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

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.


Introduction
Cold-adapted bacteria are extremophiles that are able to thrive in permanently cold environments. Some of these habitats are exposed to temperatures below 5 • C [1], and for this reason cold-adapted microorganisms have developed unique physiological tools to survive in these harsh conditions. Cold habitats are also considered as surprising reservoirs of biotechnological molecules such as antibiofilm molecules [2], surfactants [3], cold-active enzymes [4], antifreeze proteins, glycoproteins, and polysaccharides [5][6][7]. Many psychrophiles have been reported to produce outer membrane vesicles (OMVs) [8]. It has been reported that the OMVs produced by a cold-adapted bacterium contain putative proteolytic enzymes, which can serve to degrade high molecular weight molecules present in the surrounding cells [9,10] helping the bacterium in the survival of such harsh conditions.

LOS Extraction and Purification
Shewanella sp. HM13 cells were grown in Luria Bertani (LB) medium at 4 • C, as described in the Experimental section, and the LPS was isolated from dried cells using the phenol/chloroform/light petroleum (PCP) method [19], with a yield of 2.4%. As illustrated in Figure 1, sodium deoxycholatepolyacrylamide gel electrophoresis analysis (DOC-PAGE) showed, after silver nitrate gel staining, a fast migrating species typical of rough LPS (e.g., LOS). The cellular debris were also extracted by the phenol/water method [20], obtaining the same fast-migrating DOC-PAGE LOS together with proteins and nucleic acids (data not shown).

LOS Deacylation
Alkaline degradation by mild hydrazinolysis of the LOS afforded an O-deacylated LOS, named LOS-OH, which was analyzed by negative ions MALDI-TOF ( Figure 2). The spectrum revealed the presence of a cluster of ions, attributable to the LOS-OH molecule. At [21][22][23][24]. Differences of ±14 Da with respect to the main signal at m/z 2298.6 are attributable to the different lengths of fatty acids substituting the GlcN residues. A less intense signal was observed at m/z 2422.6, suggesting the presence of an additional phosphoethanolamine. Moreover, signals attributable to a core oligosaccharide and a lipid A, arising from an in-source βelimination at the glycosidic bond between the Kdo8N and the lipid A, were also displayed [25]. The signals at m/z 1360.8 and 1483.9 were both attributed to the core fragments, with the difference of 123 Da being due to the additional phosphoethanolamine. The signals of the decarboxylated core fragments were clearly visible at m/z 1316.8 and 1439.9 [25]. Finally, further fragmentation with losses of 18 u could explain the signals at m/z 1298.8 and 1421.8. The LPS-OH was de-N-acylated by strong alkaline hydrolysis, and the obtained oligosaccharide, named OS, was submitted for full 2D NMR analysis. In addition, gas chromatography-mass spectrometry (GC-MS) analysis of fatty acid methyl esters showed the presence of C12:0(3OH), C13:0(3OH), C14:0(3OH), C12:0, C13:0, C14:0, and C15:0 as the major components.

LOS Deacylation
Alkaline degradation by mild hydrazinolysis of the LOS afforded an O-deacylated LOS, named LOS-OH, which was analyzed by negative ions MALDI-TOF ( Figure 2). The spectrum revealed the presence of a cluster of ions, attributable to the LOS-OH molecule. At higher molecular masses, the signal at m/z 2298.6 was assigned the following composition: Hex 3 (Kdo8N). The appearance of the Kdo8N monosaccharide was not surprising, since it has often been reported for Shewanella LPSs [21][22][23][24]. Differences of ±14 Da with respect to the main signal at m/z 2298.6 are attributable to the different lengths of fatty acids substituting the GlcN residues. A less intense signal was observed at m/z 2422.6, suggesting the presence of an additional phosphoethanolamine. Moreover, signals attributable to a core oligosaccharide and a lipid A, arising from an in-source β-elimination at the glycosidic bond between the Kdo8N and the lipid A, were also displayed [25]. The signals at m/z 1360.8 and 1483.9 were both attributed to the core fragments, with the difference of 123 Da being due to the additional phosphoethanolamine. The signals of the decarboxylated core fragments were clearly visible at m/z 1316.8 and 1439.9 [25]. Finally, further fragmentation with losses of 18 u could explain the signals at m/z 1298.8 and 1421.8. The LPS-OH was de-N-acylated by strong alkaline hydrolysis, and the obtained oligosaccharide, named OS, was submitted for full 2D NMR analysis.

NMR Spectroscopic Analysis of OS
2D NMR spectroscopy ( 1 H, 1 H double quantum filtered-correlation spectroscopy (DQF-COSY), 1 H, 1 H total correlation spectroscopy (TOCSY), 1 H, 1 H rotating frame Overhauser enhancement spectroscopy (ROESY), 1 H, 13 C distortionless enhancement by polarization transfer-heteronuclear single quantum coherence (DEPT-HSQC), and 1 H, 13 C heteronuclear multiple bond correlation (HMBC) allowed for the assignment of all the proton and carbon chemical shifts of OS. The experiments indicated the pyranose rings for all the residues. Anomeric configurations were deduced by both proton and carbon anomeric chemical shifts, and by the 1 JC1,H1 values obtained from the coupled 1 H, 13 C DEPT-HSQC experiment ( Table 1).

NMR Spectroscopic Analysis of OS
2D NMR spectroscopy ( 1 H, 1 H double quantum filtered-correlation spectroscopy (DQF-COSY), 1 H, 1 H total correlation spectroscopy (TOCSY), 1 H, 1 H rotating frame Overhauser enhancement spectroscopy (ROESY), 1 H, 13 C distortionless enhancement by polarization transfer-heteronuclear single quantum coherence (DEPT-HSQC), and 1 H, 13 C heteronuclear multiple bond correlation (HMBC) allowed for the assignment of all the proton and carbon chemical shifts of OS. The experiments indicated the pyranose rings for all the residues. Anomeric configurations were deduced by both proton and carbon anomeric chemical shifts, and by the 1 J C1,H1 values obtained from the coupled 1 H, 13 C DEPT-HSQC experiment (Table 1). The 1 H NMR spectrum of OS displayed eight main anomeric signals, indicating at least the equivalent number of monosaccharides, as shown in Figure 3 (residues A-H). In addition, two proton signals, at δ 2.01 and 2.25 ppm, confirmed the presence of a deoxy sugar (residue I). Starting from the chemical shift of H1 of residue A, the spin system in the COSY and TOCSY spectra revealed a gluco configuration, since it showed the typical 3 J H,H vicinal coupling constant values. This residue was identified as the phosphorylated vicinal glucosamine residue (α-GlcNI), due to the multiplicity of its anomeric signal (doublet of doublets, 3 J H1,H2 = 3.6 Hz and 3 J H1,P = 7.0 Hz) and the chemical shift of its C2 (δ 55.8 ppm), as shown in Figure 4. Similarly, residue G was identified as the distal glucosamine (β-GlcNII) of the lipid A backbone. The ROESY and HMBC experiments (see below) indicated that residues G and A were linked through a linkage (1→6). Residues C, D, and E were recognized to be α-heptoses from their spin-system connectivities revealed in the COSY, TOCSY, and ROESY spectra. Indeed, in the TOCSY experiment, only the connectivities between H1 and H2 were clearly visible, indicating small values for the 3 J H1,H2 and 3 J H2,H3 . Residue C was found to be 2-substituted, since the chemical shift of its C2 was shifted downfield to 82.1 ppm (the reference value for an unsubstituted residue is 71.9 ppm) [26]. Mar. Drugs 2019, 17, x 6 of 13 The letters refer to the residues as described in Table 1.  Table 1.
Residue E did not show any downfield chemical shifts, and therefore was assigned to a terminal non-reducing α-heptose. Spin system C was identified as a 2,6,7-trisubstituted heptose, since its C2, C6, and C7 carbon chemical shifts occurred at 79.4, 78.2, and 70.8 ppm, respectively. The D,Dconfiguration for this residue was suggested based on the presence of this type of residue in other Shewanella LOSs, and from the strong similarities of the proton and carbon chemical shifts of this residue with those already reported [27]. The gluco configuration for the spin systems of residues B, F, and H was inferred from the typical 3 JH,H vicinal coupling constant values. By comparison with the 13 C chemical shifts of unsubstituted residues [28], only one low-field shifted signal was identified for the C2 of residue H, indicating that B and F were terminal non-reducing residues. Finally, I was identified as a Kdo8N residue based on the characteristic diastereotopic proton signals at δ 2.01 and 2.25 ppm, and from the high-field chemical shift of its C8 signal at δ 44.8 ppm. The α configuration  The letters refer to the residues as described in Table 1.
Mar. Drugs 2019, 17, x 6 of 13 The letters refer to the residues as described in Table 1.  Table 1.
Residue E did not show any downfield chemical shifts, and therefore was assigned to a terminal non-reducing α-heptose. Spin system C was identified as a 2,6,7-trisubstituted heptose, since its C2, C6, and C7 carbon chemical shifts occurred at 79.4, 78.2, and 70.8 ppm, respectively. The D,Dconfiguration for this residue was suggested based on the presence of this type of residue in other Shewanella LOSs, and from the strong similarities of the proton and carbon chemical shifts of this residue with those already reported [27]. The gluco configuration for the spin systems of residues B, F, and H was inferred from the typical 3 JH,H vicinal coupling constant values. By comparison with the 13 C chemical shifts of unsubstituted residues [28], only one low-field shifted signal was identified for the C2 of residue H, indicating that B and F were terminal non-reducing residues. Finally, I was identified as a Kdo8N residue based on the characteristic diastereotopic proton signals at δ 2.01 and 2.25 ppm, and from the high-field chemical shift of its C8 signal at δ 44.8 ppm. The α configuration   Table 1.
Residue E did not show any downfield chemical shifts, and therefore was assigned to a terminal non-reducing α-heptose. Spin system C was identified as a 2,6,7-trisubstituted heptose, since its C2, C6, and C7 carbon chemical shifts occurred at 79.4, 78.2, and 70.8 ppm, respectively. The D,D-configuration for this residue was suggested based on the presence of this type of residue in other Shewanella LOSs, and from the strong similarities of the proton and carbon chemical shifts of this residue with those already reported [27]. The gluco configuration for the spin systems of residues B, F, and H was inferred from the typical 3 J H,H vicinal coupling constant values. By comparison with the 13 C chemical shifts of unsubstituted residues [28], only one low-field shifted signal was identified for the C2 of residue H, indicating that B and F were terminal non-reducing residues. Finally, I was identified as a Kdo8N residue based on the characteristic diastereotopic proton signals at δ 2.01 and 2.25 ppm, and from the high-field chemical shift of its C8 signal at δ 44.8 ppm. The α configuration for this residue was inferred from the coupling constant values of 3 J H7,H8a (8 Hz) and of 3 J H7,H8b (3.2 Hz) [29]. The 1 H-31 P HSQC experiment of OS indicated three-bond correlations for the phosphorus signals, with 1 H signals as follows: H1 and H2 of residue A with 31 P signal at δ −2.1 ppm, H4 of G with 31 P signal at δ −0.2 ppm, and H4 of I with 31 P signal at δ 0.3 ppm, as shown in Figure 5. Therefore, based on these results and in agreement with other oligosaccharide structures from Shewanella species [23,24], it was possible to hypothesize that the additional phosphoethanolamine is linked through a phosphoanhydride linkage to the phosphate group at position O-4 of the Kdo8N.
The sequence of the monosaccharides and the linkage positions were obtained from the ROESY and HMBC experiments. The latter spectrum, as illustrated in Figure 6, revealed the following inter-residue correlations: H1 of G with C6 of A, H1 of D with C5 of Kdo8N (residue I), H1 of C with C6 of D, H1 of E with C2 of D, H1 of H with C7 of D, H1 of F with C2 of C, and H1 of B with C2 of H. The ROESY spectrum confirmed this sequence, since it revealed the following dipolar couplings: H1 of D with H5 of I, H1 of C with H6 of D, H1 of H with both H7s of D, H1 of E with H2 of D, H1 of B with H2 of H, and H1 of F with H2 of C. Moreover, the rotating frame Overhauser effect (ROE) contact between H5 of residue D and H3ax of I suggested a D configuration for Kdo8N [30]. Finally, ROE contacts between H1 of G and both H6s of A were found.
Mar. Drugs 2019, 17, x 7 of 13 for this residue was inferred from the coupling constant values of 3 JH7,H8a (8 Hz) and of 3 JH7,H8b (3.2 Hz) [29]. The 1 H-31 P HSQC experiment of OS indicated three-bond correlations for the phosphorus signals, with 1 H signals as follows: H1 and H2 of residue A with 31 P signal at δ −2.1 ppm, H4 of G with 31 P signal at δ −0.2 ppm, and H4 of I with 31 P signal at δ 0.3 ppm, as shown in Figure 5. Therefore, based on these results and in agreement with other oligosaccharide structures from Shewanella species [23,24], it was possible to hypothesize that the additional phosphoethanolamine is linked through a phosphoanhydride linkage to the phosphate group at position O-4 of the Kdo8N.
The sequence of the monosaccharides and the linkage positions were obtained from the ROESY and HMBC experiments. The latter spectrum, as illustrated in Figure 6, revealed the following interresidue correlations: H1 of G with C6 of A, H1 of D with C5 of Kdo8N (residue I), H1 of C with C6 of D, H1 of E with C2 of D, H1 of H with C7 of D, H1 of F with C2 of C, and H1 of B with C2 of H. The ROESY spectrum confirmed this sequence, since it revealed the following dipolar couplings: H1 of D with H5 of I, H1 of C with H6 of D, H1 of H with both H7s of D, H1 of E with H2 of D, H1 of B with H2 of H, and H1 of F with H2 of C. Moreover, the rotating frame Overhauser effect (ROE) contact between H5 of residue D and H3ax of I suggested a D configuration for Kdo8N [30]. Finally, ROE contacts between H1 of G and both H6s of A were found.   13 C HMBC spectrum of the OS oligosaccharide. The spectrum was recorded in D2O at 298 K at 600 MHz. The letters refer to the residues as described in Table 1. for this residue was inferred from the coupling constant values of 3 JH7,H8a (8 Hz) and of 3 JH7,H8b (3.2 Hz) [29]. The 1 H-31 P HSQC experiment of OS indicated three-bond correlations for the phosphorus signals, with 1 H signals as follows: H1 and H2 of residue A with 31 P signal at δ −2.1 ppm, H4 of G with 31 P signal at δ −0.2 ppm, and H4 of I with 31 P signal at δ 0.3 ppm, as shown in Figure 5. Therefore, based on these results and in agreement with other oligosaccharide structures from Shewanella species [23,24], it was possible to hypothesize that the additional phosphoethanolamine is linked through a phosphoanhydride linkage to the phosphate group at position O-4 of the Kdo8N.
The sequence of the monosaccharides and the linkage positions were obtained from the ROESY and HMBC experiments. The latter spectrum, as illustrated in Figure 6, revealed the following interresidue correlations: H1 of G with C6 of A, H1 of D with C5 of Kdo8N (residue I), H1 of C with C6 of D, H1 of E with C2 of D, H1 of H with C7 of D, H1 of F with C2 of C, and H1 of B with C2 of H. The ROESY spectrum confirmed this sequence, since it revealed the following dipolar couplings: H1 of D with H5 of I, H1 of C with H6 of D, H1 of H with both H7s of D, H1 of E with H2 of D, H1 of B with H2 of H, and H1 of F with H2 of C. Moreover, the rotating frame Overhauser effect (ROE) contact between H5 of residue D and H3ax of I suggested a D configuration for Kdo8N [30]. Finally, ROE contacts between H1 of G and both H6s of A were found.   13 C HMBC spectrum of the OS oligosaccharide. The spectrum was recorded in D2O at 298 K at 600 MHz. The letters refer to the residues as described in Table 1.  13 C HMBC spectrum of the OS oligosaccharide. The spectrum was recorded in D 2 O at 298 K at 600 MHz. The letters refer to the residues as described in Table 1. All the reported data allowed us to determine the complete structure of the saccharidic backbone of the Shewanella sp. HM13 LOS, which is shown in Figure 7.
Mar. Drugs 2019, 17, x 8 of 13 All the reported data allowed us to determine the complete structure of the saccharidic backbone of the Shewanella sp. HM13 LOS, which is shown in Figure 7.

Bacteria Growth and LPS Isolation
Shewanella sp. HM13, a psychrotrophic bacterium isolated from the intestine of horse mackerel (Trachurus japonicus), was cultured in 5 mL LB liquid medium overnight at 18 °C. Five milliliters of the culture were inoculated into 1 L LB liquid medium, and the cells were grown at 4 °C until the OD600 reached 3.0-4.0. The cells were harvested at 6800× g, 4 °C for 10 min. The cell pellets collected from 2 L cultures were freeze-dried. Dried bacterial cells (1.4 g) were extracted by the PCP method to give 34 mg of LOS (yield 2.4% w/w of dried cells), and then by the hot phenol/water method as reported previously [19,20].

DOC-PAGE Analysis
Polyacrylamide gel electrophoresis analysis (PAGE) was performed using the system of Laemmli [31] with sodium deoxycholate (DOC) as the detergent, as already described [32]. The gels were fixed in an aqueous solution of 40% ethanol and 5% acetic acid. The LOS extract bands were visualized by silver staining as previously described [33].

Sugar and Fatty Acids Analysis
The LOS sample (0.5 mg) was subjected to a methanolysis reaction with HCl/CH3OH (1.25 M, 1 mL) at 80 °C for 16 h. The obtained monosaccharides were acetylated and analyzed as acetylated methyl glycosides by GC-MS. The analysis of the fatty acids, derivatized as methyl esters, was achieved as already reported [34].

Bacteria Growth and LPS Isolation
Shewanella sp. HM13, a psychrotrophic bacterium isolated from the intestine of horse mackerel (Trachurus japonicus), was cultured in 5 mL LB liquid medium overnight at 18 • C. Five milliliters of the culture were inoculated into 1 L LB liquid medium, and the cells were grown at 4 • C until the OD 600 reached 3.0-4.0. The cells were harvested at 6800× g, 4 • C for 10 min. The cell pellets collected from 2 L cultures were freeze-dried. Dried bacterial cells (1.4 g) were extracted by the PCP method to give 34 mg of LOS (yield 2.4% w/w of dried cells), and then by the hot phenol/water method as reported previously [19,20].

DOC-PAGE Analysis
Polyacrylamide gel electrophoresis analysis (PAGE) was performed using the system of Laemmli [31] with sodium deoxycholate (DOC) as the detergent, as already described [32]. The gels were fixed in an aqueous solution of 40% ethanol and 5% acetic acid. The LOS extract bands were visualized by silver staining as previously described [33].

Sugar and Fatty Acids Analysis
The LOS sample (0.5 mg) was subjected to a methanolysis reaction with HCl/CH 3 OH (1.25 M, 1 mL) at 80 • C for 16 h. The obtained monosaccharides were acetylated and analyzed as acetylated methyl glycosides by GC-MS. The analysis of the fatty acids, derivatized as methyl esters, was achieved as already reported [34].
The absolute configurations of the glucose and glucosamine were determined by gas chromatography of the acetylated (S)-2-octyl glycosides [35]. The heptose configurations were obtained through the alditol acetates GC-MS method. Briefly, a sample of the LOS (0.5 mg), after a methanolysis reaction, was hydrolyzed at 120 • C with 2M trifluoroacetic acid (TFA) for 2 h, as already reported [36]. After neutralization, the sample was reduced with NaBD 4 and finally acetylated and injected into the GC-MS. The configuration was obtained by comparison with authentic standards.
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, Bologna, Italy 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, then 140→240 • C at 3 • C/min. Analysis of acetylated octyl glycosides was performed as follows: 150 • C for 5 min, then 150→300 • C at 6 • C/min, and finally 300 • C for 5 min. The temperature program for methyl esters of fatty acids was the following: 140 • C for 3 min, then 140→280 • C at 10 • C/min, and finally 280 • C for 20 min. The temperature program for alditol acetates was the following: 150 • C for 3 min, and then 150→330 • C at 3 • C/min.

Deacylation of the LOS
The LOS sample (30 mg) was dried over phosphorus anhydride in a vacuum chamber and then treated with hydrazine (1.5 mL) at 37 • C for 2 h. The precipitation of the LOS-OH was obtained by addition of cold acetone. The pellet was recovered after centrifugation (4 • C, 7000 rpm, 30 min), washed two times with cold acetone to remove the excess of hydrazine, suspended in water, and finally freeze-dried [37]. The LOS-OH (8 mg) was submitted to a reaction with KOH 4 M aq. (1.0 mL) for 16 h at 120 • C. The crude reaction was neutralized with HCl 2 M aq. (until pH 6) and extracted with CHCl 3 for three times. The aqueous phase of the mixture was desalted on a Sephadex G-10 column (GE Healthcare, Pittsburgh, PA, USA, 2.5 × 43 cm, 31 mL h −1 , fraction volume 2.5 mL, eluent NH 4 HCO 3 10 mm). The eluted oligosaccharide fraction was freeze-dried (1.5 mg).

Methylation Analysis
The linkage positions of the monosaccharides were obtained by the analysis of the partially methylated alditol acetates (PMAAs). The methylation reaction was achieved by incubating 1 mg of the LOS sample with CH 3 I (100 µL) and NaOH powder in dimethyl sulfoxide (DMSO, 300 µL) for 20 h [38]. The mixture was analyzed by GC-MS with the following temperature program: 90 • C for 1 min, then 90→140 • C at 25 • C/min, then 140→200 • C at 5 • C/min, then 200→280 • C at 10 • C/min, and finally 280 • C for 10 min.

Mass Spectrometry Analysis
MALDI-TOF mass spectra were acquired on a ABSCIEX TOF/TOF™ 5800 (AB SCIEX, Darmstadt, Germany) mass spectrometer equipped with an Nd:YLF laser with a λ of 345 nm, a < 500-ps pulse length, and a repetition rate of up to 1000 Hz. Approximately 2000 laser shots were accumulated for each spectrum. The calibration of the mass spectra was obtained with a hyaluronan oligosaccharides mixture. A solution of 2,5-dihydroxybenzoic acid (DHB) in 20% CH 3 CN in water (25 mg mL −1 ) was used as the matrix. The samples were desalted on a Dowex 50WX8 (H + form) and dissolved in 2-propanol/water with a 1:1 ratio. The spectra were calibrated and processed under computer control by using the Data Explorer software (v0.2.0).

Conclusions
In this paper, the complete structure of the sugar backbone of the LOS from the cold-adapted Shewanella sp. HM13 is reported. The structure has been obtained by chemical analysis, NMR spectroscopy, and MALDI-TOF mass spectrometry.
The oligosaccharide shares some structural features with those isolated from other Shewanella strains. The presence of the Kdo8N is confirmed as a hallmark of Shewanella species, having been already reported for the species oneidensis [23], algae [27], putrefaciens [40], pacifica [24], and Shewanella spp. MR-4 [21]. In addition, the D,D-configured heptose holds a central position in the oligosaccharide structure, as well as for all the other characterized LOS structures [21,23,24,27,40].
The rough nature of the Shewanella sp. HM13 LPS is not surprising, since this has also been reported for other Shewanella species [21,23,24,27,40]. Nevertheless, it is worth noting that this feature is common among the LPSs isolated from cold-adapted Gram-negative bacteria [41][42][43][44][45]. A possible explanation lies in the enhanced flexibility and stability of the outer membrane when the O-polysaccharide chain is absent [46].
Finally, to shed light on the possible structural differences among OM components of both bacterial cells and OMVs, it will be very interesting to characterize the LPS isolated from Shewanella sp. HM13 OMVs. Funding: This research was financially supported in part by JSPS KAKENHI (18K19178 to T.K. and 16K14885 to J.K.).

Conflicts of Interest:
The authors declare no conflict of interest.