N-Methylation in polylegionaminic acid is associated
with the phase-variable epitope of
Legionella pneumophila
serogroup 1 lipopolysaccharide
Identi®cation of 5-(
N
,
N
-dimethylacetimidoyl)amino- and 5-acetimidoyl
(
N
-methyl)amino-7-acetamido-3,5,7,9-tetradeoxynon-2-ulosonic acid
in the O-chain polysaccharide
Oliver Kooistra
1
, Edeltraud LuÈ neberg
2
, Yuriy A. Knirel
1,3
, Matthias Frosch
2
and Ulrich ZaÈ hringer
1
1
Research Center Borstel, Center for Medicine and Biosciences, Borstel, Germany;
2
Institute for Hygiene and Microbiology,
University of Wu
È
rzburg, Germany;
3

dimethylacetimidoyl)amino and acetimidoyl(N-methyl)
amino groups, could be allocated to the long- a nd middle-
chain O-polysaccharide species, respectively. N-Methylation
of legionaminic a cid that w as absent fro m the i sogenic
mutant 5215 and from the spontaneous phase variant 811,
correlated with the presence of the mAb 2625 epitope.
Keywords: N-methylation; lipopolysaccharide; O-chain
polysaccharide; phase variation; Legionella pneumophila.
Legionella pneumophila is a facultative intracellular parasite
and the cause o f legionellosis, a pneumonia with sometimes
fatal progression [1]. The reservoirs of legionellae are
natural or man-made water systems and their natural hosts
are various amoebae species [2]. In the human lung
L. pne umophila invades and replicates within alveolar
macrophages [ 3]. The serogroup-speci®c antigens of the
Gram-negative legionellae reside in the lipopolysaccharide
(LPS) of the outer membrane [4,5].
The chemical structure of L. pneumophila serogroup
(Sg) 1 LPS has been extensively studied [6±12] (Fig. 1).
The O-chain polysaccharide (OPS) of the LPS is an
a-( 2 ® 4)-linked homopolymer of the 5-N-acetimidoyl-
7-N-acetyl derivative of 5,7-diamino-3,5,7,9-tetradeoxynon-
2-ulosonic acid, termed legionaminic acid [7]. Initially, t he
D
-glycero-
L
-galacto con®guration was ascribed to legionam-
inic acid [7], but this was later revised ®rst to the
L
-glycero

Borstel, Germany. Fax: + 49 4537 188612, Tel.: + 49 4537 188462,
E-mail:
Abbreviations: LPS, lipop olysaccharide; OPS , O-c hain polysacc haride;
PS, polysaccharide; Sg, serogroup; GPC, gel-permeation chromatog-
raphy; HMBC, heteronuclear multiple-bond correlation; DEPT,
distortionless enhancement by polarization transfer; Kdo, 3-deoxy-
D
-manno-oct-2-ulosonic acid; Rha,
L
-rhamnose; BYCE, buered
charcoal yeast extract.
Note: part of this study was presented at the 20th International
Carbohydrate Symposium, Hamburg, Germany, August
13±September 1, 2000.
(Received 8 August 2001, revised 13 November 2001, accepted 16
November 2001)
Eur. J. Biochem. 269, 560±572 (2002) Ó FEBS 2002
linked to a terminal nonreducing
L
-rhamnose residue
(Rha
II
) of t he core oligosaccharide [ 9,10] (Fig. 1). The
LPS c ore oligosaccharide lacks heptose a nd phosphate,
contains abun dant 6-deoxy sugars and N-acetylated amino
sugars, and is highly O-acetylated [8±10,12]. Lipid A of
L. pneumophila Sg 1 consists of unusual long-chain and
branched fatty acids [6,8], which may account for its low
endotoxic potential [21±23].
Recently, phase-variable expression o f an LPS epitope of

the mAb 2625 LPS epitope was found to be associated with
alteration in virulence and serum resistance upon phase
variation [24], we were i nterested in the elucidation o f its
chemical structure. The aim of this study was to identify the
mAb 2625 epitope and to correlate it to virulence. For t his
purpose, the isogenic mutant 5215 was generated by
deletion of the LPS biosynthesis operon ORF 8±12 in the
virulent wild-type RC1. With the aid of this genetically
de®ned mutant that did not bind mAb 2625, we were able to
correlate the mAb 2625 epitope to N-methylation of the
5-acetimidoylamino group on legionaminic acid, a modi®-
cation that has not been found to date in bacterial
polysaccharides. Studies of binding of mAb 2625 to the
N-methylated legionaminic acid derivatives is described in
the accompanying paper [26].
MATERIALS AND METHODS
Bacterial strains and cultivation
L. p neumophila wild-type strain RC1 (Sg 1, subgroup
OLDA) is a virulent patient isolate that binds mAb 2625
[17,24]. The unstable phase-variant strain 811 derived from
strain RC1 is avirulent and does not bind mAb 2625 [24].
Phase variation is mediated by chromosomal insertion and
excision of a 29-kb genetic element of presumably phage
origin. It is presumed that a regulatory factor is affected
upon phase variation leading to numerous phenotypic
alterations [25].
L. pneumophila wild-type strain 5097 (Sg 1, subgroup
OLDA) was obtained from the American Type Culture
Collection, Rockville, MD ( ATCC 43109). Strain 5097 is
not virulent (presumably due to long-term passage on

-glucose (N-acetyl-
D
-quinovosamine); Rha,
L
-rhamnose; Leg and 4e -Leg,
derivatives of legionaminic and 4-epilegionaminic acid, respectively. OAc stands for O-acetyl group.
Ó FEBS 2002 N-Methylation in Legionella pneumophila LPS (Eur. J. Biochem. 269) 561
(Fig. 2 ) a 3-kb NsiI fragment (covering position 275 of ORF
8 to position 399 of ORF 11) was deleted and a kanamycin
resistance cassette was inserted inverse to the direction of
transcription of ORFs 8±12. The kanamycin cassette was a
1282 bp EcoRI restriction fragment f rom plasmid pUC4K
(Pharmacia). Orientation of the insertion w as con®rmed by
sequence analysis. The resulting construct was termed
pMH549 (Fig. 2). The entire 5.5-kb insert of pHM549
was excised with NotI±ApaI (restriction sites of the multiple
cloning site of pBC) and ligated into the EcoRV site of
plasmid vector pLAW344 [27], resulting in plasmid pMH12
(Fig. 2 ). pLAW344 harbours the sacB gene and allows
counter-selection for homologous recombination. pMH12
was introduced into L. pneumophila strainRC1byelec-
troporation and bacteria were plated on BYCE agar
supplemented with kanamycin (50 lgámL
)1
). Grown trans-
formants were harvested, suspended in sterile distilled water,
and plated on BCYE agar supplemented with kanamycin
and 5% sucrose for selection of homologous recombination
by double c ross-over. Homologous recombination w as
con®rmed b y Southern blot and PCR analysis in the

and a differential refractometer for monitoring
(Knauer). Fractions of  3 mL were collected.
SDS/PAGE and Western blot
SDS/PAGE was carried out in 14% polyacrylamide gels
using a Mini-Protean II system (Bio-Rad). LPS bands were
visualized by the silver-staining technique described else-
where [29]. For e xtracted LPS s amples, 100 lLstock-
solution (2 mgámL
)1
) was mixed with 100 lLsample
solution [30] and incubated at 95 °C for 5 min. Then,
0.75 lL(0.75lg LPS) of this solution was applied per lane.
For fractionated LPS, 1 mL of each fraction was dried in an
evaporation centrifuge, dissolved in 100 lLwater,mixed
with 100 lL sample solution [30], and incubated at 95 °C
for 5 min. The sample volume applied per lane varied from
0.25 t o 2.5 lL, depending on the refraction index o f the
corresponding fraction in GPC.
For Western blot analysis of extracted LPS samples, 2 lg
LPS per lane were applied to 12.5% polyacrylamide gels.
The sample volume of fractionated LPS applied per lane in
Western blot a nalysis varied from 0.75 to 7 .5 lLandwas
three times as high as that in silver-stained SDS/PAGE.
Fig. 2. Schematic depiction of plasmid constructs employed for generation of the ORF 8±12 mutant 5215. For detailed description see Materials and
methods. On top of the plasmids a schematic diagram of the 32-kb gene locus of L. pneumophila wild-type RC1 required for LPS biosynthesis is
depicted (adopted from [17]). The direction of the operons i s indicated by arrowheads and designation of selected ORFs are shown above gene
blocks.
562 O. Kooistra et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Western blotting onto nitrocellulose ®lter membranes was
carried out as described previously [31]. Immunostaining

total phosphate was carried out by the ascorbic acid method
[36]. Fatty acids of the lipid A portion were analysed by
GLC a nd combined GLC-MS as the methyl esters after
methanolysis (2
M
HCl/MeOH, 24 h, 120 °C) and trimethyl-
silylation with N,O-bis-(trimethylsilyl)-tri¯uoroacetamide
as described previously [6,37].
Preparation, modi®cation, and fractionation of the OPS
LPS each of wild-type RC1, mutant 5215, and phase variant
811 (750, 300, and 450 mg, respectively) was degraded at
100 °Cfor4hwith0.1
M
NaOAc/HOAc buffer (pH 4.4,
10 mg ámL
)1
LPS), and the resultant lipid A precipitate was
removed by centrifugation (5000 g, 30 min). The superna-
tant was l yophilized and desalted by G PC on a column
(2.5 ´ 50 cm; Bio-Rad) o f Sephadex G-50 (S) (Pharmacia)
using 50 m
M
pyridinium/acetate buffer (pH 4.3) and mon-
itoring with a differential refractometer (Knauer) to give the
corresponding polysaccharide (PS) portion.
PS was de-O-acetylated with 20% (v/v) aqueous NH
4
OH
at 37 °Cfor16h(10mgámL
)1

NH
4
OH/HF
was fractionated by t andem GPC on two
directly connected columns (2.5 ´ 120 cm each; Bio-Rad) of
Toyopearl TSK HW-50 (S) (Supelco) and Fractogel TSK
HW-40 (S) (Merck). Pyridinium/acetate (50 m
M
)buffer
(pH 4 .3) containing 10% (v/v) acetonitrile was used for
elution at a pump rate of 15 mLáh
)1
and a differential
refractometer (Knauer) f or m onitoring. Fractions of  3mL
were collected, appropriately combined into six pools, and
lyophilized.
NMR spectroscopy
1
HNMR,2D
1
H,
1
HNMR(COSYandNOESYwith
mixing time 300 ms), a nd H-detected
1
H,
13
CNMR
(HMQC and HMBC) spectra were recorded with a Bruker
Avance DRX-600 spectrometer.

enzymes ( e.g. SiaC of Neisseria m eningitidis [41]), and
ORF 11 showed vague homologies to plant methyl
transferases [42]. T he protei ns encoded b y t he remaining
ORFs 10 and 12 showed no homologies to known amino-
acid sequences. The only ORF 8±12 mutant accessible was
mutant 137 derived from the avirulent wild-type 5097.
Therefore, the isogenic ORF 8±12 deletion mutant 5215 was
constructed from wild-type RC1 by homologous recombi-
nation (Fig. 2) in order to compare the LPS structures of
wild-type RC1, the genetically de®ned mutant 5215, and the
phase variant 811. Unexpectedly, virulence and s erum
resistance were not affected in mutant 5215, when compared
to the parental wild-type RC1 (data not shown), which
revealed that t he mAb 2625 LPS epitope it self is not
associated with virulence of L. pne umophila strain RC1.
Characterization of LPS from wild-type RC1
and mutant 5215
In silver-stained SDS/PAGE (Fig. 3A), LPS from wild-type
RC1 and mutant 5215 displayed no difference in b anding
pattern. Both LPS showed a characteristic bimodal distri-
bution by OPS chain-length. In Western blot analysis, LPS
from mutant 5215 did not bind to mAb 2625 (Fig. 3B) but
bound to mAb LPS-1 in a different manner compared to
LPS from wild-type RC1 (Fig. 3C). MAb LPS-1 is known
to r ecognize speci®cally a conserved epitope located in the
outer core region of the LPS from L. pneumophila Sg 1
strains [12,24,43].
Chemical analy ses of both wild-type R C1 and mutant
5215 L PS revealed
L

and mutant 5215 [44]. Fatty acid composition of lipid A of
both strains was almost identical as well [44].
Investigations of the core oligosaccharide [12] and t he
lipid A backbone [44] of the LPS from phase variant 811
revealed that both were unchanged compared to wild-type
RC1. However, lipid A of phase variant 811 contained
3-hydroxylated fatty acids of different chain-length
compared to lipid A of wild-type RC1 [44].
Fractionation of LPS by GPC
To determine the location of the mAb 2625 epitope, LPS
from wild-type RC1 and mutant 5215 was fractionated by
GPC on Sephacryl S-200 HR. The refraction index elution
pro®le of the GPC of wild-type RC1 LPS (Fig. 4A)
indicated the same characteristic bimodal distribution of
long and short O-chain LPS species as in SDS/PAGE
(Fig. 3 A). A similar p ro®le showing a low amount of long
O-chain LPS species and a relatively high amount of short
O-chain L PS species w as observed f or mutant 5215 LPS
(Fig. 5 A). Silver-stained SDS/PAGE after GPC of the wild-
type RC1 LPS revealed 33 fractions (nos 96±128) containing
LPS species with different OPS chain-lengths ( Fig. 4B).
A ladder-like pattern was observed with differences of up
to 10 sugar residues within each f raction and with overlap-
ping about 6±8 r esidues in e ach p air of neighbouring
fractions.
In Western blot analysis, mAb 2625 bound only to the
wild-type LPS species from fractions 96±116, and did not
bind to LPS molecules b elow a certain size, i.e. a certain
OPS chain-length (Fig. 4C). In contrast, mAb LPS-1
exclusively bou nd to the LPS species from fractions

(wild-type R C1, m utant 5 215, and phase varia nt 811) to
cleave the lipid A moiety followed by GPC on Sephadex
G-50 (S). Based on the ®nding that the OPS is linked to the
terminal
L
-rhamnose residue of the LPS core oligosacchar-
ide (Rha
II
) [9] and the observation that 48% aqueous HF
selectively cleaves the glycosidic linkage of 6-deoxy sugars in
polysaccharides [38], a protocol was elaborated to remove
core oligosaccharide constituents from PS. When the intact
LPS w as treated with 48% aqueous HF and separated by
SDS/PAGE, Western blot analysis revealed that although
the OPS of the LPS was partially cleaved, the mAb 2625
epitope in the remaining LPS species was not affected. After
de-O-acetylation of th e LPS, the HF treatment completely
cleaved the OPS of the LPS. In this case, silver-stained
SDS/PAGE showed only low-molecular-mass molecules
resembling rough-type LPS, which did not react with mAb
2625 in Western blot. In contrast to the glycosidic linkage of
6-deoxy sugars, e.g.
L
-rhamnose [38], that of legionaminic
acid and its N-linked substituents are s table under the same
conditions [14].
Therefore, PS was de-O-acetylated, treated with 48%
aqueous HF, a nd the resultant modi®ed polysaccharide
(PS
NH

5.06 and Rha
II
H1
d
H
4.99; Fig. 9A±C), which was in acco rdance with
identi®cation of only
L
-rhamnose by GLC analysis of the
alditol a cetates derived from PS
NH4OH/HF
. T herefore, the
isolated PS
NH
4
OH/HF
species from all three strains were
composed of legionaminic acid a nd
L
-rhamnose, whereas
the major portion of the core o ligosaccharide was cleaved
by HF treatment. A
1
H,
13
C H MQC e xperiment d emon-
strated t hat PS
NH
4
OH/HF

of legionaminic acid
N-Methyl groups in bacterial polysaccharides occur rarely
[46] and published data are only scarce. Therefore, careful
NMR spectroscopic analysis was used to elucidate the
structure of N-methylated derivatives of legionaminic acid.
Comparison of the
1
H N MR spectra of the OPS of pool
III from all three investigated strains revealed four signals
between 2.9 and 3.3 p.p.m. (all singlets; Fig. 9, panels A and
D), whose presence correlated with the reactivity of mAb
2625 with LPS in Western blot. The signals were observed in
long- and middle-chain OPS from wild-type RC1, but in no
OPS from mutant 5215 (Fig. 9, panels B and E). In phase
variant 811, these signals were recognized in the OPS of the
same pools as in wild-type RC1 but were 10- to 20-fold less
intense (Fig. 9, panels C and F). Except for the region of the
four signals, the
1
H NMR spectra of the polysaccharides
from all three strains were almost identical (compare Fig. 9,
panels A±C). The middle-chain OPS of pool III from wild-
type RC1 having 15±20 residues of legionaminic acid was
the smallest one that displayed t hese signals and, moreover,
the relative intensity of these signals was the highest
compared to other pools from the same strain. Therefore,
further structural investigation was performed with this
preparation.
The
1

30.93 and 29.86, which were partially
Fig. 6. Fractionation of OPS (PS
NH
4
OH/HF
)fromL. pneumophila wild-
type RC1 by tandem gel-permeation chromatography on Toyopearl TSK
HW-50 (S) and Fractogel TSK HW-40 (S). Pools I, III, and V corre-
spond to long-, middle-, and short-chain PS
NH
4
OH/HF
, respectively;
pools II, IV, and VI correspond to intermediate fractions.
Ó FEBS 2002 N-Methylation in Legionella pneumophila LPS (Eur. J. Biochem. 269) 565
superimposed on the signals of the major higher-®eld pair in
the
1
H NMR spectrum (Fig. 10, right panel).
In the
1
H,
13
C HMBC spectrum (Fig. 10, left panel), the
proton signals of t he major lower-®eld pair at d
H
3.30 and
3.19 cross-correlated to the lower-®eld carbon signals at d
C
40.74/40.60 and 42.95/42.89. Furthermore, both proton

C
20.21 of another N-acetimidoyl group, r espectively
(Fig. 10, left panel). In addition, each proton signal
correlated to a signal of a nitrogen-bearing carbon (C5) of
legionaminic acid (d
C
55.93 and 57.32). Taken t hese data
together, it was concluded that there is present a
5-acetimidoyl(N-methyl)amino group that occurs as two
stereoisomers (Fig. 8, structure 3). An N-methylacetamido
group could b e excluded b ased on identi®cation, using 2D
NMR experiments, of a nonmethylated 7-acetamido group
of this particular legionaminic acid residue (Table 2). A
methylamino group was excluded based on published data
of the methylamino derivative of
L
-fucose i n the LPS of
Bordetella pertussis strain 1414 [48,49], in which the
N-methyl group gave a single singlet in the
1
HNMR
spectra. Similarly, the minor pair of signals at d
H
2.97 and
2.94 was a ssigned to a 5-( N-methylacetimidoyl)amino group.
A NOESY experiment (F ig. 11) was applied to stereo-
chemical analysis of the N-methylated acetimidoylamino
(acetamidine) groups in 2 and 3, which may occur as
stereoisomers due to a partial double-bond character of the
linkages at both nitrogens. A strong NOE correlation was

H
4.99, integration value set at 0.7) in the ® 3)-
a-
L
-Rha
II
-(1 ® 3)-
L
-Rha
I
disaccharide are
superimposed on the spectra. The insets show
the region between 2.8 and 3.4 p.p.m. ex-
tended 5-fold. Bold numbers refer to struc-
tures shown in Fig. 8. For abbreviations see
legend to Fig. 9.
566 O. Kooistra et al. (Eur. J. Biochem. 269) Ó FEBS 2002
2 belonged to the N-methyl groups at N
1
in trans and cis
orientation to N
2
of the acetamidine group, respectively
(Fig. 8 ; t he descriptors cis and trans for the tw o N-methyl
groups in 2 are used only to designate their positions relative
to N
2
and d o not refer to stereoisomerism). The lower-®eld
1
H NMR signal of 3 belonged to the N-methyl group at N

3.37 Z)andH3
ax
,3
eq
(axial:
d
H
1.86 E, d
H
1.89 Z, equatorial: d
H
2.48 E, d
H
2.51 Z)in
COSY, and for H7 (d
H
3.68 E, d
H
3.75 Z)andC Oofthe
7-acetamido group (d
C
175.91 E, d
C
175.74 Z)inthe
1
H,
13
C
HMBC experiment. In addition, signals for H9
(d

from wild-
type RC1 were investigated by 1D
1
H NMR spectroscopy
and signal integration was performed to calculate the
average chain-length of the PS
NH
4
OH/HF
and the distribution
of N-methylated legionaminic acid derivatives. The signal of
a-
L
-Rha
II
H1 (d
H
4.99) in the ® 3)-a-
L
-Rha
II
-(1 ® 3)-
L
-
Rha
I
disaccharide was used as a reference, because it was the
Fig. 8. Structures of 5-acetimidoylamino-7-acetamido-3,5,7,9-tetra-
deoxy-
D

d
C
(p.p.m.)
5-N-Acetimidoyl (1) 20.08 167.61
5-N-(N,N-Dimethylacetimidoyl) (2-E) 40.74 (cis) 42.95 ( trans) 16.63 167.03
40.60 (cis) 42.89 ( trans)
5-N-(N-Methylacetimidoyl) (minor) 30.93 (E) 29.86 ( Z ) ND ND 167.22 (E) 167.05 (Z)
5-N-Acetimidoyl-5-N-methyl (3) 32.56 (E) 33.73 ( Z) 20.21 (E) 20.49 ( Z) 168.23 (E) 169.02 (Z)
Ó FEBS 2002 N-Methylation in Legionella pneumophila LPS (Eur. J. Biochem. 269) 567
only anomeric proton present in a single anomeric con®g-
uration. Integration of t he signals o f 1D
1
H NMR spectra
(Fig. 7 ) indicated t hat the average chain-length of long-,
middle-, and short-chain PS
NH
4
OH/HF
(Fig. 6 , pools I, III,
and V ) i s about 40, 18, and 1 0 legionaminic acid r esidues.
Theratioofthe5-N-( N,N-dimethylacetimidoyl)-7-N-acetyl
and 5-N-a cetimidoyl-5-N-methyl-7-N-acetyl derivatives of
legionaminic acid was 1 : 1 in long-chain and 1 : 2 in
middle-chain PS
NH
4
OH/HF
, respectively. Based on the rela-
tive intensities of the proton signals it was concluded t hat
only one legionaminic acid residue is N-methylated in each

-7-N-acetyl-5-N-(Z) 1.89 2.51 3.37 ND 4.13 3.75 4.13 1.16 ND
methyl- (3)
C1 C2 C3 C4 C5 C6 C7 C8 C9 NAc
CH
3
NAc
C O
5-N-Acetimidoyl
-7-N-acetyl ( 1)
174.47 101.86 39.40 71.86 54.30 72.76 55.53 67.92 19.65 23.23 175.32
5-N-Acetimidoyl
-7-N-acetyl-5-N-
methyl- (3)
(E) ND ND 32.04x 72.66 57.32 68.41 55.01 68.41 19.44 23.71 175.91
(Z) ND ND 31.86 71.23 55.93 68.37 54.94 68.37 19.41 23.67 175.74
Fig. 9. 600-MHz
1
H NMR spectra of pool III
of the OPS (PS
NH4OH/HF
)fromthree
L. pneumophila strains. Left panel: the full
spectra of pool III from wild-type RC1 (A),
mutant 5215 (B), and phase variant 811 (C).
Assignment was made using 2D NMR
experiments and published data [7]. Right
panels: part of spectra of pool III from wild-
type RC1 (D), mutant 5215 (E), and phase
variant 811 (F). NMe
cis

1
dimension. The c orresponding
13
Cand
1
H NMR spectra are displayed along F
1
and F
2
axes. Spectra were
recorded at 600 MHz a nd 27 °C. For abbreviations see legend to Fig. 9. Cross-peaks marked by  be long to an unknown minor iso mer of
legionaminic acid.
Fig. 11. Part of a NOESY spectrum of
pool III of the OPS (PS
NH
4
OH/HF
)from
L. pneumophila wild-type RC1. The spectrum
was recorded at 600 MHz and 27 °Cusinga
mixing time 300 ms. For abbreviations see
legend to Fig. 9. Cross-pe aks marked by
 belong to an unknown minor isomer of
legionaminic acid.
Ó FEBS 2002 N-Methylation in Legionella pneumophila LPS (Eur. J. Biochem. 269) 569
mAb 2625 epitope is present in wild-type cells and is lost in
the avirulent switch mutant upon phase variation [24]. The
loss of the reactivity with mAb 2625 affects the epitope of
another LPS-speci®c monoclonal antibody, mAb LPS-1,
which is speci®c for the outer core region of the LPS from

tive analysis of the fractionated OPS from wild-type RC1
and m utant 5215 revealed min or c omponents, which
occurred only in the wild-type polysaccharide species
above a speci®c chain-length and whose presence corre-
lated with the reactivity of mAb 2625 with LPS in
Western blot.
The minor components were identi®ed as 5-N-(N,N-
dimethylacetimidoyl)-7-N-acetyllegionaminic acid (2)and
5-N-acetimidoyl-7-N-acetyl-5-N-methyllegionaminic acid
(3). In addition, 5-N-(N-methylacetimidoyl)-7-N-acetyl-
legionaminic acid was detected in a negligible amount, which
could either be a biosynthetic precursor or a degradation
product of 2. N o N -methylated a cetimidoylamino group
has been hitherto found in bacterial polysaccharides. Only
one N-meth ylated legionaminic acid residue is present in
each polysaccharide chain above a certain length. This fact
may be the reason for the failure to identify the
N-methylated derivatives in previous studies of the
nonfractionated O PS of L. pneumophila using
13
CNMR
spectroscopy [8].
Investigation of b inding af®nities by surface plasmon
resonance biomolecular i nteraction analysis revealed that
only long- and middle-chain PS
NH
4
OH/HF
from wild-type
RC1 bind to mAb 2625 but not short-chain PS

.A
lack of the mAb LPS-1 epitope due to a c ore modi®cation
can be excluded as no such modi®cation was found by
detailed structural studies of the puri®ed core oligosaccha-
rides from wild-type RC1, mutant 5215, and phase variant
811 [12]. Therefore, there is a strong indication for the
location of the N-methylated legionaminic acid residue
proximal to the LPS core oligosaccharide, though its
location at any o ther position of t he OPS c ould not be
strictly excluded.
The expression of the N-methyl-associated epitope is
suppressed in the phase variant 811. The presence of 10- to
20-fold lower amounts of 2 and 3 in strain 811 could be due
to the presence of a 10% portion of wild-type revertant cells
in the phase-variant population [24]. As mutant 5215 is as
virulent as wild-type RC1 but completely lacks the
N-methylated derivatives of legionaminic acid, it can be
excluded that the N-methyl groups are determinants of
virulence. More likely, different phenotypic alterations may
occur upon phase variation and the loss of the mAb 2625
epitope is one of them. Phase variation in L. pneumophila
was f ound to in¯uence other LPS biosynthesis pathways
involved in assembly of lipid A. Phase variant 811 expressed
a different fatty acid pro®le as compared to wild-type RC1
and mutant 5215 [44]. Although the characteristic secondary
long-chain fatty acids, such a s 2 8:0(27-oxo) and 27:0(1,27-
dioic), are present in all three s trains, positions 2 and 2¢ of
the lipid A backbone are p redominantly occupied by
16:0(3-OH) and 18:0(3-OH) in avirulent phase variant 811
but by 20:0(3-OH) in the two virulent strains [ 44].

the N-methyl groups may a djust the appropriate hydro-
phobicity and the appropriate charge to the legionaminic
acid residue, which intervenes between the highly
hydrophobic outer core region of the LPS and the OPS
having a zwitterionic character. Another putative function
of the N-methylated derivatives may be r elated to the fact
that phase variation is mediated by a genetic element o f
possibly phage origin. The N-methyl groups located close to
the core oligosaccharide may play a role o f a signal or a
receptor for phage particles, though no bacteriophage has
been yet described for L. pneumophila.
However, the most interesting question is in which way
the d ifferent phenotypic alterations that occur upon phase
variation in L. pneumophila might be connected . Our
current hypothesis is that phase variation affects a regula-
tory factor , w hich in¯uences LPS biosynthesis, virulence,
and serum resistance. Future studies will focus on the
elucidation of such factor and the mechanism of its ef®cacy.
ACKNOWLEDGEMENTS
We thank Dr B. Lindner and Mrs H. Lu
È
thje for performing MALDI-
TOF MS, H P. Cordes for running NMR spectra, and H. Moll for
expert help with GLC-MS. The skilf ul technical assistance of Mrs
K. Jakob is gratefully acknowledged. This work was supported by
grants from the Deutsche Forschungsgemeinschaft, LU 514/2±2 (E. L.
and M. F.) and ZA 149/3±2 (U. Z.), grant 436 R US 113/314 /0 from the
Deutsche Forschungsgemeinschaft (Y. A. K. and U. Z.), and grant
00-04-04009 from the Russian Foundation for Basic Research
(Y.A.K.).

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