Structural Elucidation of a Novel Lipooligosaccharide from the Cold-Adapted Bacterium OMVs Producer Shewanella sp. HM13

Abstract

Shewanella sp. HM13 is a cold-adapted Gram-negative bacterium isolated from the intestine of a horse mackerel. It produces a large amount of outer membrane vesicles (OMVs), which are particles released in the medium where the bacterium is cultured. This strain biosynthesizes a single major cargo protein in the OMVs, a fact that makes Shewanella sp. HM13 a good candidate for the production of extracellular recombinant proteins. Therefore, the structural characterization of the components of the vesicles, such as lipopolysaccharides, takes on a fundamental role for understanding the mechanism of biogenesis of the OMVs and their applications. The aim of this study was to investigate the structure of the oligosaccharide (OS) isolated from Shewanella sp. HM13 cells as the first step for a comparison with that from the vesicles. The lipooligosaccharide (LOS) was isolated from dry cells, purified, and hydrolyzed by alkaline treatment. The obtained OS was analyzed completely, and the composition of fatty acids was obtained by chemical methods. In particular, the OS was investigated in detail by 1H and 13C NMR spectroscopy and MALDI-TOF mass spectrometry. The oligosaccharide was characterized by the presence of a residue of 8-amino-3,8-dideoxy-manno-oct-2-ulosonic acid (Kdo8N) and of a d,d-heptose, with both residues being identified in other oligosaccharides from Shewanella species.

Detailed Structural Characterization of the Lipooligosaccharide from the Extracellular Membrane Vesicles of Shewanella vesiculosa HM13

“…After silver nitrate staining, the gel showed for both samples the presence of the bands at low molecular masses, thus confirming the presence of an R-type LPS. The same quickly migrating species was observed for the cells of the bacterium S. vesiculosa HM13 grown at 4 • C [14]. psychrotolerant Antarctic bacterium [8,9].…”

“…The sugar composition for both LOSs was obtained by gas chromatography-mass spectrometry (GC-MS) of the acetylated methyl glycosides. The GC-MS profiles revealed the same sugar composition as that obtained for the cells of S. vesiculosa HM13 grown at 4 °C [14]. In particular, the analysis confirmed the presence of glucose (Glc), 2-amino-2-deoxy-glucose (GlcN), and both D,D-and L-glycero-D-manno-heptoses (D,D-Hepand L,D-Hep, respectively) ( Figure 2a,b, and Table S1, Supporting Information).…”

“…Nonhydroxylated fatty acid residues were found to be C12:0, C13:0, C14:0, and C15:0. In the same spectra, signals assignable to the lipid A and to the core oligosaccharide (OS) fragments, arising from the in-source cleavage of the linkage between the GlcNII of the lipid A backbone and the 8-amino-3,8-dideoxy-manno-oct-2-ulosonic acid (Kdo8N) residue, were detected [14] (Figure 3). The mass spectra revealed that signals attributable to the core fragments were the same as those obtained at 4 °C [14].…”

“…In the same spectra, signals assignable to the lipid A and to the core oligosaccharide (OS) fragments, arising from the in-source cleavage of the linkage between the GlcNII of the lipid A backbone and the 8-amino-3,8-dideoxy-manno-oct-2-ulosonic acid (Kdo8N) residue, were detected [14] (Figure 3). The mass spectra revealed that signals attributable to the core fragments were the same as those obtained at 4 °C [14]. Indeed, the signals at m/z 1360.63 and 1483.66 can be attributed to the fragments Hex3Hep3Kdo8NP and Hex3Hep3Kdo8NPPEtN, respectively (Calculated [M − H] − m/z 1360.38 and 1483.39, respectively) (Hex, hexose; Hep, heptose; P, phosphate group; PEtN, phosphoethanolamine group).…”

“…The LPS is termed smooth (S-LPS) when the O-chain is present, whereas it is termed rough when the O-chain is absent (R-LPS or lipooligosaccharide) [12]. We previously isolated and characterized the lipooligosaccharide (LOS) from the wild sample of S. vesiculosa HM13 [14]. Usually, the small amount of EMVs' biomass does not allow for the isolation of enough amounts of LPS from EMVs to be characterized [15].…”