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CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance

ABSTRACT

Freezing temperatures are a major challenge for life at the poles. Decreased membrane fluidity, uninvited secondary structure formation in nucleic acids, and protein cold-denaturation all occur at cold temperatures. Organisms adapted to polar regions possess distinct mechanisms that enable them to survive in extremely cold environments. Among the cold-induced proteins, cold shock protein (Csp) family proteins are the most prominent. A gene coding for a Csp-family protein, cspB, was cloned from an arctic bacterium, Polaribacter irgensii KOPRI 22228, and overexpression of cspB greatly increased the freeze-survival rates of Escherichia coli hosts, to a greater level than any previously reported Csp. It also suppressed the cold-sensitivity of an E. coli csp-quadruple deletion strain, BX04. Sequence analysis showed that this protein consists of a unique domain at its N-terminal end and a well conserved cold shock domain at its C-terminal end. The most common mechanism of Csp function in cold adaption is melting of the secondary structures in RNA and DNA molecules, thus facilitating transcription and translation at low temperatures. P. irgensii CspB bound to oligo(dT)-cellulose resins, suggesting single-stranded nucleic acid-binding activity. The unprecedented level of freeze-tolerance conferred by P. irgensii CspB suggests a crucial role for this protein in survival in polar environments.

Keywords:
Cold-shock protein (Csp); Psychrophile; Cold-resistance

Introduction

Living organisms encounter several serious challenges when they are exposed to cold environments, including reduced enzyme activity, decreased membrane fluidity, reduced transport of nutrients and waste products, reduced protein movement, decreased rates of transcription and translation, slow DNA replication, retarded protein folding, cold-denaturation of proteins, and intracellular ice formation.11 Jones PG, VanBogelen RA, Neidhardt FC. Induction of proteins in response to low temperature in Escherichia coli. J Bacteriol. 1987;169:2092-2095.

2 Phadtare S. Recent developments in bacterial cold-shock response. Curr Issues Mol Biol. 2004;6:125-136.
-33 D'Amico S, Collins T, Marx J-C, Feller G, Gerday C. Psychrophilic microorganisms: challenges for life. EMBO Rep. 2006;7:385-389. Although freezing temperatures are a major environmental threat at the North and South Poles, relatively high numbers of diverse microorganisms thrive in the polar regions. In particular, α-, β-, and γ-proteobacteria and the Cytophaga-Flavobacterium-Bacteroides phyla are the most commonly found bacteria, and eukaryotes, such as yeasts and microalgae, are frequently found in polar environments.44 Brinkmeyer R, Knittel K, Jurgens J, Weyland H, Amann R, Helmke E. Diversity and structure of bacterial communities in Arctic versus Antarctic pack ice. Appl Environ Microbiol. 2003;69:6610-6619. Organisms living in the polar regions must have the ability to adapt to extremely cold temperatures. To avoid the transition from a fluid- to gel-phase cell membrane, the proportion of unsaturated and methyl-branched fatty acids in lipid membranes is increased.55 Russell NJ. Phychrophilic bacteria-molecular adaptations of membrane lipids. Comp Biochem Physiol Physiol. 1997;118:489-493. The formation of stable secondary structures in nucleic acids at low temperatures inhibits replication, transcription, and translation. Thus, nucleic acid-binding proteins, such as Csp-family proteins and RNA helicases, are induced during temperature downshifts and function as RNA chaperones,66 Jiang W, Hou Y, Inouye M. CspA, the major cold-shock protein of Escherichia coli, is an RNA chaperone. J Biol Chem. 1997;272:196-202. melting the stable secondary structures in RNA molecules, which facilitates translation and ribosome biogenesis.77 Jones PG, Krah R, Tafuri SR, Wolffe AP. DNA gyrase, CS7.4, and the cold shock response in Escherichia coli. J Bacteriol. 1992;174:5798-5802.

8 Schindler T, Graumann PL, Perl D, Ma S, Schmid FX, Marahiel MA. The family of cold shock proteins of Bacillus subtilis. Stability and dynamics in vitro and in vivo. J Biol Chem. 1999;274:3407-3413.
-99 Lim J, Thomas T, Cavicchioli R. Low temperature regulated DEAD-box RNA helicase from the Antarctic archaeon, Methanococcoides burtonii. J Mol Biol. 2000;297(3):553-567. Several chaperones, such as GroEL1010 Tosco A, Birolo L, Madonna S, Lolli G, Sannia G, Marino G. GroEL from the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125: molecular characterization and gene cloning. Extremophiles. 2003;7:1372-1381. and DnaK,1111 Yoshimune K, Galkin A, Kulakova L, Yoshimura T, Esaki N. Cold-active DnaK of an Antarctic psychrotroph Shewanella Sp. Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold temperatures. Extremophiles. 2005;9:145-150. are induced upon cold shock, possibly to cope with cold-denatured proteins. Protein folding, especially cis/trans isomerization of peptidyl prolyl bonds, occurs very slowly at low temperatures, and peptidyl prolyl isomerases play an important role in cold adaptation by facilitating the folding of functionally significant proteins.1212 Suzuki Y, Haruki M, Takano K, Morikawa M, Kanaya S. Possible involvement of an FKBP family member protein from a psychrotrophic bacterium Shewanella sp. SIB1 in cold adaptation. Eur J Biochem. 2004;271:1372-1381. Cryoprotectants, such as antifreeze proteins,1313 Jia Z, Davies PL. Antifreeze proteins: an unusual receptor-ligand interaction. Trends Biochem Sci. 2002;27:101-106. trehalose,22 Phadtare S. Recent developments in bacterial cold-shock response. Curr Issues Mol Biol. 2004;6:125-136. and exopolysaccharides,1414 Nichols CA, Guezennec J, Bowman JP. Bacterial exopolysaccharides from extreme marine environments with special consideration of the southern ocean, sea ice, and deep-sea hydrothermal vents: a review. Mar Biotechnol. 2005;7(4):253-271. have also been implicated in protection of polar organisms, either by preventing ice crystal formation or by avoiding dehydration. Genomic sequencing results suggest some other characteristics of psychrophiles, like enhanced antioxidant capacity to cope with increased production of toxic reactive oxygen species.1515 Rabus R, Ruepp A, Frickey T, et al. The genome of Desulfotalea psychrophila, a sulfate-reducing bacterium from permanently cold Arctic sediments. Environ Microbiol. 2004;6(9):887-902.

Increasing numbers of Csp homologs are being reported from psychrophiles, mostly through genomic DNA sequencing projects, but only limited functional data are available. In an effort to elucidate the detailed mechanisms that allow polar organisms to survive at low temperatures, we studied the functional roles of Csp-family proteins in polar bacteria. A psychrophilic bacterium, Polaribacter irgensii KOPRI 22228, was isolated from Arctic Sea sediment.1616 Uh J-H, Jung YH, Im H. Rescue of a cold-sensitive mutant at low temperatures by cold shock proteins from Polaribacter irgensii KOPRI 22228. J Microbiol. 2010;48:798-802. Two csp genes, cspAPi and cspCPi, were previously cloned from this bacterium and exhibited considerable homology to canonical Escherichia coli cspA (CspAEc). Overexpression of either of these two genes conferred significant cold-resistance phenotypes to their recombinant hosts, and also complemented the cold-sensitivity of a quadruple csp deletion mutant BX04 (ΔcspA, ΔcspB, ΔcspG, and ΔcspE) strain,1717 Xia B, Ke H, Inouye M. Acquirement of cold sensitivity by quadruple deletion of the cspA family and its suppression by PNPase S1 domain in Escherichia coli. Mol Microbiol. 2001;40:179-188. suggesting functional homology among those Csp-family proteins.1616 Uh J-H, Jung YH, Im H. Rescue of a cold-sensitive mutant at low temperatures by cold shock proteins from Polaribacter irgensii KOPRI 22228. J Microbiol. 2010;48:798-802. In this study, we report another Csp-homologous protein from P. irgensii KOPRI 22228, CspBPi, which contains an extra domain on its N-terminal region, in addition to the well conserved cold-shock domain (CSD) at its C-terminal region. This belongs to a subfamily of CSD-fold proteins, recently identified through metagenomic studies of psychrophilic bacteria, and no functional data is available for this kind of proteins at our best knowledge. Therefore, the function of this unique Csp protein from an arctic bacterium in conferring cold tolerance was analyzed in this study.

Materials and methods

Bacterial culture and cloning of the cspBPi gene

P. irgensii KOPRI 22228 was isolated from Arctic Sea sediments near Dasan Korean Arctic Station (Ny-Alesund, Norway), and cultured as previously reported.1616 Uh J-H, Jung YH, Im H. Rescue of a cold-sensitive mutant at low temperatures by cold shock proteins from Polaribacter irgensii KOPRI 22228. J Microbiol. 2010;48:798-802. The cspBPi gene (GenBank WP_004570868) was obtained by PCR amplification. The template DNA was extracted from P. irgensii KOPRI 22228 cells using G-spin™ bacteria genomic DNA extraction kit (Intron Co., Korea), according to the protocol suggested by the manufacturer. The forward primer sequence was CspBPi F1H, 5'-AGTAAGCTTATGGCAAAATCGCAGCAGACCT-3', and the reverse primer was CspBPi B150*B, 5'-CCGGATCCTTATATTTTGGTAACTTTAACTGCATTCATT-3' (manufactured by Bioneer Co., Daejion, Korea). The PCR mixture consisted of 5 µl of 10× PCR buffer (final concentrations: 50 mM KCl, 0.01% gelatin, 10 mM Tris-HCl, pH 9.0), 2.5 mM MgCl2, 0.2 mM of each dNTP, 200 nM of each primer, 1 µl of template DNA, and 2.5 units of Taq DNA polymerase (Takara, Japan) in the final 50 µl volume. The PCR was performed in a DNAEngine thermal cycler (Bio-Rad Laboratories Inc., USA) using a cycling condition that consisted of an initial denaturation at 95 °C for 5 min and then 30 cycles with denaturation at 94 °C for 1 min, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min. A final extension was performed at 72 °C for 5 min. The PCR products that were 0.5 kb in size were double-digested with HindIII and BamHI, and cloned using pAED4,1818 Jung CH, Im H. A recombinant human α1-antitrypsin variant, M-malton, undergoes a spontaneous conformational conversion into a latent form. J Microbiol. 2003;41:335-339. an E. coli expression vector, digested with the same enzymes. To avoid any mutations arising from error-prone Taq DNA polymerase reactions, several clones were picked for sequencing analysis. The resulting plasmids was named pAED-cspBPi.

Expression of CspBPi in E. coli

The recombinant plasmid for expression of CspBPi, pAED-cspBPi, was transformed into competent E. coli BL21(DE3) cells (Invitrogen Co., CA, USA) using the method described by Sambrook et al.1919 Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 1989. For overproduction of CspBPi, 1 ml of the overnight liquid culture was transferred to a flask containing 50 ml fresh LB medium containing 100 µg/ml ampicillin. When cell cultures had reached an OD600 of 0.4, IPTG was added to the final concentration of 0.1 mM. The cultures were incubated further at 37 °C for 2 h with vigorous shaking. Overexpression of the CspBPi was analyzed by 20% SDS-PAGE. The protein bands were visualized by Coomassie brilliant blue R250 staining.

Resistance to freezing and thawing

CspBPi protein was overexpressed in E. coli as described above. As the experimental control, E. coli transformed with a pAED4 plasmid lacking the cspBPi insert was used. One ml aliquot of liquid culture was placed at -20 °C for 2 h. The frozen cells were taken out of the freezer and put on ice for 1 h to be thawed. This process was performed in duplicates and repeated up to three cycles. Aliquots were taken at each cycle of freeze-and-thaw, and colony-forming units (CFU) were counted after incubation on LB plates at 37 °C for 24 h. The data were collected from five independent experiments and shown as an average for each point.

Purification of CspBPi

CspBPi were overexpressed at 37 °C in E. coli BL21(DE3) as described above, except that the cultures were scaled-up to 1 L liquid LB media. Cells were harvested by centrifugation, and resuspended in 40 ml of 10 mM phosphate buffer, pH 6.5. Cells were lyzed by sonication using a Bandelin Sonoplus HD2200 ultrasonic homogenizer (Berlin, Germany) as described for CspAPi.1616 Uh J-H, Jung YH, Im H. Rescue of a cold-sensitive mutant at low temperatures by cold shock proteins from Polaribacter irgensii KOPRI 22228. J Microbiol. 2010;48:798-802. The supernatant fraction was loaded on a Q-sepharose™ (Amersham Bioscience Co.) fast flow ion exchange column equilibrated with the same buffer. CspBPi protein was eluted with a 0-0.5 M NaCl gradient. Concentrations of proteins were determined using Bio-Rad DC (detergent compatible) protein assay kit, and the purity of proteins was analyzed by 20% SDS-PAGE.

Oligo(dT)-cellulose binding assays

Purified CspBPi protein was dialyzed against binding buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 50 mM KCl, and 7.4% glycerol). Fifty µl of oligo(dT)-cellulose type 7 beads (Amersham Bioscience Co.) were incubated with 50 µg of CspBPi protein at 4 °C for 4 h. The resins and bound proteins were collected by brief centrifugation, and washed twice with the binding buffer. In parallel, 50 µg of bovine serum albumin (BSA) protein, instead of CspBPi protein, was used as the experimental control. Co-precipitated proteins were analyzed by 20% SDS-PAGE and Coomassie brilliant blue R250 staining.

Rescue of cold-sensitive E. coli BX04 strain

E. coli quadruple csp deletion strain BX04 cells harboring pAED,1818 Jung CH, Im H. A recombinant human α1-antitrypsin variant, M-malton, undergoes a spontaneous conformational conversion into a latent form. J Microbiol. 2003;41:335-339. pAED-cspAPi,1616 Uh J-H, Jung YH, Im H. Rescue of a cold-sensitive mutant at low temperatures by cold shock proteins from Polaribacter irgensii KOPRI 22228. J Microbiol. 2010;48:798-802. pAED-cspBPi, or pAED-cspCPi1616 Uh J-H, Jung YH, Im H. Rescue of a cold-sensitive mutant at low temperatures by cold shock proteins from Polaribacter irgensii KOPRI 22228. J Microbiol. 2010;48:798-802. were grown in the liquid culture to an OD600 of 0.4. The cells were then streaked onto LB plates containing 0.1 mM IPTG, and incubated at temperatures ranging from 16 to 37 °C. After 15 h at 37 °C, 24 h at 25 °C, or 60 h at 16 °C, the growth of BX04 cells on the plates was observed.

Results

Cloning and sequence analysis of the cspB gene from P. irgensii KOPRI 22228

P. irgensii KOPRI 22228 was isolated from Arctic Sea sediments and grown as previously reported.1616 Uh J-H, Jung YH, Im H. Rescue of a cold-sensitive mutant at low temperatures by cold shock proteins from Polaribacter irgensii KOPRI 22228. J Microbiol. 2010;48:798-802. This bacterium is a psychrophile, with an optimum growth temperature of 10 °C. In an effort to elucidate the roles played by Csps in psychrophilic bacteria, DNA fragments from this bacterium, which contain a region encoding a conserved CSD, were cloned. A new csp-homologous gene 0.45 kb in size was obtained and named cspBPi.

A BLAST nucleotide homology search of cspBPi was performed: cspBPi exhibited low homology to the canonical cspAEc (15.8%) and E. coli cspD (12.4%). The deduced amino acid sequence of cspBPi encoded a protein 150 residues in length, and the CspBPi sequence was aligned to previously studied Csp sequences from polar bacteria: CspA from Streptomyces sp. AA8321 (CspASt),2020 Kim MJ, Lee YK, Lee HK, Im H. Characterization of cold-shock protein A of antarctic Streptomyces sp AA8321. Protein J. 2007;26:51-59. CspA from Psychromonas arctica KOPRI 22215 (CspAPa)2121 Jung YH, Yi JY, Jung H, et al. Overexpression of cold shock protein A of Psychromonas arctica KOPRI 22215 confers cold-resistance. Protein J. 2010;29:136-142. and CspAPi and CspCPi from P. irgensii KOPRI 22228.1616 Uh J-H, Jung YH, Im H. Rescue of a cold-sensitive mutant at low temperatures by cold shock proteins from Polaribacter irgensii KOPRI 22228. J Microbiol. 2010;48:798-802. All Csps have a typical β-barrel CSD composed of five β-strands, but CspBPi has a unique extra domain at its N-terminal end (Fig. 1). CSD is highly conserved among many bacterial Csps, and the three-dimensional structures of some Csps, including CspAEc and Bacillus subtilis CspB, have been reported.2222 Newkirk K, Feng W, Jiang W, et al. Solution NMR structure of the major cold shock protein (CspA) from Escherichia coli: identification of a binding epitope for DNA. Proc Natl Acad Sci USA. 1994;91:5114-5118.,2323 Schindelin H, Marahiel MA, Heinemann U. Universal nucleic acid-binding domain revealed by crystal structure of the B. subtilis major cold-shock protein. Nature. 1993;364:154-168. CSD is believed to mediate nucleic acid binding. In particular, two RNA-binding motifs, RNP1 (with consensus sequence K-G-F-G-F-I) and RNP2 (with consensus sequence V-F-V-H-F) are crucial for binding to RNA or ssDNA (boxed in Fig. 1).2424 Schröder K, Graumann P, Schnuchel A, Holak TA, Marahiel MA. Mutational analysis of the putative nucleic acid-binding surface of the cold-shock domain, CspB, revealed an essential role of aromatic and basic residues in binding of single-stranded DNA containing the Y-box motif. Mol Microbiol. 1995;16:699-708. In RNP2 of CspBPi, the first and the second Val residues were replaced with Tyr and Thr, respectively, and the last Phe residue was replaced with Val. However, all three Phe residues (Phe-15, Phe-17, and Phe-28; residue numbers according to the canonical CspAEc) in the RNA-binding motifs, which are considered to be necessary for nucleic acid-binding activity,2424 Schröder K, Graumann P, Schnuchel A, Holak TA, Marahiel MA. Mutational analysis of the putative nucleic acid-binding surface of the cold-shock domain, CspB, revealed an essential role of aromatic and basic residues in binding of single-stranded DNA containing the Y-box motif. Mol Microbiol. 1995;16:699-708.,2525 Hillier BJ, Rodriguez HM, Gregoret LM. Coupling protein stability and protein function in Escherichia coli CspA. Fold Des. 1998;3:87-93. were conserved in CspBPi. Sequence analysis of CspBPi suggested that the protein may bind to RNA or ssDNA through a canonical cold shock domain β-barrel structure. Meanwhile, the additional N-terminal domain of this protein did not show any noticeable homology to other proteins.

Fig. 1
Sequence alignment of various Csp proteins from polar organisms. Abbreviations for bacterial Csps whose amino acid sequences were analyzed here: S. sp. CspA, Streptomyces sp. AA8321 CspASt2020 Kim MJ, Lee YK, Lee HK, Im H. Characterization of cold-shock protein A of antarctic Streptomyces sp AA8321. Protein J. 2007;26:51-59.; P. irgensii CspA and CspC, Polaribacter irgensii KOPRI 22228 CspAPi and CspCPi, respectively1616 Uh J-H, Jung YH, Im H. Rescue of a cold-sensitive mutant at low temperatures by cold shock proteins from Polaribacter irgensii KOPRI 22228. J Microbiol. 2010;48:798-802.; P. irgensii CspB, P. irgensii KOPRI 22228 CspBPi from this study; Ps. arctica CspA, Psychromonas arctica KOPRI 22215 CspAPa.2121 Jung YH, Yi JY, Jung H, et al. Overexpression of cold shock protein A of Psychromonas arctica KOPRI 22215 confers cold-resistance. Protein J. 2010;29:136-142. The amino acid sequences of Csp proteins were aligned using the default settings of CLUSTAL W.2727 Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673-4680. Below the protein sequences is a key denoting conserved sequence (*), conservative mutations (:), semi-conservative mutations (.), and non-conservative mutations (). Gaps indicated by hyphens (-) were introduced to improve alignment. The RNA-binding motifs RNP1 and RNP2 are boxed.

Cold-resistance of the host overexpressing CspBPi was greatly increased

To study the roles of Csps from psychrophilic bacteria, cold resistance of the hosts harboring csp genes, was examined. Csp-overexpression was induced by the addition of IPTG to mid-log phase liquid cultures of cells carrying a csp-expression vector. When E. coli cells harboring pAED4 were frozen and thawed once, less than 1% of the original cells survived. Following repeated cycles of freezing and thawing, the number of surviving cells decreased almost exponentially. Overexpression of previously reported csp genes from polar bacteria increased freeze-survival rates of the hosts only moderately: the Ps. arctica CspAPa-expressing cells exhibited a slightly increased survival rate and the CspAPi or CspCPi-overexpressing cells showed more than five-fold increase in the survival rates in the first freeze-thaw cycle (Fig. 2).1616 Uh J-H, Jung YH, Im H. Rescue of a cold-sensitive mutant at low temperatures by cold shock proteins from Polaribacter irgensii KOPRI 22228. J Microbiol. 2010;48:798-802.,2121 Jung YH, Yi JY, Jung H, et al. Overexpression of cold shock protein A of Psychromonas arctica KOPRI 22215 confers cold-resistance. Protein J. 2010;29:136-142. Surprisingly, the CspBPi-overexpressing cells showed an extraordinary increase in freeze-tolerance: more than fifty fold the number of cells survived the first freeze-thaw cycle and the number of surviving cells increased to greater than 100 000-fold after three cycles of freezing and thawing compared to the number of surviving pAED4-carrying cells (Fig. 2).

Fig. 2
Greatly increased cold-resistance of CspBPi-overexpressing cells. The survival rates of various Csp-overexpressing cells following cycles of freeze-and-thaw are shown. The number of viable cells prior to freezing was set at 100%. ●, Control pAED4-carrying cells; ▿, CspAPa-overexpressing cells; ○, CspAPi-overexpressing cells; ■, CspCPi-overexpressing cells; ▾, CspBPi-overexpressing cells.

CspBPi binds oligo(dT)-cellulose

Since CspBPi contains CSD and the conserved Phe residues in the RNP1 and RNP2 sequence motifs, which suggest single-stranded RNA or DNA binding activity, the functionality of the single-stranded nucleic acid-binding motifs of this protein was examined. Upon incubation of E. coli BL21 cells harboring pAED-cspBPi with 0.1 mM IPTG, a protein band with an apparent molecular mass of 23 kDa on SDS-PAGE was observed. CspBPi was expressed in soluble form and purified by anion-exchange column chromatography. Partially purified CspBPi was incubated with oligo(dT)-cellulose beads at 4 °C for 4 h and subjected to a brief centrifugation. When the reaction products were analyzed by 20% SDS-PAGE, CspBPi was bound to the oligo(dT)-cellulose and co-precipitated with the resins, while neither bovine serum albumin used at the same concentration nor contaminating proteins from the CspBPi preparation were co-precipitated (Fig. 3). The result suggests that CspBPi binds ssDNA.

Fig. 3
Oligo(dT)-binding activity of CspBPi. CspBPi was incubated with oligo(dT)-cellulose at 4 °C for 4 h. Proteins bound to beads were collected by centrifugation. Lanes: MW, precision plus protein standards (Bio-Rad Laboratories Inc.; size of each protein band is shown in kDa at left of the gel); BSA, bovine serum albumin; CspB, CspBPi; bead, oligo(dT)-cellulose only. The migration position of the bound CspBPi protein is indicated with a red box.

CspBPi suppresses the cold-sensitive phenotype of the csp quadruple-deletion E. coli strain

Since overexpression of CspBPi greatly increased the cold resistance of wild-type E. coli (Fig. 2), the ability of CspBPi to suppress the cold-sensitivity of the E. coli quadruple csp deletion strain BX04 (ΔcspA, ΔcspB, ΔcspG, and ΔcspE) was also examined. Mid-log phase cultures of BX04 cells harboring pAED4, pAED-cspAPi, pAED-cspBPi, or pAED-cspCPi were streaked onto LB plates containing 0.1 mM IPTG and incubated at temperatures ranging from 16 to 37 °C. As described previously,1717 Xia B, Ke H, Inouye M. Acquirement of cold sensitivity by quadruple deletion of the cspA family and its suppression by PNPase S1 domain in Escherichia coli. Mol Microbiol. 2001;40:179-188. the growth of BX04 cells was comparable to other clones at 37 °C, but the growth was extremely retarded at 16 °C (Fig. 4). Meanwhile, overexpression of any Csps from P. irgensii complemented the cold-sensitivity of BX04 at 25 °C and at 16 °C (Fig. 4). When the growth of BX04 was followed by OD600, overexpression of CspBPi promoted the growth at low temperatures at slightly higher levels than CspAPi or CspCPi did (data not shown).

Fig. 4
Overexpression of CspBPi rescued cold-sensitive phenotype of csp quadruple-deletion E. coli strain, BX04. BX04 cells harboring pAED, pAED-cspAPi , pAED-cspBPi , or pAED-cspCPi were grown in the liquid culture to an OD600 of 0.4. The cells were then streaked on LB plates containing 0.1 mM IPTG, and incubated at temperatures ranging from 16 to 37 °C.

Discussion

Cold temperatures affect all physical-chemical parameters of living organisms, and effects include decreased solute diffusion rates, ice crystal formation, dehydration, decreased membrane fluidity, slow enzyme reaction rates, stable secondary structure formation in nucleic acids, and cold-denaturation of proteins. Psychrophilic organisms from cold ecosystems have evolved biological means to circumvent these challenges. Although transcription and translation of most genes are nearly stopped upon sudden temperature drops in mesophiles, expression of cold-shock genes are selectively induced.11 Jones PG, VanBogelen RA, Neidhardt FC. Induction of proteins in response to low temperature in Escherichia coli. J Bacteriol. 1987;169:2092-2095. Meanwhile, corresponding genes in psychrophiles are more consistently expressed, instead of being transiently induced during the cold acclimation phase,33 D'Amico S, Collins T, Marx J-C, Feller G, Gerday C. Psychrophilic microorganisms: challenges for life. EMBO Rep. 2006;7:385-389. suggesting that Csps play important roles for survival in cold environments.

In an effort to understand the mechanisms played by psychrophiles to adapt to cold biosphere, this study focused on the functions of Csps from polar bacteria. Although dozens of csp genes from psychrophiles have been identified by genomic/metagenomics approaches, their roles on cold-adaptation were elucidated only in very limited cases. Heterogeneous expression of Csps from polar microorganisms resulted in various effects on cold adaption of their recombinant hosts. Certain Csps failed to increase cold-resistance of their hosts: CspASt from an Antarctic Streptomyces neither increased the cold-resistance of wild-type E. coli, nor that of the E. coli quadruple csp deletion strain BX04.2020 Kim MJ, Lee YK, Lee HK, Im H. Characterization of cold-shock protein A of antarctic Streptomyces sp AA8321. Protein J. 2007;26:51-59. Instead overproduction of CspASt inhibited DNA replication, as non-canonical E. coli CspD does, suggesting a role for CspASt in halting DNA replication until the cell adjusts itself upon sudden temperature drops.2020 Kim MJ, Lee YK, Lee HK, Im H. Characterization of cold-shock protein A of antarctic Streptomyces sp AA8321. Protein J. 2007;26:51-59. Similarly, overexpression of a Csp from an Antarctic haloarchaeon Halorubrum lacusprofundi did not suppressed the cold sensitivity of BX04.2626 Giaquinto L, Curmi PM, Siddiqui KS, et al. Structure and function of cold shock proteins in archaea. J Bacteriol. 2007;189(15):5738-5748. Meanwhile, it has been reported that the overexpression of CspAPa increased the cold-resistance of wild-type E. coli, but not of the E. coli quadruple csp deletion strain BX04.2121 Jung YH, Yi JY, Jung H, et al. Overexpression of cold shock protein A of Psychromonas arctica KOPRI 22215 confers cold-resistance. Protein J. 2010;29:136-142. Therefore, the contribution of CspAPa to the cold survival of their recombinant hosts seemed relatively modest. Other Csps from psychrophilic organisms were more effective in increasing cold tolerance of their hosts; the overexpression of CspAPi or CspCPi not only increased the cold-survival rates of wild-type E. coli by more than five-fold following one cycle of freezing and thawing, but also rescued cold-sensitive phenotype of BX04.1616 Uh J-H, Jung YH, Im H. Rescue of a cold-sensitive mutant at low temperatures by cold shock proteins from Polaribacter irgensii KOPRI 22228. J Microbiol. 2010;48:798-802. Overexpression of Csps from a stenopsychrophilic archaeon Methanogenium frigidum or a deep sea planktonic archaeon Crenarchaeota also complemented cold susceptibility of E. coli BX04.2626 Giaquinto L, Curmi PM, Siddiqui KS, et al. Structure and function of cold shock proteins in archaea. J Bacteriol. 2007;189(15):5738-5748. Since no quantitative data on freeze-tolerance conferred by these proteins were provided, more detailed studies would be necessary to compare their effect with that of CspBPi. However, overexpression of any E. coli Csps, except CspDEc, also rescued cold-sensitive E. coli BX04,1717 Xia B, Ke H, Inouye M. Acquirement of cold sensitivity by quadruple deletion of the cspA family and its suppression by PNPase S1 domain in Escherichia coli. Mol Microbiol. 2001;40:179-188. and overexpression of CspAEc increased survival rates of the wild-type E. coli upon freezing and thawing at a comparable level to CspAPi or CspCPi (data not shown). These results show that the ability to confer cold resistance to their hosts is not the unique characteristics of Csps from psychrophiles: previous studies rather suggest that several Csps from psychrophiles have retained sufficient similarity throughout evolution to be able to function effectively in mesophiles to confer cold tolerance.

Surprisingly, overexpression of CspBPi greatly increased the cold tolerance of its recombinant host to an unprecedented level (Fig. 2). Overexpression of CspBPi not only induced a more than fifty fold increase in freeze-tolerance in wild-type E. coli (Fig. 2), but also noticeably promoted the growth of the E. coli quadruple csp deletion strain at low temperatures (Fig. 4). Elucidating the detailed mechanisms involved in freeze-tolerance conferred by CspBPi would be of great academic interest and will be pursued in a future study. CspBPi possesses cold shock domains at the C-terminal region (Fig. 1) and shares the basic characteristics of Csps, including the ability to bind single-stranded nucleic acids, as indicated by oligo(dT)-binding assays (Fig. 3). One possibility is that the CSD of CspBPi may have greatly improved activity, for example as a RNA chaperone, maintaining single stranded nucleic acid structures at extremely low temperatures and allowing efficient transcription and translation. On the other hand, CspBPi has an extra domain on its N-terminal region, and it is also possible that this unique region plays a distinct role in conferring an extraordinary increase in cold survival ability. Sequence homology search has identified several cspBPi-homologous genes, encoding both N-terminal extra domain and CSD, from evolutionarily related marine Flavobacteriaceae, including Polaribacter sp. MED152, Lacinutrix sp. 5H-3-7-4, Winogradskyella sp. PG-2, and Maribacter sp. HTCC2170. However, their functional roles on cold tolerance or transcriptional/translational regulation upon temperature downshifts have not been studied yet. It will be interesting to test freeze-tolerance of hosts overexpressing these dual domain Csp-like proteins.

Introduction of CspBPi into other organisms has potential industrial applications, including increased cold-resistance of nitrogen-fixing bacteria, such as Rhizobium and cyanobacteria, and the survival of starter cultures after storage at freezing temperatures. The long-pursued development of frozen dough may be achievable by improving the freeze-tolerance of baker's yeast. The possible application of CspBPi does not need to be limited to industrially important microorganisms and it may be introduced into plants to enhance their viability at low temperatures and increase their economic value.

Acknowledgments

The authors greatly appreciate the gift of E. coli BX04 from Dr. Sangita Phadtare and Dr. Massayori Inouye (UMDNJ). This work was supported by the Korea Research Foundation Grant funded by the Korean Government (KRF-2014-011146 and KRF-2015R1D1A1 A01058206).

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Edited by

Associate Editor: Valeria Oliveira

Publication Dates

  • Publication in this collection
    Jan-Mar 2018

History

  • Received
    26 Jan 2016
  • Accepted
    11 Apr 2017
Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
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