The Cold Shock Response

SANGITA PHADTARE* AND MASAYORI INOUYE

[SECTION EDITOR: JOHN FOSTER]

Posted January 16, 2008

Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ 08854

*Corresponding author. Mailing address: Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854. Phone: (732) 235-4116/4115, Fax: (732) 235-4559, E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it .

INTRODUCTION

Change in temperature is one of the most common stresses that an organism encounters in nature. When an organism senses downshift in temperature, it responds by eliciting cold shock response. Enterobacteria such as Escherichia coli encounter sudden drastic temperature downshift as a result of excretion from animals. Thus, the cold shock response and adaptation in this bacterium give it a selective advantage of quickly adapting to the new environment. A typical cold shock response in E. coli is exerted when a culture growing exponentially at 37°C is shifted to 15°C. When the cells encounter cold shock, there is a lag period of growth, termed acclimation phase, in which cellular synthesis of most of the proteins is inhibited as opposed to that of a select group of proteins, termed cold shock proteins. These proteins help the cells counteract various detrimental cellular changes triggered by the temperature downshift. This is followed by resumption of the cell growth with restoration of synthesis of normal cellular proteins and decrease in the rate of synthesis of cold shock proteins. Not all organisms exhibit a lag period of growth phase though. For example, bacteria such as Bacillus subtilis and Lactobacillus lactis do not have a lag stage of cold shock response (8, 115). The cold shock response is elicited in all types of bacteria: psychrophiles and psychrotrophs, mesophiles and thermophiles. It has been extensively studied by using E. coli and B. subtilis as model systems (for reviews, see references 16, 40, 82, 83, 85, 91, 114, and 118). These studies have revealed common features of the bacterial cold shock response. In this chapter, we will mainly focus on the cold shock response of E. coli.

Temperature downshift affects the cell on various levels: (i) decrease in the membrane fluidity; (ii) stabilization of the secondary structures of RNA and DNA; (iii) slow or inefficient protein folding; (iv) reduced ribosome function, affecting translation of non-cold shock proteins; (v) increased negative supercoiling of DNA; and (vi) accumulation of various sugars. Loss of membrane flexibility affects membrane-associated functions such as active transport and protein secretion. In order to adapt to low temperature, bacteria such as E. coli restore the flexibility of membranes by various mechanisms such as increasing the proportion of unsaturated fatty acids (UFAs) with low melting points and high flexibility (homeoviscous adaptation) (106). In E. coli, the UFA produced after temperature downshift is cis-vaccenic acid (cis-11-octadecenic acid). The enzyme β-ketoacyl-acyl carrier protein (ACP) synthase II converts palmitoleic acid to cis-vaccenic acid. The synthesis of this enzyme is not induced on cold shock, but the enzyme is activated at low temperature (23, 24). Stabilization of secondary structures in nucleic acids leads to impaired transcription and translation due to hindered movement of RNA polymerase or of ribosomes, respectively. Proteins such as CspA homologues act as RNA chaperones to melt the secondary structures in nucleic acids and facilitate cold shock transcription and translation. Increase in the negative supercoiling of DNA caused by temperature downshift affects DNA-related functions, such as replication, transcription, and recombination (57, 70, 112). The effect on transcription is mainly because changes in supercoiling influence the relative orientation of the −35 and −10 regions, which in turn influences recognition of some σ⁷⁰ promoters by RNA polymerase (112). Certain proteins also undergo changes such as reversible methylation of cytoplasmic signaling/adaptation domains as seen in the case of aspartate chemoreceptor (Tar) of E. coli (80). Folding of some proteins is also affected at low temperature, albeit to a lesser degree than by that by heat shock. As mentioned above, various cold shock proteins produced on temperature downshift help the cells counteract these detrimental changes. Sugars also have been shown to exert protective effect during cold shock response and adaptation.

COLD SHOCK PROTEINS

Table 1 describes various cold shock proteins induced in E. coli. In addition to these, recent DNA microarray analyses of the cold shock response of E. coli have shown that several new proteins are induced by cold shock, including several involved in sugar metabolism and transport, stress response regulators, and certain heat shock proteins (84, 93).

Table 1Cold shock-induced proteins

The CspA Family

CspA is the major cold shock protein of E. coli, and its homologues are found to be widespread among bacteria, including psychrophilic, psychrotrophic, mesophilic, and thermophilic bacteria, but are not found in archaea or cyanobacteria.

CspA homologues exhibit functional redundancy.

E. coli has a family of nine CspA homologues, four of which are induced by cold shock as shown in Table 1. Single, double, or even triple deletions of the cold shock-inducible csp genes (ΔcspAΔcspB, ΔcspAΔcspG, ΔcspBΔcspG, ΔcspAΔcspI, or ΔcspAΔcspBΔcspG) do not exert a detrimental effect on the cell’s response to cold shock. Interestingly, however, the triple deletion strain of ΔcspAΔcspBΔcspG produces larger amounts of CspE on temperature downshift (116). CspE is only mildly induced in the wild-type cells on temperature downshift. A quadruple deletion strain (ΔcspAΔcspBΔcspGΔcspE) is cold sensitive, and this defect can be overcome by the overproduction of any one of the CspA homologues except CspD. This inability of CspD may be due simply to the toxic effect exerted by its overproduction and does not necessarily mean that it lacks functional similarity with the other CspA homologues. Taken together, these observations suggest that there is a functional overlap among CspA homologues. Widespread occurrence and existence of multiple homologues of CspA suggest that these proteins play a pivotal role in the cold shock response and adaptation of bacteria.

Cells possess efficient machinery for cold shock induction of CspA homologues.

The cold shock induction of CspA homologues is presumed to be regulated at the levels of mRNA stability, translation, and transcription and does not need additional transcription factors. Significant, albeit transient, stabilization of the cspA mRNA immediately following temperature downshift is mainly responsible for its cold shock induction. The structural element that is responsible for this phenomenon is the untranslated 5' region of cspA mRNA. The half-life of cspA mRNA changes from 12 s at 37°C to >20 min at 15°C (69). Despite a promoter that is active at 37°C, the production of CspA is low at 37°C because of the extreme instability of its mRNA. A constitutive expression of cspA was observed when cspA mRNA was stabilized 150-fold due to a three-base substitution mutation within the 159-base 5'-UTR. This stabilization was found to be at least partially due to resistance against RNase E degradation (18). The second factor that contributes to the cold shock induction of CspA is its efficient translation at temperatures wherein overall transcription machinery is functioning at a suboptimal level. Another structural element in cspA mRNA plays an important role in its cold shock translation, namely the translation-enhancing element (98) located 14 bases downstream of the initiation codon (69, 75). This element is also present in other cold shock-inducible genes such as cspB, cspG, cspI, csdA, and rbfA. Because this sequence is complementary to a region in the penultimate stem of 16S rRNA, it was initially thought to enhance translation initiation by facilitating the formation of the translation preinitiation complex through binding to 16S rRNA; however, this view is now disputed and the exact mechanism of the enhancing effect on translation initiation by this element is unknown at present (69, 75, 98).

The third and possibly least significant mode of regulation of cspA induction at low temperature occurs at the level of transcription. Opinions had been divided regarding the contribution of cold shock-regulated transcription of cspA in its dramatic induction on cold shock (31). There were several observations that led researchers to believe that transcription plays a major role in cold shock induction of CspA. These included structural elements present in cspA mRNA and demonstration of its promoter activity by using various approaches. There are three structural elements present in the cspA mRNA that presumably contribute to its cold shock transcription. (i) The first is an unusually long 5'-untranslated region (5'-UTR), with a highly conserved, 11-base sequence termed the "cold box." This is a presumed transcriptional pausing site that is bypassed by RNA polymerase immediately on temperature downshift. At higher concentrations of CspA, CspA binds to its own mRNA, destabilizes the elongation complex, and thus regulates its own expression. Thus, overproduction of 5'-UTR leads to prolonged synthesis of CspA, an effect suppressed by coproduction of CspA (17, 46). (ii) The second structural element affecting cspA transcription is an AT-rich sequence (UP element) immediately upstream of the −35 region (26, 69); deletion of this region leads to decreased activity of the cspA promoter. (iii) The third structural feature of significance is an extended −10 box (a TGn motif preceding the −10 box), an important factor that may contribute to high promoter activity. It is reported that, in the presence of this motif, the −35 region is dispensable. Various approaches were used in these studies to detect cold shock induction of cspA mRNA. Moderate (~threefold) increase in activity was seen at 15°C by studying expression profiles of reporter genes such as lacZ and cat fused to the cspA promoter (26, 69). An increase in cspA transcript (four- to fivefold) on cold shock was observed by primer extension, Northern blot analysis, and DNA microarray analysis of the global transcript profile of cold-shocked cells (84). Production of CspA was also observed at 37°C during early exponential growth phase, although at a much lower level. This induction is presumed to be due to its position near oriC resulting in a higher gene-dosage effect, high concentration of its transcription activator Fis, and higher stability of its mRNA at this phase due to lower RNase activity (5). Later, this production was shown to be due to nutritional upshift (120).

All these analyses could not distinguish whether the increase in cspA transcript is due to cold-shock transcription alone or is a cumulative result of cold shock transcription and stabilization of the cspA mRNA. This question was addressed by recent studies in which a cell-free system was used to evaluate promoter activity of cspA during cold shock. It was concluded that the extended −10 motif is critical for the expression of cspA, and transcription from the cspA promoter is cold sensitive and appears to play little or no role in the low-temperature induction of cspA expression. Furthermore, no cellular factor that may act as transcription enhancer for cspA in vivo was detected (89). Thus, cspA mRNA stabilization is mainly responsible for its cold shock induction.

CspA homologues have diverse functions.

The secondary structures in RNA stabilized by temperature downshifts affect both transcription elongation and ribosomal movement on RNA and, thus, translation. CspA homologues are induced significantly immediately on temperature downshift and can bind and melt secondary structures in nucleic acids, thereby facilitating transcription and translation at low temperature. Thus CspA homologues have been termed RNA chaperones (3, 47, 88). The relative nonspecificity of binding to nucleic acids (47, 62, 87) enables these proteins to act on a wide range of mRNAs. The RNA chaperone activity also enables these proteins to act as transcription antiterminators at rho-independent terminators (3, 88). This activity is critical for the cold acclimation of cells (88) and seems to be conserved in CspA homologues from higher organisms, for example, wheat Csp protein WCSP1 (76). A similar function has also been shown for cold shock domain proteins from Arabidopsis thaliana (55). Heterologous expression of a cold shock domain protein, CSDP1, or glycine-rich RNA-binding protein, GRP7, from this bacterium complemented the cold-sensitive phenotype of a quadruple csp deletion mutant E. coli.

Recent observations suggest that, in addition to the RNA chaperone function, the CspA homologues have several other cellular roles. This is exemplified by the following observations. CspE has been shown to be involved in camphor resistance and chromosome condensation (37, 102), downregulation of poly(A)-mediated 3' to 5' exonucleolytic decay by PNPase (20), and downregulation of λ Q-mediated transcription antitermination (34), and it has an effect on UV sensitivity (66). The mechanism(s) by which CspE performs these diverse functions is not well defined. CspC and CspE have also been shown to regulate the expression of a number of RpoS-regulated stress proteins such as OsmY (osmotic stress, stationary phase), Dps (osmotic, oxidative stress and stationary phase), ProP (osmotic stress), and KatG (oxidative stress), possibly through regulating RpoS itself. These proteins also regulate expression of the universal stress protein A (UspA), a protein induced in response to numerous stresses (86). CspD, which is induced by starvation and on stationary phase (119) inhibits the initiation and elongation steps of minichromosome replication in vitro, presumably acts as a novel inhibitor of DNA replication and plays a regulatory role in chromosomal replication in nutrient-depleted cells (122). CspA homologues from other bacteria have varied roles. For example, Staphylococcus aureus CspA is involved in the susceptibility of the organism to an antimicrobial peptide derived from human neutrophil cathepsin G (53). The homologue from Lactobacillus plantarum improves adaptation of the organism to cold shock, stationary phase, and freezing stresses (14). A main hypothesis that emerges from these observations is that these proteins may play a more complex role in the stress-response network of cells (86).

Structure of CspA homologues.

The three-dimensional structure of CspA from E. coli and CspB from B. subtilis has been resolved by X-ray crystallography and NMR analysis (19, 78, 103, 104, 105). The protein consists of five antiparallel β-strands (β1 to β5) forming a β-barrel structure with two β-sheets. The two β-strands, β2 and β3, consist of the RNA-binding motifs, RNP1 and RNP2, which collectively include a surface patch of aromatic residues (W11, F18, F20, F31, H33, and F34). These residues are shown to contribute to nucleic acid binding by intercalating between DNA or RNA bases (19, 36). Of these, three residues, F18, F31, and H33, have been shown to be essential for the nucleic acid melting and transcription antitermination function of Csps and are thus pivotal for their cold-acclimation function (90). These proteins have overall negative surface charge with a positively charged aromatic patch on the surface. After binding to RNA by virtue of stacking of the aromatic side chains with RNA bases, the approach of other RNAs for intramolecular or intermolecular base pairing is prevented by charge repulsion. The structure of CspA is thus ideal for its role as an RNA chaperone (29).

Proteins Essential for Cell Growth at Low Temperature

As mentioned above, the CspA homologues have functional redundancy and thus are not individually essential for cell growth at low temperature. However, there are certain proteins, deletion of which leads to sensitivity of cells to low temperature. The DEAD-box RNA helicases CsdA and SrmB, RbfA, and PNPase belong to this group of proteins.

The DEAD-box helicases, CsdA and SrmB.

CsdA is a multifunctional, DEAD-box protein that belongs to the large family of putative RNA helicases conserved from bacteria to humans (60). These proteins play important roles in many cellular processes such as processing, transport, or degradation of RNA or ribosome biogenesis (for a review, see reference 41). CsdA is essential only at low temperature, and deletion of its gene impairs growth on cold shock (9, 50). CsdA has multiple cellular functions, such as:

• Ribosome biogenesis. The deaD/csdA gene was originally identified as a multicopy suppressor of a mutation in the rpsB gene that encodes r-protein, which suggested that CsdA may play a role in the biogenesis of the small ribosomal subunit (109), and later on it was reported that overexpression of CsdA in an S2 mutant restores the incorporation of r-proteins S2 and S1 into the 30S ribosomal subunits (74). Recently, it was shown that CsdA is involved in the biogenesis of the 50S ribosomal subunits and deletion of the csdA gene leads to a deficit in free 50S subunits and accumulation of a 40S-like particle. The authors also showed that CsdA associates with 50S precursors at low temperature and can complement the ribosome defect of a srmB deletion strain (9).

• Translation initiation. Based on the observation that CsdA has 54% identity and 87% sequence similarity to the eukaryotic initiation factor eIF4A, which catalyzes the ATP-dependent unwinding of RNA duplexes and stimulates translational initiation in eukaryotic cells, it was also suggested that CsdA assists in translation by promoting translation initiation of structured mRNAs (63).

• Stabilization and degradation of mRNAs. CsdA accumulates during the early stages of cold acclimatization and assembles into degradosomes with RNase E synthesized in cold-adapted cultures (54, 96, 121). Interestingly, CsdA was also shown to be involved in the efficient and selective degradation of Csp mRNAs by unwinding the mRNA secondary structure that impedes the processive activity of PNPase (121).

Altogether, these observations suggest that CsdA has multiple overlapping functions in several important physiological processes. It is not clear which, or whether all, of the activities of CsdA play a role in its essential cold shock function, but it appears that its role in RNA decay may be most critical for the cold acclimation of cells.

SrmB is another DEAD-box RNA helicase. The srmB gene was originally isolated as a multicopy suppressor of mutations in ribosomal protein genes rplX (encoding L24) (79), suggesting that SrmB is involved in ribosome biogenesis. Deletion of the srmB gene leads to a slow-growth phenotype at low temperature as a result of a deficit in free 50S ribosomal subunits and the accumulation of a new particle sedimenting around 40S. Thus, it was suggested that SrmB is involved in an early step of 50S assembly that is necessary for the binding of L13. This step may consist of a structural rearrangement that, at low temperature, cannot occur without the assistance of SrmB (10).

Both CsdA and SrmB can unwind duplexes with 3' or 5' extensions. Similar to CsdA, overexpression of SrmB stabilizes certain mRNAs (42) and SrmB can also bind to RNase E (54). It has been suggested that CsdA and SrmB assist 50S assembly by modulating RNA structures because their unwinding activity may be required to facilitate structural transitions within the RNA and/or to allow proper binding of r-protein(s). These proteins may act as RNA chaperones that prevent and/or resolve misfolding. For example, rRNA may become trapped in incorrect structures and require assistance to reach its active conformation (35). Despite apparent similar roles, the overexpression of SrmB does not suppress the cold sensitivity of the csdA deletion mutant and vice versa (9). Our recent data show that, in our genetic screen, another DEAD-box RNA helicase, RhlE, can complement the cold-sensitive phenotype of csdA deletion cells. We also observed that, although not detected in our genetic screen, two cold shock-inducible proteins, CspA and an exonuclease, RNase R, can also complement the cold shock function of CsdA. The data suggested that the primary role of CsdA in cold acclimation of cells is in mRNA decay and its helicase activity is pivotal for promoting degradation of mRNAs stabilized at low temperature (2). Interestingly, in B. subtilis, using fluorescent resonance energy transfer (FRET) analysis studies it was shown that the putative cold-induced helicases and the Csps work in conjunction to rescue misfolded mRNA molecules and maintain proper initiation of translation at low temperatures (39).

RbfA.

RbfA is a ribosome-binding factor and was originally identified as a multicopy suppressor of a point mutation in the 5'-end helix of the 16S ribosomal RNA. It was also shown to be associated with the 30S ribosomal subunit (12). These results suggested that RbfA might be involved in the maturation of the 16S ribosomal RNA, and indeed a later study showed it to be required for efficient 16S rRNA processing (7). Deletion of RbfA leads to constitutive cold shock response, and the cells are unable to adapt to low temperature (49). The cold sensitivity of rbfA deletion cells is directly related to their lack of translation initiation-capable 30S subunits containing mature 16S rRNA at low temperature (117).

The three-dimensional structure of RbfA, in which 25 residues have been removed from the carboxyl terminus to make it soluble, was solved by NMR spectroscopy (38). The structure of RbfA from Thermotoga maritima has also been solved (30). The analysis of E. coli RbfADelta25 showed it to contain a α+β fold containing three helices and three β-strands. The structure has type II KH-domain fold topology and is most similar to the KH domain of the E. coli Era GTPase. Its electrostatic field distribution is most similar to the KH1 domain of the NusA protein from T. maritima, another cold shock-associated RNA-binding protein. Structural and functional similarities between RbfA, NusA, and other bacterial type II KH domains suggest that these cold shock-induced proteins may be evolutionarily related.

PNPase.

The E. coli pnp gene, which encodes polynucleotide phosphorylase (PNPase) (100, 101), is essential for growth at low temperatures (64, 92). However, the exact role of PNPase in this essential function is not fully elucidated. Along with RNase II, it is the main exoribonuclease in the cell (71). It promotes the processive degradation of RNA and is also responsible for residual RNA tailing observed in E. coli mutants devoid of the main polyadenylating enzyme PAP I (72, 73). In vitro, it catalyzes the phosphorolytic degradation of RNA, releasing nucleoside 5'-diphosphate from the 3' end of the substrate, and the reverse reaction of nucleoside 5'-diphosphate polymerization with release of phosphate (61).

Expression of PNPase is posttranscriptionally autoregulated at the level of both translation and mRNA stability (45). It binds to the 5' end of the RNase III-processed transcript, leading to inhibition of translation and channeling of pnp mRNA into a degradation pathway (6, 22, 95). This regulation is temporarily relieved during cold acclimation, and the pnp mRNA becomes extremely abundant principally due to stabilization. Transcription antitermination at intercistronic intrinsic terminators also plays a role in transient induction of PNPase (3, 123). Recently, Marchi et al. showed that specific sites in the 5'-untranslated region of pnp mRNA are required for PNPase-sensitive, cold-induced suppression of Rho-dependent transcription termination. Their results suggested that suppression of Rho-dependent transcription termination within pnp and its restoration by PNPase is an autogenous regulatory circuit that modulates pnp expression during the cold acclimation phase (67).

As mentioned above, during the acclimation phase, CspA homologues are highly induced. At the end of the acclimation phase, their synthesis is reduced to new basal levels, while the non-cold shock protein synthesis is resumed, resulting in resumption of cell growth. PNPase was shown to be required to repress production of CspA homologues by degrading them selectively at the end of the acclimation phase. In a pnp mutant, expression of CspA homologues was significantly prolonged upon cold shock (93, 121). PNPase also associates with the endonuclease RNase E and other proteins in the RNA degradosome.

PNPase is a homotrimer of a 711-amino-acid polypeptide, with two RNA-binding domains, KH and S1, located at the C terminus, whereas the duplicated core of the protein is responsible for the catalytic and the homotrimerization properties (25, 99, 108). Systematic structure-function analysis of PNPase domains revealed that both first- and second-core domains are involved in the catalysis of the phosphorolytic reaction and that both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold. Thus, the α-helical domain is involved in PNPase enzymatic activity (6). Recent results also show that the KH and S1 domains of PNPase are important for proper degradosome activity of PNPase at low temperature, at which the stem-loop structures present in the target mRNAs are more stable (68).

SUGARS PLAY A PROTECTIVE ROLE DURING COLD ACCLIMATION OF CELLS

Sugars are shown to confer protection to cells undergoing cold shock. DNA microarray analysis of the cold shock response of E. coli cells showed that several genes encoding proteins involved in sugar transport and metabolism are induced by cold shock (84). These sugars transported include maltose, mannose, ribose, xylose, and trehalose. The cold shock induction of sugar transport systems and the accumulation of the sugars themselves are physiologically relevant as cold shock induction of mannose and maltose transport systems was prominently repressed in a cold-sensitive csp quadruple deletion mutant (116) that has a significantly prolonged (4 h) lag period of growth at 15°C as opposed to the normal 1-h lag period of the wild-type strain. On the other hand, trehalose is previously known to confer protection to cells against heat, cold, and osmotic stress by deterring denaturation and aggregation of proteins, by protecting against oxidative damage, and also by stabilizing the cellular membrane (51). Two genes, otsA and otsB, are involved in the synthesis of trehalose, which is increased about eightfold upon temperature downshift. Effects of trehalose deficiency are more marked at 4°C, at which temperature the cell viability is drastically reduced; this can be corrected by the expression of otsA and otsB genes.

TEMPERATURE DOWNSHIFT AFFECTS PROTEIN FOLDING

Although misfolding of proteins is not as major a problem upon cold shock as in the case of heat shock, recent reports suggest that proper folding and refolding of cold-damaged proteins is important after cold shock (52). DNA microarray analysis of the cold shock response of E. coli showed that certain molecular chaperones, such as caseinolytic proteases (Clps), trigger factor, and GroEL and GroES (84), were induced upon cold shock.

ClpB renatures and solubilizes aggregated proteins at low temperatures (94), while trigger factor (TF) (peptidyl prolyl isomerase) catalyzes the cis/trans isomerization of peptide bonds N-terminal to the proline residue, helps protein synthesis and folding to continue at low temperature (52), accelerates proline-limited steps in protein folding, and associates with ribosomes and helps folding of newly formed protein chains (65). It is probably the first chaperone to interact with the nascent polypeptide chain as it emerges from the tunnel of the 70S ribosome and thus may play an important role in cotranslational protein folding (4). It also interacts with other chaperones such as GroELS (52). Trigger factor is induced at a modest level 2 to 3 h after cold shock (51). Reduction in cellular levels of TF leads to reduced cell viability during storage at 4°C; on the other hand, its overexpression leads to increased viability. Although the GroELS system has been shown to be induced by low temperature, its exact role at low temperature has not been elucidated.

WHY STUDY THE COLD SHOCK RESPONSE?

The study of the cold shock response has implications in basic and health-related research as well as in commercial applications. The knowledge accumulated through the study of the cold shock response in E. coli and B. subtilis can be applied to other organisms. For example, it has been observed that exposing cells to cold shock prior to freezing increases cell viability due to the protective effect of cold shock proteins produced (115). This phenomenon can be exploited commercially to increase the shelf life of refrigerated foods by direct freezing, when food-spoilage bacteria are most susceptible to cellular damage, and to increase cold tolerance of bacteria, such as lactic acid bacteria, to minimize economic losses of the commercial fermentations in which these are used (8, 56). In the agriculture industry, better bio-fertilizers are made by using variants of Rhizobium that are better adapted to cold temperatures (11). A desaturase enzyme from a cyanobacterium, known to play an important role in maintaining membrane fluidity after cold shock, has also been shown to confer chilling resistance to tobacco plants (43, 81). Trehalose-producing transgenic rice plants, made by introducing otsA and otsB of E. coli, accumulate trehalose and exhibit increased tolerance to drought, salt, and cold (44).

Studies of the cold shock response have identified several genetic elements that are critical for cold shock adaptation. These include the translation enhancement element, 5' UTRs of cold-inducible genes, and cold shock proteins such as RNA chaperones that have been successfully used to create more efficient protein-synthesizing systems (110). For example, cold shock vectors use some of these novel features to produce proteins or peptides that cannot be produced by using conventional methods. This will be helpful not only for problematic proteins, such as membrane proteins, but also for producing medically important, expensive proteins and peptides in large quantities with little or no purification required (97). Several cold-adapted enzymes have been used in processes that are carried out at low temperature in the presence of biocatalysts. With these recent novel discoveries, the study of the cold shock response is rapidly gaining importance in both basic research and biotechnology.

CONCLUSION

The cold shock response is elicited by all types of bacteria and affects these bacteria at various levels, such as cell membrane, transcription, translation, and metabolism. Cold shock proteins and certain sugars play a key role in dealing with the initial detrimental effect of cold shock and maintaining the continued growth of the organism at low temperature. Widespread occurrence of CspA homologues in bacteria and the existence of multiple Csps, probably by gene duplication for adaptation to different environmental stresses, suggest that these proteins are important for cell survival. This is also consistent with the suggestion that the CspA family is one of the most ancient protein families (28). Studies of the cold shock response have provided insights into the general principles underlying cellular functions, including those of RNA, membranes, and metabolism. Structural elements critical for the cold shock response and adaptation of bacteria are now being used for biotechnological applications. In addition, cellular processes that occur during the cold shock response are being applied in the food and agriculture industries and in research. Despite recent advances in the study of cold shock response, there are several unanswered questions. It is not known why there is a need for multiple CspA homologues or whether the existence of large numbers of proteins with structurally similar domains is physiologically relevant—for example, the OB fold family proteins that include the CSD proteins as well as the S1 domain proteins. Addressing these questions in future studies should lead to a better understanding of the cold shock response of bacteria.