Characterization of
Mesorhizobium huakuii
lipid A containing both
D
-galacturonic acid and phosphate residues
Adam Choma
1
and Pawel Sowinski
2
1
Department of General Microbiology, Maria Curie-Sklodowska University, Lublin, Poland;
2
Intercollegiate NMR Laboratory,
Department of Chemistry, Technical University of Gdansk, Poland
The chemical structure of the free lipid A isolated from
Mesorhizobium huakuii IFO 15243
T
was elucidated. Lipid A
is a mixture of at least six species of molecules whose struc-
tures differ both in the phosphorylation of sugar backbone
and in fatty acylation. The backbone consists of a b (1¢fi 6)
linked 2,3-diamino-2,3-dideoxyglucose (DAG) disaccharide
that is partly substituted by phosphate at position 4¢.The
aglycon of the DAG-disaccharide has been identified as
a-
D
-galacturonic acid. All lipid A species carry four amide-
linked 3-hydroxyl fatty residues. Two of them have short
hydrocarbon chains (i.e. 3-OH-i-13:0) while the other two
have longer ones (i.e. 3-OH-20:0). Distribution of 3-hydroxyl
fatty acids between the reducing and nonreducing DAG is

endotoxic properties of lipopolysaccharide. The structure of
lipid A seems to be essential in maintaining outer membrane
integrity and flexibility and is crucial for bacterial cell
viability [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 LPS
modifications in Rhizobium [6].
The structures of Rhizobium lipid A indicate great
variation in the glycosyl component of its backbone as well
as the acylation pattern. The lipid A backbone of Sinorhizo-
bium is similar to that from enteric bacteria [7,8]. Lipids A
from Rhizobium etli and biovars of Rhizobium legumino-
sarum have identical and unusual structures. R. etli lipids A
are devoid of phosphate groups [9–11] and a galacturonic
acid residue replaces the 4¢-linked phosphate in the lipid A
backbone. The distal part (distant from the reducing end of
the backbone) of lipid A is almost the same for all lipid A
species isolated. The proximal glucosamine is partly oxi-
dized to 2-aminogluconate [12,13]. A specific deacylase
removes the ester-linked fatty acids from the C-3 position
of the lipid A precursor, thus this hydroxyl is only partially
substituted 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 to
2-aminogluconate by (1fi 6) glycoside linkage. When
compared with R. etli this lipid A lacks galacturonic acid

thedistalDAG,and(b)witha-linked galacturonic acid
at position 1 of the proximal unit. Phosphorylated and
nonphosphorylated lipid A preparations are a mixture of
three subfractions differing in acylation patterns.
Experimental procedures
Bacterial strain, growth, and isolation of
lipopolysaccharide and lipid A
Mesorhizobium huakuii IFO15243
T
strain was obtained
from the Institute for Fermentation, Osaka, Japan. Bacteria
were grown at 28 °C in liquid mannitol/yeast extract
medium 79CA [27] and were aerated by vigorous shaking.
Cells were centrifuged at 10 000 g, washed twice with saline
and once with distilled water. The wet bacterial paste was
extracted by the modified hot phenol/water procedure [28].
The water layer was dialysed firstly against tap water, then
against distilled water. The crude LPS was purified by
repeated ultracentrifugation at 105 000 g for 4 h. The LPS
solution (5 mgÆmL
)1
) in aqueous 1% (v/v) acetic acid was
kept at 100 °C for 3 h. The lipid A precipitate was collected
by centrifugation, washed twice with hot distilled water and
lyophilized.
Purification and separation of lipid A species
Crude lipid A was purified and separated into subfractions
according to a modified procedure described by Que and
coworkers [9]. Briefly, lyophilized lipid A ( 30 mg) was
dissolvedin20mLofCHCl

by adding the appropriate amount of water and chloroform.
Water layers were discarded and organic layers were
supplemented with fresh portions of the upper phase of
a freshly prepared two-phase Bligh–Dyer mixture. The
washed organic layers were separated by centrifugation and
dried. Preparations were stored at )20 °CinCHCl
3
/
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 acidic
sugars were determined by gas-liquid chromatography and
mass spectrometry. For this analysis, lipid A samples were
methanolysed (1
M
HCl, 80 °C, 18 h), N-acetylated and
trimethylsililated [29]. The content of phosphorus in lipid A
was determined according to Lowry [30].
Chemical modification of lipid A
Subfractions of lipid A (about 2 mg) were dephosphory-
lated in 48% (v/v) aqueous HF at 4 °C for 48 h [31]. HF was
removed by evaporation in the stream of nitrogen with
cooling in an ice bath. De-O-acylation of lipid A subfractions
was performed according to modified procedure of Haishima
and coworkers [31]. Preparations were treated with anhy-
drous hydrazine at 37 °C for 2 h. The reaction mixtures, after
cooling, were poured into cold acetone. The resulting lipid A
precipitates were collected, washed twice with acetone and
then gently dried in the stream of nitrogen.

about 256 laser shots.
Liquid matrix-assisted secondary ion mass spectrometry
(LSIMS) was performed using AMD 604 (AMD Intectra
GmbH) mass spectrometer operated in the negative ion
mode with primary ion beam of Cs
+
.Samplesweremixed
with 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 ion
mode. The samples were dissolved in a CHCl
3
/CH
3
OH
(2 : 1; v/v) mixture supplemented with 0.1% (v/v) concen-
trated ammonia and introduced into electrospray source
at a flow rate of 5 lLÆmin
)1
.
NMR spectroscopy
1
H-NMR experiments were performed in CDCl
3
/dimethyl-
sulfoxide-d
6
(2 : 1; v/v) mixture. 2D (DQF COSY, TOCSY,
NOESY)
1

The compositional analysis of crude lipid A preparation
obtained from M. huakuii IFO 15243
T
LPS revealed the
presence not only of 2,3-diamino-2,3-dideoxyglucose
(DAG) and a complex set of fatty acids (both ester and
amide bound), as described previously [24], but also
galacturonic acid and phosphate residues. The presence of
GalA was unequivocally confirmed by GC-MS analysis of
trimethylsilil ethers of methyl glycosides liberated from
lipid A by methanolysis (Fig. 1). The
31
P-NMR spectrum
of the crude lipid A revealed a prominent signal with
chemical shift of 1.71 p.p.m. observed at neutral pH. This
signal was shifted to 4.71 p.p.m. when the pH of the lipid A
suspension was raised to 10.6 (Fig. 2). These properties are
indicative of phosphomonoesters other than glycosyl-
1-phosphate. The location of the phosphate was directly
determined by two-dimensional heteronuclear magnetic
resonance (see below). On the basis of chemical shift value
and lack of the cross peak with protons from the lipid A
backbone on the
31
P/
1
H-HSQC spectrum, the weak signal
at 1.88 p.p.m. was attributed to inorganic phosphate
impurities of the lipid A preparation. The results of
quantitative measurements of phosphorus and DAG con-

Fig. 2.
31
P-NMR spectra of the crude lipid A from Mesorhizobium
huakuii IFO 15243
T
. 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) Ó FEBS 2004
amide- and ester-linked fatty acids was approximately 2 : 1.
Therefore, one can expect that DAG type lipid A could
contain not more than six acyl residues.
The complex mixture of the lipid A preparation was
separated into two fractions, based on DEAE gravity
column chromatography. The first fraction (designated
lipid A
–P
), which was eluted with solvent containing
250 m
M
ammonium acetate, was devoid of phosphate, as
shown by
31
P-NMR. The phosphate was detected in
the second fraction (named lipid A
+P
), successively eluted
with a solvent mixture containing 500 m
M
NH

the MALDI-TOF spectrum of lipid A
–P
treated with 48%
HF did not change significantly when compared with the
unprocessed 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 correspond
to lipid A molecules composed of two DAG, one of which
is phosphorylated, one GalA, four 3-hydroxyl fatty acids,
one (x-1) hydroxyl long chain fatty acid and one a nonpolar
fatty acyl residue. The most intense ion in this cluster (m/z
at 2357) could be attributed to the molecules of lipid A
containing two 3-OH-i-13:0 and two 3-OH-20:0 acids, as
well as two ester-bound acids (e.g. 20:0 and 27-OH-28:0).
This is merely one possible explanation due to the fact that
numerous combinations of fatty acids different to those
found in lipid A exist. However, taking into consideration
the quantities of lipid A fatty acids this proposition seems
to be the most probable. The amide-bound fatty acids
isolated from M. huakuii IFO15243
T
lipid A and from
other mesorhizobia can be separated into two clusters
[24–26]. The first contains short chain fatty acids, mainly
3-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 masses
found on the MALDI-TOF spectra it is necessary to take

originated from hexaacyl phosphorylated lipid A (pro-
minent ions at m/z 1481 and 1508). The second species
[B
1
+
(Y)
] consist of ions with masses close to that at m/z
1187. Those ions are made up of DAG, two 3-hydroxyl
fatty acyl moieties and (x-1) hydroxyl long chain fatty
acid. Those B
1
+
fragment ions support the conclusion that
the 27-hydroxyoctacosanoic acid and eicosanoic acid,
when present, are located on the distal diaminoglucosyl
residue of the lipid A. Moreover, the sugar component of
B
1
+
lacks hydroxyl groups suitable for attachment of
these fatty acids by ester bonds. The appropriate hydroxyls
are located at positions 3 of amide linked acyl of the distal
DAG. Therefore, both 27-OH-28:0 and 20:0 fatty acids are
components of acyloxyacyl residues. The predicted ions for
the third type of oxonium ions composed of DAG and
two amide acyl residues have not been registered, due to
the fact that the spectra were usually recorded from m/z
1000–3000. The correct calculation of masses for B
1
+

Table 1. Data from mass spectrometry analyses. Positive and negative ions derived from phosphorylated and nonphosphrylated lipid A fractions of Mesorhizobium huakuii IFO 1243
T
; their compositions and
proposed structures. Backbone*, trisaccharide of b-
D
-DAG-(1 fi 6)-a-
D
-DAG-(1fi1)-a-
D
-GalA; P, phosphate residue; nd, not determined.
Positive mode MS Negative mode MS Composition
MALDI-TOF
(ion type and
m/z-value)
MALDI-TOF
(ion type and
m/z-value)
ES-MS
(ion type
m/z-value)
molecular mass
calculated from
ES-MS value
DAG P GalA
3-OH-
fatty
acids
(x-1)-OH
-fatty
acids

[M-H]
–
2063
[M-2H]
)2
1030.7
2063.4 2 1 1 4 1 0 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 1 1 4 0 0 66 Backbone, phosphate,
2 · 3-OH-20:0,
2 · 3-OH-i-13:0
1640.137
B
1
+
1481
– – – 1 1 1 2 1 1 81 P-DAG,
1 · 3-OH-20:0,

1
+
1400
– – – 1 0 1 2 1 1 81 DAG
2 · 3-OH-20:0,
2 · 3-OH-i-13:0,
27-OH-28:0, 20:0
1401.297
B
1
+
1108
– – – 1 0 1 2 1 0 61 DAG
2 · 3-OH-20:0,
2 · 3-OH-i-13:0,
27-OH-28:0
1106.774
1314 A. Choma and P. Sowinski (Eur. J. Biochem. 271) Ó FEBS 2004
The B
1
+
ions from lipid A
+P
(e.g. m/z at 1187 and 1508,
Fig. 3B) differed by 80 mass units from those originating
from 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 higher

and lipid A
–P
were dissolved in a
mixture of dimethylsulfoxide (DMSO-d
6
)andchloroform
(CDCl
3
) for NMR experiments. Figure 5 shows the one-
dimentional proton spectrum of de-O-acylated lipids A
+P
.
1
Hand
13
C chemical shift assignments were based on 2D
homonuclear experiments: DQF-COSY (Fig. 6), TOCSY
(Fig. 7) and
1
H/
13
C heteronuclear single quantum coher-
ence (HSQC) experiments. The values of carbon and proton
chemical shifts are summarized in Table 2.
Three signals were identified in the anomeric region of
13
C-NMR chemical shifts for both lipid A fractions. These
data suggested that the lipid A backbone contains three
sugar residues. Four signals were found between 50 and
55 p.p.m. for each preparation. They were assigned to the

-3
showed a coupling with H
B
-4 (d
H
¼ 3.48 p.p.m.). The
remaining glycosyl proton cross-peaks were observed at
following chemical shifts: 3.48 p.p.m./3.97 p.p.m. (H
B
-4/
H
B
-5), 3.97 p.p.m./3.60 p.p.m. (H
B
-5/H
B
-6a), 3.60 p.p.m./
3.89 p.p.m. (H
B
-6a/H
B
-6b). The proton chemical shifts for
both sugar ring systems (A and B) were similar to those
published for A. pyrophilus lipid A [36]. Chemical shifts of
the distal aminosugar (sugar ring system C) in lipid A
+P
were in good agreement with those from A. pyrophilus
lipid A distal DAG, however, two shift exceptions (for H
C
-4

1
type ion clusters derived by cleavage of the glycosidic linkage in lipid A.
1316 A. Choma and P. Sowinski (Eur. J. Biochem. 271) Ó FEBS 2004
d
H
¼ 4.01 p.p.m., which was about 0.3 p.p.m. downfield
from the A. pyrophilus lipid A equivalent signal and about
0.5 p.p.m. downfield from the H-4 signal characteristic of
DAG with unsubstituted hydroxyl group at C-4 carbon
atom (d
H
for H
B
-4, Table 2). The downfield shift of H
C
-4
was caused by the presence of ester-bound phosphate
residue. Analysis of carbon chemical shifts led to the same
conclusions, since C
C
-4 (d
C
¼ 71.9 p.p.m) appeared down-
field compared to the proximal C
B
-4 unsubstituted by
phosphate (d
B
¼ 67.5 p.p.m). The location of phosphate
substituent on C

; 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 (
1
H) and 125.7 MHz (
13
C) in DMSO-d
6
/CDCl
3
(2 : 1, v/v).
Residue
(spin system)
DAG-II (C) DAG-I (B) GalA (A)
1
H d (J,[Hz])
13
C d
1
H d (J,[Hz])
13
C d
1
H d (J,[Hz])
13
C d
Lipid A

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 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. The inter- and intraresidue
signals are labeled starting from anomeric
protons. Diagnostic interresidue cross peaks
are underlined.
1318 A. Choma and P. Sowinski (Eur. J. Biochem. 271) Ó FEBS 2004
H
B
-1 of the proximal DAG. Both sugars possess a
anomeric configurations that are reflected in the small
values of J
1,2
coupling constants and the appropriate
values of chemical shifts. The downfield shift of carbon
C
B
-6 from the proximal DAG and strong cross peak
H
C
-1/H
B

Ó FEBS 2004 Structure of Mesorhizobium huakuii lipid A (Eur. J. Biochem. 271) 1319
Lipid A
–P
is deprived of phosphate. The 1D
31
P-NMR
spectrum contained only a trace signal, which in the
1
H/
31
P-
HSQC experiment gave weak intensity correlation peak
with chemical shifts almost identical as for lipid A
+P
.
Therefore, one ought to conclude that lipid A
–P
preparation
was contaminated with traces of the phosphorylated
lipid 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 distal
DAG. The absence of phosphate caused an upfield shift
of both proton H
C
-4 and carbon C

C
-2; 3.73 p.p.m./
7.46 p.p.m and H
C
-3/NH
C
-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 in
this paper. NMR data of those components for M. huakuii
IFO15243
T
lipid A are very similar to the data published
earlier for A. pyrophilus [36], R. etli CE3 [10], Rhizobium sp.
Sin-1 [16] and other lipid A preparations.
Discussion
Mesorhizobium huakuii IFO 12543
T
lipopolysaccharide
appears to posses a unique lipid A. Two backbone species
have been identified. The complete structure of the first
species was characterized as (HO)
2
PO
2
-b-
D
-DAG(1fi6)-a-
D

lipid A from bacteria belonging to the Rhizobiaceae.
According to the current knowledge, the first steps of
lipid A biosynthesis seem to be the same in all Gram-
negative bacteria and lead to a 1,4¢-bisphosphorylated
aminosugar disaccharide acylated at positions 2, 3, 2¢,3¢ by
3-hydroxyl fatty acids. Kdo disaccharide occupies position
O-6¢ on lipid A [9,42,43]. Usually, UDP-glucosamine is
a precursor for this pathway, but it is well known that
the same or very similar pathway exists within bacteria
synthesizing mixed or DAG-type lipid A [44]. Thus, com-
mon lipid A precursor molecules (Kdo)
2
lipidIV
A
,arethe
point at which the biosynthesis pathways for enterobacteria
and rhizobia diverge. In Mesorhizobium cells, the common
lipid A precursor should be processed by 1-phosphatase,
following galacturonic acid transferase, before maturation.
The predicted 4¢-O phosphatase would carry out its partial
dephosphorylation. A computer analysis of M. loti
MAFF303099 genome sequence ( />rhizobase/), a bacterium closely related to M. huakuii
[19,45], revealed sequences (mll1545, mll0630, mlr8270) with
high homology to the genes encoding key enzymes in the
lipid A biosynthesis pathway (lpxC, lpxB and lpxK, respect-
ively). Basu and coworkers [46] described a 9 kb DNA
fragment 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 a

Those fatty acids seem to be important but not essential for
symbiosis because the acpXL::kan mutant of R. legumino-
sarum lacking the 27-OH-28:0 acid in its lipid A is still able
to form nitrogen-fixing nodules [52].
Lipid A is a valuable marker in chemotaxonomic studies
of rhizobia. It is well known that the chromosomal
1320 A. Choma and P. Sowinski (Eur. J. Biochem. 271) Ó FEBS 2004
background of rhizobia varies considerably. This is also
reflected in rhizobial lipids A. The diversity in lipid A
structures indirectly confirms the hypothesis that symbiotic
N
2
-fixing bacteria evolved from nonsymbiotic and non-
related ancestors by horizontal transfer of symbiotic genes
(nod, nif, fix) as symbiotic islands. Similar conclusions were
reached when rhizobial Nod factors and the symbiotic
relationship between Rhizobium and the legumes were
compared [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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