSPs and CSHs. Crystal structure of CspB from

Bacillus subtilis in complex with ssDNA

Fluorescence microscopy of Bacillus subtilis cells

(Walid, M.E.et al. 2006)

Schematic view of the pattern of protein expression after cold shock.

Schematic representation of the Csp function duringacclimation phase.

(Horn,G.etal.2007)

.

At low temperatures, transport systems are required to counteract lower rates of diffusion and transport across the membrane and for the transport of compatible solutes (Nedwell 1999; Welsh 2000).

Two separate systems for the transport of ferric iron (AfuA and FecA), up-regulated in Psychrobacter cryohalensis K5 at low temperatures(Bakermans et al. 2007)

Transport

• During cold stress in Rhizobium the amount of malate dehydrogenase increased indicating that the glyoxylate cycle was being utilized to produce oxaloacetate for use in the pentose phosphate pathway

Glyoxylate cycle

Energy Production

Colwellia psychrerythraea produce polyhydroxy alkanoate (PHA) compounds which is linked to the degradation of fatty acids.

PHA compounds are of industrial interest for their thermoplastic and elastomeric properties.

This bacterium is known to degrade polyamides and synthesize cyanophycin .

Polyamides are of industrial interest as possible biopolymer substitutes for polyacrylates

Carbon and Nitrogen Reserves

Colwellia psychrerythraea possesses 2,4,6-trichlorophenol monooxygenase, which are involved in the degradation of pentachlorophenol, and dioxygenases which are critical to the cleavage of ring bearing and aliphatic compound degradation.

Bioremediation

Colwellia psychrerythraea synthesis extracellular enzymes, extracellular polysaccharides, sigma -70 transcription factors, glycosyl transferases for cold adaptation(Barbara et al. 2005).

Extracellular Compounds

Cold-temperature environments present several challenges, in particular reduced reactions rates, and increased viscosity .

To cope up there must be an increase in enzyme turnover (Kcat) or improvement of keff(Kcat/KM) for localised increase in enzyme Flexibility .

The rate enzymatic reactions is described by the Arrhenius equation.

Enzymatic activity

Thermodependence of activity for cold-adapted cellulase from Pseudoalteromonas haloplanktis (EGG) and its mesophilic homologue from Erwinia chrysanthemi (EGZ).

Activity of cold adapted enzymes

Molecular adaptation Explanation Genes involved

Membranes Increase the fluidity of cellular membranes

Desaturases

Freeze-protection Reduction of freezing point of cytoplasm

Antifreeze proteins and ice-binding proteins

Cold-shock and acclimationresponse

Cellular response to lowering of temperature

RNA-binding protein

Protection against reactiveoxygen species (ROS)

Increased solubility of oxygen at low temperature

Catalases, peroxidases

Proteins and enzymes Maintain catalytic efficiency at low temperatures

Reduced proline, internal hydrophobicity

Genome plasticity To increase the adaptation ability to cope with low temperatures

Transposases, prophages

Molecular adaptations in cold adapted bacteria

(Baermans C. et al 2007)

Proteins displayed significant reductions in the numbers of aspartic acid, glutamic acid, proline content and hydrophobic content, which are necessary to decrease protein structural rigidity. (Beja et al. 2002).

Genome analysis reveals that Colwellia psychrerythraea role in carbon and nutrient cycling and some useful processes like bioremediation in cold environments.

Different adaptive strategies like cell membrane synthesis PHAs (pressure adaptation), cyanophycin, glycine betaine, extracellular enzymes synthesis are well understood by genomic analysis

Adaptations to psychrophily from Metagenomic data

• High levels of non-charged polar amino acids and low hydrophobic amino acids were identified in proteins from the archaeal psychrophiles, Methanogenium frigidum .

• A reduction in the use of acidic amino acids, proline and arginine in the genome of Psychrobacter arcticus (Ayala-Del-Rı´o et al. 2010)

• Pseudoalteromonas haloplanktis suppresses ROS by upregulating the expression of dioxygen-consuming lipid desaturases (Me´digue et al. 2005)

Sequencing of ice metagenome revealed a large number of genes forcryoprotectants like glycine, betaine, choline, sarcosine and glutamate membrane fluidity desaturases

(Casanueva A. et al 2010)

In Methanococcoides burtonii, cold-adapted proteins involved in DNA replication and cell division, RNA polymerase machinery, motility, protein folding, methanogenesis, transposition and cell signalling (Goodchild et al. 2004).

When comparing the proteomes of Exiguobacterium sibiricum grown at 4 oC and 25 oC, 39 putative cold-acclimation proteins were uniquely expressed at 4oC (Qiu et al. 2006).

Adaptations to psychrophily from proteomic data

Cold adapted enzymes are used in sequential processes , where they need to be terminated before the next step is undertaken.

They offer economic benefits through energy savings

They are also used in mixed aqueous and organic solutions for organic synthesis-because of low water activity

First commercial application includes protease from novoenzyme (T.N:savinase)

Applications of psychrophiles

Microorganism or product Application

Polyunsaturated fatty acids Dietary supplements for humans, livestock and fish

Ice nucleation proteins Food industry, synthetic snow

Antifreeze proteins and solutes Cryoprotectants, cold-active catalysts

Cold-adapted bacteria and fungi Food industry, including cheese and yoghurt manufacture, meat tenderising, flavour modification and lactose removal from milk

Cold-adapted bacteria and fungi Bioremediation of ocean oil spills, contaminated ground water and toxic waste

Ice – bacteria Frost protection for plants

Methanogenic Archaea Methane production, low temperature waste treatment

Examples of applications (other than enzymes) of cold-adapted microorganisms and their products

Cavicchioli R. et al 2002

Ether-linked lipids from archaea used in the production of liposomes for vaccine and drug delivery.

Methanococcoides burtonii accumulates potassium aspartate during low-temperature growth, which decreases the Km for the binding of GTP by elongation factor 2 (EF-2)

Importance of archaeal systems

(Cavicchioli R. et al 2002)

Psychrophilic lifestyle is due to synergistic changes in overall genome, and amino acid composition, rather than the presence of a unique set of genes

Bacterial cold adaptation genes can be genetically engineered into bacteria like E. coli for the degradation of man made wastes in extreme environments.

Genomic and metagenomic data reveal the psychrophilic genome plasticity as an adaptation to cold temperatures

Conclusions

Preventing excessive secondary mRNA structures on exposure to cold due to cooperative performance of CSP and CSH.

The most temperature sensitive step due to cold adaptation is translation. Ribosomal assosiated proteins are involved in temperature sensing.

Cold active enzymes have high catalytic activity and low thermostability which are of high biotechnological potential.

Role of cryo-protectants in bacteria remains unexplored to a large extent but it is well studied in frogs, and plants.

Further investigations have to be carried out on survival at sub-zero temperatures in order to understand the life-processes at this extreme conditions

The cold adapted archaea are novel and untouched biotechnological resource.

Future prospects