Improved adaptation to cold-shock,stationary-phase,and freezing stresses in Lactobacillus plantarum overproducing cold-shock proteins

We have investigated the effect of overproducing each of the three cold shock proteins (CspL, CspP, and CspC) in the mesophilic lactic acid bacterium Lactobacillus plantarum NC8. CspL overproduction transiently alleviated the reduction in growth rate triggered by exposing exponentially growing cells to cold shock (8 degrees C), suggesting that CspL is involved in cold adaptation. The strain overproducing CspC resumed growth more rapidly when stationary-phase cultures were diluted into fresh medium, indicating a role in the adaptation and recovery of nutritionally deprived cells. Overproduction of CspP led to an enhanced capacity to survive freezing.     

RNA thermoswitches modulate Staphylococcus aureus adaptation to ambient temperatures

Thermoregulation of virulence genes in bacterial pathogens is essential for environment-to-host transition. However, the mechanisms governing cold adaptation when outside the host remain poorly understood. Here, we found that the production of cold shock proteins CspB and CspC from Staphylococcus aureus is controlled by two paralogous RNA thermoswitches. Through in silico prediction, enzymatic probing and site-directed mutagenesis, we demonstrated that cspB and cspC 5′UTRs adopt alternative RNA structures that shift from one another upon temperature shifts. The open (O) conformation that facilitates mRNA translation is favoured at ambient temperatures (22°C). Conversely, the alternative locked (L) conformation, where the ribosome binding site (RBS) is sequestered in a double-stranded RNA structure, is folded at host-related temperatures (37°C). These structural rearrangements depend on a long RNA hairpin found in the O conformation that sequesters the anti-RBS sequence. Notably, the remaining S. aureus CSP, CspA, may interact with a UUUGUUU motif located in the loop of this long hairpin and favour the folding of the L conformation. This folding represses CspB and CspC production at 37°C. Simultaneous deletion of the cspB/cspC genes or their RNA thermoswitches significantly decreases S. aureus growth rate at ambient temperatures, highlighting the importance of CspB/CspC thermoregulation when S. aureus transitions from the host to the environment.     

乳酸乳球菌中冷休克蛋白基因的高效表达及其抗冻保护作用

[目的]微生物在适应外界环境急剧降温的条件下都会发生应激反应,产生一系列蛋白质被称为冷休克蛋白.冷休克蛋白对乳酸菌适应低温环境和增强抗冻能力方面发挥着重要作用.本文目的是为了研究乳酸乳球菌中冷休克蛋白CspC、CspD的作用.[方法]将冷休克蛋白CspC、CspD基因分别重组到质粒pNZ8148,转化乳酸乳球菌NZ9000后,加入Nisin诱导,对表达产物进行SDS-PAGE电泳分析,比较重组菌与空白菌在30℃条件下菌体生长差异及反复冻融活菌数的差异.[结果]得出CspC、CspD的相对分子量分别为7.0、6.2 kDa.[结论]CspC使菌体更加迅速的恢复了生长;冷休克蛋白CspD增强了菌体的抗冻存活率(增加了30～40倍).     

A family of cold shock proteins in Bacillus subtilis is essential for cellular growth and for efficient protein synthesis at optimal and low temperatures

Like other bacteria, Bacillus subtilis possesses a family of homologous small acidic proteins (CspB, CspC and CspD, identity > 70%) that are strongly induced in response to cold shock. We show that deletion of cspC or cspD genes did not result in a detectable phenotype; in contrast, csp double mutants exhibited severe reduction in cellular growth at 15°C as well as at 37°C, including impairment of survival during the stationary phase. Two-dimensional gel analysis showed that protein synthesis was deregulated in csp double mutants and that the loss of one or two CSPs led to an increase in the synthesis of the remaining CSP(s) at 37°C and after cold shock, suggesting that CSPs down-regulate production of members from this protein family. A cspB/C/D triple mutant (64BCDbt) could only be generated in the presence of cspB in trans on a plasmid that was not lost, in spite of lack of antibiotic pressure, indicating that a minimum of one csp gene is essential for viability of B . subtilis . After cold shock, synthesis of CspB in 64BCDbt was drastically lower than in wild-type cells accompanied by cessation in growth and strong reduction in general protein synthesis. As CspB, CspC and CspD are shown to bind to RNA in a co-operative and interactive manner, CSPs are suggested to function as RNA chaperones facilitating the initiation of translation under optimal and low temperatures.     

Loss of expression of cspC, a cold shock family gene, confers a gain of fitness in Escherichia coli K-12 strains

The CspA family of cold shock genes in Escherichia coli K-12 includes nine paralogs, cspA to cspI. Some of them have been implicated in cold stress adaptation. Screening for mutations among common laboratory E. coli strains showed a high degree of genetic diversity in cspC but not in cspA and cspE. This diversity in cspC was due to a wide spectrum of variations including insertions of IS elements, deletion, and point mutation. Northern analysis of these mutants showed loss of cspC expression in all but one case. Further analysis of the loss-of-function cspC mutants showed that they have a fitness advantage in broth culture after 24 h over their isogenic wild-type derivatives. Conversely, introduction of mutated cspC alleles conferred a competitive fitness advantage to AB1157, a commonly used laboratory strain. This provides the evidence that loss of cspC expression is both necessary and sufficient to confer a gain of fitness as seen in broth culture over 24 h. Together, these results ascribe a novel role in cellular growth at 37 degrees C for CspC, a member of the cold shock domain-containing protein family.     

Characterization of Bacillus subtilis mutants resistant to cold shock-induced autolysis

Bacillus subtilis vegetative cells undergo autolysis when exposed to cold shock treatment. A mutant (CA1) resistant to cold shock was isolated, and its DNA was used for the transformation of B. subtilis 168AR. The transformant (TR1) and CA1 had almost completely lost major vegetative autolysins (CwlB and CwlG) and motility, and showed a filamentous cell morphology during the exponential phase. Expression of the sigD-lacZ fusion was reduced in TR1. But the introduction of a SigD overproducing plasmid, pHYSigD, into TR1 led to a considerable increase in the amount of autolysin, a normal cell morphology (short rod), and the cold shock-sensitive phenotype. However, motility was not restored in the transformant. The roles of pleiotropic genes in cold shock-induced autolysis are discussed.     

The synthesis of cold shock proteins and cold acclimation proteins in the psychrophilic bacteriumAquaspirillum arcticum

The synthesis of cold shock proteins (csps) in response to cold shock, and of cold acclimation proteins (caps) in response to continuous growth at low temperature, in the psychrophileAquaspirillum arcticum was investigated. With two-dimensional gel electrophoresis and computing scanning laser densitometry, cold shock treatments (10° to 0°C, 5° to 0°C, and 10° to 5°C) induced a total of 14 csps, 6 of which were induced by all three cold shocks. The production of caps in response to continuous growth at 0°C was also found. Five of the 8 caps produced were also csps which suggests that these proteins may share a common involvement in cold adaptation.     

Combined cold, acid, ethanol shocks in Oenococcus oeni: Effects on membrane fluidity and cell viability

The effects of combined cold, acid and ethanol on the membrane physical state and on the survival of Oenococcus oeni were investigated. Membrane fluidity was monitored on intact whole O. oeni cells subjected to single and combined cold, acid and ethanol shocks by using fluorescence anisotropy with 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. Results showed that cold shocks (14 and 8 °C) strongly rigidified plasma membrane but did not affect cell survival. In contrast, ethanol shocks (10-14% v/v) induced instantaneous membrane fluidisation followed by rigidification and resulted in low viability. Acid shocks (pH 4.0 and pH 3.0) exerted a rigidifying effect on membrane without affecting cell viability. Whatever the shock orders, combined cold (14 °C) and ethanol (14% v/v) shocks resulted in strong membrane rigidification. Interestingly, O. oeni survived combined cold and ethanol shocks more efficiently than single ethanol shock. Membrane rigidification was induced by ethanol-and-acid (10% v/v - pH 3.5) shock and correlated with total cell death. In contrast, O. oeni recovered its viability when subjected to cold (8 °C)-then-ethanol-and-acid shock which strongly rigidified the membrane. Our results suggested a positive short-term effect of combined cold, acid and ethanol shocks on membrane fluidity and viability of O. oeni.     

Thermal stress responses in Antarctic yeast, Glaciozyma antarctica PI12, characterized by real-time quantitative PCR

Living organisms have some common and unique strategies to response to thermal stress. However, the amount of data on thermal stress response of certain organism is still lacking, especially psychrophilic yeast from the extreme habitat. Therefore, it is not known whether psychrophilic yeast shares the common responses of other organisms when exposed to thermal stresses. In this work, the cold shock and heat shock responses in Antarctic psychrophilic yeast Glaciozyma antarctica PI12 which had an optimal growth temperature of 12 °C were determined. The expression levels of 14 thermal stress-related genes were measured using real-time quantitative PCR (qPCR) when the yeast cells were exposed to cold shock (0 °C), mild cold shock (5 °C), and heat shock (22 °C) conditions. The expression profiles of the 14 genes at these three temperatures varied indicating that these genes had their specific roles to ensure the survival of the yeast. Under cold shock condition, the afp4 and fad genes were over-expressed possibly as a way for the G. antarctica PI12 to avoid ice crystallization in the cell and to maintain the membrane fluidity. Under the heat shock condition, hsp70 was significantly up-regulated possibly to ensure the proteins fold properly. Among the six oxidative stress-related genes, MnSOD and prx were up-regulated under cold shock and heat shock, respectively, possibly to reduce the negative effects caused by oxidative stress. Interestingly, it was found that the trehalase gene, nth1 that plays a role in degrading excess trehalose, was down-regulated under the heat shock condition possibly as an alternative way to accumulate trehalose in the cells to protecting them from being damaged.     

Restart of exponential growth of cold-shocked Yersinia enterocolitica occurs after down-regulation of cspA1/A2 mRNA

The cellular content of major cold shock protein (MCSP) mRNA transcribed from the tandem gene duplication cspA1/A2 and growth of Yersinia enterocolitica were compared when exponentially growing cultures of this bacterium were cold shocked from 30 to 20, 15, 10, 5, or 0 degrees C, respectively. A clear correlation between the time point when exponential growth resumes after cold shock and the degradation of cspA1/A2 mRNA was found. A polynucleotide phosphorylase-deficient mutant was unable to degrade cspA1/A2 mRNA properly and showed a delay, as well as a lower rate, of growth after cold shock. For this mutant, a correlation between decreasing cspA1/A2 mRNA and restart of growth after cold shock was also observed. For both wild-type and mutant cells, no correlation of restart of growth with the cellular content of MCSPs was found. We suggest that, after synthesis of cold shock proteins and cold adaptation of the cells, MCSP mRNAs must be degraded; otherwise, they trap ribosomes, prevent translation of bulk mRNA, and thus inhibit growth of this bacterium at low temperatures.     

Improved adaptation to heat,cold, and solvent tolerance in <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>

The effect of overproducing each of the three small heat shock proteins (Hsp; Hsp 18.5, Hsp 18.55, and Hsp 19.3) was investigated in Lactobacillus plantarum strain WCFS1. Overproduction of the three genes, hsp 18.5, hsp 18.55, and hsp 19.3, translationally fused to the start codon of the ldhL gene yielded a protein of approximately 19 kDa, as estimated from Tricine sodium dodecyl sulfate–polyacrylamide gel electrophoresis in agreement with the predicted molecular weight of small Hsps. Small Hsp overproduction alleviated the reduction in growth rate triggered by exposing exponentially growing cells to heat shock (37 or 40°C) and cold shock (12°C). Moreover, overproduction of Hsp 18.55 and Hsp 19.3 led to an enhanced survival in the presence of butanol (1% v/v) or ethanol (12% v/v) treatment suggesting a potential role of L. plantarum small Hsps in solvent tolerance.