An introduction to Psychrophiles

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1.1. Psychrophiles

Psychrophiles are extremophilic microorganisms that are cold-loving, with an optimal temperature for growth at about 15or lower, a maximal and minimal growth temperature being about 20°C and 0°C or lower, respectively (Moyer and Morita, 2007; Morita, 1975). The term, psychrotroph, is used to denote those microorganisms that are cold-tolerant with the ability to grow at low temperatures, but have optimal and maximal growth above 15°C and 20°C respectively. Many terms were coined to designate psychrophiles. These terms were cryophile, rhigophile, psychrorobe, thermophobic bacteria, Glaciale, Bakterien, facultative psychrophile, psychrocartericus, psychrotrophic and psychrotolerant (Morita, 1975). The term “psychrophile” (cold loving) was first given by Schmidt-Nielsen in 1902 to describe bacteria capable of growing at 0°C (Pikuta et. al., 2007). Psychrophiles are widely found among different bacterial taxa. They belong to such genera as Pseudomonas, Vibrio, Alcaligenes, Bacillus, Arthrobacter, Moritella, Photobacterium and Shewanella (Prescott, 2008).

Psychrophiles and psychrotrophs are extremophiles capably adapted to these extensive cold environmental conditions (Moyer and Morita, 2007). Both groups of microorganisms have evolved a range of structural and functional adaptations that enable them to survive in extremely cold environmental conditions: (i) increased fluidity of cellular membranes, (ii) the ability to accumulate compatible solutes (iii) the expression of cold shock, antifreeze and ice-nucleating proteins, (iv) production of cold-active enzymes (Dziewit and Bartosik, 2014; Casanueva et al., 2010).

The ability to sense changes in temperature greatly affects adaptation to low temperature. The cell membrane acts as a primary sensor, which turns more rigid at cold temperatures, activating a membrane-associated sensor. The sensor transmits the signal to a response regulator, which induces up regulation of numerous genes responsible for modulation of membrane fluidity. This contributes to cold adaptation of bacteria (Margesin and Miteva, 2010; Shivaji and Prakash, 2010). At current, the functional low temperature limits of psychrophiles are -12°C for reproduction and -20°C for metabolism (Margesin and Miteva, 2010; Bakermans, 2008).

Microorganisms employ various strategies to enhance membrane fluidity. The major change in fatty acid composition in response to lowered temperature is in the extent of unsaturation, increase in methyl-branched fatty acids, in polar carotenoids, decrease in the average chain length of fatty acids and in the ratio of sterol/phospholipids (Margesin and Miteva, 2010; Shivaji and Prakash, 2010; Deming, 2009; Russell, 2008).

Psychrophiles generate cold-active enzymes, which are highly active even at low temperatures. These enzymes are heat-labile and can get inactivated at temperatures favourable for their mesophilic counterparts. However, cold-active enzymes are able to maintain their flexibility and functions of the active site even at lowered temperatures (Margesin and Miteva, 2010; Feller, 2007; D’Amico et al., 2006). Cold-shock response is observed in psychrophiles, the number of cold-shock proteins increases with the severity of the cold-shock. Cold-acclimation proteins are also produced continuously during growth at low temperature (Margesin and Miteva, 2010; Phadtare, 2004). Various compounds are produced by the psychrophilic microorganisms to protect themselves against intracellular freezing (Margesin and Miteva, 2010; Christner, 2010; Deming, 2009). They accumulate compatible solutes (polyamines, polyols, sugars, amino acids). They also produce antifreeze proteins (AFPs), which are ice-binding proteins, capable of modifying the ice crystal structure and inhibiting the growth of ice (Margesin and Miteva, 2010; Gilbert et al., 2004).

1.2. Sources of Psychrophiles

Most of the Earth’s surface is cold. The oceans, which cover three quarters of the Earth’s surface, comprise the largest low-temperature ecosystem, with an average temperature of 5ËšC, and the depths at 1-3ËšC (Brock, 2012). There are ecosystems in Earth with permanently low temperatures that include the regions of the Arctic and Antarctic with polar ice-sheets, glaciers, permafrost, the snow-caps and glaciers of high mountains, and the deep water and marine sediments of the oceans. Enormous areas of permafrost are formed in the polar regions (Pikuta et. al., 2007). Survival of a particular organism strongly depends on temperature, either indirectly, through its influence on water, or directly, through its effect on the organic molecules that make up the living cells (Margesin and Miteva, 2010; Poindexter, 2009). Cold environments are not sterile.

A wide variety of microorganisms, including bacteria, archaea, yeasts, filamentous fungi and algae can be found growing at any low-temperature environment which contains some liquid. Psychrophiles can be readily isolated from Arctic and Antarctic habitats; they constitute an enormous habitat for psychrophiles because 90% of the ocean is 5ËšC or colder (Brock,2011). In polar regions, bacteria and algae making up psychrophilic microbial communities grow in dense masses within and under sea ice, and even on the surfaces of glaciers and snowfields. A snowfield or a glacier can appear pink owing to the bright red spores of a snow algae Chlamydomonas nivalis. Within glaciers there occurs a network of liquid water channels where solutes are concentrated. In this, prokaryotes thrive and reproduce. Several psychrophilic bacteria have been isolated from sea ice and marine sediments (Brock, 2011). A psychrophilic archaeon, Methanogenium, has been isolated from Ace Lake in Antarctic (Prescott, 2007).The psychrophiles that inhabit the deep sea oceans (with a constant temperature of 4â-¦C below a depth of 1,000 m) are true extremophiles as they are adapted not only to low temperatures, but also to further environmental constraints (Pikuta et. al., 2007; Feller and Gerday 2003). Major challenges to microorganisms in cold ecosystems are reduced enzymatic reaction rates, limited nutrient bioavailability, extreme pH and salinity, as well as limited water activity (Margesin and Miteva, 2010).

1.3. Glacier and Its Importance

“Any large mass of snow and ice on the land that persists for many years may be called a glacier” (Meier and Post, 1849). When more snow falls than melts, over a number of years, glaciers are formed. This snow accumulates and grows thicker, becomes compressed and is changed into dense, solid ice. On Earth, most of glacial ice is contained within vast ice sheets in the polar regions. Apart from that, glaciers can be found in mountain ranges on every continent. Glaciers are categorized by their morphology, thermal characteristics, and behaviour. They are of many forms. They may be Alpine or Cirque glaciers which form on the crests and slopes carved out of mountainsides by erosion, or slope glaciers formed on exposed slopes, those formed on the ridges where snow is deposited by wind are drift glaciers. Large mountain glaciers that flow down hills form valley glaciers, piedmont glaciers form when ice from glaciers in the mountains spread out at the foot of a mountain range (Meier and Post, 1849).

1.4. Plasmid and Its Importance

Plasmids are autonomously self-replicating covalently bonded, circular, extra-chromosomal DNA molecules that are found in many cells. They can be found as single or multiple copies and may carry a few to several hundred genes. Plasmids can only multiply inside a host cell. Around 50% of bacteria found in the wild contain one or more plasmids (Clark, 2005).The size of plasmids varies widely, from several kilo bases to hundreds of kilo bases (Feinbaum, 1998).

Plasmids carry genes for managing their own life cycles and some plasmids carry genes that specify a wide variety of properties to the host cell. These properties vary from plasmid to plasmid, that include: resistance to various antibiotics, resistance to heavy metals, sensitivity or resistance to bacteriophages, sensitivity to mutagens, production of restriction enzymes, production of toxins, catabolism of complex organic molecules, determination of virulence, and ability to transfer DNA (Feinbaum, 1998). However, some plasmids are of no use as they exhibit special mechanisms to protect their own survival at the expense of the host cell. Nonetheless, most plasmids carry genes beneficial to their host cells (Clark, 2005). Plasmids often carry genes for antibiotic resistance. This defends bacteria from human medicines as well as from antibiotics that occur naturally. Plasmids carrying genes for resistance to toxic heavy metals like lead, cadmium or mercury provide extra defense for bacteria. Some plasmids can help bacteria to grow utilizing complex chemicals, while other plasmids provide virulence and colonization factors to infectious bacteria thus aiding in counteracting the host immune system (Clark, 2005).

A wide variety of plasmids, modified for different purposes, is used in molecular biology research and is often used to carry genes during genetic engineering. Plasmids can be widely used as vectors to move genes between organisms. A plasmid typically possesses three basic features: a replicator, a selectable marker, and a cloning site (Feinbaum, 1998). The replicator is a stretch of DNA containing the origin of replication and genes encoding replication proteins. The selectable marker is generally a gene encoding resistance to some antibiotic, essential for maintaining the presence of the plasmid in cells. The cloning site is a cleavage site for restriction endonuclease where foreign DNA can be inserted without interfering with the plasmid’s normal function (Feinbaum, 1998). Plasmids may be of two types, based on the copy number of a plasmid i.e. number of plasmid molecules maintained per bacterial cells.

High-copy-number plasmids may be defined as those which occur in ≥20 copies per bacterial cell, and low-copy-number plasmids as those which occur in <20 copies per cell (Feinbaum, 1998). High-copy-number plasmids are smaller in size than low-copy-number plasmids, and hence are more frequently used in molecular biological techniques because of their convenient size and ease of preparation in large quantities from cells that bear them. Plasmids were originally classified according to incompatibility. The inability of two different plasmids that belong to same family to co-exist in the same host cell is known as incompatibility. Plasmid families can be known as incompatibility groups and plasmids of the same incompatibility group exhibit very similar DNA sequences in their replication genes (Clark, 2005). Plasmids are incompatible if they share any function necessary for the regulation of plasmid replication. For instance, ColE1- and Pmb1- derived plasmids are incompatible with one another but are compatible with p15A plasmids (Feinbaum, 1998).Some of the particular bacterial plasmids are: F, R, and Col plasmids.

F Plasmid: A plasmid called the fertility or F factor is a circular DNA molecule of 94.5 kilobase pairs (Freifelder, 2008). This plasmid plays an important role in conjugation as it carries genes responsible for cell attachment and plasmid transfer between specific bacterial strains. The tra operon contains most of the information required for plasmid transfer. The genes contained in this operon encode for sex pili that attach the F+ cell (donor) to an F- cell. The F factor also contains ‘insertion sequences’ that help in plasmid integration into the host cell chromosome to generate an Hfr cell. F can integrate in either ways, clockwise and anticlockwise (Freifelder, 2008).

R plasmids: These plasmids confer drug-resistance. They carry genes that encode for enzymes capable of inhibiting or modifying antibiotics (Prescott, 2008). Most R plasmids comprise of two adjoining segments of DNA called RTF (resistance transfer factor) and r determinant. RTF bear genes that regulate DNA replication and copy number, the transfer genes and occasionally genes for tetracycline resistance. The other segment carries other genes for resistance to antibiotics like penicillin, ampicillin, chloramphenicol, streptomycin, kanamycin and sulphonamide (Freifelder, 2008).

Col plasmids: The colicinogenic or col plasmids are borne by E. coli that has the ability to produce colicins. They carry genes for the synthesis of colicins, which are proteins capable of inhibiting the growth of other bacterial strains. Two types of colicins are seen, true colicins and defective phage particles. ColE1 is the best studied Col plasmid, which is mobilizable but nonconjugative (Freifelder, 2008). However, some Col plasmids are conjugative and may carry resistance genes (Prescott, 2008).

1.5. Psychrophilic plasmids and their importance

Survival in extremely cold ecosystem is a challenge for any kind of organism. Such an environment is highly susceptible to the damaging effects of external factors and is prone to undergo frequent alterations. Mostly psychrophilic and psychrotolerant bacteria inhabitate such harsh environments, by adopting various strategies to cope with the cold. It is very crucial for the organism to adjust rapidly with the changing environmental conditions in order to survive. Such an adaptation is often facilitated by plasmids- extrachromosomal replicons chiefly involved in horizontal gene transfer. Over the last decade, the genomic sequences of numerous cold-active bacteria have been obtained and plasmids of many psychrophilic and psychrotolerant bacteria have been identified and characterized. These plasmids have been named pPSYCHplasmids.

Analyses of the larger plasmids have revealed the presence of numerous genes, which may contribute to increased phenotypic flexibility of the host strains. These genes encode enzymes possibly involved in (i) protection against cold and ultraviolet radiation, (ii) scavenging of reactive oxygen species, (iii) metabolism of amino acids, carbohydrates, nucleotides and lipids, (iv) energy production and conversion, (v) utilization of toxic organic compounds, and (vi) resistance to heavy metals and antibiotics. Some plasmids contain type II restriction-modification systems involved in both plasmid stabilization and protection against foreign DNA. Plasmids increase the flexibility of bacterial genomes enhancing the genetic diversity of their hosts (Dziewit and Bartosik, 2014; Siefert, 2009). Apart from carrying a set of basic structural genetic modules involved in plasmid replication, maintenance and spread, these mobile genetic elements also carry additional genetic information. These additional genes can confer certain advantages to the host strains under particular environmental conditions (Dziewit and Bartosik, 2014; Heuer and Smalla, 2012; Wiedenbeck and Cohan, 2011).

A number of plasmid-bearing bacterial strains have been isolated from permanently cold environments comprising of Arctic (Moller et al., 2014; Dziewit et al., 2013), Antarctica (Dziewit et al., 2013; Kobori et al., 1984) and Siberia (Petrova et al., 2014). The majority of the identified plasmids belong to Gram-negative bacteria. The size of the plasmids range from 1.3 kb (pTA144 Dw of Moraxella sp. TA144) to 417 kb (pBWB401 of Bacillus weihenstephanensis KBAB4). Some of the completely sequenced pPSYCHplasmids are: pVSAL43 isolated from Allivibrio salmonicide LFI1238, pCP1 from Flavobacterium psychrophilum D12, pWNCR9 from Carnobacterium gilichinskyi WN13592, pP60P2 from Psychrobacter sp. DAB AL60, pSFKW33 from Shewanella sp. 33B etc. (Dziewit and Bartosik, 2014). It has been strongly suggested that plasmids of psychrophilic and psychrotolerant bacteria may determine various metabolic properties of the host cell and hence the overall functioning of bacterial cells.

Plasmids of cold-active bacteria encode for cold shock protein (CSP) which is one of the primary responses of bacteria to low temperatures. Synthesis of CSP counteracts the harmful effects of lowered temperature. The proteins included in this group are helicases, nucleases, along with other ribosome-associated components associated with DNA and RNA. Plasmids of cold-active bacteria also encode DNA-repair proteins allowing the cells to cope with increased UV radiation. Along with that, the plasmids encode a number of proteins responsible for generation of energy during nutrient deprivation, genes providing resistance to phage infection, heavy metals or antibiotics. Therefore, the pPSYCH plasmid may play an important role in the adaptation of psychrophilic and psychrotolerant bacteria to low temperatures and rapid environmental changes (Dziewit and Bartosik, 2014).