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O-specific polysaccharide structure isolated from the LPS of the Antarctic bacterium
Pseudomonas ANT_J38B

Rossella Di Guida, Angela Casillo, Maria Michela Corsaro

PII: S0008-6215(20)30312-8
DOI: https://doi.org/10.1016/j.carres.2020.108125
Reference: CAR 108125

To appear in: Carbohydrate Research

Received Date: 20 May 2020

Revised Date: 27 July 2020
Accepted Date: 6 August 2020

Please cite this article as: R. Di Guida, A. Casillo, M.M. Corsaro, O-specific polysaccharide structure
isolated from the LPS of the Antarctic bacterium Pseudomonas ANT_J38B, Carbohydrate Research
(2020), doi: https://doi.org/10.1016/j.carres.2020.108125.

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 O-specific polysaccharide structure isolated from the LPS of the Antarctic bacterium
Pseudomonas ANT_J38B

Rossella Di Guida, 1 Angela Casillo,1 Maria Michela Corsaro1*

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Department of Chemical Sciences, University of Naples Federico II, Complesso Universitario
Monte S. Angelo, Via Cintia 4, 80126 Naples, Italy. rossella.diguida@unina.it (RDG);
angela.casillo@unina.it (AC)

*Corresponding author. Tel. +39 081674149 Fax +39 081674393

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E-mail address: corsaro@unina.it (Maria Michela Corsaro)

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Abstract
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Pseudomonas ANT_J38B is a Gram-negative bacterium isolated from an Antarctic island. LPS

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was extracted using the phenol/chloroform/petroleum ether method. A mild acid hydrolysis
followed by a gel filtration purification afforded the O-chain. The polysaccharide was
characterized by means of chemical analyses and NMR spectroscopy. The O-chain displays a
disaccharide repeating unit with the following backbone: →4)-α-L-GulpNAc3OAcAN-(1→3)-β-
D-QuipNAc-(1→.

Keywords: Lipopolysaccharide; NMR; Psychrotolerant; Pseudomonas; Gulopyranuronamide

1. Introduction

Pseudomonas sp. ANT_J38B is a psychrotolerant Gram-negative bacterium isolated from a

sample soil collected at the Jardine Peak (King George Island, Antarctica), at a depth of 15-20 cm
[1]. Psychrotolerant bacteria, able to grow at a temperature ranging from low to mild
temperatures, have developed many strategies to survive in these harsh conditions, such as the
structural modification of macromolecules belonging to the external layer including
lipopolysaccharides (LPSs). Lipopolysaccharides are amphiphilic macromolecules, representing
about 75% of the outer membrane of Gram-negative bacteria [2]. These molecules share a
common general structure subdivided into three distinct regions covalently linked, according to
their different chemical structure and biosynthesis: a glycolipid domain (lipid A), an
oligosaccharide region (core) and a hydrophilic O-specific polysaccharide (O-chain). The LPS is

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anchored in the outer membrane by the lipid A moiety, whereas the O-chain is placed at the
interface between the bacterium and its environment. The latter LPS portion can be absent or

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partly truncated in some Gram-negative strains. The presence of the O-chain gives to the bacterial
colonies a smooth morphology and, for this reason, the LPS has been named smooth or S-LPS.
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Instead, when the polysaccharidic portion is lacking the bacterial colonies show a rough
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morphology, and the LPS has been termed rough or R-LPS (lipooligosaccharide, LOS) [3].
Usually, the LPSs from a single cultivation represent a family of macromolecules, differing in the
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length of their O-chains, which also comprise a fraction of R-LPS [4].

The O-chain is the most variable portion of the LPS, even within a given bacterial species.
Therefore, it constitutes the chemical basis for the serological classification of individual wild-
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type bacterial strains. The repeating unit can be linear or branched and can contain many non-
carbohydrate substituents such as phosphate, amino acids, and acetyl groups [5]. Only a few
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bacterial strains or species contain homopolymeric O-chains [6-8]. The O-polysaccharide is also
often referred to as the O-antigen because is the major antigen targeted by host antibody
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responses [9]. Indeed, is one of the most important virulence factors in the interaction of bacteria
with humans and animals [10]. The highly diversity in O-chain structure and composition may
have developed during evolution in order to escape the host's immune system and to hide the
common units, i.e. the lipid A and the inner core region attached to it. It has also an important
biological role in the protection of the bacterial cells [11-13].
In this paper, the LPS from Pseudomonas sp. ANT_J38B grown at 4 °C was isolated and its O-
chain was characterized through chemical and spectroscopic analyses.
2. Results and discussion

2.1 LPS extraction and chemical analyses

Pseudomonas sp. ANT_J38B cells grown at 4 °C in LB (Luria Bertani) growth medium was
extracted by the PCP method [14], followed by the phenol/water extraction [15]. The PCP extract
was analysed by 14% DOC-PAGE electrophoresis and visualized after silver staining. The DOC-
PAGE experiment allowed establishing the smooth nature of LPS since the typical “ladder-like”
pattern was visible (Figure 1). The aqueous extract only showed a very low amount of S-LPS by
the DOC-PAGE analysis (results not shown); therefore, all the analyses were performed on the
LPS from the PCP extraction.
Sugar and fatty acids of the LPS sample derivatized as acetylated methyl glycosides (MGA) and
fatty acids methyl esters (FAME), respectively, were analysed by GC-MS, and recognized from

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the fragmentation pattern and the retention time, by comparison with those of authentic standards.
The FAME chromatogram revealed the presence of 3-hydroxydecanoic [C10:0(3-OH)], 2-

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hydroxydodecanoic [C12:0(2-OH)], 3-hydroxydodecanoic [C12:0(3-OH)], dodecanoic (C12:0),
tetradecanoic (C14:0), and pentadecanoic acids (C15:0). Instead, sugar analysis suggested the
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presence of quinovosamine (QuiN), mannose (Man), glucose (Glc), gulosaminuronic acid
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(GulNA), glucosamine (GlcN), and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). Sugar analysis
performed after HF treatment revealed the additional presence of a heptose, thus suggesting its
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phosphorylation [16].

2.2 O-antigen polysaccharide purification and NMR analysis

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The LPS was hydrolysed under mild acid conditions to cleave the glycosidic linkage between the
Kdo and the GlcNII of the lipid A moiety. After centrifugation, the supernatant containing the
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saccharidic portion of the LPS was separated from the lipid A. The obtained supernatant was
purified through a P-10 Biogel gel filtration chromatography, using water as eluent. The fraction
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containing the O-chain, named OPS, was characterized by chemical analysis and 1D and 2D NMR
spectroscopic experiments.
The GC-MS of the acetylated methyl glycosides of OPS revealed that only the QuiN and the
GulNA were the constituents of the O-chain. The absolute configuration of these monosaccharides
was deduced to be D for QuiN and L for the GulNA, as demonstrated by the GC-MS of acetylated
(R)-2-octyl glycosides.
Based on data of 1D and 2D NMR experiments two spin systems were assigned. The 1H NMR
spectrum showed in the anomeric region (4.4-5.5 ppm) several signals (Figure 2), whereas the 1H,13C
DEPT-HSQC spectrum (Figure 3) revealed that only the proton signals at δ 5.07 and 4.49 ppm
correlated with anomeric carbon signals at δ 97.0 and 102.1 ppm, respectively. Furthermore, the up-
field region of the 1H spectrum showed signals attributable to N- and O-acetyl groups in the range
1.8-2.2 ppm, and a signal at ẟ 1.29 ppm attributable to a methyl group of the Qui2N residue.
Residue A was recognized as a 2-acetamido-2-deoxy-3-O-acetylgulopyranuronamide
(GulpNAc3OAcAN) as follows: in the COSY spectrum starting from the H-1 at δ 5.07 ppm, the H-2
proton at δ 4.52 ppm was found; this last was correlated in the DEPT-HSQC experiment with a C-2
resonance occurring at δ 44.6 ppm. The last value was indicative of the gulo configuration as already
reported [17]. Following the COSY experiment, H-2 proton signal was correlated to the proton
signal at δ 5.37 ppm (H-3), which is in turn correlated with the carbon signal at δ 68.7 ppm
attributable to a carbon substituted with an O-acetyl group. Successively, in the COSY spectrum,
from the H-3 the correlations with the proton H-4 at δ 4.12 ppm and the H-5 at δ 4.94 ppm were
found. Finally, in the HMBC experiment the H-5 proton signal at δ 4.94 ppm showed a long-range
scalar connectivity with the C-6 signal at δ 173.9 ppm, thus confirming the presence of an uronic
acid residue. The values of the 13C chemical shifts (Table 1) definitively confirmed a gulo configured
hexopyranose [17]. In addition, the carbon chemical shifts were indicative of a substitution at C-4, as
demonstrated by a downfield shift of this carbon signal (δ 76.0 ppm) together with an upfield shift of
both C-3 and C-5 values, when compared with those of the α-GulpNAc [18]. Since the chemical shift
value of the C-6 signal suggested the occurrence of an amide, we checked it by performing the

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proton spectrum and the HMBC experiment at two different pD values, namely at pD 2 and pD 7. No
differences were observed for 1H and 13C chemical shifts at the two pD values, thus confirming

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amidation at the carboxyl group of the GulNAcA [19].
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The correlations present in COSY and TOCSY spectra suggested the gluc oconfiguration for residue
B. In particular, residue B was identified as a β-N-acetylquinovosamine, since in the 1H,13C DEPT-
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HSQC experiment its H-2 at δ 3.88 ppm correlated with a nitrogen bearing carbon at δ 55.8 ppm and
since the 13C chemical shift values of each carbon of this monosaccharide were comparable to those
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already reported [20]. Furthermore, the C-3 resonance of B at δ 79.1 ppm was shifted downfield with
respect to that of an unsubstituted quinovosamine residue [21] suggesting a substitution at this
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position. The β configuration for this monosaccharide was confirmed by H-1, H-3 and H-1, H-5
NOE contacts in the NOESY experiment.
The N-acetyl substitution of C-2 of residues A and B was proven by the ROESY spectrum. For
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residues A the methyl signal at δ 1.97 ppm showed a ROE contact with H-2 at δ 4.52 ppm. In the
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same spectrum, a ROE contact between H-2 of B at δ 3.88 and methyl signal at δ 1.95 ppm was
clearly visible.
Finally, the O-acetyl substitution at position 3 of residue A was confirmed by the HMBC
spectrum, where the methyl signal at δ 2.17 ppm showed a long-range scalar connectivity with the
carbonyl group at δ 174.0 ppm, which is in turn correlated with the H-3 of A at δ 5.37 ppm.
The sequence of the disaccharide repeating unit of the polysaccharide was deduced trough the
inter-residues NOE correlations: H-1 of A with H-3 of B, and H-1 of B with H-4 of A.
Based on the above data, the structure of the O-chain from Pseudomonas sp. ANT_J38B was
identified as reported in the scheme:
3. Conclusions

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In conclusion, in this note the structure of the O-chain from the LPS of the psychrotolerant
Gram-negative bacterium Pseudomonas sp. ANT_J38B, isolated from an Antarctic island, is

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reported. The smooth LPS was unusually recovered from the PCP extraction, suggesting the
presence of a high hydrophobic LPS. The O-chain consists of a disaccharide repeating unit
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built up of 2-acetamido-2,6-di-deoxyglucopyranose (β-D-3-QuipNAc) and 2-acetamido-2-
deoxy-gulopyranuronamide, which is acetylated at O-3 position (α-L-4-GulpNAc3OAcAN).
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The O-chain reported in this note has already been found for the LPS isolated from the
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mushroom’s pathogenic bacterium Pseudomonas tolasii. In the last an uncomplete acetylation

at the position O-3 of GulNAcAN residue was observed [19].
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The finding of the O-chain in the lipopolysaccharide of cold adapted bacteria is quite
uncommon, since up to now the majority of the LPSs from psychrophiles grown at low
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temperatures characterised showed to be rough [16, 22-26]. The lack of the O-chain portion
could reflect a strategy for the bacterium to enhance the functionality of membranes at freezing
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or sub-freezing growth temperatures. Examples of O-chain from psychrophilic bacteria display

as a common feature the presence of both uronic acids and amino sugars [27, 28] and of acyl
substituents, that gives hydrophobicity to the residues. The presence of hydrophobic
monosaccharides may suggest the ability to counteract the ice crystal formation around the cell,
as demonstrated for some capsular and extracellular polysaccharides [29-30].
4. Experimental section

4.1 Cell growth and LPS isolation

Pseudomonas sp. ANT_J38B cells were grown at 4 °C in Luria Bertani medium until the
exponential phase and separated from the supernatant by centrifugation (15 min, 8000 rpm, 4 °C).
Dried cells (5.4 g) were extracted by PCP (phenol/chloroform/petroleum ether, 2:5:8 v/v/v) [14].
The extract was freeze-dried to obtain 30 mg of crude LPS (yield 1.1 %). The cellular debris were
also extracted by the hot phenol water method [15]. The PCP precipitate and the aqueous extract
were screened by 14% DOC-PAGE (Sodium Deoxycholate Polyacrylamide Electrophoresis) and
stained by Tsai’s procedure [31].

4.2 Chemical analysis

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Monosaccharides were analysed as acetylated methyl glycosides (MGA) by GC-MS, as previously

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reported [32]. The absolute configuration of the sugars was determined by gas-chromatography
analysis of their acetylated (R)-2-octyl glycosides [33]. All the samples were analysed on an
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Agilent Technologies gas chromatograph 7820A equipped with a mass selective detector 5977B
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and a HP-5ms capillary column (Agilent, Italy 30 m x 0.25 mm i.d., flow rate 1 mL/min, He as
carrier gas). MGA were analysed using the following temperature program: 140 °C for 3 min, then
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140→240 °C at 3 °C/min. The temperature program for octyl glycosides was performed at 150 °C
for5 min, then 150 °C→240 °C at 6 °C min-1, and 240 °C for 5 min.
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4.3 OPS purification

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The LPS (50 mg) was hydrolysed with 5% aqueous CH3COOH (5 mL, 100 °C, 3 hours). The
obtained suspension was then centrifuged at 4 °C (7000 rpm for 30 minutes). The precipitate was
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washed twice with water and the supernatant layer was lyophilized. The obtained supernatant was
fractioned on a Biogel P-10 column (Biorad, 0.75 x 95 cm, flow rate 13 mL/h, fraction volume 2.5
mL) eluted with water and, monitored with a Knauer refractive index. Finally, a high-molecular-
mass OPS fraction (6 mg) and low molecular fractions (3.5 mg) were obtained.

4.4 NMR spectroscopy

1 13
H and C NMR spectra were recorded using a Bruker Avance 600 MHz spectrometer (Milano,
Italy) at 298 K, at pD 7, by using internal sodium 3-trimethylsilylpropanoate-2,2,3,3-d4 (δH 0.0
ppm) and external acetone (δC 31.45 ppm) as references. 2D experiments (1H, 1H DQF-COSY; 1H,
1
H TOCSY; 1H, 1H ROESY; 1H, 1H NOESY; 1H, 13
C DEPT-HSQC; 1H, 13
C HMBC) were
performed using standard Bruker software, and Bruker TopSpin 2.1 program was used to acquire
and processed the NMR data. A mixing time of 100 ms was used in 1H, 1H TOCSY, 1H, 1H
ROESY, and 1H, 1H NOESY experiments.
1
H and 1H, 13
C HMBC NMR spectra were also recorded at pD 2 and 298 K, using the same
spectrometer and references.

Acknowledgements

Authors thank Lukasz Dziewit, from the University of Warsaw, for kindly providing the bacterial
strain, Anella Saggese from the Biology Department of University of Naples for the bacterial
growth. Finally, authors thank Valeria Costantino and Germana Esposito from Pharmacy
Department of University of Naples for kindly supporting in the NMR experiments.

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Table 1. 1H (600 MHz) and 13C NMR (125 MHz) chemical shift (δ, ppm) assignments of OPS
from Pseudomonas ANT_J38B.

H1 H2 H3 H4 H5 H6 CH3CO CH3CO CH3CO CH3CO

Residue
C1 C2 C3 C4 C5 C6 N-2 N-2 O-3 O-3

A 5.07 4.52 5.37 4.12 4.94 - 1.97 - 2.17 -

α-D-4-GulpNAc3OAcAN 97.0 44.6 68.7 76.0 66.7 173.9 22.4 174.0 20.8 173.9
B 4.49 3.88 3.62 3.31 3.51 1.29 1.95 -
β-L-3-QuipNAc 102.1 55.8 79.1 74.5 72.4 17.8 22.4 174.4

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Figures

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Fig. 1
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ppm 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5

Fig. 2
 ppm

B6 20
OAc
NAc 30

A2 40

B2 50

A5 60
A3
B5
A4 70
B4
80
B3
A1 90

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B1 100

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ppm 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5
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Fig. 3
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Fig. 1. 14% DOC-PAGE analysis of the PCP extract (lane a) from Pseudomonas sp. ANT_J38B,
and LPS from Escherichia coli 055: B5 (lane b), used as standard.

Fig. 2. 1H NMR spectrum of the OPS from Pseudomonas ANT_J38B. Spectrum was recorded at
600 MHz in D2O, at 298K.

Fig. 3. Selected region of the 1H,13C DEPT-HSQC spectrum of the OPS from Pseudomonas
ANT_J38B. Arabic numerals refer to the H/C pairs in the monosaccharide residues denoted as
indicated in Table 1. Spectrum was recorded in D2O at 298 K, and 600 MHz.

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Highlights

 The Antarctic bacterium Pseudomonas ANT_J38B produced a smooth-LPS.

 The O-specific polysaccharide from Pseudomonas ANT_J38B was extracted and purified.
 The OPS structure was completely characterized by 1D and 2D NMR spectroscopy.
 The OPS consists of a disaccharide repeating unit containing β-D-Qui2NAc and α-L-
GulNAc3OAcAN
Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:

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