A considerable fraction of life develops in the sea at temperatures lower than 15°C. Little is known about the adaptive features selected under those conditions. We present the analysis of the genome sequence of the fast growing Antarctica bacterium Pseudoalteromonas haloplanktis TAC125. We find that it copes with the increased solubility of oxygen at low temperature by multiplying dioxygen scavenging while deleting whole pathways producing reactive oxygen species. Dioxygen-consuming lipid desaturases achieve both protection against oxygen and synthesis of lipids making the membrane fluid. A remarkable strategy for avoidance of reactive oxygen species generation is developed by P. haloplanktis, with elimination of the ubiquitous molybdopterin-dependent metabolism. The P. haloplanktis proteome reveals a concerted amino acid usage bias specific to psychrophiles, consistently appearing apt to accommodate asparagine, a residue prone to make proteins age. Adding to its originality, P. haloplanktis further differs from its marine counterparts with recruitment of a plasmid origin of replication for its second chromosome.[Supplemental material is available online at www.genome.org. The sequence data from this study have been submitted to EMBL under accession nos. CR954246 and CR954247.

Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes.

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.

ABSTRACT:The low temperatures of polar regions and high altitude environments, especially icy habitats, present challenges for many microorganisms. Their ability to live under subfreezing conditions implies the production of compounds conferring cryotolerance. Colwellia psychrerythraea 34H, a -proteobacterium isolated from subzero Arctic marine sediments, provides a model for the study of life in cold environments. We report here the identification and detailed molecular primary and secondary structures of capsular polysaccharide from C. psychrerythraea 34H cells. The polymer was isolated in the water layer when cells were extracted by phenol/water and characterized by one-and two-dimensional NMR spectroscopy together with chemical analysis. Molecular mechanic and dynamic calculations were also performed. The polysaccharide consists of a tetrasaccharidic repeating unit containing two amino sugars and two uronic acids bearing threonine as substituent. The structural features of this unique polysaccharide resemble those present in antifreeze proteins and glycoproteins. These results suggest a possible correlation between the capsule structure and the ability of C. psychrerythraea to colonize subfreezing marine environments.

The chemical structural variations induced by different growth temperatures in the lipooligosaccharide and exopolysaccharide components extracted from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC 125 are described. The increase in phosphorylation with the increase in growth temperature seems to be general, because it happens not only for the lipooligosaccharide but also for the exopolysaccharide. Structural variations in the lipid components of lipid A also occur. In addition, free lipid A is found at both 25 and 4°C but not at 15°C, which is the optimal growth temperature, suggesting a incomplete biosynthesis of the lipooligosaccharide component under the first two temperature conditions. Lipopolysaccharides (LPSs) are amphiphilic molecules contained in the outer leaflet of the external membrane of gramnegative bacteria. They are anchored in the membrane by the lipid part (lipid A), which is covalently linked to an oligosaccharide fragment (core) that, in turn, is bonded to a polysaccharide part (O antigen, or O side chain). Due to their outward location, the LPSs are involved in mechanisms of interaction with the surroundings. Despite the fact that gram-negative bacteria colonize very different organisms and environments, LPSs show a common architectural structure (17). This suggests that the molecular structures of the LPS components can play an important role in host or environment specificity. In this context, the structures of LPSs of extremophilic bacteria evoke much interest owing to the extreme conditions under which they live (14). The cold adaptation of psychrophilic bacteria, which enables them to thrive in environments below 5°C, necessitates the acquisition of unique structural features for membrane components, so that membrane fluidity and effective transport of nutrients under cold conditions are guaranteed. The exopolysaccharides (EPSs) that many bacteria are able to produce may also be involved in interaction with the environment, in addition to having rheological properties of potential economical interest (6,19,21).Recently we have been interested in structural elucidation of both the saccharide backbones (5) and the lipid A moieties (4) of the LPS components of Pseudoalteromonas haloplanktis TAC 125, a cold-adapted bacterium isolated from Antarctic seawater (1) and grown at 15°C. In the first paper (5), Corsaro et al. showed that the LPS fraction consists of two lipooligosaccharides (LOSs); that is, it lacks the O chains. The major component possesses the following sugar backbone structure:The latter two units are both acylated at positions 2 and 3, with 3-hydroxydodecanoyl residues (3-OH-12:0) linked both as esters and as amides (4). The hydroxyl of the (3-OH-12:0) residue linked at position 3 of the nonreducing glucosamine is esterified by a dodecanoyl residue (12:0). Here we describe the variations that occur in the LOS structures when the bacterium is grown at two temperatures other than 15°C: one lower (4°C) and one higher (25°C). Since this bacterium is also able to produ...