Temperature has been deemed one of the most important factors for growth and modulates selection and distribution of microorganisms throughout the Earth. Microorganisms can be classified according to the range of temperatures in which they can grow: hyperthermophiles (temperature range of 80–115 °C, with optimum growth at >80 °C), thermophiles (40–110 °C, optimum ∼60 °C), mesophiles (10–50 °C, optimum ∼37 °C), psychrotrophs (0–30 °C, optimum ∼22–25 °C) and psychrophiles (0–20 °C, optimum ≤15 °C) [1].

Adaptation of microorganisms that live in extreme temperatures (either high or low) is gaining particular interest among scientists, due to the importance of understanding the molecular basis of adaptation and biotechnological applications [1]. In contrast to thermophilic and/or hyperthermophilic microorganisms, only a few studies have been conducted so far to address the adaptation to low temperatures (cold adaptation). This might be due to commercial reasons in which thermostable microorganisms or enzymes are highly valuable for biotechnological applications [2,3]. However, some cold-adapted enzymes were reported to be promising and have recently been widely applied in biotechnology [4–8]. Therefore, understanding the adaptation of microorganisms to low temperature, including the machinery enzymes, is also important for industrial purpose. The information may be also ecologically important since approximately 70% of the earth’s biosphere has a constant temperature of 4–5 °C [4,9] the reason why it is populated predominantly by psychrophilic and psychrotropic bacteria. It is noted also that some pathogenic microorganisms survive in low temperature, therefore the insight on this mechanism would be useful for controlling pathogens.

Once microorganisms are exposed to low temperatures they may face a series of problems that affect their growth. Primarily, problems encountered at low temperatures are associated with membrane viscosity and permeability, synthesis of macromolecules (e.g., replication, transcription and translation), slower metabolic rates due to lower kinetics of enzymatic reactions, and the ability to sense and transmit temperature signals to the regulatory networks of cells for effective responses [1].

Protein folding can be given as an example of problems associated with the synthesis of macromolecules and a slower metabolic rate. Folding of proteins, especially for cis-proline containing proteins, is suggested to be a rate-limiting step for bacterial growth in cold environments [10]. The conformations of folded proteins are usually compatible with only one isomer of a peptide bond, in which the trans form is preferred to the cis form due to energetic reasons [11]. However, in the case of peptide bonds preceding proline residues (Xaa-Pro), some of them correctly form cis peptide bonds in folded proteins [12]. Consequently, interconversion of cis-trans Xaa-Pro peptide bonds take place during folding processes and are termed as peptidyl prolyl cis-trans isomerizations.

However, the kinetics of cis-trans isomerization is intrinsically slow because it involves the rotation about a partial double bond. Isomerization is much slower at low temperatures, as are other chemical reactions, since it is thermodynamically linked [11]. Therefore, cis-trans isomerization is regarded as a rate-limiting step of protein folding. Peptidyl prolyl cis-trans isomerases (PPIases; EC 5.2.1.8) catalyze cis-trans isomerization of Xaa-Pro peptide bonds (Figure 1) and assist proteins to achieve correct native conformations. Due to this ability, PPIases are considered to be foldases, together with protein disulfide isomerases (PDI) [13,14]. Currently, at least three structurally unrelated families of PPIase have been indentified: FK506-binding proteins (FKBP), cyclophilins, and parvulins [15,16].

Psychrophilic and psychrothrophic bacteria produce a cold shock response, which is defined as the sum of cellular reactions to overcome the problems associated with decreases in temperature [1]. Cold shock responses are indicated by the expression of cold shock proteins (CSPs) or class 1 proteins, which are involved in the adaptation of microorganisms to low-temperature environments. Foldases and chaperones have been classified as CSPs, which are important for overcoming a series of problems in protein folding at low temperatures, including slow cis-trans isomerization [1].

Despite PPIases being clearly classified as foldases, multidomain PPIases have also been reported to exhibit chaperone functions as indicated by their ability to bind incompletely folded proteins and prevent aggregation [17–23]. In addition, Wang and Tsou [13] noted that foldases may also act as chaperones and vice versa. Therefore, the dual functions of PPIases would be an interesting topic for in-depth study in relation to their involvement in cold-adaptation.

However, to our knowledge, studies pertaining to this issue are limited. Despite some PPIases from cold-adapted organisms have been reported and linked to cold adaptation [10,24,25], these reports are based on proteomic analysis without further structural and functional studies. Our group has been extensively studying FKBP22 from the psychrotrophic bacterium, Shewanella sp. SIB1 (SIB1 FKBP22). Expression of SIB1 FKBP22 was significantly increased when Shewanella sp. SIB1 was grown at low temperatures, indicating that it is involved in cold-adaptation [26]. SIB1 FKBP22 is thought to have dual functions, since it exhibits PPIase activity (foldase) and is able to bind to a folding intermediate protein (chaperone). This review focuses on structural and functional studies of SIB1 FKBP22 and its possible involvement in cold adaptation through its dual function as a foldase and a chaperone.

Shewanella sp. SIB1 was previously isolated from reservoir water at the Shibugaki petroleum processing plant (Teikoku Oil, Niigata, Japan). Physiological and morphological characterizations were examined using an API 20NE Kit (bioMérieux, Durham, NS, USA) and scanning electron microscopy, respectively [27]. Physiologically, strain SIB1 is able to assimilate glucose, d-mannitol, maltose and malic acid. In addition, this strain possesses cytochrome oxidase and β-glucosidase enzymes and exhibits protease activity as indicated by its ability to degrade gelatin substrates. Further biochemical characterization has shown that the SIB1 strain is consistently able to degrade a catechol (a hydroxylated aromatic hydrocarbon), although in small amounts. On the contrary, the SIB1 strain cannot degrade two other hydrocarbon compounds, namely alkenes (aliphatic carbon) and naphtalene (an aromatic hydrocarbon). Morphologically, strain SIB1 is rod-shaped with a diameter of 0.3 μm and a length of 1.0–1.3 μm (Figure 2). The 16S RNA gene of strain SIB1 shows a 99.9% homology to the 16S RNA gene of psychrotrophic Shewanella sp. AC10 [28], which obviously classify strain SIB1 in the genus Shewanella.

Growth temperature profiles of strain SIB1 have been identified [27]. Results show that the SIB1 strain does not grow at temperatures above 30 °C, while the maximum growth rate was at 20 °C. However, maximum cell density of this strain is reached at 4 °C (Figure 3). This consideration prompted us to confidently classify this strain as a psychrophile (previously classified as psychotrophic bacterium [27]). Shewanella sp. SIB1 is an appropriate model to decipher the mechanisms for cold-adaptation. To achieve this goal, we have studied several proteins isolated from Shewanella sp. SIB1, including RNase HI [29], FKBP22 [26], alkaline phosphatase [8], RNase HII [30] and CutA [31]. However, this review focuses on SIB1 FKBP22 since, among the proteins mentioned above, only this protein is involved in cold shock responses of strain SIB1 and therefore is thought to be important for cold adaptation [26].

Responses of psychrotrophic and psychrophilic bacteria to low temperatures are characterized by changes in their profile of cellular contents. Some proteins that are associated with adaptation to low temperatures are highly expressed. Hence, we have investigated proteins that are important for cold adaptation of Shewanella sp. SIB1 by employing two-dimensional electrophoresis [26]. As a part of proteome analysis, 2D-PAGE has been widely used in order to identify putative proteins/enzymes involved in cold adaptation of various microorganisms [24,32–35].

The 2D-PAGE maps were obtained from extracted soluble fractions of Shewanella sp. SIB1, which were grown at 4 and 20 °C. By comparing the intensity of spots in the gel at these two temperatures, we found that the cellular content of several proteins were greatly increased at 4 °C as compared to 20 °C (Figure 4). One of these proteins is referred to as P28, which has an apparent mass of 28 kDa and an isoelectric point of 4.0. Suzuki et al. [26] reported that when Shewanella sp. SIB1 is grown at 10 and 0 °C, the cellular content of P28 is increased as compared to those grown at 20 °C. However, this protein did not significantly increase at 15 °C. Collectively, P28 is thought to be greatly increased when Shewanella sp. SIB1 is grown at temperatures below 10 °C. This evidence prompted us to classify P28 as a cold shock protein of Shewanella sp. SIB1 which might be involved in cold adaptation.

Further analysis indicates that P28 shares 56% amino acid sequence identity to Escherichia coli FKBP22 (accession no. AAC77164) [36], 43% to E. coli FkpA (accession no. AAC76372) [37] and 41% to Legionella pneumophila MIP (accession no. S42595) [38]. These proteins are members of the PPIase family which exhibit activity towards peptide (PPIase_pep) and protein (PPIase_pro) substrates. Electrospray ionization mass spectrometry (ESI-MS) analysis revealed that the molecular mass of P28 was 23.9 kDa, which is comparable to that calculated from the amino acid sequence. According to the aforementioned data, P28 is defined as SIB1 FKBP22, which indicates that this protein is a member of FKBP from SIB1 with a molecular mass of approximately 22 kDa.

Oligomeric state analysis by sedimentation equilibrium analytical ultracentrifugation indicated that SIB1 FKBP22 assumes a homodimeric protein of 48 kDa, about twice as large as those calculated [26]. L. pneumophila MIP, E. coli FkpA and E. coli FKBP22 were also reported to be homodimeric proteins [36–38]. Therefore, SIB1 FKBP22, as well as E. coli FkpA, was classified to the (MIP)-like FKBP protein subfamily. However, when gel filtration was employed to analyze the oligomeric state of SIB1 FKBP22, the deduced molecular mass was 3–4 times larger than calculated, indicating that SIB1 FKBP22 forms a trimeric or tetrameric structure. This discrepancy might be caused by the shape of SIB1 FKBP22, since it is apparently a non globular protein that migrates through the gel filtration column faster than other globular proteins that were used for the calibration of molecular mass.

Due to the lack of a crystal structure image of SIB1 FKBP22, a three-dimensional model of this protein has been constructed based on the crystal structure of L. pneumophila MIP [39]. Based on this model, these proteins are predicted to assume a non-globular V-shaped homodimeric structure, in which two monomers interact with each other at their N-domains (Figure 5). Each monomer assumes a dumbbell-like structure, in which the N-domain, consisting of α1 and α2 helices, and the C-domain, consisting of six β strands (β1–β6) and an α4 helix, are linked by an α3 helix, which is 40 residues long. The C-domains, which are located at both ends of a V-shaped structure, face each other across the cleft of this structure. The interface of two monomers, which is located at the bottom of the V-shaped structure, is stabilized by the hydrophobic interactions between the α1 helix of one monomer and the α2 helix of the other.

It is important to confirm whether SIB1 FKBP22 is a typical cold-adapted enzyme. In order to be involved in cold adaptation, a protein must adapt to low temperatures and remain fully functional. For this purpose, we have analyzed PPIase activity of SIB1 FKBP22 at various temperatures and determined its relationship to the stability of the protein.

The role of SIB1 FKBP22 in protein folding at low temperatures remains to be clarified. Two groups of proteins, chaperones and foldases, have been recognized as groups of accessory proteins that assist the maturation of nascent polypeptides into functional proteins within cells [13]. Chaperones are defined as a group of proteins that assist correct folding without covalent changes. They recognize and selectively bind nonnative proteins to prevent aggregation. In contrast, foldases catalyze necessary covalent reactions directly involved in protein folding. The foldases, therefore, differ from chaperones due to their involvement in covalent changes. Only two families of foldases have been characterized thus far: namely PDI and PPIases.

Wang and Tsou [13] provided interesting evidence that in some cases, foldases may act as chaperones. Likewise, chaperones may exhibit catalytic activity that is involved in folding protein. Therefore, a protein may act as foldase and chaperone to assist protein folding. In this respect, SIB1 FKBP22 may behave as a foldase and chaperone to overcome problems in protein folding at low temperatures as faced by Shewanella sp. SIB1.

SIB1 FKBP22 has been shown to be overproduced when the cells are grown at low temperatures and has shown typical features of cold-adapted enzymes. These features prompted us to propose that SIB1 FKBP22 is involved in cold adaptation of Shewanella sp. SIB1. However, it remains to be clarified exactly how FKBP22 is involved in the cold-adaptation of Shewanella sp. SIB1. According to the aforementioned results, we propose that SIB1 FKBP22 is important for folding proteins in the cells of Shewanella sp. SIB1 when grown at low temperatures. This might be due to the ability of SIB1 FKBP22 to act as a foldase and chaperone at low temperatures.

We believed that, as for other bacteria [1], exposing Shewanella sp. SIB1 to low temperatures causes a series of cellular problems, including the folding of proteins. In respect to the folding of proteins containing a cis-proline residue, cis-trans isomerization becomes much slower at low temperatures, since it is thermodynamically linked [11]. Moreover, slow folding, which might be caused by slow cis-trans isomerization, promotes others problems, which include the misfolding or degradation of protein substrates.

To address this problem, SIB1 FKBP22 was significantly increased when Shewanella sp. SIB1 was grown at low temperatures. This evidence was obviously shown by our 2D-PAGE analysis, as previously described. Once expressed, SIB1 FKBP22 will act as foldase through its PPIase_pro activity to catalyze a rate-limiting step of folding proteins containing a cis-proline residue. This response is part of strategy used for a cold adaptation process through proper folding of proteins and/or maintenance of the quaternary structure of proteins [59].

The ability of SIB1 FKBP22 to act as a foldase is supported by its chaperone activity that facilitates the binding affinity to a folding intermediate protein substrate. In the absence of chaperone activity, the PPIase_pro activity of SIB1 FKBP22 was significantly decreased and therefore could not act as an efficient foldase. Moreover, chaperone activity of SIB1 FKBP22 may also be important to prevent aggregation of proteins without a cis-proline residue. It has been reported that low temperature caused denaturation and aggregation of proteins [1]. However, the ability of SIB1 FKBP22 to prevent aggregation of proteins remains to be shown experimentally.

It is also possible for SIB1 FKBP22 to have other roles for the cold adaptation of Shewanella sp. SIB1. It has been reported that PPIase is involved in various biological processes including cell signaling, cell cycling, immune responses, and neural function [60]. However, the involvement of SIB1 FKBP22 in other biological functions of Shewanella sp. SIB1 and its relationship with cold adaptation remains to be clarified.

Requirements of PPIases for cold adaptation have been observed in various organisms, regardless of whether they are hyperthermophiles, mesophiles or psychrophiles. These findings also revealed that all members of PPIases may be involved in cold adaptation with unclear mechanism.

Apart from SIB1 FKBP22, other FKBP members were also reported to be involved in cold adaptation in various organisms. FKBP18 from a hyperthermophilic archea, Thermococcus sp. KS-1, has been thought to be important at lower growth temperatures than the optimum in Thermococcus sp. KS-1 cells [61]. In the mesophilic bacterium, E. coli, the cold inducible trigger factor (EcTF) has been reported to be important for cold adaptation of E. coli to low temperatures. Inactivation of the gene for EcTF resulted in decreased viability of E. coli in cold temperatures [62]. Pyshcrophilic trigger factor of Antartic bacterium Pseudoalteromonas haloplanktis TAC125, PhTF, was also reported to be a primary chaperone for growth in the cold [10]. It is interesting to note that these cytoplasmic proteins exhibit both PPIase (either toward peptide and protein substrates) and chaperone activities as indicated by the ability to prevent aggregation. It is reminiscent of the ability of SIB1 FKBP22 and therefore FKBP18, EcTF and PhTF may also be involved in cold adaptation through their foldase and chaperone activities.

Other PPIases that have been previously linked to low-temperature adaptation include cell-associated PpiB of Bacillus subtilis [24] and RotA of Erwinia chrysanthemi [63]. Based on the sequence similarities, these proteins belong to cyclophilin-like protein. However, there is no further information on the foldase and chaperone activities of these proteins. It is noted that cyclophilin is also considered as foldase based on its ability to accelerate the refolding of RNase T₁ in vitro. Considering that PpiB and RotA are periplasmic proteins, it is suspected that these proteins are involved in cold adaptation, by facilitating protein folding in the periplasm, as other periplasmic PPIases do [64]. In addition, perisplasmic PPIase has also been reported to participate in the assembly of the outer membrane proteins [65]. However, it remains unclear whether this function has any relation to the cold adaptation mechanism.

CsPinA, a parvulin member of psychrophilic archaeon Cenarchaeum symbiosum, is thought to have an important role in the survival of this archaeon at low temperature [25]. It is commonly known that parvulin exhibits foldase and chaperone activities, which seems to be also exhibited by CsPinA. Both activities may be utilized for overcoming cellular problems that occur at low temperature. Interestingly, parvulin is unique in comparison to other PPIase members since parvulin prefers phosphorylated serine or threonine N-terminal to proline residue [60]. Since phosphorylated substrates are usually related to various biological processes (signal transduction, cell cycle, apoptosis), parvulin is believed to be involved in such biological processes in the cells [60]. However, experimental evidences are necessary to clarify the relationship between these biological roles with cold adaptation mechanism.

Noteworthy, it was found that some PPIases are secreted out of the cells, such as HP0175 of Helicobacter pylori and MIP of L. pneumophila [66,67]. But, these proteins have also been reported to be important for the growth of these organisms at low temperatures. Since these proteins are secreted out of the cell, the mechanism of their involvement in cold adaptation may differ to that of unsecreted PPIases, which are active inside the cell. L. pneumophila MIP is reported to contribute in facilitating protein secretion. L. pnemumophila MIP interacts with a newly secreted protein and, with its PPIase and/or chaperone activities, causes changes that convert the protein from enzymaticaly inactive to active prior or upon being secreted out the cell [67]. It is reported that the abundance of protein presents in wild-type culture supernatants changes upon the decrease in growth temperature of L. pneumophila [68]. Although there is no further information for the involvement of HPO175 in cold adaptation, it is speculated that this protein employs a similar mechanism with L. pneumophila MIP, as other secreted PPIase.

Previous studies have reported that PPIases are involved in cold adaptation of various organisms as part of their cold shock responses and adaptive mechanisms. However, there is no detailed explanation for the mechanism of involvement of PPIase in cold adaptation. In this review, we used SIB1 FKBP22 as a model protein, which was involved in the cold-shock responses of Shewanella sp. SIB1. Our extensive studies on SIB1 FKBP22 have revealed that this protein may be involved in cold adaptation through its ability to assist the folding of proteins at low temperatures, either as a foldase or a chaperone. As a foldase, SIB1 FKBP22 catalyzes the folding of proteins containing a cis-proline residue, where its folding rate is significantly restricted by cis-trans isomerizaton. As a chaperone, SIB1 FKBP22 was able to bind nonnative protein substrates, which included proteins containing cis-proline residues, during the folding reaction. Moreover, SIB1 FKBP22 may prevent the aggregation of proteins during folding through its chaperone activity. To our knowledge, SIB1 FKBP22 is the only PPIase from psychrophilic bacteria that has been extensively studied so far.

Apparently, foldase and chaperone activities are common mechanisms of PPIase which is involved in cold adaptation. However, additional mechanisms could be attributed for some periplasmic and secreted PPIases. Periplasmic PPIases are apparently not only involved in folding of various proteins in the periplasm, but also for assembling of outer membrane proteins. Meanwhile, secreted PPIases facilitate secretion of proteins that are necessary for the survival of microorganisms at low temperature. More extensive work to broaden our knowledge on these additional mechanisms of PPIases for cold adaptation is necessary.