Adaptive response to cold temperatures and characterization of cspA in Salmonella typhimurium LT2

Salmonella typhimurium is a major foodborne microbial pathogen which primarily contaminates poultry products causing salmonellosis in humans. S. typhimurium LT2 cultures, when transferred from 37 °C to 5 °C or 10 °C, showed an initial lag period in growth with an approximate generation time of 10–25 h. Western blot assay using E. coli CS7.4 antibody and analysis of radiolabeled total cellular proteins from S. typhimurium cultures after exposure to 10 °C or 5 °C showed elevated expression of a major cold shock protein, CS7.4. Identification of a decreased level of CS7.4 at 37 °C suggests that the expression of this protein may require a large temperature downshift. Putative regulatory protein binding segment on the 5-untranslated region referred as Fragment 7 in S. typhimurium exhibited a 90.6% and a 56.25% nucleotide sequence identity when compared with the Fragment 7 of E. coli and S. enteritidis, respectively. The differences in the nucleotide sequence within the Fragment 7 between S. typhimurium and S. enteritidis may explain the differential expression of CspA at 37 °C. The nucleotide sequence of the open reading frame of S. typhimurium cspA gene showed a single base difference at 816 bp position from a G to a C which altered the amino acid residue from a glycine to an alanine. In addition to CspA, an elevated expression of a 105 kDa, and decreased expression of 6 proteins were evidenced when cultures of S. typhimurium were exposed to 10 °C or 5 °C. Differential expression of the CspA and other proteins in S. typhimurium following exposure to cold temperatures suggest that adaptation and continued growth and survival at cold temperatures in this pathogen may be aided by these cold-responsive proteins.     

Identification of a Cold Shock Gene in Lactic Acid Bacteria and the Effect of Cold Shock on Cryotolerance

When Lactic Acid Bacterial cultures were frozen at −20°C for 24 h, the cell viability decreased drastically, but when they were cold shocked at 10°C for 2 h prior to freezing, viability improved significantly for the Lactococcus lactis subsp. lactis strains (25–37%) and Pediococcus pentosaceus PO2 (18%), but not for the Lactococcus lactis subsp. cremoris strains tested or for one strain of Lactobacillus helveticus LB1 and Streptococcus thermophilus TS2. When the period for cold shock was extended to 5 h, the viability increased even further for those strains that displayed cold shock cryotolerance. Use of degenerate PCR primers based on the major cold shock protein (csp) of both Escherichia coli and Bacillus subtilis resulted in PCR products from all strains tested. The PCR product from Lactococcus lactis ssp. lactis M474 was cloned and sequenced, and the deduced amino acid sequence displayed a high sequence similarity to other csp's. Use of PCR primers based on the M474 sequence resulted in PCR products being produced only from the lactococcal strains studied and not from the Lactobacillus helveticus, Streptococcus thermophilus, or Pediococcus pentosaceus strains tested. Received: 18 October 1996 / Accepted: 28 January 1997     

Family of the major cold-shock protein, CspA (CS7.4), of Escherichia coli, whose members show a high sequence similarity with the eukaryotic Y-box binding proteins

The cspA is a gene of Escherichia coli, whose expression is specifically induced at low temperatures to a level of 13% of total protein synthesis. The CspA protein consisting of 70 amino add residues has high sequence similarity with eukaryotic Y-box DNA-binding proteins. We found two independent clones from the Kohara miniset phage collection, which hybridized with a DNA fragment containing cspA. DNA sequencing of these clones confirmed that the two genes are highly homologous to cspA. One designated cspB is mapped at 35 min on the E. coli chromosome and encodes a 71-residue protein with 79% identity to CspA, while the other, cspC, is mapped at 40 min and encodes a 69-residue protein with 70% identity. In addition, a DNA sequence upstream of the clpA gene at 19 mm published elsewhere contains an open reading frame for a 74-residue protein with 45% identity to CspA. All csp genes were fused in the coding regions with the lacZ gene, and the expression of β-galactosidase was examined for these hybrid genes upon cold shock. A similar cold-shock induction to cspA was observed for cspB but not cspC and cspD. These results Indicate that E. coli has a family of the cspA gene, some of which are induced by cold shock.     

Adaptive Response to Cold Temperatures in Vibrio vulnificus

The effectiveness of rapid chilling or freezing of oysters to reduce Vibrio vulnificus levels in shellfish may be compromised by product handling procedures that permit cold adaptation. When a V. vulnificus culture was shifted from 35°C to 6°C conditions, it underwent transition to a non-culturable state. Cells adapted to 15°C prior to change to 6°C condition, however, remain viable and culturable. In addition, cultures adapted to 15°C were able to survive better upon freezing at −78°C compared with cultures frozen directly from 35°C. Inhibition of protein synthesis by addition of chloramphenicol in a V. vulnificus culture immediately prior to the exposure to the adaptive temperature eliminated inducible cold tolerance. These results suggest that cold-adaptive “protective” proteins may enhance survival and tolerance at cold temperatures. In addition, removal of iron from the growth medium by adding 2,2′-Dipyridyl prior to cold adaptation decreased the viability by approximately 2 logarithm levels. This suggests that iron plays an important role in adaptation at cold temperatures. Analysis of total cellular proteins on an SDS polyacrylamide gel electrophoresis, labeled with ³⁵S-methionine during exposure at 15°C, showed elevated expressions of a 6-kDa and a 40-kDa protein and decreased expression of an 80-kDa protein. These results suggest that, for V. vulnificus, survival and tolerance at cold temperatures could be due to the expression of cold-adaptive proteins other than previously documented major cold shock proteins such as CS7.4 and CsdA. In this study, for the first time we have shown that exposure to an intermediate cold temperature (15°C) causes a cold adaptive response, helping this pathogen remain in culturable state when exposed to a much colder temperature (6°C). This adaptive nature to cold temperatures could be important for shellfish industry efforts to reduce the risk of V. vulnificus infection from consuming raw oysters. Received: 30 July 1998 / Accepted: 1 October 1998     

Overexpression of Cold Shock Protein A of <Emphasis Type="Italic">Psychromonas arctica</Emphasis> KOPRI 22215 Confers Cold-Resistance

A polar bacterium was isolated from Arctic sea sediments and identified as Psychromonas artica, based on 16S rDNA sequence. Psychromonas artica KOPRI 22215 has an optimal growth temperature of 10 °C and a maximum growth temperature of 25 °C, suggesting this bacterium is a psychrophile. Cold shock proteins (Csps) are induced upon temperature downshift by more than 10 °C. Functional studies have researched mostly Csps of a mesophilic bacterium Escherichia coli, but not on those of psychrophilic bacteria. In an effort to understand the molecular mechanisms of psychrophilic bacteria that allow it withstand freezing environments, we cloned a gene encoding a cold shock protein from P. artica KOPRI 22215 (CspA_Pa) using the conserved sequences in csp genes. The 204 bp-long ORF encoded a protein of 68 amino acids, sharing 56% homology to previously reported E. coli CspA protein. When CspA_Pa was overexpressed in E. coli, it caused cell growth-retardation and morphological elongation. Interestingly, overexpression of CspA_Pa drastically increased the host’s cold-resistance by more than ten times, suggesting the protein aids survival in polar environments.     

Cold-tolerant alkane-degrading Rhodococcus species from Antarctica

Bioremediation is a possible mechanism for clean-up of hydrocarbon-contaminated soils in the Antarctic. Microbes indigenous to the Antarctic are required that degrade the hydrocarbon contaminants found in the soil, and that are able to survive and maintain activity under in situ conditions. Alkane- degrading bacteria previously isolated from oil-contaminated soil from around Scott Base, Antarctica, grew on a number of n-alkanes from hexane (C6) through to eicosane (C20) and the branched alkane pristane. Mineralization of ¹⁴C-dodecane was demonstrated with four strains. Representative isolates were identified as Rhodococcus species using 16S rDNA sequence analysis. Rhodococcus spp. strains 5/14 and 7/1 grew at −2°C but numbers of viable cells declined when incubated at 37°C. Both strains appear to have the major cold-shock gene cspA. Partial nucleotide sequence analyses of the PCR-amplified cspA open reading frame from Rhodococcus spp. strains 5/14 and 7/1 were approximately 60% identical to cspA from Escherichia coli. Accepted: 6 September 1999     

Effect of Different Temperature Upshifts on Protein Synthesis by the Psychrotrophic Bacterium Pseudomonas fragi

Pseudomonas fragi, a psychrotroph bacterium involved in meat product spoilage, was shifted either from 5° to 20°C or 30°C and from 28° to 34°C. The heat-shocked cells in the mid-log phase rapidly reached the characteristic growth rate of the postshock temperature. The patterns of synthesized proteins were compared by autoradiography of two-dimensional gel electrophoregrams. The rates of synthesis, after transfer of cells from 5° to 30°C, 5° to 20°C, and 28° to 34°C, changed for 30, 26, and 21 proteins respectively, of which 19, 17, and 12 were increased respectively. Thirteen proteins changed similarly for the three treatments, and two of the seven overexpressed proteins were immunologically related to the Escherichia coli DnaK and GroEL heat shock proteins. From the four low-molecular-mass proteins, belonging to the family of DNA-binding cold shock proteins (CSPs) such as CS7.4, the major E. coli CSP [15], the amounts of C7.0 and C8.0 decreased rapidly after the upshifts, whereas that of E7.0 and E8.0 increased greatly. Received: 22 November 1995 / Accepted: 22 December 1995     

The oatmeal nematode <Emphasis Type="Italic">Panagrellus redivivus</Emphasis> survives moderately low temperatures by freezing tolerance and cryoprotective dehydration

The cold tolerance abilities of only a few nematode species have been determined. This study shows that the oatmeal nematode, Panagrellus redivivus, has modest cold tolerance with a 50% survival temperature (S ₅₀) of −2.5°C after cooling at 0.5°C min⁻¹ and freezing for 1 h. It can survive low temperatures by freezing tolerance and cryoprotective dehydration; although freezing tolerance appears to be the dominant strategy. Freezing survival is enhanced by low temperature acclimation (7 days at 5°C), with the S ₅₀ being lowered by a small but significant amount (0.42°C). There is no cold shock or rapid cold hardening response under the conditions tested. Cryoprotective dehydration enhances the ability to survive freezing (the S ₅₀ is lowered by 0.55°C, compared to the control, after 4 h freezing at −1°C) and this effect is in addition to that produced by acclimation. Breeding from survivors of a freezing stress did not enhance the ability to survive freezing. The cold tolerance abilities of this nematode are modest, but sufficient to enable it to survive in the cold temperate environments it inhabits.     

Rates of chilling to 0°C: implications for the survival of microorganisms and relationship with membrane fluidity modifications

The effects of slow chilling (2°C min⁻¹) and rapid chilling (2,000°C min⁻¹) were investigated on the survival and membrane fluidity of Escherichia coli, of Bacillus subtilis, and of Saccharomyces cerevisiae. Cell death was found to be dependent on the physiological state of cell cultures and on the rate of temperature downshift. Slow temperature decrease allowed cell stabilization, whereas the rapid chilling induced an immediate loss of viability of up to more than 90 and 70% for the exponentially growing cells of E. coli and B. subtilis, respectively. To relate the results of viability with changes in membrane physical state, membrane anisotropy variation was monitored during thermal stress using the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene. No variation in the membrane fluidity of all the three microorganisms was found after the slow chilling. It is interesting to note that fluorescence measurements showed an irreversible rigidification of the membrane of exponentially growing cells of E. coli and B. subtilis after the instantaneous cold shock, which was not observed with S. cerevisiae. This irreversible effect of the rapid cold shock on the membrane correlated well with high rates of cell inactivation. Thus, membrane alteration seems to be the principal cause of the cold shock injury.     

Thermal tolerance limits in six weevil species (Coleoptera, Curculionidae) from sub-Antarctic Marion Island

Supercooling points, lower lethal temperatures, and the effect of short-term exposures to low temperatures were examined during both winter and summer in the adults of six weevil species from three different habitats on Marion Island. Upper lethal limits and the effects of short-term exposure to high temperatures were also examined in summer-acclimatized adult individuals of these species. Bothrometopus elongatus, B. parvulus, B. randi, Ectemnorhinus marioni, and E. similis were freeze tolerant, but had high lower lethal temperatures (−7 to −10°C). Seasonal variation in these parameters was not pronounced. Physical conditions of the habitat appeared to have little effect on cold hardiness parameters because the Ectemnorhinus species occur in very wet habitats, whereas the Bothrometopus species inhabit drier areas. The adults of these weevil species are similar to other high southern latitude insects in that they are freeze tolerant, but with high lower lethal temperatures. In contrast, Palirhoeus eatoni, a supra-littoral species, avoided freezing and had a mean supercooling point of −15.5 ± 0.94°C (SE) in winter and −11.8 ± 0.98°C in summer. Survival of a constant low temperature of −8°C also increased in this species from 6 h in summer to 27 h in winter. It is suggested that this strategy may be a consequence of the osmoregulatory requirements imposed on this species by its supra-littoral habitat. Upper lethal temperatures (31–34°C) corresponded closely with maximum microclimate temperatures in all of the species. This indicates that the pronounced warming, accompanied by the increased insolation that has been recorded at Marion Island, may reduce survival of these species. These effects may be compounded as a consequence of predation by feral house mice on the weevils. Received: 4 February 1997 / Accepted: 3 May 1997     

Cold shock proteins improve E. coli cell-free synthesis in terms of soluble yields of aggregation-prone proteins

Protein folding is usually slowed-down at low temperatures, and thus low-temperature expression is an effective strategy to improve the soluble yield of aggregation-prone proteins. In this study, we investigated the effects of a variety of cold shock proteins and domains (Csps) on an Escherichia coli cell extract-based cell-free protein synthesis system (CF). Most of the 12 Csps that were successfully prepared dramatically improved the protein yields, by factors of more than 5 at 16°C and 2 at 23°C, to levels comparable to those obtained at 30°C. Their stimulatory effects were complementary to each other, while CspD and CspH were inhibitory. The Csps’ effects correlated well with their Pfam CSD family scores (PF00313.22). All of the investigated Csps, except CspH, similarly possessed RNA binding and chaperon activities and increased the messenger RNA amount irrespective of their effect, suggesting that the proper balance between these activities was required for the enhancement. Unexpectedly, the 5′-untranslated region of cspA was less effective as the leader sequence. Our results demonstrated that the use of the Csps presented in this study will provide a simple and highly effective strategy for the CF, to improve the soluble yields of aggregation-prone proteins.     

Freezing tolerance of ectomycorrhizal fungi in pure culture

The ability to survive freezing and thawing is a key factor for the existence of life forms in large parts of the world. However, little is known about the freezing tolerance of mycorrhizal fungi and their role in the freezing tolerance of mycorrhizas. Threshold temperatures for the survival of these fungi have not been assessed experimentally. We grew isolates of Suillus luteus, Suillus variegatus, Laccaria laccata, and Hebeloma sp. in liquid culture at room temperature. Subsequently, we exposed samples to a series of temperatures between +5°C and −48°C. Relative electrolyte leakage (REL) and re-growth measurements were used to assess the damage. The REL test indicated that the lethal temperature for 50% of samples (LT₅₀) was between −8.3°C and −13.5°C. However, in the re-growth experiment, all isolates resumed growth after exposure to −8°C and higher temperatures. As many as 64% of L. laccata samples but only 11% in S. variegatus survived −48°C. There was no growth of Hebeloma and S. luteus after exposure to −48°C, but part of their samples survived −30°C. The fungi tolerated lower temperatures than was expected on the basis of earlier studies on fine roots of ectomycorrhizal trees. The most likely freezing tolerance mechanism here is tolerance to apoplastic freezing and the concomitant intracellular dehydration with consequent concentrating of cryoprotectant substances in cells. Studying the properties of fungi in isolation promotes the understanding of the role of the different partners of the mycorrhizal symbiosis in the freezing tolerance.     

Cold hardening reduces photoinhibition of Eucalypts nitens and E. pauciflora at frost temperatures

Photoinhibition of photosynthesis at low temperatures was investigated in two species of subalpine eucalypt, Eucalypts nitens (Deane and Maiden) Maiden and E. pauciflora Sieb. ex Spreng. Imposition of an artificial cold-hardening treatment increased the frost tolerance of leaf tissue and increased tolerance to excess light. Cold-hardened seedlings of both species had a higher photosynthetic capacity than non-hardened seedlings at 6 and 16°C and lower levels of non-photochemical quenching (NPQ) at 20 and 5°C. Furthermore, hardened seedlings had faster rates of NPQ development at 5 and −3.5°C. An increase in minimal fluorescence, which indicates slowly reversible photoinhibition, was evident in all seedlings at −1.5 and −3.5°C but was less pronounced in hardened seedlings, with a threefold faster rate of development of NPQ, at −3.5°C than non-hardened seedlings. Hardened seedlings also recovered faster from photoinhibition at −3.5°C. Thus cold hardening increased tolerance to high light in these species. Differences between E. nitens and E. pauciflora in their response to excess light were small and significant only at −3.5°C. Faster recovery from photoinhibition of E. pauciflora was consistent with its occurrence in colder habitats than E. nitens. Received: 27 April 1997 / Accepted: 9 September 1997     

Chilling and freezing stress in live oaks (Quercus section Virentes): intra- and inter-specific variation in PS II sensitivity corresponds to latitude of origin

Sensitivity to cold and freezing differs between populations within two species of live oaks (Quercus section Virentes Nixon) corresponding to the climates from which they originate. Two populations of Quercus virginiana (originating from North Carolina and north central Florida) and two populations of the sister species, Q. oleoides, (originating from Belize and Costa Rica) were grown under controlled climate regimes simulating tropical and temperate conditions. Three experiments were conducted in order to test for differentiation in cold and freezing tolerance between the two species and between the two populations within each species. In the first experiment, divergences in response to cold were tested for by examining photosystem II (PS II) photosynthetic yield (ΔF/F _m′) and non-photochemical quenching (NPQ) of plants in both growing conditions after short-term exposure to three temperatures (6, 15 and 30°C) under moderate light (400 μmol m⁻² s⁻¹). Without cold acclimation (tropical treatment), the North Carolina population showed the highest photosynthetic yield in response to chilling temperatures (6°C). Both ecotypes of both species showed maximum ΔF/F _m′ and minimum NPQ at their daytime growth temperatures (30°C and 15°C for the tropical and temperate treatments, respectively). Under the temperate treatment where plants were allowed to acclimate to cold, the Q. virginiana populations showed greater NPQ under chilling temperatures than Q. oleoides populations, suggesting enhanced mechanisms of photoprotective energy dissipation in the more temperate species. In the second and third experiments, inter- and intra-specific differentiation in response to freezing was tested for by examining dark-adapted F _v/F _m before and after overnight freezing cycles. Without cold acclimation, the extent of post-freezing declines in F _v/F _m were dependent on the minimum freezing temperature (0, −2, −5 or −10°C) for both populations in both species. The most marked declines in F _v/F _m occurred after freezing at −10°C, measured 24 h after freezing. These declines were continuous and irreversible over the time period. The North Carolina population, however, which represents the northern range limit of Q. virginiana, showed significantly less decline in F _v/F _m than the north central Florida population, which in turn showed a lower decline in Fv/F _m than the two Q. oleoides populations from Belize and Costa Rica. In contrast, after exposure to three months of chilling temperatures (temperate treatment), the two Q. virginiana populations showed no decline in F _v/F _m after freezing at −10°C, while the two Q. oleoides populations showed declines in F _v/F _m reaching 0.2 and 0.1 for Costa Rica and Belize, respectively. Under warm growth conditions, the two species showed different F ₀ dynamics directly after freezing. The two Q. oleoides populations showed an initial rise in F ₀ 30 min after freezing, followed by a subsequent decrease, while the Q. virginiana populations showed a continuous decrease in F ₀ after freezing. The North Carolina population of Q. virginiana showed a tendency toward deciduousness in response to winter temperatures, dropping 58% of its leaves over the three month winter period compared to only 6% in the tropical treatment. In contrast, the Florida population dropped 38% of its leaves during winter. The two populations of the tropical Q. oleoides showed no change in leaf drop during the 3-months winter (10% and 12%) relative to their leaf drop over the same timecourse in the tropical treatment. These results indicate important ecotypic differences in sensitivity to freezing and cold stress between the two populations of Q. virginiana as well as between the two species, corresponding to their climates of origin.     

Thermotolerance and molecular chaperone function of the small heat shock protein HSP20 from hyperthermophilic archaeon, <Emphasis Type="Italic">Sulfolobus solfataricus P2</Emphasis>

Small heat shock proteins are ubiquitous in all three domains (Archaea, Bacteria and Eukarya) and possess molecular chaperone activity by binding to unfolded polypeptides and preventing aggregation of proteins in vitro. The functions of a small heat shock protein (S.so-HSP20) from the hyperthermophilic archaeon, Sulfolobus solfataricus P2 have not been described. In the present study, we used real-time polymerase chain reaction analysis to measure mRNA expression of S.so-HSP20 in S. solfataricus P2 and found that it was induced by temperatures that were substantially lower (60°C) or higher (80°C) than the optimal temperature for S. solfataricus P2 (75°C). The expression of S.so-HSP20 mRNA was also up-regulated by cold shock (4°C). Escherichia coli cells expressing S.so-HSP20 showed greater thermotolerance in response to temperature shock (50°C, 4°C). By assaying enzyme activities, S.so-HSP20 was found to promote the proper folding of thermo-denatured citrate synthase and insulin B chain. These results suggest that S.so-HSP20 promotes thermotolerance and engages in chaperone-like activity during the stress response.     

Functional expression of anti-hepatitis B virus (HBV) preS2 antigen scFv by <Emphasis Type="Italic">cspA</Emphasis> promoter system in <Emphasis Type="Italic">Escherichia coli</Emphasis> and application as a recognition molecule for single-walled carbon nanotube (SWNT) field effect transistor (FET)

The preS2 antigens of hepatitis B virus (HBV), which causes a serious health problem in the world, have been implicated in hepatocyte cell binding and viral penetration. Therefore, the importance of antibody production against preS2 antigen for early diagnosis of HBV has been well established. In this study, the recombinant HBV preS2 single chain variable fragment (scFv) antibody was successfully expressed in E. coli with the novel cold shock vector (pCold) under the cspA promoter, and its expression level was compared with the pET vector under the T7 promoter. Additionally, a host with an oxidizing cytoplasm, E. coli trxB/gor double mutant, was used to improve the soluble expression. The anti-HBV preS2 scFv using pCold vector was successfully expressed in a soluble and functional form in both wild type and double mutant E. coli, while the scFv using the pET vector was expressed in an insoluble form in spite of using a double mutant providing an oxidizing environment. The induction with 0.05 mM IPTG showed a 2-fold higher functional expression compared to induction with 1 mM IPTG, and the functional expression at the induction temperature (15°C), which is optimal temperature for pCold vector, was improved 2-fold and 3- fold at 4 and 25°C, respectively. The efficacy of anti-HBV preS2 scFv for detecting HBV preS2 antigen was tested and verified by using Ni-decorated single-walled carbon nanotube (SWNT) field effect transistors.