Báo cáo khoa học: N-Methylation in polylegionaminic acid is associated with the phase-variable epitope of Legionella pneumophila serogroup 1 lipopolysaccharide pptx

Eur. J. Biochem. 269, 560±572 (2002) Ó FEBS 2002

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 Kooistra1, Edeltraud Luneberg2, Yuriy A. Knirel1,3, Matthias Frosch2 and Ulrich Zahringer1
È
È
1

Research Center Borstel, Center for Medicine and Biosciences, Borstel, Germany; 2Institute for Hygiene and Microbiology,
University of WuÈrzburg, Germany; 3N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia

Previously, a phase-variable epitope was detected in the
virulent wild-type strain RC1 of Legionella pneumophila
serogroup 1 subgroup OLDA using a lipopolysaccharidespeci®c monoclonal antibody, mAb 2625 [Luneberg, E.,
È
Zahringer, U., Knirel, Y. A., Steinmann, D., Hartmann, M.,
È
Steinmetz, I., Rohde, M., Kohl, J. & Frosch, M. (1998)
J. Exp. Med. 188, 49±60]. In the present study, an isogenic
mutant strain, termed 5215, was constructed by deletion of
genes involved in the biosynthesis of the mAb 2625 epitope.
Mutant 5215 was as virulent as the parental wild-type RC1
but did not bind mAb 2625. The two strains showed no
di€erence in the core oligosaccharide and lipid A but in
the O-chain polysaccharide structure, which is a homopolymer of 5-acetimidoylamino-7-acetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (a derivative

of legionaminic acid). NMR spectroscopic studies revealed a
hitherto unknown modi®cation of bacterial polysaccharides
in the wild-type strain, namely N-methylation of the 5-acetimidoylamino group on a single legionaminic acid residue
that is located, most likely, proximal to the core oligosaccharide. Two major N-methylated substituents, the (N,Ndimethylacetimidoyl)amino and acetimidoyl(N-methyl)
amino groups, could be allocated to the long- and middlechain O-polysaccharide species, respectively. N-Methylation
of legionaminic acid that was absent from the isogenic
mutant 5215 and from the spontaneous phase variant 811,
correlated with the presence of the mAb 2625 epitope.

Legionella pneumophila is a facultative intracellular parasite
and the cause of 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. pneumophila 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-acetimidoyl7-N-acetyl derivative of 5,7-diamino-3,5,7,9-tetradeoxynon2-ulosonic acid, termed legionaminic acid [7]. Initially, the
D-glycero-L-galacto con®guration was ascribed to legionaminic acid [7], but this was later revised ®rst to the L-glycero
-D-galacto con®guration [13,14] and, ®nally, to the
D-glycero-D-galacto con®guration [15,16]. Similarities in
the biosynthesis pathway of legionaminic acid and neuraminic acid (5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2ulosonic acid) have been described previously [17]. In strains
belonging to the Pontiac group, e.g. Philadelphia 1 [5,18],
polylegionaminic acid is quantitatively 8-O-acetylated
[7,19], but in other Sg 1 strains of the non-Pontiac group,

including those of subgroup OLDA [5,18], it is only
speci®cally 8-O-acetylated at a few legionaminic acid
residues [12]. In the Pontiac group, the 8-O-acetyl has been
identi®ed as a part of the epitope of LPS-speci®c monoclonal antibodies mAb 2 and mAb 3/1 [18,19], and the 8-Oacetyl transferase-encoding gene lag-1 has been described
previously [20]. In L. pneumophila Sg 1 LPS, the OPS is

Correspondence to U. Zahringer, Forschungszentrum Borstel,
È
Zentrum fur Medizin und Biowissenschaften, Parkallee 22, D-23845
È
Borstel, Germany. Fax: + 49 4537 188612, Tel.: + 49 4537 188462,
E-mail:
Abbreviations: LPS, lipopolysaccharide; OPS, O-chain polysaccharide;
PS, polysaccharide; Sg, serogroup; GPC, gel-permeation chromatography; HMBC, heteronuclear multiple-bond correlation; DEPT,
distortionless enhancement by polarization transfer; Kdo, 3-deoxyD-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)

Keywords: N-methylation; lipopolysaccharide; O-chain
polysaccharide; phase variation; Legionella pneumophila.


Ó FEBS 2002

N-Methylation in Legionella pneumophila LPS (Eur. J. Biochem. 269) 561

Fig. 1. Schematic representation of the L. pneumophila serogroup 1 lipopolysaccharide structure. Adapted from [6±12,15,16,20]. Sugar abbreviations:
GlcN3N, 2,3-diamino-2,3-dideoxy-D-glucose; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; Man, D-mannose; GlcNAc, 2-acetamido-2-deoxy-Dglucose (N-acetyl-D-glucosamine); QuiNAc, 2-acetamido-2,6-dideoxy-D-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.

linked to a terminal nonreducing L-rhamnose residue
(RhaII) of the core oligosaccharide [9,10] (Fig. 1). The
LPS core oligosaccharide lacks heptose and phosphate,
contains abundant 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 of an LPS epitope of
L. pneumophila has been reported [24]. The LPS phasevariant strain 811 was isolated from the virulent wild-type
strain RC1 (Sg 1, subgroup OLDA), and found to be
avirulent and serum sensitive [24]. The altered LPS phenotype could be distinguished with the aid of the LPS-speci®c
monoclonal antibody mAb 2625, which bound to wild-type
RC1 but not to phase variant 811. Revertants from strain
811 retain all phenotypic characteristics of the wild-type
[24]. We have recently shown that phase variation in
L. pneumophila depends on chromosomal excision and
insertion of an unstable 29-kb genetic element of presumably phage origin [25]. The 29-kb sequence is located in a
de®ned and conserved site within the chromosome of the
virulent wild-type strain RC1. In contrast, in the phasevariant strain 811, the 29-kb element is excised from the
chromosome and replicates as a high copy plasmid resulting
in the avirulent phenotype [25].
In another study, we characterized a 32-kb gene locus
responsible for LPS biosynthesis in L. pneumophila [17].
Complementation of the spontaneous stable LPS mutant
strain 137 that did not bind mAb 2625 was achieved with a

gene from the avirulent parental wild-type strain 5097. The
gene, designated ORF 8, was able to restore mAb 2625
binding and exhibited homologies to bacterial methyl
transferase genes [17].
Until now, the structure of the mAb 2625 epitope was not
known, because in previous studies no structural difference
could be detected between the LPS of wild-type RC1 and
phase variant 811 [24] or between the LPS of the avirulent
wild-type 5097 and the corresponding mutant 137 [17]. As
the mAb 2625 LPS epitope was found to be associated with
alteration in virulence and serum resistance upon phase
variation [24], we were interested in the elucidation of its
chemical structure. The aim of this study was to identify the
mAb 2625 epitope and to correlate it to virulence. For this

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. pneumophila 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
arti®cial media) and binds mAb 2625. The spontaneous
stable mutant strain 137 that does not bind mAb 2625 was
derived from strain 5097 [17]. A point mutation in ORF 8
resulting in a frame shift with presumably polar effects is
responsible for loss of the mAb 2625 epitope in mutant
strain 137 [17]. All strains were grown on buffered charcoal
yeast extract (BCYE) agar with a-growth supplement
(Merck).
Construction of the L. pneumophila ORF 8±12 mutant
strain 5215 from wild-type strain RC1
For deletion of the ORF 8±12 operon, a 7150 bp HpaI±SpeI
restriction fragment from pEL28 (covering position 456 of
ORF 7 to position 710 of ORF 13) was ligated into the
HincII site of pBC. From the resulting plasmid pMH546


562 O. Kooistra et al. (Eur. J. Biochem. 269)

Ó FEBS 2002

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 is indicated by arrowheads and designation of selected ORFs are shown above gene
blocks.

(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 from plasmid pUC4K
(Pharmacia). Orientation of the insertion was 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 strain RC1 by electroporation and bacteria were plated on BYCE agar
supplemented with kanamycin (50 lgámL)1). Grown transformants 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 cross-over. Homologous recombination was
con®rmed by Southern blot and PCR analysis in the
resulting mutant strain, termed 5215.
Isolation and fractionation of LPS
LPS from wild-type RC1, mutant 5215, and phase variant
811 was extracted from enzyme-digested cells by a modi®ed
phenol/chloroform/petroleum ether procedure as described
previously [6,7]. The LPS yields from dry cells were 9, 16,

and 6% (w/w), respectively.
Fractionation of the intact LPS by OPS chain-length was
achieved by gel-permeation chromatography (GPC) in

10 mM Tris buffer (pH 8.0) containing 1 mM EDTA,
0.25% (w/w) sodium deoxycholate, 0.2 M NaCl, and
0.02% (w/w) NaN3 as described previously [28]. Brie¯y,
5 mg LPS were dissolved under ultrasonication in 3.5 mL
5 mM EDTA and 2% (w/w) sodium deoxycholate.
Non-soluble material was removed by centrifugation and
discarded. The sample was applied to a column
(2.5 ´ 120 cm; Bio-Rad) of Sephacryl S-200 HR (Pharmacia) using the buffer mentioned above at a pump rate of
10 mLáh)1 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 elsewhere [29]. For extracted LPS samples, 100 lL stocksolution (2 mgámL)1) was mixed with 100 lL sample
solution [30] and incubated at 95 °C for 5 min. Then,
0.75 lL (0.75 lg 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 lL water, 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 to 2.5 lL, depending on the refraction index of 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 analysis varied from 0.75 to 7.5 lL and was

three times as high as that in silver-stained SDS/PAGE.


Ó FEBS 2002

N-Methylation in Legionella pneumophila LPS (Eur. J. Biochem. 269) 563

Western blotting onto nitrocellulose ®lter membranes was
carried out as described previously [31]. Immunostaining
was performed subsequently with mAb 2625 [24] or mAb
LPS-1 (Progen) and alkaline phosphatase-conjugated goat
anti-(mouse Ig) Ig (Dianova) [24].
Chemical analysis
GLC was performed with a Hewlett-Packard Model 5890
Series II instrument equipped with a 30-m capillary column
of SPB-5 (Supelco) using a temperature gradient
150 ® 320 °C at 5 °Cámin)1. Monosaccharides were
analysed by GLC as the alditol acetates after hydrolysis
with 0.1 M HCl for 48 h at 100 °C for neutral sugars [32] or
with 10 M HCl for 30 min at 80 °C for amino sugars [33].
Following hydrolysis, sugars were dried, reduced with
NaBH4, and acetylated with acetic anhydride in pyridine
(30 min, 85 °C). Total hexosamine was determined by the
Morgan±Elson reaction after acid hydrolysis (4 M HCl,
16 h, 100 °C) [34]. 3-Deoxy-D-manno-oct-2-ulosonic acid
(Kdo) was determined by the thiobarbituric acid assay
according to the modi®ed method [35]. Quanti®cation of
total phosphate was carried out by the ascorbic acid method
[36]. Fatty acids of the lipid A portion were analysed by
GLC and combined GLC-MS as the methyl esters after

methanolysis (2 M HCl/MeOH, 24 h, 120 °C) and trimethylsilylation 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 °C for 4 h with 0.1 M NaOAc/HOAc buffer (pH 4.4,
10 mgámL)1 LPS), and the resultant lipid A precipitate was
removed by centrifugation (5000 g, 30 min). The supernatant was lyophilized and desalted by GPC on a column
(2.5 ´ 50 cm; Bio-Rad) of Sephadex G-50 (S) (Pharmacia)
using 50 mM pyridinium/acetate buffer (pH 4.3) and monitoring with a differential refractometer (Knauer) to give the
corresponding polysaccharide (PS) portion.
PS was de-O-acetylated with 20% (v/v) aqueous NH4OH
at 37 °C for 16 h (10 mgámL)1 PS); the following lyophilization and desalting as described above yielded
PSNH4OH. To obtain PSNH4OH devoid of core sugars,
PSNH4OH was treated with 48% (v/v) aqueous hydro¯uoric
acid (HF) at 4 °C for 7 days [38] (10 mgámL)1 PSNH4OH),
the reagent was removed under a stream of nitrogen, and
the resultant PSNH4OH/HF was desalted as above.
PSNH4OH/HF was fractionated by tandem 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 mM) 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) for monitoring. Fractions of  3 mL
were collected, appropriately combined into six pools, and
lyophilized.
NMR spectroscopy
1
H NMR, 2D 1H,1H NMR (COSY and NOESY with

mixing time 300 ms), and H-detected 1H,13C NMR

(HMQC and HMBC) spectra were recorded with a Bruker
Avance DRX-600 spectrometer. 13C NMR and DEPT-135
NMR spectra were recorded with a Bruker Avance
DPX-360 spectrometer. Standard Bruker software was used
to acquire and process the NMR data. Samples were
lyophilized three times from 2H2O and measured in 2H2O
(2H, 99.996%; Cambridge Isotope Laboratories) at 27 °C.
Chemical shifts were referenced to external acetone (dH
2.225 p.p.m.; dC 31.45 p.p.m.).

RESULTS
Construction and characterization of the ORF 8±12
mutant strain 5215 from wild-type strain RC1
Genes involved in formation of the LPS epitope bound by
mAb 2625 were identi®ed on a LPS biosynthesis gene cluster
and assigned to the ORF 8±12 operon [17]. ORF 8 showed
homologies to bacterial methyl transferases [39,40], ORF 9
exhibited sequence similarities to Neu5Ac condensing
enzymes (e.g. SiaC of Neisseria meningitidis [41]), and
ORF 11 showed vague homologies to plant methyl
transferases [42]. The proteins encoded by the remaining
ORFs 10 and 12 showed no homologies to known aminoacid 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 recombination (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 serum
resistance were not affected in mutant 5215, when compared

to the parental wild-type RC1 (data not shown), which
revealed that the mAb 2625 LPS epitope itself is not
associated with virulence of L. pneumophila 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 banding
pattern. Both LPS showed a characteristic bimodal distribution 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 recognize speci®cally a conserved epitope located in the
outer core region of the LPS from L. pneumophila Sg 1
strains [12,24,43].
Chemical analyses of both wild-type RC1 and mutant
5215 LPS revealed L-rhamnose, D-mannose, Kdo, and
amino sugars, i.e. total hexosamine according to the
Morgan±Elson reaction after acid hydrolysis (total of
2-amino-2-deoxy-D2,3-diamino-2,3-dideoxy-D-glucose,
glucose, and 2-amino-2,6-dideoxy-D-glucose), in the molar
ratios of  2 : 2 : 2 : 5. According to GLC data, the ratio of
2-amino-2-deoxy-D-glucose and 2-amino-2,6-dideoxy-Dglucose was the same in the LPS from wild-type RC1 and
mutant 5215. Comprehensive analysis by NMR spectroscopy and MALDI-TOF MS revealed no difference in
composition and structure of the core oligosaccharides
isolated from wild-type RC1 and mutant 5215 [12]. NMR
spectroscopy revealed that the lipid A backbone is a


564 O. Kooistra et al. (Eur. J. Biochem. 269)

Ó FEBS 2002

Fig. 3. Silver-stained SDS/PAGE (A) and Western blot with mAb 2625
(B) and mAb LPS-1 (C) of L. pneumophila LPS from wild-type RC1
(lane 1) and mutant 5215 (lane 2). In A, 0.75 lg LPS per lane was
applied, and 2 lg LPS in B and C.

1,4Â-bisphosphorylated (1Â đ 6)-linked disaccharide of
2,3-diamino-2,3-dideoxy-D-glucose in both wild-type RC1
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 the
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].

Fig. 4. Fractionation of LPS from L. pneumophila wild-type RC1 by
gel-permeation chromatography on Sephacryl S-200 HR. Part of
refraction index elution pro®le of LPS (A). Silver-stained SDS/PAGE
(B) and Western blot with mAb 2625 (C) and mAb LPS-1 (D) of
fractionated LPS. The dotted lines in the top panel indicate fractions
and the dashed lines in the lower panels indicate the borders of
di€erent, appropriately aligned SDS/polyacrylamide gels or Western
blots.

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. 3A). A similar pro®le showing a low amount of long
O-chain LPS species and a relatively high amount of short
O-chain LPS species was observed for mutant 5215 LPS
(Fig. 5A). Silver-stained SDS/PAGE after GPC of the wildtype 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 fraction and with overlapping about 6±8 residues in each pair 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 below a certain size, i.e. a certain
OPS chain-length (Fig. 4C). In contrast, mAb LPS-1
exclusively bound to the LPS species from fractions
113±126, and did not bind to LPS molecules above a
certain size (Fig. 4D). This rather sharp margin clearly

Fig. 5. Fractionation of LPS from L. pneumophila mutant 5215 by gelpermeation chromatography on Sephacryl S-200 HR. Part of refraction
index elution pro®le of LPS (A). Western blot with mAb 2625 (B) of
fractionated LPS. The dotted lines in the top panel indicate fractions
and the dashed lines in the lower panel indicate the borders of di€erent,
appropriately aligned Western blots.

showed that the mAb 2625 epitope is only present or
accessible in LPS species having a speci®c OPS chain-length
of  15 and more residues of legionaminic acid.

Ó FEBS 2002

N-Methylation in Legionella pneumophila LPS (Eur. J. Biochem. 269) 565

As followed from Fig. 3, all LPS-containing fractions
(nos 96±128) from mutant 5215 bound mAb LPS-1
(Fig. 5B).
Preparation and characterization of OPS
An OPS attached to the core oligosaccharide (PS) was
prepared by mild acid hydrolysis of LPS from each strain
(wild-type RC1, mutant 5215, and phase variant 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 oligosaccharide (RhaII) [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 was 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 the 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 stable under the same
conditions [14].

Therefore, PS was de-O-acetylated, treated with 48%
aqueous HF, and the resultant modi®ed polysaccharide
(PSNH4OH/HF) was fractionated by tandem GPC on Toyopearl TSK HW-50 (S) and Fractogel TSK HW-40 (S) to
separate long-, middle-, and short-chain OPS species. The
middle-chain species were present in a low amount (e.g. see
pool III in Fig. 6). Only a negligible difference in the elution
pro®les of PSNH4OH/HF from wild-type RC1, mutant 5215,
and phase variant 811 was observed.

Fig. 6. Fractionation of OPS (PSNH4OH/HF) from L. pneumophila wildtype RC1 by tandem gel-permeation chromatography on Toyopearl TSK
HW-50 (S) and Fractogel TSK HW-40 (S). Pools I, III, and V correspond to long-, middle-, and short-chain PSNH4OH/HF, respectively;
pools II, IV, and VI correspond to intermediate fractions.

OPS from each strain separated into pools I to VI (Fig. 6)
were investigated by 1H NMR spectroscopy. The 1H NMR
spectra (only pools I, III, and V are shown in Fig. 7)
indicated that legionaminic acid with its major substituents,
5-N-acetimidoyl and 7-N-acetyl groups (Fig. 8, structure 1)
remained intact (see also Tables 1 and 2). The only
anomeric proton signals present in the spectra were those
of two L-rhamnose residues (RhaI H1 dH 5.06 and RhaII H1
dH 4.99; Fig. 9A±C), which was in accordance with
identi®cation of only L-rhamnose by GLC analysis of the
alditol acetates derived from PSNH4OH/HF. Therefore, the
isolated PSNH4OH/HF species from all three strains were
composed of legionaminic acid and L-rhamnose, whereas
the major portion of the core oligosaccharide was cleaved
by HF treatment. A 1H,13C HMQC experiment demonstrated that PSNH4OH/HF contained at the reducing end
either an Rha disaccharide ® 3)-a-L-RhaII-(1 ® 3)-L-RhaI
( 70%) or a single Rha residue ® 3)-L-RhaII ( 30%), in

both cases the reducing Rha residue being predominantly,
but not exclusively, a-con®gured. The disaccharide was
identi®ed by the H1 chemical shift of RhaII (compare
published data [9]) and a characteristic down®eld
displacement of the C1 signal from dC 95.17 in nonlinked
Rha to dC 103.21 in a-linked RhaII (compare to published
data in [45]).
Identi®cation of N-methylated derivatives
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 1H NMR 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 1H 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 wildtype RC1 having 15±20 residues of legionaminic acid was
the smallest one that displayed these 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 1H NMR chemical shifts and the grouping of the
signals in pairs (one pair at dH 3.30 and 3.19, and the other at
dH 3.03 and 2.95; see below) resembled those of N-methyl
groups in stereoisomers of N-acetimidoyl-N-methylglycine
(N-acetimidoylsarcosine) at dH 3.70 (E) and dH 3.60 (Z) [47].
The corresponding 13C NMR chemical shifts (one pair at dC
42.95/42.89 and 40.74/40.60, and the other at dC 33.73 and
32.56, respectively) and a DEPT-135 NMR experiment
con®rmed N-linked methyl groups. The 1H,13C HMQC
experiment revealed also a third, minor pair of signals at dH
2.97 and 2.94 and dC 30.93 and 29.86, which were partially


566 O. Kooistra et al. (Eur. J. Biochem. 269)

Ó FEBS 2002

Fig. 7. 1D 1H NMR spectra of the long-,
middle-, and short-chain PSNH4OH/HF (pools I,
III, and V; panels A±C) from wild-type RC1.
The integral traces for selected signals with the
corresponding integration values referenced to
the anomeric proton signal of RhaII (H1 dH
4.99, integration value set at 0.7) in the ® 3)a-L-RhaII-(1 ® 3)-L-RhaI disaccharide are
superimposed on the spectra. The insets show
the region between 2.8 and 3.4 p.p.m. extended 5-fold. Bold numbers refer to structures shown in Fig. 8. For abbreviations see
legend to Fig. 9.

superimposed on the signals of the major higher-®eld pair in

the 1H NMR spectrum (Fig. 10, right panel).
In the 1H,13C HMBC spectrum (Fig. 10, left panel), the
proton signals of the major lower-®eld pair at dH 3.30 and
3.19 cross-correlated to the lower-®eld carbon signals at dC
40.74/40.60 and 42.95/42.89. Furthermore, both proton
signals correlated to the signals of the same nonprotonated
carbon (dC 167.03) and the same C-methyl group (dC 16.63)
of an N-acetimidoyl group. In a NOESY experiment, the
two N-methyl signals correlated to each other. These data
suggested that both N-methyl groups are linked to the same
nitrogen of an N-acetimidoyl group, i.e. that an
(N,N-dimethylacetimidoyl)amino group is present (Fig. 8,
structure 2). This group is linked to C5 of a legionaminic
acid residue, which is substituted with a nonmethylated
N-acetimidoyl group in the other legionaminic acid residues
of the OPS.
The proton signals of the major higher-®eld pair at dH
3.03 and 2.95 correlated in the 1H,13C HMBC spectrum to
different nonprotonated carbon signals at dC 169.02 and dC
168.23 and different C-methyl carbon signals at dC 20.49
and dC 20.21 of another N-acetimidoyl group, respectively
(Fig. 10, left panel). In addition, each proton signal
correlated to a signal of a nitrogen-bearing carbon (C5) of

legionaminic acid (dC 55.93 and 57.32). Taken these 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 be excluded based 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 in the LPS of
Bordetella pertussis strain 1414 [48,49], in which the
N-methyl group gave a single singlet in the 1H NMR
spectra. Similarly, the minor pair of signals at dH 2.97 and
2.94 was assigned to a 5-(N-methylacetimidoyl)amino group.
A NOESY experiment (Fig. 11) was applied to stereochemical 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
observed between the lower-®eld signal from each major
pair of the N-methyl signals (dH 3.30 in 2 and dH 3.03 in 3)
and the corresponding C-methyl signals of the N-acetimidoyl group, but only a weak or no cross-peak could be
detected for the higher-®eld signal (dH 3.19 in 2 and dH 2.95
in 3). Hence, the lower- and higher-®eld 1H NMR signals of


Ó FEBS 2002

N-Methylation in Legionella pneumophila LPS (Eur. J. Biochem. 269) 567

Fig. 8. Structures of 5-acetimidoylamino-7-acetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (5-N-acetimidoyl-7-Nacetyllegionaminic acid, 1) and its N-methylated derivatives,
5-N-(N,N-dimethylacetimidoyl)-7-N-acetyllegionaminic acid (2-E) and
two stereoisomers of 5-N-acetimidoyl-7-N-acetyl-5-N-methyllegionaminic acid (3-E and 3-Z).

2 belonged to the N-methyl groups at N1 in trans and cis
orientation to N2 of the acetamidine group, respectively
(Fig. 8; the descriptors cis and trans for the two N-methyl

groups in 2 are used only to designate their positions relative
to N2 and do not refer to stereoisomerism). The lower-®eld
1
H NMR signal of 3 belonged to the N-methyl group at N2
in the E isomer and the higher-®eld signal to that in the
Z isomer, in which N1 is in trans and cis orientation to C5 of
legionaminic acid, respectively (Fig. 8).
Like 3, 2 may theoretically occur as two stereoisomers,
E and Z, with regard to the partial double bond at N2, while
the NMR spectra showed the presence of only one isomer.
Molecular modelling data (not shown) suggested that this
isomer is 2-E with trans orientation of N1 to C5 as 2-Z
would be sterically hindered by interaction of one of the
N-methyl groups at N1 (that in cis orientation to N2) with
the pyranose ring of legionaminic acid.
The N-methyl signal in 3 gave strong NOE correlations
to three more proton signals, which were assigned to H4,
H7, and either H6 or, less likely, H8 of legionaminic acid.
The assignment was performed by correlations between
signals for H4 (dH 3.33 E, dH 3.37 Z) and H3ax,3eq (axial:
dH 1.86 E, dH 1.89 Z, equatorial: dH 2.48 E, dH 2.51 Z) in
COSY, and for H7 (dH 3.68 E, dH 3.75 Z) and CˆO of the
7-acetamido group (dC 175.91 E, dC 175.74 Z) in the 1H,13C
HMBC experiment. In addition, signals for H9
(dH 1.17 E, dH 1.16 Z) were found by a weak NOE
correlation with the N-methyl group, whereas signals for H6
and H8 appeared to coincide (dH 4.19 E, dH 4.13 Z). A
signal for H5 of 3 could not be reliably identi®ed, most
likely, owing to its coincidence with the H5 signal of
the major, non-N-methylated derivative 1. The NOESY

data suggested that in the predominant conformation of
both 3-E and 3-Z the N-methyl group at N2 has an axial
(or close to axial) orientation related to the pyranose ring
of legionaminic acid and lies on or close to a plane
formed by H4, H6, and H7. In contrast, in 2 no NOE
correlation was observed between the N-methyl groups
and any proton of legionaminic acid, evidently owing to
the remoteness of the N-methyl groups at N1 from the
pyranose ring.
Long-, middle-, and short-chain PSNH4OH/HF from wildtype RC1 were investigated by 1D 1H NMR spectroscopy
and signal integration was performed to calculate the
average chain-length of the PSNH4OH/HF and the distribution
of N-methylated legionaminic acid derivatives. The signal of
a-L-RhaII H1 (dH 4.99) in the ® 3)-a-L-RhaII-(1 ® 3)-LRhaI disaccharide was used as a reference, because it was the

Table 1. 1H and 13C NMR chemical shifts for N-methylated acetimidoylamino groups in pool III of the OPS from wild-type RC1. NMe, N-methyl
group; NAmCH3 and NAmCˆN, C-methyl group and nonprotonated carbon of the 5-acetimidoylamino group, respectively. ND, not determined.
Derivative of legionaminic acid
dH (p.p.m.)
5-N-Acetimidoyl (1)
5-N-(N,N-Dimethylacetimidoyl) (2-E)
5-N-(N-Methylacetimidoyl) (minor)
5-N-Acetimidoyl-5-N-methyl (3)
dC (p.p.m.)
5-N-Acetimidoyl (1)
5-N-(N,N-Dimethylacetimidoyl) (2-E)
5-N-(N-Methylacetimidoyl) (minor)
5-N-Acetimidoyl-5-N-methyl (3)

NAmCH3

NMe

3.19 (cis)
2.97 (E)
2.95 (E)

3.30 (trans)
2.94 (Z)
3.03 (Z)

40.74 (cis)
40.60 (cis)
30.93 (E)
32.56 (E)

42.95
42.89
29.86
33.73

(trans)
(trans)
(Z)
(Z)

NAmCˆN

2.24
2.19

2.25 (E)
2.30 (E)

ND
2.19 (Z)
20.08
16.63

ND
20.21 (E)

167.61
167.03
ND
167.22 (E)
20.49 (Z) 168.23 (E)

167.05 (Z)
169.02 (Z)


568 O. Kooistra et al. (Eur. J. Biochem. 269)

Ó FEBS 2002

Table 2. 1H and 13C NMR chemical shifts for 1 and 3 in the OPS pool III from wild-type RC1. NAcCH3 and NAcCˆO, C-methyl group and
nonprotonated carbon of the 7-acetamido group, respectively. ND, not determined.
Derivative of
legionaminic acid
5-N-Acetimidoyl

-7-N-acetyl (1)
5-N-Acetimidoyl (E)
-7-N-acetyl-5-N- (Z)
methyl- (3)
5-N-Acetimidoyl
-7-N-acetyl (1)
5-N-Acetimidoyl (E)
-7-N-acetyl-5-N- (Z)
methyl- (3)

d (p.p.m.)
H3ax
1.61

H3eq
2.62

H4
4.02

H5
3.53

H6
3.95

H7
3.89

H8

3.89

H9
1.20

NAcCH3
2.08

1.86
1.89

2.48
2.51

3.33
3.37

ND
ND

4.19
4.13

3.68
3.75

4.19
4.13

1.17

1.16

2.17
ND

C1
174.47

C2
101.86

C3
39.40

C4
71.86

C5
54.30

C6
72.76

C7
55.53

C8
67.92

C9

19.65

NAcCH3 NAcCˆO
23.23
175.32

ND
ND

ND
ND

32.04x
31.86

72.66
71.23

57.32
55.93

68.41
68.37

55.01
54.94

68.41
68.37

19.44
19.41

23.71
23.67

only anomeric proton present in a single anomeric con®guration. Integration of the signals of 1D 1H NMR spectra
(Fig. 7) indicated that the average chain-length of long-,
middle-, and short-chain PSNH4OH/HF (Fig. 6, pools I, III,
and V) is about 40, 18, and 10 legionaminic acid residues.
The ratio of the 5-N-(N,N-dimethylacetimidoyl)-7-N-acetyl
and 5-N-acetimidoyl-5-N-methyl-7-N-acetyl derivatives of
legionaminic acid was 1 : 1 in long-chain and 1 : 2 in
middle-chain PSNH4OH/HF, respectively. Based on the relative intensities of the proton signals it was concluded that

175.91
175.74

only one legionaminic acid residue is N-methylated in each
polysaccharide chain above a speci®c length.

DISCUSSION
Phase variation in L. pneumophila has dramatic effects on
the virulence of the bacteria in various in vitro and in vivo
models. Phase variation can be monitored with the aid of
mAb 2625 that is speci®c for an epitope in the LPS of
L. pneumophila Sg 1 subgroups OLDA and Oxford. The

Fig. 9. 600-MHz 1H NMR spectra of pool III
of the OPS (PSNH4OH/HF) from three

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 wildtype RC1 (D), mutant 5215 (E), and phase
variant 811 (F). NMecis and NMetrans,
N-methyl groups in 2-E (the descriptors cis
and trans designate the positions of the
N-methyl groups relative to N2); NMe,
N-methyl group in the stereoisomers 3-E and
3-Z; NAcCH3 and NAmCH3 , C-methyl groups
of 7-acetamido and 5-acetimidoylamino
substituents, respectively. Bold numbers refer
to structures shown in Fig. 8.


Ó FEBS 2002

N-Methylation in Legionella pneumophila LPS (Eur. J. Biochem. 269) 569

Fig. 10. Sections of 2D 1H,13C HMBC (left panel) and HMQC (right panel) spectra of pool III of the OPS (PSNH4OH/HF) from L. pneumophila wildtype RC1. Spectra are aligned in F1 dimension. The corresponding 13C and 1H NMR spectra are displayed along F1 and F2 axes. Spectra were
recorded at 600 MHz and 27 °C. For abbreviations see legend to Fig. 9. Cross-peaks marked by   belong to an unknown minor isomer of
legionaminic acid.

Fig. 11. Part of a NOESY spectrum of
pool III of the OPS (PSNH4OH/HF) from
L. pneumophila wild-type RC1. The spectrum
was recorded at 600 MHz and 27 °C using a
mixing time 300 ms. For abbreviations see

legend to Fig. 9. Cross-peaks marked by
  belong to an unknown minor isomer of
legionaminic acid.


570 O. Kooistra et al. (Eur. J. Biochem. 269)

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
L. pneumophila Sg 1 strains [12,24,43]. In Western blot, the
mAb LPS-1 binding pattern of LPS from wild-type RC1
was different from that of phase variant 811. A similar
alteration has been observed for LPS from the spontaneous
mutant 137 derived from wild-type strain 5097 [17]. LPS of
the mutants did not bind to mAb 2625, but showed a
stronger binding to mAb LPS-1 compared to the corresponding wild-type LPS.
It was suggested that an alteration in the LPS structure
enhances the accessibility of the mAb LPS-1 epitope and
that the altered structure may be located in the core
oligosaccharide [24]. However, the latter could not be
con®rmed by chemical studies. Comprehensive analysis,
using NMR spectroscopy and MALDI-TOF MS, of the
core oligosaccharides from all three strains (wild-type RC1,
mutant 5215, and phase variant 811) revealed no structural
deviation from each other and from the strain Philadelphia
1 core oligosaccharide studied earlier [9,10,12].
Fractionation of LPS by GPC and subsequent Western

blot analysis enabled us to determine the location of the
mAb 2625 epitope in the long and middle O-chain LPS
species of wild-type strain RC1. Remarkably, mAb LPS-1
bound only to LPS species lacking the mAb 2625 epitope,
thus suggesting that the mAb 2625 epitope interferes with
the binding of mAb LPS-1.
The OPS of L. pneumophila Sg 1 is a polylegionaminic
acid (Fig. 8, structure 1), which could not be depolymerized to monomers [7]. Therefore, detailed NMR spectroscopic studies were performed with selectively degraded
polysaccharides fractionated by tandem GPC. Comparative analysis of the fractionated OPS from wild-type RC1
and mutant 5215 revealed minor components, which
occurred only in the wild-type polysaccharide species
above a speci®c chain-length and whose presence correlated with the reactivity of mAb 2625 with LPS in
Western blot.
The minor components were identi®ed as 5-N-(N,Ndimethylacetimidoyl)-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-acetyllegionaminic acid was detected in a negligible amount, which
could either be a biosynthetic precursor or a degradation
product of 2. No N-methylated acetimidoylamino group
has been hitherto found in bacterial polysaccharides. Only
one N-methylated 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 OPS of L. pneumophila using 13C NMR
spectroscopy [8].
Investigation of binding af®nities by surface plasmon
resonance biomolecular interaction analysis revealed that
only long- and middle-chain PSNH4OH/HF from wild-type
RC1 bind to mAb 2625 but not short-chain PSNH4OH/HF.
The same holds true for any PSNH4OH/HF from phase

variant 811 or mutant 5215 [26]. As binding to mAb LPS-1
is dependent on O-acetylation [12], de-O-acetylated
PSNH4OH/HF does not bind to mAb LPS-1 at all.

Ó FEBS 2002

However, interference of the mAb 2625 epitope with
binding of the core-speci®c mAb LPS-1 in Western blot
suggested that the N-methylated legionaminic acid residue is
located in the OPS close to or, most likely, next to the core,
i.e. is linked to the terminal L-rhamnose residue (RhaII) of
the core oligosaccharide of long- and middle-chain LPS
species. Unfortunately, this link could not be proved
directly, as no interresidue NOE correlation could be
observed between any proton of either 2 or 3 and RhaII. A
lack of the mAb LPS-1 epitope due to a core modi®cation
can be excluded as no such modi®cation was found by
detailed structural studies of the puri®ed core oligosaccharides 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 other position of the OPS could 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 found 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 as 28:0(27-oxo) and 27:0(1,27dioic), are present in all three strains, positions 2 and 2¢ of
the lipid A backbone are predominantly 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].
Similar studies of avirulent wild-type 5097 and the
derived spontaneous mutant 137 revealed N-methylation
in the OPS of the wild-type but not of the mutant strain. In
mutant 137, a point mutation inactivated the ORF 8 gene
encoding methyl transferase [17], which is probably, at least
in part, responsible for N-methylation. N-Methyl groups
were also found in the OPS from strain Philadelphia 1, in
which their 1H NMR chemical shifts are in¯uenced by
8-O-acetylation of the N-methylated legionaminic acid
residue [12]. Interestingly, the 8-O-acetyl group masks the
mAb 2625 epitope, which has become accessible to the
antibody only after chemical de-O-acylation of the LPS [12].
Previously, at least four epitopes have been described on the
de-O-acetylated LPS from strain Philadelphia 1, which were
recognized by monoclonal antibodies produced against
non-Philadelphia 1 strains of L. pneumophila Sg 1 [19]. The
N-methyl groups are presumably present in all and unique
to Sg 1 strains of L. pneumophila, as the ORF 8±12 operon

occurs in Sg 1 strains, but neither in strains from other
L. pneumophila serogroups, nor in non-pneumophila strains
[17].
Although present as minor components, the N-methylated residues of legionaminic acid contribute to the
immunospeci®city of L. pneumophila. The investigation of
binding of mAb 2625 to the N-methylated legionaminic acid


Ó FEBS 2002

N-Methylation in Legionella pneumophila LPS (Eur. J. Biochem. 269) 571

derivatives is described in the accompanying paper [26].
Being hydrophobic and having a signi®cant electronic effect,
the N-methyl groups may adjust the appropriate hydrophobicity 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 related to the fact
that phase variation is mediated by a genetic element of
possibly phage origin. The N-methyl groups located close to
the core oligosaccharide may play a role of 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 different phenotypic alterations that occur upon phase
variation in L. pneumophila might be connected. Our
current hypothesis is that phase variation affects a regulatory factor, which 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. Luthje for performing MALDIÈ
TOF MS, H.-P. Cordes for running NMR spectra, and H. Moll for
expert help with GLC-MS. The skilful 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 RUS 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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