SummaryThe proteomes expressed at 4°C and 18°C by the psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis have been compared using two-dimensional differential in-gel electrophoresis, showing that translation, protein folding, membrane integrity and anti-oxidant activities are upregulated at 4°C. This proteomic analysis revealed that the trigger factor is the main upregulated protein at low temperature. The trigger factor is the first molecular chaperone interacting with virtually all newly synthesized polypeptides on the ribosome and also possesses a peptidyl-prolyl cis-trans isomerase activity. This suggests that protein folding at low temperatures is a rate-limiting step for bacterial growth in cold environments. It is proposed that the psychrophilic trigger factor rescues the chaperone function as both DnaK and GroEL (the major bacterial chaperones but also heat-shock proteins) are downregulated at 4°C. The recombinant psychrophilic trigger factor is a monomer that displays unusually low conformational stability with a Tm value of 33°C, suggesting that the essential chaperone function requires considerable flexibility and dynamics to compensate for the reduction of molecular motions at freezing temperatures. Its chaperone activity is strongly temperaturedependent and requires near-zero temperature to stably bind a model-unfolded polypeptide.

The proteomes expressed at 4°C and 18°C by the psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis were compared using two-dimensional differential in-gel electrophoresis with special reference to proteins repressed by low temperatures. Remarkably, the major cold-repressed proteins, almost undetectable at 4°C, were heat shock proteins involved in folding assistance.The Gram-negative bacterium Pseudoalteromonas haloplanktis is a typical representative of the Gammaproteobacteria found in cold marine environments, and strain TAC125 has been isolated from seawater sampled along the Antarctic ice shelf. Such strains thrive permanently in seawater at about Ϫ2°C to ϩ4°C but are also anticipated to endure long-term frozen conditions when entrapped in the winter ice pack. The genome of P. haloplanktis TAC125 has been fully sequenced (11). This work has further allowed a proteomic study of its cold acclimation proteins (CAPs), i.e., proteins that are continuously overexpressed at a high level during growth at low temperatures (14). This has demonstrated that protein synthesis and protein folding are the main upregulated functions, suggesting that both cellular processes are limiting factors for bacterial development in cold environments. Here we report a proteomic survey of cold-repressed proteins at 4°C in order to complete the metabolic pattern of the bacterium's growth at low temperature.Temperature dependence of growth. The Antarctic bacterium maintains a doubling time of ϳ4 h at 4°C in a marine broth, with an extrapolated generation time of 5 h 15 min at 0°C (Fig. 1a). When the culture temperature is raised to 20°C, the generation time decreases moderately (e.g., 1 h 40 min at 18°C) with a concomitant increase in the biomass produced at the stationary phase (Fig. 1b). At temperatures higher than 20°C, a drastic reduction in cell density at the stationary phase is recorded (Fig. 1b), indicating heat-induced stress on the cell. P. haloplanktis fails to grow above 29°C. According to this growth behavior, the temperatures of 4°C and 18°C were selected here for the differential comparison of the proteomes, as 18°C does not induce excessive stress as far as growth rate and biomass are concerned.Cold-induced versus cold-repressed proteins. The proteomes expressed by the Antarctic bacterium at 4°C and 18°C during the logarithmic phase of growth were compared by two-dimensional (2D) differential in-gel electrophoresis as described previously (14). In a typical single 2D gel (see Fig. S1 in the supplemental material), 142 protein spots were more abundant at 4°C (CAPs), whereas 309 protein spots were less intense at 4°C than at 18°C. This unexpectedly large number of cold-repressed proteins already indicates that numerous cellular functions are downregulated during growth at low temperature. The repression factors (or induction factors for CAPs), given by relative spot abundance between 4°C and 18°C, are illustrated in Fig. 2. This distribution shows that 21% of coldrepressed proteins display a downregulation facto...

SummaryThe protein folding process in psychrophiles is impaired by low temperature, which exerts several physicochemical constraints, such as a decrease in the folding rate, reduced molecular diffusion rates and increased solvent viscosity, which interfere with conformational sampling. Furthermore, folding assistance is required at various folding steps according to the protein size. Recent studies in the field have provided contrasting and sometimes contradictory results, although protein folding generally appears as a rate-limiting step for the growth of psychrophiles. It is proposed here that these discrepancies reflect the diverse adaptive strategies adopted by psychrophiles in order to allow efficient protein folding at low temperature. Cold adaptations apparently superimpose on pre-existing cellular organization, resulting in different adaptive strategies. In addition, microbial lifestyle further modulates the properties of the chaperone machinery, which possibly explains the occurrence of cold-adapted and non-cold-adapted protein chaperones in psychrophiles.

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The growth behavior and the proteomic response after a cold shock were investigated in the psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis. Remarkably, no cold-induced proteins were observed in the proteome, whereas some key proteins were repressed. This suggests noticeable differences in the cold shock response between a true psychrophile and mesophiles.