Dried cells from haloalkaliphilic bacterium Salinivibrio sharmensis BAG^T were extracted by the PCP method [14]. The obtained lipopolysaccharidic fraction was hydrolyzed under mild acidic condition, with 5% acetic acid. The gas chromatography mass spectrography (GC-MS) analysis of the fatty acids methyl esters derivative, obtained after the LPS methanolysis, showed the presence of 3-hydroxy-dodecanoic [C12:0(3-OH)] and 3-hydroxy-tetradecanoic acids [C14:0(3-OH)], as the most abundant species, together with dodecanoic (C12:0), tetradecanoic (C14:0) and hexadecenoic (C16:1) acids.

Lipid A was analyzed by ESI FT-ICR mass spectrometry. The charge deconvoluted negative ions ESI FT-ICR mass spectrum showed the presence of nine signals (M1–M9, Figure 1, Table 1) corresponding to a number of glycoforms differing in their acylation as well as their phosphorylation degree. In particular, they ranged from hexa-acylated to di-acylated glycoforms and, except for M9, were mono or bis-phosphorylated.

In particular M1, M3, M5 and M7 corresponded to the bis-phosphorylated glycoforms. The following composition was attributed to the species M1, with the mass 1741.14 Da, GlcN₂P₂[C12:0(3-OH)]₂[C14:0(3-OH)]₂(C12:0)(C14:0) (calculated molecular mass: 1741.17 Da). Species M3 was identified as penta-acylated lipid A since it carries no C12:0 fatty acid, while the M5 species was missing a C12:0 and 3-hydroxy dodecanoic acid. The tri-acylated species M7 contained only one 3-hydroxy dodecanoic and two 3-hydroxy tetradecanoic acids. Finally M9 was the only di-acylated species corresponding to the following composition GlcN₂P[C14:0(3-OH)]₂. Less abundant species, due to the replacement of the C14:0 with a C16:1 (+26 u) or a C12:0 (−28 u) fatty acid, were also present. In order to obtain the detailed information on the distribution of fatty acids on the disaccharidic backbone, MS and MS/MS spectra in the positive ion mode were generated.

The FT-ICR-MS (positive ion mode) of lipid A showed the presence of the adduct ion [M + Et₃N + H]⁺ at m/z 1843.31, which corresponded to the hexa-acylated, bis-phosphorylated glycoform (calculated molecular mass: m/z 1843.303). The MS/MS spectrum of this species, obtained by infrared multiphoton dissociation (IRMPD) (see experimental section), displayed a B⁺ fragment ion [15,16] at m/z 1058.76, to which the following composition was attributed GlcNP[C12:0(3-OH)][C14:0(3-OH)](C12:0)(C14:0) (calculated mass m/z 1058.77). These results proved that both the secondary fatty acids (C12:0 and C14:0) were linked to the distal non-reducing end glucosamine.

By treating the sample with NH₄OH [17], the lipid A was deprived of primary O-linked fatty acids. The product was analyzed again in the positive ion mode. The mass spectrum revealed the presence of four main signals corresponding to [M + H]⁺ adducts with one or two Et₃N molecules, followed by the signals of the corresponding monophosphorylated species at 80 u lower (N1–N6, Figure 2, Table 2).

The [M + 2Et₃N + H]⁺ adduct ion at 1365.91 m/z (N1) was selected for fragmentation. The MS/MS spectrum showed the presence of a B⁺ fragment ion at m/z 678.4 (GlcNP[C14:0(3-OH)]C14:0, calculated m/z 678.3, Figure 3a), thus revealing that C14:0 was linked as acyloxyamide to the GlcN II. As a consequence, C12:0 resulted to be linked as acyloxyacyl to the GlcN II. In Figure 2, starting from the N1 signal, a species at 26 u higher masses was found (m/z 1391.90), which corresponded to the replacement of the C14:0 with C16:1, as already found.

The MS/MS spectrum of the signal at m/z 1391.9 (Figure 3b) displayed the presence of a B⁺ fragment ion at m/z 704.4 confirming that C16:1 occupies the same position of C14:0. The MS/MS spectrum of N3 showed the presence of a B⁺ fragment ion at 678.4 m/z, the same as obtained by N1 fragmentation (data not shown). This pointed out that the phosphate group on the proximal glucosamine was not stoichiometric. The lack of the phosphate group was a consequence of the 5% acetic acid hydrolysis. In fact, in a recently published paper [18], where the oligosaccharidic portion of the LPS from Salinivibrio sharmensis was characterized after mild and strong alkaline treatment of the LPS, the phopshorylation was stoichiometric. On the basis of all the data obtained from the mass spectra as well as from the chemical analysis, the structure of the lipid A from the S. sharmensis strain BAG^T is depicted in Scheme 1.

S. sharmensis strain BAG^T (=ATCC BAA-1319^T = DSM 18182^T), isolated from samples collected in a small permanent saline lake in Ras Mohammed Park, located at Sharm el-Sheikh (Egypt) [13], was grown at 35 °C and at pH 9.0, using a medium containing the following components (g/L): yeast extract 10.0, NaCl 100.0, Na₃-citrate 3.0, Na₂CO₃ 3.0, KCl 2.0, MgSO₄·7H₂O 1.0, MnCl₂·4H₂O 0.00036, FeSO₄ 0.05 (NaCl and Na₂CO₃ were autoclaved separately). The medium was autoclaved for 20 min at 121 °C. The inoculum was prepared using a dilution of 1:50 v/v. Bacterial growth was directly monitored in an UV/Vis spectrophotometer DU 730 (Beckman Coulter) by utilizing the change in optical density at 540 nm. After incubation for 24 h at an absorbance of 2.0, the cells were collected by centrifugation at 10,000× g and lyophilized. Dried bacteria cells (7 g) were extracted by PCP method [14], obtaining 103 mg of LPS (yield 1.5% of dried cells).

The LPS (30 mg) was incubated with 5% aqueous CH₃COOH (3 mL) for 4 h at 100 °C. The sample was then centrifuged at 10,000× g for 15 min and two fractions were recovered: the lipid A (5.3 mg) and the saccharidic portion (22.7 mg).

A sample of the lipid A fraction (0.5 mg) was dried and methanolyzed, as already described elsewhere [12]. Briefly, 1 mL of 1 M HCl/CH₃OH was added to the sample and the reaction was carried out at 80 °C for 20 h. The crude mixture was extracted twice with hexane. The methanol layer, containing the methyl glycosides, was dried and the residue acetylated by treating with pyridine (200 μL) and acetic anhydride (100 μL) at 100 °C for 30 min. Then both the hexane layer, containing the fatty acids as methyl esters derivatives, and the acetylated methyl glycosides were analyzed by GC-MS. The analyses were performed on a Agilent Technologies gas chromatograph 6850A equipped with a mass selective detector 5973 N and a Zebron ZB-5 capillary column (Phenomenex, 30 m × 0.25 mm i.d., flow rate 1 mL/min, He as carrier gas). The following temperature program was used for lipid analysis—140 °C for 3 min, 140 °C→280 °C at 10 °C/min. Acetylated methyl glycosides were analyzed with the following temperature programs—150 °C for 3 min, 150 °C→240 °C at 3 °C/min.

Lipid A (0.6 mg) was incubated with conc. NH₄OH (100 μL) as reported [17]. The sample was dried and analyzed by ESI FT-ICR mass spectrometry.

Electrospray ionization Fourier transform ion cyclotron (ESI FT-ICR) mass spectrometry was performed in the negative and positive ion mode using an APEX QE (Bruker Daltonics) equipped with a 7 Tesla actively shielded magnet. For the negative-ion spectra, samples (~10 µg) were dissolved in a 50:50:0.001 (v/v/v) mixture of 2-propanol, water, and triethylamine (pH 8.5). The samples were sprayed at a flow rate of 2 µL min⁻¹. Capillary entrance voltage was set to 3.8 kV and drying gas temperature to 200 °C. The instrument was externally calibrated with appropriate standards. The negative ion mass spectra were charge deconvoluted, and the mass numbers given refer to the monoisotopic mass of the neutral molecules.

The MS/MS lipid species were analyzed in the positive ion mode using a 50:50:0.03 (v/v/v) mixture of 2-propanol, water, and 30 mM ammonium acetate (pH 4.5). Small amounts of triethylamine were added to generate triethylamine adduct ions, which are favorable as precursor ions for MS/MS [16]. Precursor ions were isolated in the ICR-cell and then fragmented by infrared multiphoton dissociation (IRMPD). For this, the unfocused beam of a 35-watt, CO₂ laser (Synrad) was directed through the center of the trap. The duration of laser irradiation was adapted for optimal fragmentation and varied between 50 and 200 ms, and fragment ions were detected after a delay of 0.5 ms.