Characterization of Mesorhizobium huakuii lipidA containing both

Characterization of Mesorhizobium huakuii lipid A containing bothD-galacturonic acid and phosphate residues

Adam Choma1 and Pawel Sowinski2

1Department of General Microbiology, Maria Curie-Sklodowska University, Lublin, Poland; 2Intercollegiate NMR Laboratory,

Department of Chemistry, Technical University of Gdansk, Poland

The chemical structure of the free lipid A isolated fromMesorhizobium huakuii IFO 15243T was elucidated. Lipid Ais a mixture of at least six species of molecules whose struc-tures differ both in the phosphorylation of sugar backboneand in fatty acylation. The backbone consists of a b (1¢fi 6)linked 2,3-diamino-2,3-dideoxyglucose (DAG) disaccharidethat is partly substituted by phosphate at position 4¢. Theaglycon of the DAG-disaccharide has been identified asa-D-galacturonic acid. All lipid A species carry four amide-linked 3-hydroxyl fatty residues. Two of them have shorthydrocarbon chains (i.e. 3-OH-i-13:0) while the other twohave longerones (i.e. 3-OH-20:0).Distributionof 3-hydroxyl

fatty acids between the reducing and nonreducing DAG issymmetrical. The nonpolar as well as (x-1) hydroxyl longchain fatty acids are components of acyloxyacyl moieties.Two acyloxyacyl residues occur exclusively in the non-reducingmoiety of the sugar backbone but their distributionhas not been established yet. The distal DAG amide-boundfatty acid hydroxyls are not stoichiometrically substitutedby ester-linked acyl components.

Keywords: Mesorhizobium huakuii; lipid A; 2,3-diamino-2,3-dideoxy-D-glucose; MALDI-TOF; 2D-NMR.

Lipopolysaccharides (LPS) are characteristic components ofthe outer leaflet of the outer membranes of Gram-negativebacteria. Those glycoconjugates have a common generalarchitecture. They contain three distinct regions: lipid A, anonrepeating oligosaccharide core and an O-polysaccharidecomposed of a varying number of repeating units. TheO-polysaccharide chain is the major target of animalimmune responses, thus it is also referred to as theO-antigen. The core oligosaccharide is a spacer betweenthe O-chain and lipid A and is linked to the latter by an acidlabile ketosidic bond. Lipids A in many Gram-negativebacteria (especially in animal pathogens) have a conservedstructure. In the majority of cases, their backbones arecomposed of a b-1,6-D-glucosamine disaccharide with twophosphate residues attached at positions 1 and 4¢. Up tofour fatty acids are bound by ester or amide linkagesto the backbone glucosamines. Lipid A is responsible for theendotoxic properties of lipopolysaccharide. The structure oflipid A seems to be essential inmaintaining outermembraneintegrity and flexibility and is crucial for bacterial cellviability [1–3].Lipopolysaccharide is important in the process of sym-

biotic interaction between Rhizobium and the host plant[4,5]. Environmental conditions (in planta and ex planta) as

well as plant-derived molecular signals induce entire LPSmodifications in Rhizobium [6].The structures of Rhizobium lipid A indicate great

variation in the glycosyl component of its backbone as wellas the acylation pattern. The lipid A backbone of Sinorhizo-bium is similar to that from enteric bacteria [7,8]. Lipids Afrom Rhizobium etli and biovars of Rhizobium legumino-sarum have identical and unusual structures. R. etli lipids Aare devoid of phosphate groups [9–11] and a galacturonicacid residue replaces the 4¢-linked phosphate in the lipid Abackbone. The distal part (distant from the reducing end ofthe backbone) of lipid A is almost the same for all lipid Aspecies isolated. The proximal glucosamine is partly oxi-dized to 2-aminogluconate [12,13]. A specific deacylaseremoves the ester-linked fatty acids from the C-3 positionof the lipid A precursor, thus this hydroxyl is only partiallysubstituted by an acyl residue in the matured lipid A [14].The symbiont of Sesbasnia, Rhizobium sp. Sin-1 [15],has lipid A composed of b-D-glucosamine attached to2-aminogluconate by (1fi 6) glycoside linkage. Whencompared with R. etli this lipid A lacks galacturonic acidat position 4¢ [16].In contrast to the abovementioned lipid A structures, the

mesorhizobial and bradyrhizobial lipids A have not beenfully chemically characterized to date. Bradyrhizobiumlipid A backbones are composed exclusively of 2,3-di-amino-2,3-dideoxyglucose with mannose as a subsituent insome of them [4,5,17,18]. No data about Allorhizobium(renamed Rhizobium undicola [19]) and scant informationaboutAzorhizobium [20,21] lipopolysaccharides and lipidsAare available. Mesorhizobium loti lipids A contain DAGand phosphate residues [22,23] and M. huakuii also posses-ses DAG-type lipid A [24]. Mesorhizobium lipids A areknown to carry a number of b-hydroxyl fatty acidsaccompanied by small amounts of 4-oxo fatty acids.

Correspondence to A. Choma, Department of General Microbiology,

Maria Curie-Sklodowska University, 19 Akademicka St.,

20–033 Lublin, Poland.

Fax: + 48 81 5375959, Tel.: + 48 81 5375981,

E-mail: [email protected]

Abbreviations: DAG, 2,3-diamino-2,3-dideoxyglucose; LPS,

lipopolysaccharides.

(Received 12 September 2003, revised 6 February 2004,

accepted 16 February 2004)

Eur. J. Biochem. 271, 1310–1322 (2004) � FEBS 2004 doi:10.1111/j.1432-1033.2004.04038.x

Numerous ester-linked nonpolar and (x-1) hydroxyl longchain fatty residueswere found in those preparations [25,26].In this report, we describe the structural investigation of a

unique lipid A isolated from Mesorhizobium huakuii. Weshow that DAG-type lipid A backbone is double decorated:(a) nonstoichiometrically, with phosphate at position 4¢ ofthe distal DAG, and (b) with a-linked galacturonic acidat position 1 of the proximal unit. Phosphorylated andnonphosphorylated lipid A preparations are a mixture ofthree subfractions differing in acylation patterns.

Experimental procedures

Bacterial strain, growth, and isolation oflipopolysaccharide and lipid A

Mesorhizobium huakuii IFO15243T strain was obtainedfrom the Institute for Fermentation, Osaka, Japan. Bacteriawere grown at 28 �C in liquid mannitol/yeast extractmedium 79CA [27] and were aerated by vigorous shaking.Cells were centrifuged at 10 000 g, washed twice with salineand once with distilled water. The wet bacterial paste wasextracted by the modified hot phenol/water procedure [28].The water layer was dialysed firstly against tap water, thenagainst distilled water. The crude LPS was purified byrepeated ultracentrifugation at 105 000 g for 4 h. The LPSsolution (5 mgÆmL)1) in aqueous 1% (v/v) acetic acid waskept at 100 �C for 3 h. The lipid A precipitate was collectedby centrifugation, washed twice with hot distilled water andlyophilized.

Purification and separation of lipid A species

Crude lipid A was purified and separated into subfractionsaccording to a modified procedure described by Que andcoworkers [9]. Briefly, lyophilized lipid A (� 30 mg) wasdissolved in 20 mL of CHCl3/methanol/H2O (2 : 3 : 1;v/v/v) and loaded onto a DEAE column (1 cm · 7 cm,Whatman DE23). The column was washed with 30 mL ofthe same solvent and that eluate was collected as a singlefraction. Next, the lipid material was eluted by a two stepgradient of ammonium acetate: first with 30 mL of CHCl3/methanol/250 mM NH4Ac (2 : 3 : 1; v/v/v), and then with30 mL of CHCl3/CH3OH/500 mM NH4Ac (2 : 3 : 1;v/v/v). The presence of organic substances in the eluatewasmonitored by spotting 10 lL of each fraction on a silicaplate and visualized by spraying the plate with 10% (v/v)sulfuric acid in methanol followed by charring. Separatedfractions were converted to two-phase Bligh–Dyer systemby adding the appropriate amount of water and chloroform.Water layers were discarded and organic layers weresupplemented with fresh portions of the upper phase ofa freshly prepared two-phase Bligh–Dyer mixture. Thewashed organic layers were separated by centrifugation anddried. Preparations were stored at )20 �C in CHCl3/methanol (1 : 1; v/v).

Glycosyl composition analysis

Lipid A samples were analysed for fatty acids and amino-sugars as described previously [24]. Neutral and acidicsugars were determined by gas-liquid chromatography and

mass spectrometry. For this analysis, lipid A samples weremethanolysed (1 M HCl, 80 �C, 18 h), N-acetylated andtrimethylsililated [29]. The content of phosphorus in lipid Awas determined according to Lowry [30].

Chemical modification of lipid A

Subfractions of lipid A (about 2 mg) were dephosphory-lated in 48% (v/v) aqueousHFat 4 �C for 48 h [31].HFwasremoved by evaporation in the stream of nitrogen withcooling inan icebath.De-O-acylationof lipid Asubfractionswasperformedaccording tomodifiedprocedureofHaishimaand coworkers [31]. Preparations were treated with anhy-droushydrazineat37 �Cfor2 h.The reactionmixtures, aftercooling,were poured into cold acetone. The resulting lipid Aprecipitates were collected, washed twice with acetone andthen gently dried in the stream of nitrogen.

Gas chromatography-mass spectrometry

GC-MS was carried out on a Hewlett-Packard gaschromatograph (model HP5890A) equipped with a capil-lary column (HP-5MS, 30 m · 0.25 mm) and connected toa mass selective detector (MSD model HP 5971). Heliumwas the carrier gas. The temperature program for fatty acidmethyl esters and for alditol acetates analysis was as follows:initially 150 �C for 5 min, then raised to 310 �C at a ramprate of 3 �C min)1, final time 20 min. The temperatureprogram for trimethylsililo derivatives of methyl glycosideswas, accordingly: initially 80 �C for 2 min, then raised to310 �C at a ramp rate of 4 �C min)1, final time 5 min.

Mass spectrometry

Matrix-assisted laser desorption ionization-time of flight(MALDI-TOF) mass spectrometry was performed on aVoyager-Elite (PE Biosystems) instrument using delayedextraction, in both positive and negative ion modes. Thesamples were desorbed with a nitrogen laser and extractionvoltage of 20 kV. Lipid A samples were dissolved inCHCl3/CH3OH (2 : 1; v/v). The analysed compounds (0.5 lL)were mixed with 50% (v/v) 2,5-dihydrobenzoic acid inacetonitril as matrix. Each spectrum was the average ofabout 256 laser shots.Liquid matrix-assisted secondary ion mass spectrometry

(LSIMS) was performed using AMD 604 (AMD IntectraGmbH) mass spectrometer operated in the negative ionmode with primary ion beam of Cs+. Samples were mixedwith a matrix of meta-nitrobenzyl alcohol (m-NBA).Lipid A was analysed by ESI-MS using Finnigan Mat

TSQ 700 mass spectrometer operated in the negative ionmode. The samples were dissolved in a CHCl3/CH3OH(2 : 1; v/v) mixture supplemented with 0.1% (v/v) concen-trated ammonia and introduced into electrospray sourceat a flow rate of 5 lLÆmin)1.

NMR spectroscopy1H-NMR experiments were performed in CDCl3/dimethyl-sulfoxide-d6 (2 : 1; v/v) mixture. 2D (DQFCOSY, TOCSY,NOESY) 1H-NMR and 1H/13C as well as 1H/31P-HSQCexperiments were carried out on Varian Unity plus 500

� FEBS 2004 Structure of Mesorhizobium huakuii lipid A (Eur. J. Biochem. 271) 1311

instrument at 48 �C using standard VARIAN software. 1D31P-NMR spectra were registered on a Bruker 300 spectro-meter, operating at 121.58 MHz at 40 �C. For this analysis,the lipid A was dissolved in D2O containing 2% deoxycho-late and 5 mM Na2EDTA. The pH of lipid A solutions wasadjusted withNaOH to 7.3 and 10.6, respectively. Phospho-rous chemical shifts were measured relative to an externalstandard of 85% (v/v) phosphoric acid at 0.00 p.p.m.

Results

Chemical analyses

The compositional analysis of crude lipid A preparationobtained from M. huakuii IFO 15243T LPS revealed thepresence not only of 2,3-diamino-2,3-dideoxyglucose(DAG) and a complex set of fatty acids (both ester andamide bound), as described previously [24], but alsogalacturonic acid and phosphate residues. The presence ofGalA was unequivocally confirmed by GC-MS analysis oftrimethylsilil ethers of methyl glycosides liberated fromlipid A by methanolysis (Fig. 1). The 31P-NMR spectrumof the crude lipid A revealed a prominent signal withchemical shift of 1.71 p.p.m. observed at neutral pH. Thissignal was shifted to 4.71 p.p.m. when the pH of the lipid Asuspension was raised to 10.6 (Fig. 2). These properties areindicative of phosphomonoesters other than glycosyl-1-phosphate. The location of the phosphate was directlydetermined by two-dimensional heteronuclear magneticresonance (see below). On the basis of chemical shift valueand lack of the cross peak with protons from the lipid Abackbone on the 31P/1H-HSQC spectrum, the weak signalat 1.88 p.p.m. was attributed to inorganic phosphateimpurities of the lipid A preparation. The results ofquantitative measurements of phosphorus and DAG con-tent showed that no more than half of the lipid Amoleculesbear phosphate residues.Fatty acids found in the IFO 15243T lipid A (Fig. 1) can

be divided into two groups. The first one, easily liberated bymild alkali or acid solvolysis, contains all saturated andunsaturated nonpolar as well as (x-1) hydroxyl, and (x-1)

oxo long chain fatty acids. These are ester-linked to thelipid A. The second group of fatty acids needs strongliberation conditions [32]. This group comprises all3-hydroxyl and 4-oxo fatty acids, which are connecteddirectly to the lipid A backbone via amino groups [24]. Themolar proportions among fatty acids isolated from lipid Awere almost the same as described earlier for the total LPS[24]. The main amide-bound fatty acids identified were asfollows: 3-OH-12:0, 3-OH-i-13:0, 3-OH-20:0 and 3-OH-21:0. Among them, 3-OH-i-13:0 and 3-OH-20:0 clearlypredominated. The types of ester-bound fatty acids werealso numerous, but only four of them, namely i-17:0, 20:0,22:1 and particularly 27-OH-28:0 fatty acid, predomi-nated (Fig. 1, [24]). The calculated proportion between

Fig. 1. GC-MS profile of trimethylsilyl ether derivatives of N-acetylated methyl glycosides and fatty acid methyl esters obtained by methanolysis of

dephosphorylated lipid A fromMesorhizobium huakuii IFO 15243T. Peaks were identified by their mass spectra and by comparison of retention times

with standards. GalA, galacturonic acid; DAG, 2,3-diamino-2,3-dideoxyglucose; p.e. impurity-ester of phtalic acid; *, unidentified compound. For

more details about the fatty acid composition of IFO 15243T see [24].

Fig. 2.31P-NMR spectra of the crude lipid A from Mesorhizobium

huakuii IFO 15243T. The signal at 1.71 p.p.m. (A) recorded at pH 7.3,

shifted to 4.71 p.p.m. (B) at pH 10.60, and represents ester-bound

monophosphate residue.

1312 A. Choma and P. Sowinski (Eur. J. Biochem. 271) � FEBS 2004

amide- and ester-linked fatty acids was approximately 2 : 1.Therefore, one can expect that DAG type lipid A couldcontain not more than six acyl residues.The complex mixture of the lipid A preparation was

separated into two fractions, based on DEAE gravitycolumn chromatography. The first fraction (designatedlipid A–P), which was eluted with solvent containing250 mM ammonium acetate, was devoid of phosphate, asshown by 31P-NMR. The phosphate was detected inthe second fraction (named lipid A+P), successively elutedwith a solvent mixture containing 500 mM NH4Ac.

MALDI-TOF analysis of lipid A preparations

Both subfractions of lipid A were investigated by massspectrometry. Ions representative of each species of lipid A,recorded on the negative and positive ion MALDI-TOFand negative ion ES-MS spectra, their correspondingcomposition and the theoretically calculated masses arelisted in Table 1.Lipid A+P is a complex mixture of individual molecules.

The two major species (Z and Y) could be easilydistinguished from mononegatively charged pseudomole-cular ions on MALDI-TOF spectrum (Fig. 3A). The third(X) cluster of ions, which were less intensive, was visiblebetweenm/z at 1600 and 1700. All those ions correspond tolipid A that posseses a backbone with a monophosphateresidue accompanied by six, five and four acyl moieties,respectively. Dephosphorylation procedure, to whichlipid A+P was submitted, led to a downshift of eachmolecular ion by 80 mass units. The spectrum of thedephosphorylated lipid A+P is almost identical to thatobtained for the lipid A–P preparation (Fig. 4). Moreover,the MALDI-TOF spectrum of lipid A–P treated with 48%HF did not change significantly when compared with theunprocessed preparation (data not shown).Species Z of lipid A+P (Fig. 3A) contained ions within the

range from 2287 to 2478 mass units. Those ions correspondto lipid A molecules composed of two DAG, one of whichis phosphorylated, one GalA, four 3-hydroxyl fatty acids,one (x-1) hydroxyl long chain fatty acid and one a nonpolarfatty acyl residue. The most intense ion in this cluster (m/zat 2357) could be attributed to the molecules of lipid Acontaining two 3-OH-i-13:0 and two 3-OH-20:0 acids, aswell as two ester-bound acids (e.g. 20:0 and 27-OH-28:0).This is merely one possible explanation due to the fact thatnumerous combinations of fatty acids different to thosefound in lipid A exist. However, taking into considerationthe quantities of lipid A fatty acids this proposition seemsto be the most probable. The amide-bound fatty acidsisolated from M. huakuii IFO15243T lipid A and fromother mesorhizobia can be separated into two clusters[24–26]. The first contains short chain fatty acids, mainly3-OH-12:0 and 3-OH-i-13:0, whereas the second is repre-sented by 3-OH-20:0 and other fatty acids similar in length.For correct calculation of the pseudomolecular ion massesfound on the MALDI-TOF spectra it is necessary to takeinto account the masses of two 3-OH short chain fattyacyls (e.g. 3-OH-i-13:0) and two longer 3-OH fatty acylresidues (e.g. 3-OH-20:0).The ions from species Y are usually 295 mass units

lighter than the respective ions from species Z. That

corresponds to a loss of eicosanoyl residue from hexaacyllipid A. Therefore, the Y species comprise ions represent-ing lipid A molecules carrying five acyl residues (four3-OH fatty acids and one (x-1) hydroxyl long chain fattyacid). The ion at m/z 1640 and those close to m/z 1640,designated as species X, correspond to tetraacyl lipid Amolecules with all acyl residues directly linked to the sugarbackbone by amide bonds. De-O-acylation of lipid Afractions led to decay of species Z and Y and resulted inincrease of signals corresponding to ions of species X(data not shown). The total decrease of mass due to de-O-acylation of phosphorylated as well as of nonphospho-rylated lipid A was the same and equalled 717 Da (loss ofboth 294 and 423 mass units).The positive ion MALDI-TOF mass spectra of the

lipid A+P (Fig. 3B) showed two additional species gener-ated after laser-induced cleavage of glycosidic linkagesbetween 2,3-diamino-2,3-dideoxyglucoses within the lipidA backbone. The first species [B1

+(Z)] of oxonium ions

originated from hexaacyl phosphorylated lipid A (pro-minent ions at m/z 1481 and 1508). The second species[B1

+(Y)] consist of ions with masses close to that at m/z

1187. Those ions are made up of DAG, two 3-hydroxylfatty acyl moieties and (x-1) hydroxyl long chain fattyacid. Those B1

+ fragment ions support the conclusion thatthe 27-hydroxyoctacosanoic acid and eicosanoic acid,when present, are located on the distal diaminoglucosylresidue of the lipid A. Moreover, the sugar component ofB1

+ lacks hydroxyl groups suitable for attachment ofthese fatty acids by ester bonds. The appropriate hydroxylsare located at positions 3 of amide linked acyl of the distalDAG. Therefore, both 27-OH-28:0 and 20:0 fatty acids arecomponents of acyloxyacyl residues. The predicted ions forthe third type of oxonium ions composed of DAG andtwo amide acyl residues have not been registered, due tothe fact that the spectra were usually recorded from m/z1000–3000. The correct calculation of masses for B1

+ typeions requires taking into account the appropriate amide-linked fatty acids. That group of acyl residues consists offatty acid pairs. The first acid in each pair is shorter (e.g.3-OH-i-13:0) while the second one is longer (e.g. 3-OH-20:0). Analysis of lipid A by means of LSI mass spectro-metry revealed negative ions m/z at 862.7, 876.8 and 890.7(data not shown). The most intensive ion (m/z at 876.8)corresponds to a lipid A fragment composed of DAG,GalA, 3-OH-i-13:0 and 3-OH-20:0. A similar ion wasobserved for P. gingivalis and F. meningosepticum lipids Aon negative ion FAB-MS/MS spectra [33,34]. In conclu-sion, these data point to the symmetrical localization ofamide-bound acyl residues in M. huakuii lipid A. The2,3-diacylamido-2,3-dideoxyglucose, obtained by mildsolvolysis [35] followed by mild hydrolysis of the dephos-phorylated lipid A, was reduced with NaBD4, thansubjected to Smith oxidation, again reduced with NaBH4

and after acetylation, the four-carbon fragments of DAGcarrying amide-bound fatty acids were analysed by meansof GC-MS. Preliminary data from those experimentsindicate that N-2 position in distal and proximal DAG isoccupied mainly by 3-hydroxyleicosanoic acids. The shor-ter acids were found to be bound at N-3 position of theamino sugar ring. The fatty acid distribution will beverified during further studies.


Table1.Datafrommassspectrometryanalyses.Positiveandnegativeionsderived

fromphosphorylatedandnonphosphrylatedlipid

AfractionsofM

esor

hizo

bium

huak

uiiIFO1243

T;theircompositionsand

proposedstructures.Backbone*,trisaccharideofb-

D-D

AG-(1fi

6)-a-D-D

AG-(1fi

1)-a-D-G

alA;P,phosphateresidue;nd,notdetermined.

PositivemodeMS

NegativemodeMS

Composition

MALDI-TOF

(iontypeand

m/z-value)

MALDI-TOF

(iontypeand

m/z-value)

ES-M

S

(iontype

m/z-value)

molecularmass

calculatedfrom

ES-M

Svalue

DAG

PGalA

3-OH-

fatty

acids

(x-1)-OH

-fatty

acids

Nonpolar

fatty

acids

Totalnumber

offattyacid

carbonatoms

Proposed

composition

ofthemolecule

Predicted

molecular

mass

Phosphorylatedlipid

Afraction

[M+

Na]+

2380

[M-H

]–

2357

[M-2H])2

1177.9

2357.8

21

14

11

114

Backbone*,phosphate,

2·3-OH-20:0,

2·3-OH-i-13:0,

27-OH-28:0,20:0

2357.398

[M+

Na]+

2085

[M-H

]–

2063

[M-2H])2

1030.7

2063.4

21

14

10

94

Backbone,phosphate,

2·3-OH-20:0,

2·3-OH-i-13:0,

27-OH-28:0

2062.875

[M+

Na]+

1663

[M-H

]–

1640

[M-2H])2

nd

–2

11

40

066

Backbone,phosphate,

2·3-OH-20:0,

2·3-OH-i-13:0

1640.137

B1+

1481

––

–1

11

21

181

P-D

AG,

1·3-OH-20:0,

1·3-OH-i-13:0,

27-OH-28:0,20:0

1481.276

B1+

1187

––

–1

11

21

061

P-D

AG,

1·3-OH-20:0,

1·3-OH-i-13:0,

27-OH-28:0

1186.753

Unphosphorylatedlipid

Afraction

[M+

Na]+

2299

––

–2

01

41

1114

Backbone,

2·3-OH-20:0,

2·3-OH-i-13:0,

27-OH-28:0,20:0

2277.419

[M+

Na]+

2004

––

–2

01

41

094

Backbone,

2·3-OH-20:0,

2·3-OH-i-13:0,

27-OH-28:0,20:0

1982.896

B1+

1400

––

–1

01

21

181

DAG

2·3-OH-20:0,

2·3-OH-i-13:0,

27-OH-28:0,20:0

1401.297

B1+

1108

––

–1

01

21

061

DAG

2·3-OH-20:0,

2·3-OH-i-13:0,

27-OH-28:0

1106.774


The B1+ ions from lipid A+P (e.g. m/z at 1187 and 1508,

Fig. 3B) differed by 80 mass units from those originatingfrom lipid A–P (e.g. m/z at 1108 and 1428, Fig. 4).Comparing Figs 3B and 4, it is easy to notice that

the phosphate deprived lipid A appears to have a highernumber of connected fatty acids. On the spectrum,shown in Fig. 4, the signals for hexaacyl lipid A areconsiderably more intensive than others. Pentaacyl

lipid A molecules dominate in the case of the phos-phorylated lipid A preparation. Possibly, a weak acidhydrolysis (the procedure used for lipid A liberation)causes a partial de-O-acylation of the native lipid Amolecules.In contrast to R. etli, R. leguminosarum and S. melilotii

[8–10], we did not find lipid A molecules containing3-hydroxylbutyrate or 3-metoxylbutyrate.

Fig. 3. Negative (A) and positive (B) ion MALDI-TOF mass spectra of the phosphorylated subfraction of lipid A from M. huakuii IFO 15243T.

Lipid A yields three ion clusters (Z, Y, X). They differ by the degree of acylation. Species X contains four amide-bound fatty acids. Species Y is

pentaacyl lipid A (with 27-OH-28:0 fatty acid residue). Species Z is hexaacyl lipid A. The proposed formulas and masses of pseudomolecular ions

([M ) H]– and [M + Na]+) are summarized in Table 1. The individual ions in the clusters differ by 14 units (acyl chain length differences). Positive

ion spectrum contains two B+1 type ion clusters derived from cleavage of the glycosidic linkage in lipid A. Unidentified ions are marked with

asterisks (*).


NMR spectroscopy of lipid A preparations

De-O-acylated lipids A+P and lipid A–P were dissolved in amixture of dimethylsulfoxide (DMSO-d6) and chloroform(CDCl3) for NMR experiments. Figure 5 shows the one-dimentional proton spectrum of de-O-acylated lipids A+P.1H and 13C chemical shift assignments were based on 2Dhomonuclear experiments: DQF-COSY (Fig. 6), TOCSY(Fig. 7) and 1H/13C heteronuclear single quantum coher-ence (HSQC) experiments. The values of carbon and protonchemical shifts are summarized in Table 2.Three signals were identified in the anomeric region of

13C-NMR chemical shifts for both lipid A fractions. Thesedata suggested that the lipid A backbone contains threesugar residues. Four signals were found between 50 and55 p.p.m. for each preparation. They were assigned to theC-2 and C-3 carbon atoms linked directly with the aminogroups. The remaining sugar ring carbon signals wereobserved in the region from 60 to 78 p.p.m. TOCSY andDQF-COSY spectra revealed three glycosyl ring systems.

The anomeric proton (HA-1) at 4.98 p.p.m. was assigned toa-linked galacturonic residue. Its spin system (A) consists offive protons for which all the cross peaks have been tracedand marked on Fig. 6 and resulting chemical shifts listed inTable 2. Analysis of the sugar proton system B (Fig. 7) wasinitiated at the anomeric proton (HB-1, dH ¼ 4.87 p.p.m.,J1,2 ¼ 2.8Hz). That proton showed an evident correlationto HB-2 (dH ¼ 3.84 p.p.m.), which showed a strong corre-lation to HB-3 (dH ¼ 4.08 p.p.m.). Furthermore, HB-3showed a coupling with HB-4 (dH ¼ 3.48 p.p.m.). Theremaining glycosyl proton cross-peaks were observed atfollowing chemical shifts: 3.48 p.p.m./3.97 p.p.m. (HB-4/HB-5), 3.97 p.p.m./3.60 p.p.m. (HB-5/HB-6a), 3.60 p.p.m./3.89 p.p.m. (HB-6a/HB-6b). The proton chemical shifts forboth sugar ring systems (A and B) were similar to thosepublished for A. pyrophilus lipid A [36]. Chemical shifts ofthe distal aminosugar (sugar ring system C) in lipid A+P

were in good agreement with those from A. pyrophiluslipid A distal DAG, however, two shift exceptions (forHC-4and HC-3) were observed. The HC-4 signal appeared at

Fig. 5. Proton NMR spectrum of de-O-acylated lipids A+P fraction. The sample was dissolved in DMSO-d6/CDCl3 (1 : 2, v/v). The spectrum was

recorded at 500 MHz, at 48 �C. Some signals from sugar backbone are indicated. The letters refer to the carbohydrate spin systems as was described

in the text and shown in Table 2. The numerals next to the letters indicate the protons in the respective residues. Signal positions from olefinic

protons, terminal methyl protons, bulk methylene protons and protons from a, b and c positions of 3-hydroxy fatty acids are marked with;

-CH ¼ CH-, -CH3, and -CH2-, a, b and c, respectively. CHCl3, DMSO and H2O represent signals from solvents and absorbed water.

Fig. 4. Positive ion MALDI-TOF mass spectrum of unphosphorylated subfraction of lipid A fromM. huakuii IFO 15243T. This lipid A subfraction

yields the three ion clusters X1, Y1 and Z1. They differ in the degree of acylation pattern and contain four, five and six acyl residues, respectively.

The spectrum contains two B+1 type ion clusters derived by cleavage of the glycosidic linkage in lipid A.


dH ¼ 4.01 p.p.m., which was about 0.3 p.p.m. downfieldfrom the A. pyrophilus lipid A equivalent signal and about0.5 p.p.m. downfield from the H-4 signal characteristic ofDAG with unsubstituted hydroxyl group at C-4 carbonatom (dH for HB-4, Table 2). The downfield shift of HC-4was caused by the presence of ester-bound phosphateresidue. Analysis of carbon chemical shifts led to the sameconclusions, since CC-4 (dC ¼ 71.9 p.p.m) appeared down-

field compared to the proximal CB-4 unsubstituted byphosphate (dB ¼ 67.5 p.p.m). The location of phosphatesubstituent on CC-4 was established upon HC-4/

31P(4.01 p.p.m./1.35 p.p.m) correlation observed in 1H/31PHSQC spectrum.The sequence of the monosaccharides was established

by NOESY experiment (Fig. 8). A strong interresidueNOE signal was observed between HA-1 of GalA and

Fig. 6. A partial DQF-COSY spectrum of de-O-acylated phosphorylated subfraction of lipid A. The spectrum was recorded at 500 MHz, at 48 �C.The letters refer to the carbohydrate spin systems as was described in the text and shown in Table 2. The numerals next to the letters indicate the

protons in the respective residues.

Fig. 7. A partial TOCSY spectrum of de-O-acylated phosphorylated subfraction of lipid A. The spectrum was recorded at 500 MHz, at 48 �C. Theletters refer to the carbohydrate spin systems as was described in the text and shown in Table 2. The numerals next to the letters indicate the protons

in the respective residues.


Table 2. 1H- and 13C-NMR chemical shifts and coupling constants of sugar backbones of lipid A fractions. DAG-I proximal 2,3-diamino-2,3-

dideoxyglucose moiety in the lipid A from M. huakuii IFO 15243T, DAG-II distal 2,3-diamino-2,3-dideoxyglucose moiety in the lipid A from

M. huakuii IFO 15243T; lipid A+P, phosphorylated fraction of lipid A; lipid A–P, unphosphorylated lipid A; nd, not determined; J, coupling

constant. Spectra were recorded at 500 MHz (1H) and 125.7 MHz (13C) in DMSO-d6/CDCl3 (2 : 1, v/v).

Residue

(spin system)

DAG-II (C) DAG-I (B) GalA (A)

1H d (J,[Hz]) 13C d 1H d (J,[Hz]) 13C d 1H d (J,[Hz]) 13C d

Lipid A+P

H-1 4.39

(8.2)

C-1 103.1 H-1 4.87

(2.8)

C-1 92.8 H-1 4.98

(2.8)

C-1 94.9

H-2 3.72 C-2 54.5 H-2 3.84 C-2 52.0 H-2 3.78 C-2 67.9

H-3 3.94 C-3 54.7 H-3 4.08 C-3 51.3 H-3 3.94 C-3 68.8

H-4 4.01 C-4 71.9 H-4 3.48 C-4 67.5 H-4 4.08 C-4 70.8

H-5 3.23 C-5 77.8 H-5 3.97 C-5 71.8 H-5 4.42 C-5 71.1

H-6a 3.53 C-6 60.8 H-6a 3.60 C-6 68.7 C-6 nd

H-6b 3.79 H-6b 3.89

NH-2 7.23 NH-2 7.26

NH-3 7.26 NH-3 7.24

Lipid A–P

H-1 4.35

(� 8)

C-1 102.8 H-1 4.89

(� 2)

C-1 92.4 H-1 5.02

(� 3)

C-1 94.5

H-2 3.73 C-2 53.8 H-2 3.86 C-2 52.0 H-2 3.78 C-2 68.2

H-3 3.77 C-3 53.8 H-3 4.13 C-3 51.7 H-3 3.95 C-3 71.7

H-4 3.34 C-4 68.8 H-4 3.52 C-4 70.3 H-4 4.12 C-4 70.5

H-5 3.22 C-5 nd H-5 3.93 C-5 71.8 H-5 4.42 C-5 71.0

H-6a 3.58 C-6 61.4 H-6a 3.62 C-6 68.8 C-6 nd

H-6b 3.71 H-6b 3.93

NH-2 7.46 NH-2 7.42

NH-3 7.38 NH-3 7.41

Fig. 8. A partial NOESY spectrum of de-

O-acylated phosphorylated subfraction of

lipid A. The spectrum was recorded at

500 MHz and at 48 �C. The letters refer to thecarbohydrate spin systems as was described in

the text and shown in Table 2. The numerals

next to the letters indicate the protons in the

respective residues. The inter- and intraresidue

signals are labeled starting from anomeric

protons. Diagnostic interresidue cross peaks

are underlined.


HB-1 of the proximal DAG. Both sugars possess aanomeric configurations that are reflected in the smallvalues of J1,2 coupling constants and the appropriatevalues of chemical shifts. The downfield shift of carbonCB-6 from the proximal DAG and strong cross peakHC-1/HB-6a (4.39 p.p.m./3.60 p.p.m), as well as lessintensive cross peak at 4.39 p.p.m./3.89 p.p.m. (HC-1/HB-6b) on NOESY spectrum, unequivocally indicate the

presence of (1fi 6) glycosidic linkage between the twoDAG residues. Chemical shifts: CC-1 (103.1 p.p.m), HC-1(4.39 p.p.m) and large (� 8Hz) coupling constants J1,2measured for the distal DAG confirmed its b-anomericconfiguration.Putting all the presented data together, we propose the

chemical structures for lipid A+P (species Z, Y, X) as shownin Fig. 9.

Fig. 9. Tentative structures of lipid A species fromMesorhizobium huakuii IFO 12543T.The proposition of the positions of 3-hydroxyl acyls is based

on preliminary chemical degradation of lipid A. The predicted positions of ester bound fatty acids were elicited from literature data and specificity

of LpxXl acyltransferase [46]. The proposed structures corresponds to [M + Na]+ ions atm/z 2380 (Z), 2085 (Y), 1663 (X) in Fig. 2B and to ions at

m/z 2299 (Z1), 2004 (Y1), 1583(X1) in Fig. 3B.


Lipid A–P is deprived of phosphate. The 1D 31P-NMRspectrum contained only a trace signal, which in the 1H/31P-HSQC experiment gave weak intensity correlation peakwith chemical shifts almost identical as for lipid A+P.Therefore, one ought to conclude that lipid A–P preparationwas contaminated with traces of the phosphorylatedlipid A variety.Chemical shifts (for protons and carbons) assigned to

lipid A–P were almost identical to those of lipid A+P

(Table 2) with the exception of H-4 and C-4 in distalDAG. The absence of phosphate caused an upfield shiftof both proton HC-4 and carbon CC-4. These values ofchemical shifts became similar to the corresponding valuesfrom the proximal DAG (Table 2). Three 13C signals in therange of 50–55 p.p.m. were observed. Two of them wereassigned to CB-2 and CB-3 (52.0 p.p.m and 51.7 p.p.m.,respectively) in the proximal DAG. The third signal wasfound to represent CC-2 and CC-3 overlapped resonancesof the distal DAG. Similarly, chemical shifts of HC-2 andHC-3 of the distal DAG (lipid A–P) were almost identical.They were distinguished upon two separate cross peakswith different amide protons (HC-2/NHC-2; 3.73 p.p.m./7.46 p.p.m and HC-3/NHC-3; 3.77 p.p.m./7.38 p.p.m.)observed in DQF-COSY spectrum.The chemical shifts and cross peak positions derived from

remaining N-acyl residue protons are not being discussed inthis paper. NMR data of those components for M. huakuiiIFO15243T lipid A are very similar to the data publishedearlier forA. pyrophilus [36],R. etliCE3 [10], Rhizobium sp.Sin-1 [16] and other lipid A preparations.

Discussion

Mesorhizobium huakuii IFO 12543T lipopolysaccharideappears to posses a unique lipid A. Two backbone specieshave been identified. The complete structure of the firstspecies was characterized as (HO)2PO2-b-D-DAG(1fi6)-a-D-DAG(1fi1)-a-D-GalA. Thus, the diaminosugar back-bone is flanked at both sides with the negatively chargedresidues D-GalA and phosphate. In relation to this struc-ture, the second species of lipid A is devoid of phosphateand therefore possesses nonsymmetrically located andweaker negative charge. Lipid A with DAG disaccharidebackbone, without phosphate at position 1 has originallybeen identified in the extremely termophilic bacterium A.pyrophilus. Its backbone is substituted from both sides (atposition 1 and 4¢) with a-D-GalA [36]. Within the Rhizo-biaceae, other lipids A without phosphate were isolatedfrom R. etli, R. leguminosarum biovars and Rhizobium sp.Sin-1. Negative charges in those molecules originated fromgalacturonic acid attached at 4¢ and/or from a proximalglucosamine oxidized to the form of 2-aminogluconic acid[10,16]. Membrane associated oxidase responsible for thisprocess has been recently detected within OM preparationfrom R. leguminosarum [12,13]. DAG–DAG disaccharidehas been found in the lipid A from Bradyrhizobium sp.(Lupinus) [17], M. loti [22], A. pyrophilus [36], B. abortus[37], L. pneumophila [38,39] and other bacteria, but struc-tural studies have been performed only for a few lipid Apreparations [36,40,41]. Here, we describe the first completestructure of 2,3-diamino-2,3-dideoxyglucose backbonelipid A from bacteria belonging to the Rhizobiaceae.

According to the current knowledge, the first steps oflipid A biosynthesis seem to be the same in all Gram-negative bacteria and lead to a 1,4¢-bisphosphorylatedaminosugar disaccharide acylated at positions 2, 3, 2¢, 3¢ by3-hydroxyl fatty acids. Kdo disaccharide occupies positionO-6¢ on lipid A [9,42,43]. Usually, UDP-glucosamine isa precursor for this pathway, but it is well known thatthe same or very similar pathway exists within bacteriasynthesizing mixed or DAG-type lipid A [44]. Thus, com-mon lipid A precursor molecules (Kdo)2lipidIVA, are thepoint at which the biosynthesis pathways for enterobacteriaand rhizobia diverge. In Mesorhizobium cells, the commonlipid A precursor should be processed by 1-phosphatase,following galacturonic acid transferase, before maturation.The predicted 4¢-O phosphatase would carry out its partialdephosphorylation. A computer analysis of M. lotiMAFF303099 genome sequence (http://www.kazusa.or.jp/rhizobase/), a bacterium closely related to M. huakuii[19,45], revealed sequences (mll1545, mll0630, mlr8270) withhigh homology to the genes encoding key enzymes in thelipid A biosynthesis pathway (lpxC, lpxB and lpxK, respect-ively). Basu and coworkers [46] described a 9 kb DNAfragment from R. leguminosarum that encodes C28 acyl-transferase (LpxXL) and related proteins that may partici-pate in the biosynthesis of (x-1)-28:0 and similar fatty acids.Among the genes identified, a structural gene encoding ahighly specific acyl carrier protein (acpXL) [42,46] capable oflong chain fatty acid (C28–C30) incorporation as thesecondary substituent of amide-bound 3-hydroxyl fattyacyls has been found. Chromosomal fragments fromM. lotiMAFF303099 have very similar sequence to genes encodingthose enzymes [46]. Moreover, another DNA fragmentfrom M. loti contains a sequence corresponding to lpxEof R. leguminosarum. This gene, when expressed in E. coli,yields a product with a 1-phosphatase activity [47].Therefore, it is very likely that lpxE gene is present withinM. huakuii that produces lipid A where phosphate atposition 1 is entirely replaced by galacturonic acid.Long chain fatty acids with a hydroxyl group at the (x-1)

position are present in lipids A of almost all bacteria fromthe Rhizobiaceae [18,25] and in lipids A of facultativeintracellular pathogens [48–50]. Low endotoxin activity oflipopolysaccharides from those bacteria has been observed.For example, the lethal toxicity in galactose-sensitized micewas 1000-fold lower for M. loti LPS when compared toS. typhimurium LPS [23]. It was shown that the lipopoly-saccharides from R. etli, R. galegae, and Rhizobium sp.Sin-1 display a largely reduced capacity to induce TNFa inhuman monocytes [51]. Moreover, Sin-1 LPS preparationantagonized an E. coli-induced synthesis of TNFa by thesemonocytes. The inhibition of the plant innate immunesystem should be achieved for the proper development ofsymbiosis. The experimental data suggest that the presenceof long chain (x-1) hydroxyl fatty acids influencing theimmunogenicity of lipid A could be one of the factorsresponsible for such immune system response inhibition.Those fatty acids seem to be important but not essential forsymbiosis because the acpXL::kan mutant of R. legumino-sarum lacking the 27-OH-28:0 acid in its lipid A is still ableto form nitrogen-fixing nodules [52].Lipid A is a valuable marker in chemotaxonomic studies

of rhizobia. It is well known that the chromosomal


background of rhizobia varies considerably. This is alsoreflected in rhizobial lipids A. The diversity in lipid Astructures indirectly confirms the hypothesis that symbioticN2-fixing bacteria evolved from nonsymbiotic and non-related ancestors by horizontal transfer of symbiotic genes(nod, nif, fix) as symbiotic islands. Similar conclusions werereached when rhizobial Nod factors and the symbioticrelationship between Rhizobium and the legumes werecompared [53].

Acknowledgements

The authors are grateful to I. Komaniecka for help with some

experiments and to Dr T. Urbanik-Sypniewska for critical reading of

the manuscript and helpful discussion.

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Characterization of Mesorhizobium huakuii lipidA containing both