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The cryosphere, covering about one-fifth of the surface of the Earth, comprises several components: snow, river and lake ice, sea ice, ice sheets, ice shelves, glaciers and ice caps, and frozen ground which exist, both on land and beneath the oceans (Vaughan DG, et al. 2013). All these habitats, combining the low temperature and the low liquid water activity, are challenging for all the forms of life (Casanueva et al., 2010). These extreme environments are inhabited by microorganisms of all three domains of life; in particular, cold-adapted microorganisms belong to Archea and Bacteria domains. To survive in these harsh life conditions, these microorganisms have developed many adaptation strategies, including the over-expression of cold-shock and heat-shock proteins, the presence of unsaturated and branched fatty acids that maintain membrane fluidity (Chattopadhyay et al., 2006), the different phosphorylation of membrane proteins and lipopolysaccharides (Ummarino et al., 2003; Corsaro et al., 2004; Carillo et al., 2013; Casillo et al., 2015), and the production of cold-active enzymes (Huston et al., 2004), antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs), and cryoprotectants (Deming et al., 2009). Among cryoprotectants, carbohydrate-based extracellular polymeric substances (EPS) have a pivotal role in cold adaptation, as they form an organic network within the ice, modifying the structure of brine channels and contributing in the enrichment and retention of microrganisms in ice (Krembs et al., 2002; Krembs et al., 2011; Ewert et al., 2013). Macromolecules belonging to the external layer are fundamental in adaptation mechanisms, as for example the lipopolysaccharides (LPSs), which constitute the 75% of the outer membrane. LPSs have a structural role since increase the strength of bacterial cell envelope and mediate the contacts with the external environment. The general structure of an LPS is characterized by three distinct portions: the lipid A, composed of the typical glucosamine disaccharide backbone with different pattern of acylation on the two sugar residues, the core oligosaccharide, distinguishable in a inner core and a outer core, and the O-chain polysaccharide built up of oligosaccharide repeating units. This latter moiety can be absent, and in that case LPSs are named lipooligosaccharides (LOSs). Schematic representation of a lipopolysaccharide. Since the outer membrane of Gram-negative bacteria is constituted mainly by LPSs, it is reasonable to assume that structural changes could be present in these macromolecules isolated from cold-adapted bacteria. This work has been focused especially on three different psychrophilic microorganisms, that are considered models for the study of adaptive strategies to subzero lifestyle: Colwellia psychrerythraea strain 34H Psychrobacter arcticus 273-4 Pseudoalteromonas haloplanktis TAC125 In particular, LPS molecules from C. psychrerythraea 34H grown in different conditions, and from P.arcticus, have been purified and analyzed by NMR spectroscopy and mass spectrometry. By comparing the structures obtained, especially for core oligosaccharides, it is possible to speculate that all of them are characterized by high negative charge density. This negative charge is furnished either by phosphate groups, usually linked to Kdo and lipid A saccharidic residues, or by uronic acids. These characteristics have been already found in other LPSs from psychrophilic microorganisms (Corsaro et al., 2004; Corsaro et al., 2008; Carillo et al., 2011), suggesting that such structural elements contribute to the tightness of the outer-membrane and to the association of LPS molecules through divalent cations (Ca2+ and Mg2+). LOS structure from C.psychrerythraea 34H. LOS from P.arcticus 273-4. Starting from the core region of LOS from C. psychrerythraea, previously characterized (Carillo et al., 2013), the structure of lipid A was totally elucidated. The high heterogeneity of this structure, showed by the fatty acids analysis, was confirmed by the complexity of MS and MS/MS spectra. These experiments, indicated a variable state of acylation ranging from tetra- to hepta-acylated glycoforms. The lipid A moiety displayed a structure that is quite new among the LPSs. In fact, it shows the presence of unsaturated 3-hydroxy fatty acids, a feature that up to now is reported only for Agrobacterium tumefaciens (Silipo et al., 2004) and Vibrio fischeri (Philips et al., 2011). In particular, the structure of lipid A from Colwellia psychrerythraea 34H is very similar to that of Vibrio fischeri; in both structures, very intriguing is the presence of an unusual set of modifications at the secondary acylation site of the position 3 of GlcNI consisting of phosphoglycerol (GroP) differently substituted. The structural characterization of different exopolysaccharides produced by Colwellia psychrerythraea have also been reported. The capsular polysaccharide structure from C. psychrerythraea is composed of a tetrasaccharidic repeating unit containing two amino sugars and two uronic acids. The unique characteristic of the capsular polysaccharide is the presence of the α-aminoacid, threonine as substituent (Carillo et al., 2015). The decoration of the polysaccharide with threonines is particularly intriguing to consider. In fact, amino acid motifs are common and crucial for the interaction with ice in several different kinds of antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) (Graether et al., 2000). Then, the molecular mechanic and dynamic calculations were performed, in collaboration with Prof. Randazzo of Department of Pharmacy; the computed model shows that the CPS seems to assume in the space a zig-zag" flexible arrangement and that the overall structure can be imagined like a spatial repetition of an hairpin-like substructure, where the threonines are placed externally and available to interact with the ice. These results, the resemblance of our CPS structure to that of AFGPs, and the lack of sequence coding for a known AFP in the genome of C. psychrerythraea 34H prompted us to assay the purified polymer for ice recrystallization inhibition activity. This analysis, performed by Dr. Bayer-Giraldi, suggest that CPS interacts with ice and that it has an effect on recrystallization (Carillo et al., 2015). Colwellia psychrerythraea is also involved in the production of other two different exopolysaccharides (EPSs) with cryoprotectant activity: an acidic polysaccharide, named EPS, and a mannan. The EPS structure consists of a trisaccharidic repeating unit containing two galacturonic acids and one residue of 2-acetamido-2,6-dideoxy-D-glucose (Qui2NAc). Again, this structure shows the presence of an α-aminoacid, but in this case the decoration is represented by an alanine linked to the galacturonic acid residue. The chemical nature of the EPS is similar to that of the CPS, as it shows both galacto- and gluco-configured monosaccharides and aminoacids. Ice recrystallization inhibition activity, performed by Prof. Matthew Gibson, has been tested also for the EPS
Note:
Dissertation 2016
Language:
Italian
