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...

Pseudoalteromonas haloplanktis TAB 23 is a Gram-negative psychrophilic bacterium isolated from the Antarctic coastal sea. To survive in these conditions psychrophilic bacteria have evolved typical membrane lipids and "antifreeze" proteins to protect the inner side of the microorganism. As for Gram-negative bacteria, the outer membrane is mainly constituted by lipopoly- or lipooligosaccharides (LPS or LOS, respectively), which lean towards the external environment. Despite this, very little is known about the peculiarity of LPS from Gram-negative psychrophilic bacteria and what their role is in adaptation to cold temperature. Here we report the complete structure of the LOS from P. haloplanktis TAB 23. The lipid A was characterized by MALDI-TOF MS analysis and was tested in vitro showing a significant inhibitory effect on the LPS-induced pro-inflammatory cytokine production when added in culture with LPS from Escherichia coli. The product obtained after de-O-acylation of the LPS was analyzed by MALDI-TOF MS revealing the presence of several molecular species, differing in phosphorylation degree and oligosaccharide length. The oligosaccharide portion released after strong alkaline hydrolysis was purified by anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) to give three main fractions, characterized by means of 2D NMR spectroscopy, which showed a very short highly phosphorylated saccharidic chain with the following general structure. α-Hepp3R,6R,4R'-(1→5)-α-Kdop4P-(2→6)-β-GlcpN4R-(1→6)-α-GlcpN1P (R=-H(2)PO(3) or -H; R'=α-Galp-(1→4)-β-Galp-(1→ or H-).

Many cold habitats contain plenty of microorganisms that represent the most abundant cold-adapted life forms on earth. These organisms have developed a wide range of adaptations that involve the cell wall of the microorganism. In particular, bacteria enhance the synthesis of unsaturated fatty acids of membrane lipids to maintain the membrane fluidity, but very little is known about the adaptational changes in the structure of the lipopolysaccharides (LPSs), the main constituent of the outer leaflet of the outer membrane of Gram-negative bacteria. The aim of this study was to investigate the chemical structure of these LPSs for insight into the temperature-adaptation mechanism. For this objective, the cold-adapted Psychromonas arctica bacterium, which lives in the arctic sea-water near Spitzbergen (Svalbard islands, Arctic) was cultivated at 4 degrees C. The lipooligosaccharides (LOSs) were isolated and analysed by means of chemical analysis and electrospray ionisation high-resolution Fourier transform mass spectrometry. The LOS was then degraded either by mild hydrazinolysis (O-deacylation) or with hot 4 M KOH (N-deacylation). Both products were investigated in detail by using 1H and 13C NMR spectroscopy and mass spectrometry. The core consists of a mixture of species that differ because of the presence of nonstoichiometric D-fructose and/or D-galacturonic acid units.

Cold‐adapted bacteria are microorganisms that thrive at very low temperatures in permanently cold environments (0–10 °C). Their ability to survive under these harsh conditions is the result of molecular evolution and adaptations, which include the structural modification of the phospholipid membrane. To give insight into the role of the membrane in the mechanisms of adaptation to low temperature, the characterization of other cell‐wall components is necessary. Among these components, the lipopolysaccharides are complex amphiphilic macromolecules embedded in the outer leaflet of the external membrane, of which they are the major constituents. The cold‐adapted Colwellia psychrerythraea 34H bacterium, living in deep sea and Arctic and Antarctic sea ice, was cultivated at 4 °C. The lipooligosaccharide (LOS) was isolated and analysed by means of chemical analysis. Then it was degraded either by mild hydrazinolysis (O‐deacylation) or hot KOH (4 M; N‐deacylation). Both products were investigated in detail by 1H and 13C NMR spectroscopy and by ESI FT‐ICR mass spectrometry. The oligosaccharide portion consists of a unique and very short species with the following general structure: α‐L‐Col‐(1→2)‐α‐D‐GalA‐(1→2)‐α‐D‐Man‐[3‐P‐D‐Gro]‐(1→5)‐α‐D‐Kdo‐4‐P‐Lipid‐A.