2.1. Chemical Modification of the Cellular Membrane
The cell membrane of prokaryotes becomes more rigid in low temperatures, and some chemical changes occur in the membrane fatty acids to prevent cellular damage. New lipid molecules are synthesized or modified to produce lipids with a low gel-liquid crystalline phase transition to maintain membrane fluidity [
15]. The main changes observed in the membrane fatty acids include an increase in the number of unsaturations and methyl groups, a decrease in the chain length, and an increased rate of anteiso chemical ramifications compared to the iso ramifications [
15]. This process of membrane adaptation is commonly termed homeoviscous adaptation [
16]. Polyunsaturated fatty acids (PUFAs) have a much lower melting temperature compared to monounsaturated fatty acids. Thus, PUFAs are responsible to maintain membrane fluidity even in temperatures below 0 °C. The unsaturated branched-chain fatty acids are generated by anaerobic (de novo synthesis) or aerobic pathways (post-synthesis modification) [
15].
An anaerobic pathway is commonly found in Gram-positive bacteria of the
Bacillales order, where unsaturated branched-chain fatty acids are synthesized from simpler molecules such as acetyl-CoA [
17]. First, acetyl-CoA is converted to malonyl-CoA by acetyl-CoA caboxylase and subsequently linked to an acyl carrier protein (ACP), forming malonyl-ACP. This molecule undergoes successive rounds of elongation of its fatty acid chain through a cyclic pathway whose reactions are catalyzed by enzymes encoded by the genes
fabF,
fabG,
fabI, and
fabH. The newly synthesized fatty acid molecule is then linked to glycerol-3-phosphate to form phosphatidic acid, which is a key intermediate molecule of all membrane glycerolipids [
17].
In the aerobic pathway, the unsaturations are introduced directly into the membrane phospholipids by desaturase enzymes through dehydrogenation reactions. In
Bacillus subtilis, the expression of Δ5-fatty acid desaturase is activated by a two-component system called DesR-DesK [
18]. It has been suggested that a change in membrane fluidity caused by low temperatures result in conformational changes in DesK, triggering autokinase activity [
19]. Once activated, DesK phosphorylates DesR, which binds to DNA, inducing the expression of desaturase genes [
20,
21].
Omic studies have allowed a better understanding of the microbial cold adaptation mechanisms through the identification of differentially expressed proteins. In a genomic study of
Colwellia psychrerytharea the proteins involved in the synthesis, ramification, and cis-isomerization of polyunsaturated fatty acids were described [
22]. Subsequently, the authors identified differentially expressed genes of polyunsaturated fatty acid synthases (
pfaC,
pfaA, and
pfaD) [
23]. To date, these synthase enzymes have been described only in marine bacteria [
23,
24]. In
Sphingopyxis alaskensis, the enzymes involved in the de novo synthesis of fatty acids were described using quantitative proteomic approaches [
25]. However, it was not possible to determine whether the bacterium produces new fatty acid chains or desaturates the existing membrane lipids. Recent studies have shown that two psychrotrophic species—
Exiguobacterium sibiricum 255-15 and
Psychrobacter arcticus 273-4—repress the expression of their genes associated with fatty acid biosynthesis while upregulating the genes associated with desaturation at low temperatures [
9,
26].
Interestingly,
E. sibiricum 255-15 exhibited an increase in the expression of genes involved in peptidoglycan biosynthesis. An increase in cell wall density can protect bacteria against cell disruption that may be caused by ice formation and osmotic pressure at low temperatures [
9]. The same behavior was observed in
Planococcus halocryophilus Or1 [
27]. In contrast, other studies have demonstrated that the species
P. arcticus represses the expression of genes involved in peptidoglycan biosynthesis and enhances the expression of genes involved in the autolytic cleavage of the cell wall [
26]. In
Sphingopyxis alaskensis, a high abundance of proteins involved in cell wall biogenesis was described at 10 °C including a membrane structural lipoprotein OmpA which acts in the optimization of the structure and function of the membrane [
25]. Recently, a transcriptomic analysis of
Listeria monocytogenes cultivated under low temperatures and osmotic stress revealed the upregulation of genes associated with the biosynthesis of peptidoglycan and fatty acid molecules [
28].
It is also important to note that the activity of membrane carriers is directly influenced by the lipidic state of the membrane [
29]. The transport and diffusion through the membrane are also compromised at low temperatures. To balance this deficit, proteins of the transport system are upregulated. Despite the different mechanisms observed in the Bacteria domain, the molecular modifications at low temperatures have one single purpose: increase the number of membrane polyunsaturated branched-chain fatty acids to maintain membrane fluidity and the correct transport and diffusion of substances through this important biological barrier.
2.2. Cold-Adapted Enzymes
Microbial adaptation to extreme temperatures requires the evolution of enzymes to work with a high catalytic efficiency under these extreme conditions. Such extremophilic enzymes are valuable tools for studying the relationships between protein stability, dynamics, and function [
30]. Low temperatures markedly reduce the k
cat of nearly all enzymatic reactions in a cell [
31]. However, because this may not seem to be a significant barrier to microbial physiological processes, it is very clear that psychrophilic and psychrotrophic enzymes have adapted to efficiently operate at low temperatures. This enzymatic efficiency depends on the ratio between K
cat/K
m. K
cat measures how many substrate molecules are converted in products in a unit of time under optimal catalytic conditions. The K
cat constant is commonly called the “turnover number.” The constant K
m measures the substrate concentration that drives the reaction to half of its maximum velocity.
A high value of K
cat (fast turnover) and a low value of K
m (high affinity for a given substrate) increase the enzymatic efficiency. This enzymatic efficiency is directly dependent on the conformational dynamics of the enzyme. Using proteomic, molecular modeling, X-ray crystallography, and Nuclear Magnetic Resonance (NMR), it was observed that a low level of conformational stability allows cold-adapted enzymes to have high rates of enzymatic turnover at low temperatures [
5,
32,
33]. These analyses led to the concept of “flexibility”, which describes the capacity of an enzyme to exhibit increased catalytic activity due to the loss of conformational stability. High flexibility occurs as a result of a reduction in the number of chemical interactions between the amino acids of the protein. This low molecular rigidity allows better complementarity between the active site and the substrate at a low energy cost. Many chemical factors of the enzyme contribute to increased catalysis in cold, including a decrease in the hydrophobicity of the protein core, a decrease in the number of aliphatic amino acids and protein residues forming salt bridges, and increased entropy. Not all of these characteristics are present in the same cold-adapted enzyme, but this list represents some of the changes observed by comparing psychrophilic enzymes to their mesophilic counterparts [
34,
35].
Amino acid composition seems to be an important characteristic to cold adaptation in several microorganisms. An α-amilase from the psychrophilic ciliated protozoon
Euplotes focardii showed large modifications in amino acid composition when compared to an α-amilase of the mesophilic congeneric species
Euplotes crassus. This modification consequently alters the types of intramolecular and surface chemical bonds [
36]. Psychrophilic enzyme of
E. focardii avoided charged, aromatic, and hydrophobic residues on its surface [
36]. The genome of
Psychrobacter arcticus 273-4 shows a statistically significant modification of amino acid composition compared to the mesophilic microorganisms, which can facilitate the flexibility of the proteins at low temperatures and consequently maintain cell viability in cold habitats [
35]. Another example of altered amino acid composition is described in the genus
Vibrionaceae. The psychrophilic species of the genus have proteins with a reduced number of proline residues [
37]. Proline decreases the flexibility of the protein due to the rigidity of its nitrogen–carbon bond [
38]. Thus, proline substitution in psychrophilic proteins increases flexibility of the molecule and consequently decreases the energy required to interact with the substrate. Arginine is also considered an amino acid that promotes structural rigidity since it forms salt bridges and hydrogen bonds with side chains of the protein structure [
39]. A low amount of arginine has been observed in a thermolysin of the psychrophilic
Antarctic bacterium [
39].
2.3. Cold Shock and Cold Acclimation Proteins
One of the most prominent responses of microorganisms to cold environments is the expression of cold shock or cold acclimation proteins. It is important to note that psychrophilic and psychrotrophic as well as mesophilic and thermophilic microorganisms express cold shock proteins to neutralize the effects of temperature reduction. A cold shock response occurs when the microorganism is transferred from an optimal growth temperature to a cold temperature, triggering an immediate and transient molecular response. However, the acclimation process occurs when the bacteria remain exposed to cold for a long period, leading to a late and continuous molecular response [
40]. CSPs are expressed by homologous genes that exhibit RNA chaperone activity and thus act to destabilize secondary structures of RNA erroneously formed due to exposure to cold. The activity of these proteins maintains the correct flux of the transcription and translation process in prokaryotes [
41].
The first bacterial cold shock protein reported was CspA of
Escherichia coli [
42]. Subsequently, several other CSPs were described in a large range of Gram-positive and Gram-negative bacteria. In
E. coli, cold shock proteins can be divided into two major groups: I and II. CSPs belonging to group I (CspA, CspB, CspG, CspI, CsdA, RbfA, NusA, and PNPase) are drastically induced at low temperatures compared to the CSPs of group II (RecA, IF-2, H-NS, GyrA, Hsc66, and HscB). CspA, CspB, CspG, and CspI act as RNA chaperones [
41]. After cold shock, the expression of CSPs of group I is dramatically decreased while other proteins are expressed during the acclimation phase to maintain cell function. CsdA is a DEAD-box RNA helicase that increases septation, resulting in the formation of coccobacilli shape at low temperatures [
43]. CsdA also acts as a RNA chaperone [
43]. RbfA is a ribosome binding factor that is involved in ribosome maturation at cold temperatures [
44]. Finally, PNPase is an enzyme that catalyzes the phosphorolysis of single-stranded polyribonucleotides and is the major factor responsible for the reduction of CSPs in bacterial cells after cold shock response [
45].
Currently, several studies have reported other molecular functions of CSP homologues such as osmotic balance, protection to oxidative stress, starvation, and other types of stress, showing that this protein family has a greater importance than previously thought to the process of microbial adaptation to extreme conditions [
46].
2.4. Other Important Aspects
In addition to the mechanisms of cold adaptation mentioned above, the production of carotenoids markedly contributes to bacterial survival in cold environments. Carotenoids are tetraterpenoids, pigments found naturally occurring in microorganisms, plants, and even animals. Carotenoids are synthesized by several species of bacteria, algae, and fungi in response to several environment stresses [
47]. Prokaryotic organisms that produce carotenoids have been summarized by Takano and colleagues [
48]. However, since 2006 several carotenoid-producing bacterial species were discovered in cold environments [
49]. Carotenoids are detected in the membrane of psychrophilic [
49], psychrotrophic [
27], and mesophilic [
50] bacterial species. The high frequency of pigment production in strains isolated from cold environments suggests that these pigments play an important role in the adaptation to this ecological niche [
51]. At low temperatures, the production of polar carotenoids suppresses the production of non-polar carotenoids. This chemical modification was observed in
Arthrobacter agilis,
Sphingobacterium antarcticus, and
Micrococcus roseus [
50,
52,
53].
In addition, carotenoids protect free-living bacteria from high levels of UV radiation and promote resistance to cellular oxidative stress [
49]. Several genes involved in carotenoid biosynthesis, such as
idi,
crtE,
crtB,
crtI,
crtEB,
crtYe, and
crtYf, were described in bacterial species of the
Arthrobacter genus isolated from Antarctic soils [
49]. Recently, the Prokaryotic Carotenoid Database (ProCarDB) was created by using 304 unique carotenoids synthesized through 50 biosynthetic pathways distributed in 611 prokaryotes [
54].
Additionally, thermal stress affects the osmotic balance of the microbial cell, resulting in a large efflux of cytoplasmatic water. Therefore, to prevent water loss and intracellular ice formation, bacterial cells accumulate compatible solutes in the cell cytoplasm. Examples of such cryo-protectant molecules are glucose, trehalose, glycogen, fructose, alanine, betaine, mannitol, and glycerol. These substances also prevent protein aggregation by stabilizing cytoplasmic macromolecules [
55].
Figure 1 summarizes the main molecular modifications that occur in bacterial cells adapted to low temperatures, as described above.