Structural investigation on the lipooligosaccharide fraction ofpsychrophilic Pseudoalteromonas haloplanktis TAC 125 bacterium

M. Michela Corsaro, Rosa Lanzetta, Ermenegilda Parrilli, Michelangelo Parrilli and M. Luisa Tutino

Dipartimento di Chimica Organica e Biochimica, Universita di Napoli Federico II, Complesso Universitario di Monte S. Angelo, Napoli,

Italy

The core structure of the cell-wall lipooligosaccharide

(LOS) fraction of an Antarctic Gram-negative bacterium,

Pseudoalteromonas haloplanktis TAC 125 strain, was deter-

mined to be deacetylated alditols. These were obtained from

native LOS fraction by O-deacylation, dephosphorylation,

reduction and finally N-deacylation. Two novel structures were

detected, the more highly represented molecule consisting of

the following hexasaccharide chain: a-D-ManpNH2-(1!3)-b-

D-Galp-(1!4)-a-L-glycero-D-manno-Hepp-(1!5)-a-D-Kdo-

(2!6)-b-D-GlcpNH2-(1!6)-D-GlcNH2(ol) while the

corresponding pentasaccharide, lacking the ManpNH2

residue, was less abundant.

To the best of our knowledge, the structural investigation

presented here, mainly performed by NMR and MS

methods, is the first report of the lipopolysaccharide

fraction of a psychrophilic bacterium.

Keywords: psychrophilic; lipopolysaccharide; Pseudo-

alteromonas haloplanktis.

Psychrophilic bacteria are microorganisms which live incold conditions, generally below 5 8C. In order to surviveand grow in cold environments, they have evolved via acomplex range of adaptations to all of their cellular andextra-cellular components. Most interest in this field hasbeen addressed to the protein and lipid components [1,2],whereas much less attention has been given to thelipopolysaccharide component, which, being located onthe periphery of the external membrane of Gram-negativebacteria, is the nearest macromolecule to the outside environ-ment. It has been suggested that acetylation [3] and/orphosphorylation [4] of the polysaccharide moiety of lipo-polysaccharide (LPS) might have a role in its psychrophilicproperties.

This oversight perhaps arises from the great interest inpotential biotechnological utilization of these bacteria’senzymes [5]. Indeed, a substantial saving of energy can beexpected from the use of such enzymes, that are very activein the temperature range 0–20 8C. In this regard, LPSs alsoseem to play a role: lipases from psychrophilic Moraxella

spp. have been found to be active in association with LPSs,forming high molecular mass complexes [2].

Therefore, the investigation on the structure of LPS frompsychrophilic microorganisms may give insight into theircold-adaptation mechanisms and also result in an in-depthknowledge of the optimas use of their enzymes.

This paper reports the isolation of the lipooligosaccharide(LOS) fraction, and the structural analysis of its saccharidepart, from a wild-type strain of the Gram-negative Pseudo-alteromonas haloplanktis TAC 125 bacterium which hasbeen isolated from the Antarctic ocean [6].

M A T E R I A L S A N D M E T H O D S

Bacterial strain, growth conditions, and LOS extraction

P. haloplanktis TAC 125 was isolated from Antarctic oceanwater as previously reported [6]. The bacterium wasaerobically grown at 15 8C in TYP medium (16 g:L21

Bactotryptone, Difco; 16 g:L21 yeast extract Difco;16 g:L21 sea salt, pH 7.5). This temperature can be con-sidered the optimal growth temperature as the doubling timeis 35 min, while at 4 8C, which is the actual environmentaltemperature, the doubling time is 100 min. When the liquidculture reached the late exponential phase (D600 ¼ 12),cells were harvested by centrifugation for 15 min, 2700 g,4 8C and lyophilized. LOS was extracted from lyophilizedbacteria according to the PCP method [7]. The LOS yieldresulted to be 0.8%.

GC-MS analyses

All the analyses were performed on a Hewlett Packard 5890instrument equipped with a RTX-5 capillary column(Restek, 30 m � 0.25 internal diameter, flow rate 1 mLper min, He as carrier gas). Acetylated methyl glycosidesanalysis was performed with the following temperatureprogram: 150 8C for 5 min, 150!250 8C at 3 8C:min21,250 8C for 10 min. The alditol acetates mixture was

Correspondence to M. M. Corsaro, Dipartimento di Chimica Organica e

Biochimica, Universita di Napoli Federico II Complesso Universitario

di Monte S. Angelo, via Cynthia, 4 80126 Napoli, Italy.

Fax: 1 39 081674393, Tel.: 1 39 081674149,

E-mail: corsaro@unina.it

(Received 16 March 2001, revised 2 July 2001, accepted 3 August

2001)

Abbreviations: COSY, correlation spectroscopy; ESI-MS,

electrospray ionization-mass spectrometry, Hepp,

L-glycero-D-manno-heptopyranose; HMBC, heteronuclear multiple

bond correlation; HOHAHA, homonuclear Hartmann-Hahn

correlation; HPAEC, high-performance anion-exchange

chromatography; HSQC, heteronuclear single quantum coherence;

Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; LPS, lipopolysaccharide;

LOS, lipooligosaccharide; ManpNH2,

2-amino-2-deoxy-D-mannopyranose; OS, oligosaccharide; ROESY,

rotating-frame nuclear Overhauser enhancement.

Eur. J. Biochem. 268, 5092–5097 (2001) q FEBS 2001

analysed with the following temperature program: 150 8Cfor 5 min, 150!300 8C at 3 8C:min21. For partiallymethylated alditol acetates, the temperature program was:90 8C for 1 min, 90!140 8C at 25 8C:min21, 140!200 8Cat 5 8C:min21, 200!280 8C at 10 8C:min21, 280 8C for10 min. Acetylated octyl glycosides were analysed asfollows: 150 8C for 5 min, 150!240 8C at 6 8C:min 21,240 8C for 5 min.

NMR spectroscopy

Spectra were recorded on a Bruker DRX400 Avancespectrometer using a 5-mm multinuclear inverse Z-gradprobe. 13C and 1H chemical shifts were measured in D2Ousing 1,4-dioxane (d 67.4) and sodium 3-trimethylsilyl-(2,2,3,3-2H4)propionate (d 0.00), respectively, as internalstandards. 2D homonuclear experiments (COSY, HSQC,HMBC, ROESY, HOHAHA) were performed usingstandard pulse sequences available in the Bruker software.

Mass spectrometry

Fast-atom bombardment mass spectra of methylated OS1and OS2 were recorded on a VG ZAB HF instrument.

Electrospray mass spectra were performed on a Bio-Qtriple quadrupole instrument (Micromass). LOS sample(100 pmol) was deionized on Dowex H1 resin (Fluka) anddissolved in 10% triethylamine in 50% acetonitrile andinjected into the ion source at a flow rate of 10 mL:min21.All spectra were acquired in negative mode.

Determination of absolute configurations ofmonosaccharides

A sample of LOS (2 mg) was hydrolysed with a mixture of2 M H2SO4/AcOH and analysed as acetylated octyl glycosidesas already described [8]. The sample was analysed by GC-MS.

Glycosyl analysis

A sample of LOS (1 mg) was dried under vacuum over P2O5

for 16 h and then subjected to methanolysis by adding 1 M

HCl/MeOH (1 mL) for 20 h at 80 8C. The methyl glyco-sides obtained were acetylated with pyridine (200 mL) andacetic anhydride (100 mL) for 30 min at 100 8C andanalysed by GC-MS. Another sample of LOS (1 mg) washydrolysed with 4 M trifluoroacetic acid for 1 h at 100 8C,reduced with NaBD4 and acetylated and analysed byGC-MS.

Preparation of oligosaccharides

The LOS fraction (70 mg) was stirred in anhydrous hydrazine(3.5 mL) at 37 8C for 1.5 h [9]. The reaction mixture wascooled in an ice bath and cold acetone was added in smallportions until the precipitation was complete. The samplewas then centrifuged (8000 g, 4 8C, 15 min), the pellet(LOS-OH) was washed twice with cold acetone and finallydissolved in water for lyophilization (32 mg). The samplewas then transferred to a polypropylene tube and dephos-phorylated by aqueous HF treatment (1 mL, 48 8C, 48 h).After this, 2 mL of cold water was added and the solutionwas dialysed (Spectrapore, cut-off 3500 Da) until the

outside water was neutral. The inside solution was reducedwith NaBH4 (8 mg) for 20 h and ultrafiltrated by an Amicon(cut-off 1000 Da). The lyophilized residue (28 mg) wasthen treated with 2 mL of 4 M KOH for 16 h at 120 8C.After neutralization at pH 6, obtained by adding 2 M HCl,extraction with CH2Cl2 was performed. The oligosaccharides,contained in the aqueous phase, were purified from salts bygel filtration chromatography on a Sephadex G 10 column(Pharmacia, 1.5 � 44 cm, 10 mM NH4HCO3 as eluant, flow22 mL:h21) giving after lyophilization a residue of 5 mg(OS).

HPAEC purification of OSs

Anion-exchange chromatography of OS fraction (5 mg) wasperformed using a Dionex DX 500 instrument, equippedwith a gradient pump, pulsed amperometric detector, andCarboPak PA 100 column (Dionex, 4 � 50 mm, flow0.8 mL:min21). The OSs were eluted with the followinggradient: 80 mM NaOH for 10 min, linear to 300 mM

NaOAc in 30 min in 80 mM NaOH, linear to 500 mM in80 mM NaOH. The samples were neutralized with HCl afterelution from the column. Two fractions were obtained: OS1(1 mg) and OS2 (500 mg).

Methylation analysis

OS samples were methylated as reported previously [10].Linkage analysis was performed as follows: the methylatedsample was carboxymethyl reduced with lithium triethyl-borohydride (Aldrich), hydrolysed mildly to cleaveketosidic linkage, reduced by means of NaBD4, then totallyhydrolysed, reduced with NaBD4 and finally acetylated asreported previously [11].

R E S U L T S A N D D I S C U S S I O N

P. haloplanktis TAC 125 was grown as reported in Materialsand methods. As the method of LPS extraction from smoothbacteria, i.e. the hot phenol/water method [12], was notsuccessful in this case, the LPS was isolated from themembrane by the phenol/chloroform/light petroleum etherprocedure [7]. This suggested a highly lipophilic characterof the LPS fraction. Its electrophoretic behaviour onSDS/PAGE showed a very high mobility suggesting a shortsaccharide moiety with respect to the lipid part, and in fact,the ESI-MS spectrum of the LOS fraction (Fig. 1) showed alow molecular mass indicating its LOS nature. In particular,the spectrum revealed two molecular species at m/z 2134and 2332, respectively, indicating the presence of two LOSsdiffering in a C-12OH fatty acid residue.

The sugar composition was performed by gas chromato-graphic analysis of the acetylated methyl glycosides andgave galactose, Kdo, 2-amino-2-deoxymannose, 2-amino-2-deoxyglucose and heptose. This last was identified, asalditol acetate, to be L-glycero-D-manno-heptose by com-parison with an authentic sample.

Mild hydrazinolysis of LOS resulted in the O-deacylatedproduct (LOS-OH) whose ESI-MS spectrum showed twosignals, at m/z 1754 and 1551, indicating the presence of twoLOS-OH components differing in an amino sugar residue.This structural difference did not appear in the ESI-MSspectrum of LOS, but the peak would be expected to occur at

q FEBS 2001 LOS structures from P. haloplanktis (Eur. J. Biochem. 268) 5093

m/z 2129, which is obscured by the peak at m/z 2134. Inaddition, this spectrum revealed the presence of threephosphate groups, which were eliminated by treatment withaqueous HF. This was followed by the reduction of theanomeric centre of the terminal residue to give the alditolLOS-OH(ol) whose ESI-MS spectrum showed two signals,at m/z 1514 and 1311, confirming the presence of twosaccharide chains differing in one amino sugar residue.Complete delipidation was achieved by KOH hydrolysis,which removed all the N-acyl fatty acid residues. Thistreatment afforded a mixture of OSs which was resolved byHPAEC chromatography into two fractions, OS1 and OS2.The more abundant fraction (OS1) showed a pseudomole-cular ion peak at m/z 1076 in an ESI-MS spectrum thatcorresponds to two amino sugars, one aminoalditol, onehexose, one heptose and one Kdo unit. The methylationanalysis indicated, in particular, the presence of terminal2-amino-2-deoxymannose, 3-linked galactose, 4-linkedheptose, 5-linked Kdo and 6-linked glucosamine. The 1HNMR (Fig. 2) showed four anomeric signals of the sameintegral intensity occurring at d 5.09 (1.9 Hz), 4.89(2.4 Hz), 4.58 (8.2 Hz) and 4.41 (7.7 Hz), which werecorrelated to the signals at d 92.7, 101.1, 99.6 and 102.7,respectively, in the carbon spectrum where the anomericsignal of Kdo occurs at d 99.9. Starting from the anomericsignals, from the signals of the nitrogen-bearing carbons andfrom methylene signals of Kdo, 2D homonuclear andheteronuclear experiments (COSY, HSQC, HMBC, ROESY,HOHAHA) allowed us to identify the chemical shifts of therelevant signals of each monosaccharide (Table 1). Fromthese data, the presence of glucosaminitol, which wassuggested by the ESI-MS spectrum but not shown bymethylation analysis, was also revealed.

The determination of the absolute configuration for allmonosaccharides was determined to be D by the octylglycoside method [13].

The b anomeric configurations of Gal and GlcN wereinferred by proton and carbon chemical shift and 3JH,H

values of their anomeric centres (Fig. 3). The a configur-ations were suggested by the low value of chemical shiftdifference between methylene protons, for Kdo [14], and byproton and carbon chemical shifts of anomeric atoms forManpNH2 and Hepp residues (see below). The sequencewas deduced from the long-range 1H,13C heterocorrelatedexperiment, which showed connectivity between theanomeric protons of ManpNH2 (d 5.09), Hepp (d 4.89)and Galp (d 4.41) and C3 (d 76.7) of Galp, C5 (d 75.9) ofKdo and C4 (d 77.8) of Hepp, respectively. The low-fieldshifts of these carbon signals with respect to theunsubstituted reference compounds [15] confirmed theattachment points obtained from methylation analysis.

In addition to confirming the saccharide sequence, theROESY experiment provided evidence to support theassignment of an a-D-configuration of the ManpN residue,as its anomeric proton showed intraresidue NOE with onlyH2 and interresidue NOE with both H3 and H4 of the b-D-galactose unit [16]. Also in the case of heptose, its anomericproton showed an intraresidue NOE only with H2, inagreement with its a configuration, whereas both theanomeric protons of b-D-galactose and of b-D-glucosamineshowed an intraresidue NOE with H3 and H5.

On the basis of the above data, structure 1 below issuggested for the more abundant fraction OS1: a-D-ManpNH2-(1!3)-b-D-Galp-(1!4)-a-L-glycero-D-manno-Hepp-(1!5)-a-D-Kdo-(2!6)-b-D-GlcpNH2-(1!6)-D-GlcNH2(ol).

The ESI-MS spectrum of the smaller fraction OS2showed two pseudomolecular ion peaks, the first at m/z1076, attributed to OS1, and the second at m/z 915 indicatinga saccharide chain devoid of an amino sugar unit.Accordingly its 1H-NMR spectrum lacks the anomericsignal of ManpNH2.

Fig. 1. ESI-MS spectrum of the LOS fraction.

5094 M. M. Corsaro et al. (Eur. J. Biochem. 268) q FEBS 2001

Confirmation of the structure came from the 1% AcOHhydrolysis of the LOS fraction, which gave a mixture oftetra- and tri-saccharide whose fast-atom bombardment MSspectrum of permethylated derivatives showed the pseudo-molecular ion peaks at m/z 991 and 746, in agreement witha terminal anhydro-Kdo unit [17] (structures A and B).Therefore the structure 1 devoid of the ManpNH2 residuecould be attributed to the smaller component OS2.

In addition in the same fast-atom bombardment MSspectrum, all the fragmentation peaks indicated in structureA were also present, which supported the monosaccharidesequence of 1.

The product of alkaline treatment of the core oligo-saccharide sample arising from acetic acid hydrolysis

Fig. 2. 400-MHz 1H-NMR spectrum at 30 8C in D2O of OS1.

Table 1. 1H and 13C chemical shifts (d) and coupling constants of relevant signals of OS1 in D2O at 30 8C. ND, not determined.

Sugar

unit

H1

C1

H2

C2

H3

C3

H4

C4

H5

C5

H6

C6

H7

C7

H8

C8

t-ManpN 5�.09[1.9] 3�.55 4�.05 3�.49 3�.85 3�.58-3.66

92�.7 53�.9 67�.1 ND 72�.3 60�.1

3-Galp 4�.41 [7.7] 3�.51 3�.67 4�.01 4�.01 3�.56

102�.7 ND 76�.7 69�.7 ND 60�.9

4-Hepp 4�.89 [2.4] 3�.97 3�.93 3�.82 3�.98 3�.59 3�.63-3.80

101�.1 69�.3 69�.3 77�.8 72�.1 72�.0 60�.9

5-Kdo – – 1�.74-1.97 3�.98 3�.98 3�.54 3�.73 3�.97

174�.5 99�.9 35�.0 65�.8 75�.9 72�.0 69�.7 63�.4

6-GlcpN 4�.58 [8.2] 2�.91 3�.48 3�.36 3�.46 3�.72-3.82

99�.6 55�.7 74�.5 70�.0 ND 61�.7

6-GlcNol 3�.75-3.62 3�.38 3�.96 3�.51 3�.83 ND

58�.8 55�.3 65�.9 ND ND ND

q FEBS 2001 LOS structures from P. haloplanktis (Eur. J. Biochem. 268) 5095

allowed us to exclude the presence of O-acetyl groups inthe molecule because its 1H NMR spectrum retained theacetyl signal at d 2.06 due to the N-acetyl of 2-acetamido-2-deoxy-mannose.

This investigation which, to our knowledge, is the firstreported on the structure of an LOS fraction from apsychrophilic bacterium, led us to define a novel core [18]structure characterized by a very short linear chaincontaining only one unit of both heptose and Kdo. Thislast feature suggests the possibility of a phosphate group atthe 4 position of Kdo, as occurs in the case of Haemophilusinfluenzae [17,18] and in Vibrio cholerae [18]. The forma-tion of the anhydro-Kdo might support the presence ofphosphate at its C4 position [17]. Two phosphate groupswere inferred from low field chemical shifts of the GlcNanomeric signal at d 5.36 and of H-4 of the Kdo unit at d4.62 in a COSY spectrum of LOS-OH. As for the thirdphosphate we were not able to identify the H-40 of the distalGlcN, due to the crowding of the pertinent 1H NMR region.

All the above data led us to suggest the structure depictedin Fig. 4 as the saccharide backbone of the major componentof the LOS fraction.

Interestingly, LOS represents 0.8% of the total dry cellweight only. This evidence suggests a high glycero-phospholipids content of the outer leaflet of P. haloplanktisTAC 125 outer membrane. This hypothesis together with the

above described structural characteristics of the LOS, i.e.the linear nature of the inner core and the lack of theO-antigen side chain, suggests that this bacterium may bethermosensitive [19]. The data presented in this paperrepresent the first step towards the in-depth analysis of themolecular mechanisms that underlie the cold-adaptationof this psychrophilic bacterium. Finally, the lack of theO-specific chain and the very short core of LOS suggest adeep, rough nature of P. haloplanktis TAC 125 strain LPS.

A C K N O W L E D G E M E N T S

We thank the Centro di Metodologie Chimico-Fisiche of the University

Federico II of Naples for the NMR and CNR for financial support. We

are grateful to Professor C. Gerday for his support, frequent exchange

of ideas and valuable advice. This work was supported by grants of the

European Union (program ‘COLDNET’ contract ERB FMRX

CT97 0131), of Ministero dell0 Universita e della Ricerca Scientifica

(Progetti di Rilevante Interesse Nazionale 1999) and of Consiglio

Nazionale delle Ricerche (CNR contract 97.01138.PF49 Progetto

Finalizzato ‘Biotecnologie’).

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