Structural Investigation of the Oligosaccharide Portion Isolated from the Lipooligosaccharide of the Permafrost Psychrophile Psychrobacter arcticus 273-4

Psychrophilic microorganisms have successfully colonized all permanently cold environments from the deep sea to mountain and polar regions. The ability of an organism to survive and grow in cryoenviroments depends on a number of adaptive strategies aimed at maintaining vital cellular functions at subzero temperatures, which include the structural modifications of the membrane. To understand the role of the membrane in the adaptation, it is necessary to characterize the cell-wall components, such as the lipopolysaccharides, that represent the major constituent of the outer membrane. The aim of this study was to investigate the structure of the carbohydrate backbone of the lipooligosaccharide (LOS) isolated from the cold-adapted Psychrobacter arcticus 273-4. The strain, isolated from a 20,000-to-30,000-year-old continuously frozen permafrost in Siberia, was cultivated at 4 °C. The LOS was isolated from dry cells and analyzed by means of chemical methods. In particular, it was degraded either by mild acid hydrolysis or by hydrazinolysis and investigated in detail by 1H and 13C NMR spectroscopy and by ESI FT-ICR mass spectrometry. The oligosaccharide was characterized by the substitution of the heptose residue, usually linked to Kdo in the inner core, with a glucose, and for the unusual presence of N-acetylmuramic acid.

genus for permafrost and other polar environments [25], suggesting that many of its members are adapted to low temperatures and have evolved molecular-level changes that aid survival at low temperatures.
Psychrobacter arcticus 273-4 is a Gram-negative bacterium isolated from a 20,000-to-30,000-year-old continuously frozen permafrost horizon in the Kolyma region in Siberia that was not exposed to temperatures higher than 4 °C during isolation [5].
In this paper, we report the structural characterization of the carbohydrate backbone of the LOS of Psychrobacter arcticus 273-4 grown at 4 °C.
The lipooligosaccharide was degraded both by mild hydrazinolysis (O-deacylation) and by acetic acid hydrolysis. The products were investigated by means of chemical analysis, by 1 H and 13 C NMR spectroscopy and by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS).

LPS Extraction and Purification
Psychrobacter arcticus strain 273-4 cells were grown at 4 °C and removed from the medium by centrifugation. Dried bacteria cells were extracted using a phenol/chloroform/light petroleum (PCP) mixture to obtain the crude LPS. Due to the very low amount of LPSPCP (0.03%), cells were extracted by phenol/water method, and the aqueous phase was dialyzed and freeze-dried. In order to purify LPSw from other cell contaminants, the sample was treated with DNase, RNase, and protease followed by dialysis (LPSW, 3.1%). The purified sample (LPSW) was analyzed by DOC-PAGE electrophoresis, and the silver nitrate staining showed bands at low molecular masses, thus revealing a rough LPS (LOS, Figure 1). The sugar composition of the intact LOS was obtained by GC-MS analysis of the acetylated methyl glycosides and revealed the occurrence of rhamnose (rha), galactose (gal), glucose (glc), N-acetylmuramic acid (NAM), and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). Methylation analysis  indicated the presence of 3-substituted Rha, terminal Glc, 4-substituted Glc, 3-substituted Gal, terminal  NAM, 3,4,6-trisubstituted Glc, 3,4-disubstituted Glc, terminal Kdo, and 4,5-disubstituted Kdo. The methylation data also revealed a pyranose ring for all the residues. The absolute configurations of the sugar residues were determined by GC-MS analysis of the corresponding acetylated 2-octyl glycosides; all the hexoses were founded to be in the D-configuration, while rhamnose residue in the L-configuration. The absolute configuration of N-acetylmuramic acid was supported by the NMR data (see below).

Deacylation of the LPS
The LOSW was O-deacylated with anhydrous hydrazine and the product obtained (LOS-OH) was analyzed by ESI FT-ICR mass spectrometry. The charge deconvoluted mass spectrum showed various K-adducts [M + n(K − H)] of four main ion populations M1-M4 (Figure 2), the composition of which is reported in Table 1. The most abundant ion population with a mass of 2633.927 u was attributed to the following composition: DeoxyHexHex5Kdo2NAMHexN2P2 [14:0(3OH)] [12:0(3OH)] ([M1 + (K − H)], calculated monoisotopic mass: 2633. 934 u). The signal of M3, occurring at 162.052 u lower than M1, suggested the presence of ion populations containing one hexose less. In addition, the intensity of the signal of M3 suggests very low abundance of this glycoform. The ion populations M2 and M4 were attributed the same sugar composition as M1 and M3, respectively, whereas the mass difference of 28.03 u is due to a 3-hydroxy dodecanoic in place of the 3-hydroxy tetradecanoic acid.
In addition, the methylation data revealed that the lack of the hexose residue for the ion populations M3 and M4 was from the position O-6 of the 3,4,6-trisubstituted glucose.

Mild Acid Hydrolysis of the LPS
The well-known ability of the LOS to form micellar aggregates in aqueous solution did not allow the direct structural NMR analysis. Thus, the LOS was hydrolyzed under mild acidic conditions to cleave the unstable Kdo glycosidic linkage between the lipid A and the saccharidic region. After centrifugation, the supernatant containing the core oligosaccharidic portion of the LOS was separated from a precipitate constituted by the lipid A. The supernatant was analyzed by ESI FT-ICR MS. The charge deconvoluted mass spectrum displayed the presence of two main ion populations (N1 and N2, Figure 3). As expected, for the most abundant N1, occurring at 1469.501 u (calculated monoisotopic mass: 1469.48 u), it was found the following composition: NAMDeoxyHexHex5Kdo1. Again, the difference of 162.056 u with N2 confirmed the presence of an ion population lacking one hexose residue. No peaks with two Kdo residues were found, since the ketosidic bond is much more acid-labile than the common aldosidic bonds. Signals at 46.00 and 18.01 u lower mass values with respect to N1 were both assignable to Kdo artifacts [26].
The supernatant mixture was further purified on a Bio-Gel P-10 chromatography column (Bio-Rad Laboratories S.r.l, Milano, Italy ), using pyridinium acetate buffer as eluent. The main obtained fraction, named OS, was studied by two-dimensional NMR spectroscopy.
The 1 H-NMR spectrum of the OS fraction, recorded at 310 K, is shown in Figure 4. Seven anomeric proton signals (A-G), attributable to core monosaccharide residues, were present in the region between δ 4.5 and δ 5.4 ppm ( Table 2).
The 1 H-NMR spectrum of OS was also recorded at 318 K (data not shown) in order to reduce the anomeric signals overlapping. In this experiment, the anomeric proton signal of E was clearly visible. Moreover, the integration of all anomeric signals showed a relative ratio of 1:1 except for the signal at 4.51 ppm. In fact, the peak area for this signal was twice the amount of every other proton anomeric signal, thus indicating the coincidence of H-1 of F with H-1 of G chemical shifts.
By considering all the two-dimensional NMR experiments, the spin systems of all the monosaccharides were identified ( Table 2). The spectrum was recorded in D2O at 310 K at 600 MHz. The letters refer to the residues as described in Table 2 and Scheme 1. Residue A with H-1/C-1 signals at δ 5.36/95.1 ppm was identified as a 3-O-(1-carboxyethyl) ether of 2-acetamido-2-deoxy glucopyranosyl residue (namely N-acetylmuramic acid (NAM)), with an α-anomeric configuration, as suggested by the low 3 JH-1,H-2 value (3.1 Hz). Moreover, its H-2 proton at δ 3.72 ppm was correlated, in the DEPT-HSQC experiment ( Figure 5), with a C-2 resonance occurring at δ 55.0 ppm, thus indicating a nitrogen-bearing carbon atom. In addition, the HMBC spectrum showed a long range scalar coupling between the signal of H-3 at δ 3.78 ppm with the signal at δ 79.8 ppm, attributed to C-2′ of 1-carboxyethyl substituent. The same experiment also revealed a correlation between the signal at δ 4.43 ppm, attributed to H-2′, with both the signals of C-1′ (δ 183.2 ppm) and C-3′ (δ 20.0 ppm), respectively, of 1-carboxyethyl substituent. Finally, a correlation between H-2 signal at δ 3.72 ppm and the carbonyl signal of NAc group at δ 175.5 ppm was also identified.
The correlations of each H-1 to H-6 with all other protons of residues B, D, E, and G in the TOCSY spectrum provided evidence for the gluco configuration of all these ring systems.
Residue B with H-1/C-1 signals at δ 5.16/100.9 ppm was assigned to a 3,4,6 trisubstituted α-glucose unit on the basis of the small anomeric coupling constant value ( 3 JH-1,H-2 = 3.7 Hz). The downfield shift of C-3, C-4, and C-6 values of this unit at δ 78.0, 75.2, and 68.8 ppm, respectively [27], identified its substitution. This residue was linked to Kdo residue at the O-5 position, as shown by the correlation between H-1 B and C-5 of H in the HMBC spectrum ( Figure 6, Table 3).    The lack of heptose residue, usually linked in the inner core to the Kdo, has been found so far in the Moraxellaceae [28] and Rhizobiaceae families [29,30]. The only example of a heptose-deficient core region among lipopolysaccharides from psychrophiles was found in Colwellia psychrerythraea strain 34H [22].

Anomeric Atom in Sugar Residue (δ) Correlations to Atom in Sugar Residue (δ) ROESY HMBC
Residues D and G with H-1/C-1 signals at δ 4.95/102.9 and δ 4.51/103.8 ppm, respectively, were identified as terminal β-glucoses, since none of their carbons were shifted by glycosylation. For both residues the β configuration was inferred by the high 3 JH-1,H-2 values (8.1 and 8.0 Hz for D and G, respectively). Intra-residue NOE (Nuclear Overhauser Effect) contacts of H-1 with H-3 and H-5 (δ 3.52 and 3.47 ppm, and δ 3.52 and 3.46 ppm, for D and G, respectively) were in agreement with β-anomeric configurations.
A 3 JH-1,H-2 coupling constant of 8.0 Hz for residue E indicated a β-configuration, which was also confirmed by intra-residue NOEs. The C-4 of residue E was downfield shifted at δ 79.7 ppm with respect to the unsubstituted value [31], thus evidencing that this position was glycosylated. The residue F with H-1/C-1 signals at δ 4.52/104.2 was identified as a galacto configured residue since the TOCSY experiment showed correlations only from H-1 to H-4; in particular, it was identified as a β-galactose ( 3 JH-1,H-2 = 8.0 Hz). Moreover, the downfield shift of proton resonance of C-3 at δ 81.6 ppm instead of δ 73.8 ppm of an unsubstituted residue [31] indicated glycosylation at this position.
The residue C with H-1/C-1 signals at δ 5.06/103.5 ppm was recognized as an α-rhamnose residue, since the TOCSY spectrum showed scalar correlations of the ring protons with methyl signal in the up-field region at δ 1.30 ppm. Its α configuration was suggested by the 3 JH-1,H-2 value (<3 Hz) and by the value of its C-5 chemical shift [32]. The downfield shift of carbon resonance of C-3 at δ 77.2 ppm with respect to the value of δ 71.0 ppm [31] indicated glycosylation at this position.
The Kdo H-5 proton was identified by vicinal scalar coupling with H-4 in the COSY spectrum. Moreover, the residue resulted to be glycosylated at O-5 position, as suggested by the downfield shift of its C-5 carbon signal at δ 77.0 ppm with respect to the value of δ 67.5 ppm for an unsubstituted Kdo [33].
The sequence of the residues was deduced from the HMBC experiment ( Figure 6, Table 3 Inter-residue NOE contacts, obtained from ROESY experiments (Table 3), confirmed this sequence, since dipolar couplings were observed between: H-1

of B and H-5 of H, H-1 of G and both H-6 of B, H-1 of E and H-4 of B, H-1 of F and H-4 of E, H-1 of D and H-3 of B, H-1 of A and H-3 of C, H-1 of C and H-3 of F.
The absolute configuration of residue A is based on NMR considerations. The chemical shift of C-1 at δ 95.1 ppm indicates that the N-acetylmuramic acid has the opposite configuration of L-rhamnose, since a value of near 103 ppm would be expected for the same absolute configuration of residue C [34]. As for 1-carboxyethyl substituent, the configuration of (R) for C-2′ was deduced by comparing both 1 H and 13 C NMR chemical shifts of residue A with those of N-acetylisomuramic acid [35,36], characterized by a (S) configuration at C-2′.
In conclusion, the complete structure of the core oligosaccharide of the LOS from Psychrobacter arcticus 273-4 is reported in Scheme 1.

Bacteria Growth and LPS Isolation
P. arcticus strain 273-4, isolated from permafrost soil located in Siberia. Shake flask cultivation were performed in Luria-Bertani broth [37] at 4 °C in aerobic condition. When the liquid cultures reached late exponential phase (about 90 h, OD 600nm 4) cells were collected by centrifugation for 15 min at 7000 rpm at 4 °C.
Dried bacteria cells (3.1 g) were extracted first by PCP method to give very poor yield of LOS, LPSPCP (yield 0.03% w/w of dried cells) and then by hot phenol/water method [38,39]. A 240 mg amount of water extract was dialyzed (cut-off 3500 Da) and then digested with proteases, DNases, and RNases to remove contaminating proteins and nucleic acids. The sample was dialyzed (cut-off 3500 Da) in order obtaining 96 mg of sample (LPSW, yield 3.1% w/w of dried cells).

Sugar and Fatty Acids Analysis
LOS (1 mg) was treated with HCl/CH3OH (1.25 M, 1 mL) and the methanolysis was performed at 80 °C for 16 h. The monosaccharides obtained were acetylated and analyzed as acetylated methyl glycosides by GC-MS. The fatty acids were analyzed as methyl esters [11].
The absolute configuration of the sugars was determinated by gas chromatography of the acetylated (S)-2-octyl glycosides [40]. All the sample derivatives were analyzed on an Agilent Technologies gas chromatograph 6850A equipped with a mass selective detector 5973N and a Zebron ZB-5 capillary column (Phenomenex, 30 m × 0.25 mm i.d., flow rate 1 mL/min, He as carrier gas). Acetylated methyl glycosides were analyzed using the following temperature program: 140 °C for 3 min, 140 °C → 240 °C at 3 °C/min. Analysis of acetylated octyl glycosides was performed as follows: 150 °C for 5 min, 150 °C → 300 °C at 6 °C/min, 300 °C for 5 min. The temperature program for methyl esters of fatty acids is the following: 140 °C for 3 min, 140 °C → 280 °C at 10 °C/min, 280 °C for 20 min.

Methylation Analysis
The linkage positions of the monosaccharides were determined by GC-MS analysis of the partially methylated alditol acetates (PMAAs).
To identify the Kdo, the sample was then treated for the reduction of the carboxymethyl groups with sodium boro deuteride NaBD4, mildly hydrolyzed (0.1 M trifluoroacetic acid TFA, 100 °C, 30 min) to cleave ketosidic linkages, followed by a reduction (NaBD4) of hemiketal group. The product was totally hydrolyzed with 2 M TFA at 120 °C for 2 h, reduced with NaBD4, and acetylated with Ac2O and pyridine (50 µL each, 100 °C for 30 min). The mixture was analyzed by GC-MS with the following temperature program: 90 °C for 1 min, 90 °C → 140 °C at 25 °C/min, 140 °C → 200 °C at 5 °C/min, 200 °C → 280 °C at 10 °C/min, at 280 °C for 10 min.

Deacylation of the LOS
The LOS (70 mg) was dried over phosphorus anhydride under vacuum and then incubated with hydrazine (3.5 mL, at 37 °C for 2 h). To precipitate the LOS-OH, cold acetone was added; the pellet was recovered after centrifugation at 4 °C and 7000 rpm for 30 min, washed two times with acetone, and finally suspended in water and lyophilized (55 mg) [43].

Mild Acid Hydrolysis
The LOS (20 mg) was hydrolyzed with 1% aqueous CH3COOH (2 mL, 100 °C for 4 h). The resulting suspension was then centrifuged (7500 rpm, 4 °C, 30 min) and the pellet was washed twice with water. The supernatant layers obtained were combined and lyophilized. The mixture of oligosaccharides was then fractionated on a Bio-Gel P-10 column (Biorad, 1.5 × 110 cm, flow rate 15 mL/h, fraction volume 2 mL) and eluted with water buffered with 0.05 M pyridine and 0.05 M AcOH, obtaining the oligosaccharide fraction named OS (6 mg).

Mass Spectrometry Analysis
Electrospray ionization Fourier transform ion cyclotron (ESI FT-ICR) mass spectrometry was performed in negative ion mode using an APEX QE (Bruker Daltonics GmbH, Bremen, Germany) equipped with a 7 Tesla actively shielded magnet. The LOS sample was dissolved at a concentration of ~10 ng/μL, sprayed at a flow rate of 2 μL/min, and analyzed as described previously [44]. Mass spectra obtained were charge-deconvoluted and the mass numbers given refer to the monoisotopic masses of the neutral molecules.

NMR Spectroscopy
1 H and two-dimensional NMR spectra were performed using a Bruker Avance 600 MHz spectrometer equipped with a cryoprobe (Bruker Italia, Milano, Italy). Two-dimensional homo-and heteronuclear experiments (COSY, TOCSY, ROESY, DEPT-HSQC, and HMBC) were performed using standard pulse sequences available in the Bruker software. 1 H was measured at 310 K and 318 K while two-dimensional NMR spectra were recorded at 310 K and the mixing time for TOCSY and ROESY experiments was 100 ms. The 13 C NMR spectrum was recorded in D2O at 298 K Bruker Avance 400 MHz spectrometer (data not shown).

Conclusions
In this paper, the complete structure of the sugar backbone of the LPS from the permafrost isolate Psychrobacter arcticus 273-4 is reported.The structure shows a particular inner core region, with a residue of glucose linked to the Kdo in place of a manno-heptose. This structural feature has been found only in another psychrophile, namely Colwellia psychrerythrae 34H, which showed a mannose residue linked to the Kdo.
Generally, the oligo-and polysaccharides produced by marine bacteria are distinguished by the acidic character [45] and by the occurrence of unusual sugars [46], non-sugar substituents [22,[47][48][49] or structures that are highly phosphorylated [10]. Although P. arcticus 273-4 was isolated from Arctic permafrost, it displays similar characteristics of cold-adapted marine isolates, due to the presence of the unusual residue of NAM. N-acetylmuramic acid, commonly encountered as a component of bacterial cell-wall peptidoglycan, has been already found in the O-specific polysaccharide of Yersinia ruckerii [50] and Proteus penneri [51], but to the best of our knowledge, this is the first time that it has been found in a core oligosaccharide.
It is well known that cold-adapted microorganisms are able to modify the fluidity of the cellular membrane in response to a lowering of temperature by producing a higher content of unsaturated, polyunsaturated, and methyl-branched fatty acids [52,53]. Instead, how bacteria modify the LPS structures in response to the cold stress is still poorly understood.
Even though only few LPS structures from cold-adapted bacteria have been characterized [11,12,22,23], their attractive feature is the production of rough lipopolysaccharides. Moreover, it is worth noting that Psychrobacter arcticus 273-4, a permafrost isolate, shares this feature with marine isolates. To the best of our knowledge, only two examples of smooth lipopolysaccharides isolated from psychrophiles have been reported so far [54,55], even if the isolates were grown at 24 °C.
By increasing the number of characterized LPS structures from psychrophiles, it will be conceivable in the future to find a connection between the lack of the polysaccharidic portion and the Gram-negative membrane cold adaptation.