Role In Cold Adaptation Research Articles

Over-activation of cold tolerance in arabidopsis causes carbohydrate shortage compared with Chorispora bungeana

Many plants cope with cold stress by developing acquired freezing tolerance (AFT) through cold acclimation (CA), and some species have strong basal freezing tolerance (BFT) independent of CA. Although CA has been extensively studied, its potential in agricultural applications is still unclear. Here, carbohydrate metabolism and transcriptome in AFT plant Arabidopsis and BFT plant Chorispora bungeana were compared with each other. The results showed that, although both species were able to accumulate soluble sugars during CA, leaf starch accumulation in the daytime was almost blocked in Arabidopsis while it was greatly enhanced in C. bungeana, revealing that Arabidopsis experienced carbohydrate shortage during CA. Transcriptome and pathway enrichment analysis found that genes for photosynthesis antenna proteins were generally repressed by cold stress in both species. However, cold-up-regulated genes were enriched in protein translation in Arabidopsis, whilst they were enriched in carotenoid biosynthesis, flavonoid biosynthesis, and beta-amylases in C. bungeana. Furthermore, weighted gene co-expression network analysis (WGCNA) showed that the inhibition of starch accumulation was associated with down-regulation of genes for photosynthesis antenna proteins and up-regulation of genes for protein translation, DNA repair, and proteasome in Arabidopsis but not in C. bungeana. Taken together, our results revealed that over-activation of common tolerant mechanisms resulted in insufficient carbohydrate supplies in Arabidopsis during CA, and photoprotective mechanisms played important roles in cold adaptation of C. bungeana. These findings uncovered the drawback of CA in improving freezing tolerance and highlighted photoprotection as a possible solution for agricultural applications.

Novel mitochondrial genome rearrangements including duplications and extensive heteroplasmy could underlie temperature adaptations in Antarctic notothenioid fishes

Mitochondrial genomes are known for their compact size and conserved gene order, however, recent studies employing long-read sequencing technologies have revealed the presence of atypical mitogenomes in some species. In this study, we assembled and annotated the mitogenomes of five Antarctic notothenioids, including four icefishes (Champsocephalus gunnari, C. esox, Chaenocephalus aceratus, and Pseudochaenichthys georgianus) and the cold-specialized Trematomus borchgrevinki. Antarctic notothenioids are known to harbor some rearrangements in their mt genomes, however the extensive duplications in icefishes observed in our study have never been reported before. In the icefishes, we observed duplications of the protein coding gene ND6, two transfer RNAs, and the control region with different copy number variants present within the same individuals and with some ND6 duplications appearing to follow the canonical Duplication-Degeneration-Complementation (DDC) model in C. esox and C. gunnari. In addition, using long-read sequencing and k-mer analysis, we were able to detect extensive heteroplasmy in C. aceratus and C. esox. We also observed a large inversion in the mitogenome of T. borchgrevinki, along with the presence of tandem repeats in its control region. This study is the first in using long-read sequencing to assemble and identify structural variants and heteroplasmy in notothenioid mitogenomes and signifies the importance of long-reads in resolving complex mitochondrial architectures. Identification of such wide-ranging structural variants in the mitogenomes of these fishes could provide insight into the genetic basis of the atypical icefish mitochondrial physiology and more generally may provide insights about their potential role in cold adaptation.

Novel rRNA transcriptional activity of NhaR revealed by its growth recovery for the bipA-deleted Escherichia coli at low temperature.

The BipA protein is a universally conserved GTPase in bacterial species and is structurally similar to translational GTPases. Despite its wide distribution, BipA is dispensable for growth under optimal growth conditions but is required under stress conditions. In particular, bipA-deleted cells (ESC19) have been shown to display a variety of phenotypic changes in ribosome assembly, capsule production, lipopolysaccharide (LPS) synthesis, biofilm formation, and motility at low temperature, suggesting its global regulatory roles in cold adaptation. Here, through genomic library screening, we found a suppressor clone containing nhaR, which encodes a Na+-responsive LysR-type transcriptional regulator and whose gene product partially restored the growth of strain ESC19 at 20°C. The suppressed cells showed slightly reduced capsule production and improved biofilm-forming ability at 20°C, whereas the defects in the LPS core and swimming motility were not restored but aggravated by overexpression of nhaR. Notably, the overexpression partially alleviated the defects in 50S ribosomal subunit assembly and rRNA processing of ESC19 cells by enhancing the overall transcription of rRNA. Electrophoretic mobility shift assay revealed the association of NhaR with the promoter of seven rrn operons, suggesting that NhaR directly regulates rRNA transcription in ESC19 at 20°C. The suppressive effects of NhaR on ribosomes, capsules, and LPS were dependent on its DNA-binding activity, implying that NhaR might be a transcriptional factor involved in regulating these genes at 20°C. Furthermore, we found that BipA may be involved in adaptation to salt stress, designating BipA as a global stress-responsive regulator, as the deletion of bipA led to growth defects at 37°C and high Na+ concentrations without ribosomal defects.

Structural basis for the cold activation and adaptation of an α-agarase from marine bacterium Catenovulum agarivorans STB13

Enzymes have high activity at low temperatures are defined as cold-adapted enzymes, but few enzymes show an activation effect after low-temperature incubation. In this study, we cloned an agarase from the marine bacterium Catenovulum agarivorans STB13 (Ca-AGA) and expressed the protein in Bacillus subtilis. The enzyme exhibited a peculiar cold activation, which was enhanced by 5-fold after incubation at −20 °C or 4 °C for 6 h. Fluorescence and CD spectra studies confirmed the secondary and tertiary structural changes in Ca-AGA at low temperature, which formed compact protein structures after low-temperature treatment. The enzyme also showed outstanding cold adaptation, retaining approximately 60% of its maximum activity at 10 °C. Based on a bioinformatics analysis, the carbohydrate-binding modules (CBMs) and long linker region were presumed to play a crucial role in cold adaptation due to a relatively high percentage of acidic residues and a negative charge density compared to the catalytic domain. A mutation that truncated the linker-CBM region resulted in a 59.97% reduction of the activity at 10 °C. This study describes an α-agarase of cold activation and adaptation in detail, which may have great potential for producing functional agaro-oligosaccharides in food and pharmaceutical industrial applications at ambient temperature.

Genome-wide characterization of the Elovl gene family in Gymnocypris przewalskii and their potential roles in adaptation to cold temperature.

The elongase of the very long-chain fatty acids (Elovls) gene family in fish has more diversity than in other vertebrates, which plays several critical roles in fatty acid synthesis and low-temperature stress adaptation. Gymnocypris przewalskii settles in plateau lakes with cold and resource-poor settings, and the evolution and function of Elovl genes in this fish are unknown. In the study, to identify the Elovl genes in G. przewalskii, the genome-wide identification and phylogenetic analysis of the gene members have been conducted with the expression profile of different tissues under cold stress. Fatty acid compositions, meanwhile, were detected in both the hepatopancreas and skeletal muscle during cold adaptation. A total of 21 Elovl members have been identified from the genome of G. przewalskii, belonging to Elovl1, Elovl2, Elovl4, Elovl5, Elovl6, Elovl7, and Elovl8 subgroups, with conserved ELO domain and four common motifs. Phylogenetic analysis revealed that subfamilies Elovl1 and Elovl7, Elov2, and Elovl5 have a closer genetic relationship, while the Elovl6 class was classed into an independent clade. Synteny analysis showed that whole-genome duplication, tandem duplicates, and gene conversion could drive the Elovls family expansion in G. przewalskii. The Ka/Ks and RELAX analysis showed distinguishing positive selection traces in ORF sequences of gpElovl2. Transcriptional data showed that different gpElovl subtypes exhibited a tissue-specific expression. Subtypes gpElovl1a, gpElovl2 and gpElovl6l were highly expressed induced by cold stress, as well as fatty acid metabolism-related genes, including Acyl-CoA synthetase long-chain gene (Ascl1a-1) and Stearyl-CoA desaturase gene (Scd1a-1). In addition, monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) contents of the hepatopancreas and skeletal muscle were significantly increased under 15-day cold stress. These results provide a better understanding of fish Elovl genes and their roles in cold adaptation.

In Silico Structural and Functional Analysis of Cold Shock Proteins in Pseudomonas fluorescens PF08 from Marine Fish

Pseudomonas fluorescens is a specific spoilage microorganism of refrigerated marine fish, and is highly adapted to low temperature. Cold shock proteins (CSPs) play an important role in cold adaptation of bacteria. In this study, CSP genes were identified from the genome of P. fluorescens PF08 by search of the conserved domain of CSPs with HMMER software, and the CSP physicochemical properties, structures, and functions were analyzed through bioinformatics. Five typical CSPs were identified in the P. fluorescens PF08 genome (PfCSPs). All five PfCSPs are small hydrophilic acidic proteins with a molecular mass of ca. 7.4 kDa. They are located in the cytoplasm and are nonsecretory and nontransmembrane proteins. Multiple sequence alignment analysis indicated that the CSPs are highly conserved between species, especially in DNA-binding sites and RNA-binding motifs that can bind to single-stranded DNA and RNA. The five PfCSPs clustered with CspD from Escherichia coli and Salmonella Typhimurium, which suggests a close homology and high functional similarity among the five PfCSPs and CspD. The secondary and tertiary structures of the PfCSPs are in accordance with the characteristics of the CSP family, and ligand binding sites with higher likelihood were found in PfCSPs. The five PfCSPs were predicted to interact with some of the same proteins that are involved in virulence, stress responses (including to low temperature), cell growth, ribosome assembly, and RNA degradation. The results provide further elucidation of the function of CSPs in adaptation to low temperatures by P. fluorescens.

A Novel Cold-adapted Methylovulum species, with a High C16:1ω5c Content, Isolated from an Arctic Thermal Spring in Spitsbergen.

A novel cold-adapted methane-oxidizing bacterium, termed TFB, was isolated from the thermoglacial Arctic karst spring, Trollosen, located in the South Spitsbergen National Park (Norway). The source water is cold and extremely low in phosphate and nitrate. The isolate belongs to the Methylovulum genus of gammaproteobacterial methanotrophs, with the closest phylogenetic affiliation with Methylovulum miyakonense and Methylovulum psychrotolerans (96.2 and 96.1% 16S rRNA gene sequence similarities, respectively). TFB is a strict aerobe that only grows in the presence of methane or methanol. It fixes atmospheric nitrogen and contains Type I intracellular membranes. The growth temperature range was 2–22°C, with an optimum at 13–18°C. The functional genes pmoA, mxaF, and nifH were identified by PCR, whereas mmoX and cbbL were not. C16:1ω5c was identified as the major fatty acid constituent, at an amount (>49%) not previously found in any methanotrophs, and is likely to play a major role in cold adaptation. Strain TFB may be regarded as a new psychrotolerant or psychrophilic species within the genus Methylovulum. The recovery of this cold-adapted bacterium from a neutral Arctic thermal spring increases our knowledge of the diversity and adaptation of extremophilic gammaproteobacterial methanotrophs in the candidate family “Methylomonadaceae”.

Microevolution and Adaptive Strategy of Psychrophilic Species Flavobacterium bomense sp. nov. Isolated From Glaciers.

Numerous mountain glaciers located on the Tibetan Plateau are inhabited by abundant microorganisms. The microorganisms on the glacier surface are exposed to the cold, barren, and high-ultraviolet radiation environments. Although the microbial community composition on glaciers has been revealed by high-throughput sequencing, little is known about the microevolution and adaptive strategy of certain bacterial populations. In this study, we used a polyphasic approach to determine the taxonomic status of 11 psychrophilic Flavobacterium strains isolated from glaciers on the Tibetan Plateau and performed a comparative genomic analysis. The phylogenetic tree based on the concatenated single-copy gene sequences showed the 11 strains clustered together, forming a distinct and novel clade in the genus Flavobacterium. The average nucleotide identity (ANI) values among these strains were higher than 96%. However, the values much lower than 90% between them and related species indicated that they represent a novel species and the name Flavobacterium bomense sp. nov. is proposed. The core and accessory genomes of strains in this new Flavobacterium species showed diverse distinct patterns of gene content and metabolism pathway. In order to infer the driving evolutionary forces of the core genomes, homologous recombination was found to contribute twice as much to nucleotide substitutions as mutations. A series of genes encoding proteins with known or predicted roles in cold adaptation were found in their genomes, for example, cold-shock protein, proteorhodopsin, osmoprotection, and membrane-related proteins. A comparative analysis of the group with optimal growth temperature (OGT) ≤ 20°C and the group with OGT > 20°C of the 32 Flavobacterium type strains and 11 new strains revealed multiple amino acid substitutions, including the decrease of the proline and glutamine content and the increase of the methionine and isoleucine content in the group with OGT ≤ 20°C, which may contribute to increased protein flexibility at low temperatures. Thus, this study discovered a novel Flavobacterium species in glaciers, which has high intraspecific diversity and multiple adaptation mechanisms that enable them to cope and thrive in extreme habitats.

Barley cold-induced CISP proteins contribute to the accumulation of heavy metals in roots

Cold-induced CISP genes are widely distributed in Poaceae species such as barley, rye, and wheat. In our previous study, we suggested that barley CISP proteins encoded by CISP genes, CISP1 and CISP2, serve as an RNA chaperone and play a vital role in cold adaptation of barley. However, we found that the expression of barley CISP genes are also induced by other abiotic stresses such as heavy metal stresses. Their expression was rapidly upregulated in roots by treatment with an excess of Cu, Zn and Mn. Furthermore, we confirmed that the barley CISP proteins are mainly localized to the root apical meristems where the heavy metals are accumulated. These data suggest that the barley CISPs should play an important role in the adaptation to the heavy metal stresses. We then investigated the influence of CISP2 overexpression on Cu stress tolerance of plants. The results showed that the overexpression of CISP2 inhibits growth rate of rice and increase the accumulation of Cu in roots. Based on these findings, we proposed that the barley CISPs are multifunctional protein and involved in the accumulation of heavy metals to regulate the balance of metal ions.

The bZIP73 transcription factor controls rice cold tolerance at the reproductive stage.

SummaryCold temperature during the reproductive stage often causes great yield loss of grain crops in subtropical and temperate regions. Previously we showed that the rice transcription factor bZIP73Jap plays an important role in cold adaptation at the seedling stage. Here we further demonstrate that bZIP73Jap also confers cold stress tolerance at the reproductive stage. bZIP73 Jap was up‐regulated under cold treatment and predominately expressed in panicles at the early binucleate and flowering stages. bZIP73Jap forms heterodimers with bZIP71, and co‐expression of bZIP73 Jap and bZIP71 transgenic lines significantly increased seed‐setting rate and grain yield under natural cold stress conditions. bZIP73Jap:bZIP71 not only repressed ABA level in anthers, but also enhanced soluble sugar transport from anthers to pollens and improved pollen grain fertility, seed‐setting rate, and grain yield. Interestingly, bZIP73Jap:bZIP71 also regulated the expression of qLTG3‐1 Nip, and qLTG3‐1 Nip overexpression lines greatly improved rice tolerance to cold stress during the reproductive stage. Therefore, our work establishes a framework for rice cold stress tolerance through the bZIP71‐bZIP73Jap‐qLTG3‐1Nip‐sugar transport pathway. Together with our previous work, our results provide a powerful tool for improving rice cold stress tolerance at both the seedling and the reproductive stages.

Roles of Active-Site Aromatic Residues in Cold Adaptation of Sphingomonas glacialis Esterase EstSP1.

The aromatic amino acids, Tyr or Trp, which line the active-site walls of esterases, stabilize the catalytic His loop via hydrogen bonding. A Tyr residue is preferred in extremophilic esterases (psychrophilic or hyperthermophilic esterases), whereas a Trp residue is preferred in moderate-temperature esterases. Here, we provide evidence that Tyr and Trp play distinct roles in cold adaptation of the psychrophilic esterase EstSP1 isolated from an Arctic bacterium Sphingomonas glacialis PAMC 26605. Stern–Volmer plots showed that the mutation of Tyr191 to Ala, Phe, Trp, and His resulted in reduced conformational flexibility of the overall protein structure. Interestingly, the Y191W and Y191H mutants showed increased thermal stability at moderate temperatures. All Tyr191 mutants showed reduced catalytic activity relative to wild-type EstSP1. Our results indicate that Tyr with its phenyl hydroxyl group is favored for increased conformational flexibility and high catalytic activity of EstSP1 at low temperatures at the expense of thermal stability. The results of this study suggest that, in the permanently cold Arctic zone, enzyme activity has been selected for psychrophilic enzymes over thermal stability. The results presented herein provide novel insight into the roles of Tyr and Trp residues for temperature adaptation of enzymes that function at low, moderate, and high temperatures.

Transcriptomic analyses on muscle tissues of Litopenaeus vannamei provide the first profile insight into the response to low temperature stress.

The Pacific white shrimp (Litopenaeus vannamei) is an important cultured crustacean species worldwide. However, little is known about the molecular mechanism of this species involved in the response to cold stress. In this study, four separate RNA-Seq libraries of L. vannamei were generated from 13°C stress and control temperature. Total 29,662 of Unigenes and overall of 19,619 annotated genes were obtained. Three comparisons were carried out among the four libraries, in which 72 of the top 20% of differentially-expressed genes were obtained, 15 GO and 5 KEGG temperature-sensitive pathways were fished out. Catalytic activity (GO: 0003824) and Metabolic pathways (ko01100) were the most annotated GO and KEGG pathways in response to cold stress, respectively. In addition, Calcium, MAPK cascade, Transcription factor and Serine/threonine-protein kinase signal pathway were picked out and clustered. Serine/threonine-protein kinase signal pathway might play more important roles in cold adaptation, while other three signal pathway were not widely transcribed. Our results had summarized the differentially-expressed genes and suggested the major important signaling pathways and related genes. These findings provide the first profile insight into the molecular basis of L. vannamei response to cold stress.

Polymorphism in the TRP8 gene in Kyrgyz population: Putative association with highland adaptation

The human TRPM8 gene encodes a receptor mediating cold sensitivity, and this points to its putative role in cold adaptation. The structural variability of the TRPM8 gene for five single-nucleotide polymorphisms (SNPs) has been studied in the Kyrgyz population. The SNPs are located in the coding regions of the gene, and three of them are confined to a 20-bp segment in exon 7. The frequencies of minor SNP alleles are as follows: rs13004520 G/C = 0.06; rs28901637 A/T = 0.13; rs11562975 G/C = 0.27; rs7593557 G/A = 0.21; rs11563071 C/G = 0.12. The analyzed sample of Kyrgyz population includes 275 individuals living at different altitudes and under drastically different climatic conditions. The frequency of the minor rs11562975 allele in the highlanders (living above 3200 m ASL and higher) is lower by a factor of 1.5 than in the residents of lower regions (760–3000 m ASL; p < 0.01). This result indicates a selective role of rs11562975 in adapting to cold. A comparison of haplotype frequencies in the Kyrgyz population with Europeans, East Asians, and Africans shows a clear narrowing of genotype variation in Europeans in comparison to all the others. Probably, this phenomenon is related to a decline in the population size (bottleneck effect) during evolution. We consider the exon–intron structure of the TRPM8 gene. The epigenetic markers in the vicinity of the gene have been analyzed. Two strong binding sites for insulator ctcf proteins are present there. They are likely to be associated with chromatin conformation and alternative splicing regulation. A structural–functional characterization of genes for the TRP protein family is provided.

The Role of the Sympathoadrenal System in Adaptation to Cold

Noradrenaline released from sympathetic nerve endings and adrenaline secreted by the adrenal cortex have been shown to play roles in cold adaptation. The calorigenic effect of noradrenaline increases during acclimation. Catecholamines stimulate noncontractile thermogenesis in brown fat tissue, white fat tissue, and skeletal muscle. Adrenergic increases in cold tolerance are associated with increases in UCP expression in the tissues. The following signal pathway operates in mediating the calorigenic effect of catecholamines in prolonged cold exposure: catecholamines → β-adrenergic receptors → adenylate cyclase, cAMP → protein kinase A → p38 kinase → transcription factors → increased UCP expression. The following signal pathway operates on acute exposure to cold: catecholamines → β-adrenergic receptors → adenylate cyclase → cAMP → protein kinase A → hormone-sensitive lipase → free fatty acids → UCP → uncoupling of oxidative phosphorylation. The calorigenic effect of stimulation of α1-adrenoreceptors is mediated by the following mechanism: noradrenaline → α1-adrenergic receptors → phospholipase C → inositol-1,4,5-triphosphate → [Ca2+]i → increased calorigenic effect of catecholamines.

Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus

Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (Tm). Mutation of His299 to Tyr increased the optimal temperature, the Tm, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures.

Polymorphism in the TRP8 gene in Kyrgyz population: putative association with highland adaptation

The human TRPM8 gene encodes a receptor mediating cold sensitivity, and this fact points to its putative role in cold adaptation. The structural variability of the TRPM8 gene for five single-nucleotide polymorphisms (SNPs) has been studied in Kyrgyz population. The SNPs are located in coding regions of the gene, and three of them are confined to a segment of 20 bp in exon 7. The frequencies of minor SNP alleles are: rs13004520 G/C = 0.06; rs28901637 А/Т = 0.13; rs11562975 G/C = 0.27; rs7593557 G/A = 0.21; rs11563071 С/G = 0.12. The analyzed sample of Kyrgyz population includes 275 individuals living at different altitudes and under drastically different climatic conditions. The frequency of the minor rs11562975 allele in highlanders (living above 2 800 m A. S. L.) is one-third lower than in residents of lower regions (760–2 800 m A. S. L.; p &lt; 0.01). This result presumes a selective role of rs11562975 in cold adaptation. Comparison of haplotype frequencies in Kyrgyz people with Europeans, East Asians, and Africans shows a clear narrowing of genotype variation in Europeans in comparison to all others. Probably, this phenomenon is related to a population size decline (bottleneck effect) during the evolution. We consider the exon– intron structure of the TRPM8 gene. Epigenetic markers in the vicinity of the gene have been analyzed. Two strong binding sites for insulator ctcf proteins are present there. They are likely to be associated with chromatin conformation and alternative splicing regulation. A structure–functional characterization of genes for the TRP protein family is provided.

A Mutant Chaperonin That Is Functional at Lower Temperatures Enables Hyperthermophilic Archaea To Grow under Cold-Stress Conditions.

Thermococcus kodakarensis grows optimally at 85°C and possesses two chaperonins, cold-inducible CpkA and heat-inducible CpkB, which are involved in adaptation to low and high temperatures, respectively. The two chaperonins share a high sequence identity (77%), except in their C-terminal regions. CpkA, which contains tandem repeats of a GGM motif, shows its highest ATPase activity at 60°C to 70°C, whereas CpkB shows its highest activity at temperatures higher than 90°C. To clarify the effects of changes in ATPase activity on chaperonin function at lower temperatures, various CpkA variants were constructed by introducing single point mutations into the C-terminal region. A CpkA variant in which Glu530 was replaced with Gly (CpkA-E530G) showed increased ATPase activity, with its highest activity at 50°C. The efficacy of the CpkA variants against denatured indole-3-glycerol-phosphate synthase of T. kodakarensis (TrpCTk), which is a CpkA target, was then examined in vitro. CpkA-E530G was more effective than wild-type CpkA at facilitating the refolding of chemically unfolded TrpCTk at 50°C. The effect of cpkA-E530G on cell growth was then examined by introducing cpkA-E530G into the genome of T. kodakarensis KU216 (pyrF). The mutant strain, DA4 (pyrF cpkA-E530G), grew as well as the parental KU216 strain at 60°C. In contrast, DA4 grew more vigorously than KU216 at 50°C. These results suggested that the CpkA-E530G mutation prevented cold denaturation of proteins under cold-stress conditions, thereby enabling cells to grow in cooler environments. Thus, a single base pair substitution in a chaperonin gene allows cells to grow vigorously in a new environment. Thermococcus kodakarensis possesses two group II chaperonins, cold-inducible CpkA and heat-inducible CpkB, which are involved in adaptation to low and high temperatures, respectively. CpkA might act as an "adaptive allele" to adapt to cooler environments. In this study, we compared the last 20 amino acids within the C termini of the chaperonins and found a clear correlation between the CpkA-type chaperonin gene copy number and growth temperature. Furthermore, we introduced single mutations into the CpkA C-terminal region to clarify its role in cold adaptation, and we showed that a single base substitution allowed the organism to adapt to a lower temperature. The present data suggest that hyperthermophiles have evolved by obtaining mutations in chaperonins that allow them to adapt to a colder environment.

Cold adaptation of a psychrophilic chaperonin from Psychrobacter sp. and its application for heterologous protein expression.

A chaperonin, PsyGroELS, from the Antarctic psychrophilic bacterium Psychrobacter sp. PAMC21119, was examined for its role in cold adaptation when expressed in a mesophilic Escherichia coli strain. Growth of E. coli harboring PsyGroELS at 10°C was increased compared to the control strain. A co-expression system using PsyGroELS was developed to increase productivity of the psychrophilic enzyme PsyEst9. PsyEst9 was cloned and expressed using three E. coli variants that co-expressed GroELS from PAMC21119, E. coli, or Oleispira antarctica RB8(T). Co-expression with PsyGroELS was more effective for the production of PsyEst9 compared tothe other chaperonins. PsyGroELS confers cold tolerance to E. coli, and shows potential as an effective co-expression system for the stable production of psychrophilic proteins.

Involvement of the CasK/R two-component system in optimal unsaturation of the Bacillus cereus fatty acids during low-temperature growth

Bacillus cereus sensu lato is composed of a set of ubiquitous strains including human pathogens that can survive a range of food processing conditions, grow in refrigerated food, and sometimes cause food poisoning. We previously identified the two-component system CasK/R that plays a key role in cold adaptation. To better understand the CasK/R-controlled mechanisms that support low-temperature adaptation, we performed a transcriptomic analysis on the ATCC 14579 strain and its isogenic ∆casK/R mutant grown at 12°C. Several genes involved in fatty acid (FA) metabolism were downregulated in the mutant, including desA and desB encoding FA acyl-lipid desaturases that catalyze the formation of a double-bond on the FA chain in positions ∆5 and ∆10, respectively. A lower proportion of FAs presumably unsaturated by DesA was observed in the ΔcasK/R strain compared to the parental strain while no difference was found for FAs presumably unsaturated by DesB. Addition of phospholipids from egg yolk lecithin rich in unsaturated FAs, to growth medium, abolished the cold-growth impairment of ΔcasK/R suggesting that exogenous unsaturated FAs can support membrane-level modifications and thus compensate for the decreased production of these FAs in the B. cereus ∆casK/R mutant during growth at low temperature. Our findings indicate that CasK/R is involved in the regulation of FA metabolism, and is necessary for cold adaptation of B. cereus unless an exogenous source of unsaturated FAs is available.

Development- and cold-regulated accumulation of cold shock domain proteins in wheat

Cold shock domain (CSD) proteins, or Y-box proteins, are nucleic acid-binding proteins that are widely distributed from bacteria to higher plants and animals. Bacterial CSD proteins play an essential role in cold adaptation by destabilizing RNA secondary structures. WHEAT COLD SHOCK DOMAIN PROTEIN 1 (WCSP1) shares biochemical functions with bacterial CSD proteins and is possibly involved in cold adaptation. In this study, the temporal and spatial distribution of the wheat cold shock domain protein family (WCSPs) was serologically characterized with regard to plant development and cold adaptation. Four WCSP genes were identified through database analysis and were classified into three classes based on their molecular masses and protein domain structures. Class I (20 kD) and class II (23 kD) WCSPs demonstrated a clear pattern of accumulation in root and shoot meristematic tissues during vegetative growth. In response to cold, marked increases in WCSP levels were observed but the pattern of accumulation differed by tissue. Accumulation of WCSPs in crown tissue during cold acclimation was observed in the winter cultivar ‘Chihokukomugi’ but not in the spring cultivar ‘Haruyutaka’, suggesting a possible function for WCSPs in cold acclimation. During flower and seed development, protein levels of class I and class II WCSPs remained high. The class III WCSP (27 kD) was detected only during seed development. The highest level of class III WCSP accumulation was observed at the milky seed stage. Together, the results of this study provide a view of CSD protein accumulation throughout the life cycle of wheat and suggest that WCSPs function differentially in plant development and cold adaptation.