Microbial adaptations to low temperature activity

The ability to grow at low, but not at moderate, temperatures is proposed to be dependent on adaptive changes in the microorganisms. It is supposed that the main adaptations appear in proteins and lipids. Adaptation to cold envi- ronments can be genotypic or phenotypic. Genotypic adaptations are changes that have occurred over an evolutionary time scale, while phenotypic adaptation occurs within the lifetime of an organism (Russell 1990). Cold-adapted changes in enzymes and protein structures are fixed in the genome. Furthermore, although phenotypic changes in microbial lipid composition are not genetic, the ability to adjust lipid composition (e.g. genes encoding desaturases) is.

5.3.2.1 Cold-adapted proteins

In general, enzymes from CAMs have higher specific activity and cat- alytic efficiency at lower temperatures than their counterparts from mesophiles and thermophiles. This is generally achieved by synthesizing enzymes that are more flexible at low temperatures, which may be associated with reduced sta- bility at higher temperatures. The thermostability of a protein is dependent on many non-covalent interactions (e.g. hydrogen bonds, salt bridges, van der Waals interactions, and hydrophobic bonds) between the peptide backbone and the amino acid side groups. Only recently have the structural changes in cold-active enzymes been studied (Russell 2000). The present knowledge on structural data comes from studies of soluble cytoplasmic enzymes. Different adaptations are used to achieve conformational flexibility in the active site at low temperatures,

and the changes are not necessarily the opposite of those that confer thermosta- bility. Hydrogen bonds and electrostatic interactions are formed exothermically, so they are stronger at low temperatures. In contrast, hydrophobic bonds are formed endothermically and will be weaker at low temperatures. According to Russell (2000), some general adaptations that confer the necessary conforma- tional flexibility to the active site in cold-active enzymes include:

r residues with greater polarity and less hydrophobicity

r additional glycine and low arginine/lysine ratio in the polypeptides r fewer hydrogen bonds, aromatic interactions, and ion pairs

r lack of salt bridges

r additional surface loop(s) with increased polar residues and decreased proline content

r modified α-helix dipole interactions

r reduced hydrophobic interactions between enzyme sub-units.

Berchet et al. (2000) performed structural analysis of elongating factor G (EF- G), an essential protein involved in protein synthesis. EF-G was isolated from the

bacterium Arthrobacter globiformis SI55, which grows between −5◦C and 32◦C.

Several structural and conformational changes were supposed to be important for low-temperature flexibility and activity when the EF-G from the cold-adapted strain was compared to EF-Gs from two related mesophilic bacterial strains.

Presently our knowledge about membrane-bound cold-active enzymes and their mechanisms is lacking. Enzymes involved in redox reactions, such as in oxidation of hydrocarbons and electron transfer, are located in lipid membrane structures. Russell (2002) suggests that enzymes embedded in the cytoplasmic membrane also need to be cold adapted in order to function at low temperature, and that this adaptation will depend not only on the intrinsic protein structure of the enzyme, but also on the physical properties of the surrounding membrane lipids. Interactions between the membrane lipids and embedded proteins are probably also important in microbial temperature sensing.

5.3.2.2 Lipids and membranes at low temperature

The cytoplasmic membrane in bacteria is predominately proteins and lipids. The lipid fraction accounts for about 25% of the total dry weight of the membrane in E. coli, and phospholipids dominate the lipid fraction (>90%). The fatty acids in the phospholipids vary in chain length, and in number and type of double bonds and alkyl side groups. The chemical structure of the fatty acids determines to a large extent the thermostability and flexibility of the cytoplas- mic membrane.

Since mesophilic bacteria often have very similar lipid composition to psy- chrotolerant microorganisms, the membrane composition does not correlate with the microbial classification in growth temperature ranges. Phenotypic changes in membrane lipids are observed as responses to changes in growth tem- perature. This mechanism is not restricted to CAM but is found in mesophiles and thermophiles as well. Bacteria adjust the fatty acid composition of mem- brane phospholipids in response to changes in the growth temperatures. To maintain a normal fluid state of the membrane at low temperatures, modifica- tion of the fatty acids occurs. Membrane fluidity is necessary for survival and growth, and when the growth temperature falls the membrane fluidity will decrease, and membrane-associated metabolic processes mediated by enzymes, cytochromes, and permeases will be slower. Several lipid changes, such as short- ening of fatty acid chains, trans- and cis-desaturation, and branching, increase the membrane fluidity at low temperatures. The rate of lipid modifications in response to transfer to and from cold temperatures will depend on the biosyn- thetic mechanisms involved. Changes in the degree of desaturation are normally fast, since the modification occurs directly on the lipids in the existing mem- brane. Changes in methyl-branching normally take more time, since de novo synthesis of the fatty acid molecules is necessary (Gounot and Russell 1999).

5.3.2.3 Microbial response to temperature reduction

Sudden changes of temperature may induce the synthesis of stress pro- teins in bacteria. If the temperature is shifted to just beyond the lower or upper growth limits, then cold shock or heat shock will occur. Cold-shock response (CSR) is observed in many bacterial strains. In mesophilic bacteria, cold shock results in a growth lag or acclimation phase. In this period, the number of polysomes decreases while 70 S monosomes accumulate. Genes for several dif- ferent cold-shock proteins (CSP) are also expressed in mesophiles (Thieringer et al. 1998). He suggested that CSP are synthesized to enable gene expression and as noted by Cavicchioli et al. (2000) to continue protein synthesis at low temperatures. Many of these proteins seem to be involved in stabilizing ribo- somes and protein synthesis at low temperatures. CSP probably contribute to ensuring that balanced microbial growth can continue. At present, we do not have a complete picture of CSP in different microorganisms and little is known about their specific function.

One of the most significant differences between mesophilic and cold-adapted bacteria is that ribosomes retain their ability to form polysomes at temperatures

as low as 5◦C (Broeze et al. 1987). The relative rate of synthesis of most cellular

proteins is also maintained after cold shock (Whyte and Inniss 1992; Michel et al. 1997). The findings show that cold-adapted bacteria sustain their activity at low

temperatures, and that temperature-mediated inhibition in mesophiles might be responsible for their inability to grow at low temperatures (Gounot and Russell 1999).

Another bacterial response to low temperature exposure is the onset of cry- oprotective mechanisms. Trehalose is found at high concentrations in many organisms that naturally survive dehydration. Accumulation of this disaccha- ride up to 500 mM in response to heat shock, osmotic stress, and during the stationary phase is found in bacteria and yeasts. Recent studies indicate that this sugar plays a major role in cell protection against harsh environmental conditions (Panicker et al. 2002; Bej et al. 2000). Kandror et al. (2002) demon- strated that synthesis and accumulation of trehalose is essential for viability at low temperatures. The enzymes for trehalose synthesis are induced in wild-type E. coli under cold-shock conditions, and the resulting trehalose accumulation increased viability when temperatures fell to near freezing.

Although at present we do not know much about these temperature-induced effects on hydrocarbon-degrading bacteria, it is nevertheless important to con- sider temperature relative to nutrient uptake when laboratory experiments with soil samples and field bioremediation schemes are planned, performed, and explained. Better understanding of the response of hydrocarbon-degrading soil bacteria to temperature reduction will help us in future efforts to optimize biodegradation rates at low temperatures.