Structural characterization of the lipopolysaccharide
O
-polysaccharide
antigen produced by
Flavobacterium columnare
ATCC 43622
Leann L. MacLean
1
, Malcolm B. Perry
1
, Elizabeth M. Crump
2
and William W. Kay
2
1
Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada;
2
Department of Biochemistry and
Microbiology, University of Victoria, Victoria, British Columbia, Canada
The structure of the antigenic O-chain polysaccharide of
Flavobacterium columnare ATCC 43622, a Gram-negative
bacterium that causes columnaris disease in warm water fish,
was determined by high-field 1D and 2D NMR techniques,
MS, and chemical analyses. The O-chain was shown to be
an unbranched linear polymer of a trisaccharide repeat-
ing unit composed of 2-acetamido-2-deoxy-
D
-glucuronic
acid (
D
-GlcNAcA), 2-acetamidino-2,6-dideoxy-

Experimental procedures
Bacterial culture
F. columnare (ATCC 43622, NRCC 6160) was grown at
16 °C in a 52-L fermentor in medium of composition:
tryptone, 4 g; yeast extract, 0.4 g; MgSO
4,
0.5 g; CaCl
2
,
0.5 g; sodium acetate, 0.2 g; maltose, 10 gÆL
)1
;pHwas
adjusted to 7.00 with 0.1
M
NaOH. A 2.5-L inoculum
grown at 22 °C was used, with stirring at 200 r.p.m. and
dissolved oxygen at 20%. Cells were killed with 1% phenol
(final concentration, 2 h at 4 °C) in late exponential phase at
25.5hgrowth(A
600
¼ 3.34). After acidification with acetic
acid to pH 4 at 0 °C to break the gel-like constitution, the
suspended cells were harvested by centrifugation (yield
 300 g wet paste).
Preparation of LPS and
O
-PS
F. columnare cells (300 g wet paste) were extracted for
15 min at 65 °C with vigorously stirred 50% (w/v)
aqueous phenol (1.2 L), and, after cooling (4 °C) and

2-acetamidino-2,6-dideoxy-
L
-galactose (2-acetamidino-2-deoxy-
L
-fucose); LPS, lipopolysaccharide; O-PS, O-polysaccharide;
CPS, capsular polysaccharide.
(Received 21 May 2003, revised 25 June 2003,
accepted 27 June 2003)
Eur. J. Biochem. 270, 3440–3446 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03736.x
dissolved in water and lyophilized to yield LPS (1.68 g),
which was used in all further studies.
The addition of acetone (6 vol.) to the supernatant from
the above ultracentrifugate remaining after the collection of
LPS afforded a precipitate (94 mg), which, on Sephadex G-
50 column chromatography, yielded a void-volume elution
product (55 mg) tentatively identified as capsular polysac-
charide (CPS).
O
-PS
LPS (1.40 g) was delipidated by treatment with 1% (v/v)
acetic acid (100 mL) at 100 °C for 2 h and, after removal of
precipitated lipid A (170 mg), the lyophilized water-soluble
products were fractionated by Sephadex G-50 column
chromatography to yield an O-PS fraction (K
av
0.03–0.12,
390 mg) and a low-molecular-mass putative core oligosac-
charide fraction (K
av
0.75, 110 mg).

pyridinium
acetate (pH 4.5) as the mobile phase, and the eluate was
continuously monitored using a Waters R403 differential
refractometer.
LPS samples (2 lg) were electrophoresed in 14% poly-
acrylamide in the presence of deoxycholate. Bands were
detected using the silver-staining directions of Tsai & Frasch
[7].
NMR spectroscopy
1
Hand
13
C NMR spectra were recorded on a Varian
Inova 400 spectrometer with samples in 99% D
2
Oat
55 °C, and internal acetone standard (2.225 p.p.m. for
1
H and 31.07 p.p.m. for
13
C) employing standard
COSY, TOCSY (mixing time 80 ms), NOESY (mixing
time 200 ms), heteronuclear single quantum correlation
(HSQC), and heteronuclear multiple-bond correlation
(gHMBC) (optimized for 5 Hz long-range coupling
constant).
Chemical procedures
Quantitative conversion in the O-PS of the acetamidino
function into an acetamido function was effected by
treatment of the O-PS with 5% aqueous triethylamine

O) of isolated aldoses, as
previously described [8]. The absolute configuration of
derived 2-acetamido-2-deoxyhexoses was confirmed by
GLC analysis of their acetylated 2-(S)-butyl glycosides,
prepared under previously described conditions [9].
Optical rotations were measured at 20 °C in 10-cm
microtubes, using a Perkin-Elmer 243 polarimeter.
Results
Fermenter-grown cells of F. columnare were extracted by a
modified hot phenol/water method [10], and a S-type LPS,
found almost exclusively in the phenol phase of the cooled
extract, was obtained in 12% yield by ultracentrifugation of
the concentrated dialyzed extract. Deoxycholate/PAGE
analysis of the LPS gave a typical ladder-like banding
pattern in which the step separations suggested that the LPS
was composed of repeating trisaccharide units [11]. On
treatment with 6 vol. acetone, the ultracentrifugate afforded
a precipitate which, on Sephadex G-50 gel filtration, gave a
void-volume fraction ( 2% yield) of a glycan tentatively
identified as CPS. A lower-molecular-mass fraction (K
av
0.7,
180 mg) which gave a strong colorimetric (phenol/H
2
SO
4
)
reaction for carbohydrate contained glycopeptides in which
the oligosaccharide moieties had similar structure and
composition (results not reported) to those previously

1,2
 3Hz)and4.70(J
1,2
8.8 Hz) p.p.m. with
J
1,2
couplings indicative of two a-linkage and one b-linkage,
respectively; two methyl signals at 1.21 and 1.17 p.p.m. (6H)
characteristic of two 6-deoxyhexose residues; an N-acyl
substituent at 2.25 p.p.m. (3H); and four signals (2.10–1.93
p.p.m.) characteristic of methyl signals of two N-acetyl and
two O-acetyl substituents.
The
13
C-NMR spectrum of the O-PS (Fig. 1) showed
inter alia three anomeric signals at 102.6 (J
C-1,H-1
164 Hz),
97.1 (J
C-1,H-1
172 Hz) and 97.0 (J
C-1,H-1
180 Hz) p.p.m.
having J
C-1,H-1
coupling constants indicative of one b-link-
age and two a-linkages, respectively, together with a sharp
singlet at 93.9 p.p.m. subsequently identified as the C4
resonance of a 4-ketohexose residue. Also present were
two sharp singlets at 15.8 and 11.9 p.p.m. characteristic

1
H and
13
C NMR chemical shifts of the native LPS O-PS from F. columnare ATCC 43622. Spectra run in D
2
Oat55°C with internal
acetone reference (2.225 p.p.m. for
1
H and 31.07 p.p.m. for
13
C). Coupling constants (Hz) are given in parentheses. Tentative assignments for
residue A: N-H7 (8.83 p.p.m.) and N-H7
1
(8.57 p.p.m.) at 35 °C(10%D
2
O/90% H
2
O, v/v).
Glycose
residue
Chemical shift (p.p.m.)
H1/C1 H2/C2 H3/C3 H4/C4 H5/C5 H6/C6
A 5.14 (2.2) 4.28 (10.2) 5.16 (nr) 4.13 (2) 4.57 1.17
97.1 (172) 51.1 71.5 78.8 67.9 15.8
B 4.97 (3) 4.24 (3.2) 3.78 (nr) – 3.91 1.21
97.0 (180) 52.7 77.7 93.9 70.1 11.9
C 4.70 (8.8) 3.93 (10.0) 5.26 (9.8) 4.08 (10.0) 3.79 –
102.6 (164) 55.4 76.5 74.1 77.8 175.4
3442 L. L. MacLean et al.(Eur. J. Biochem. 270) Ó FEBS 2003
glycose residues were arbitrarily labeled A–C in order of

3,4
and J
4,5
( 10 Hz), and
from its anomeric coupling constants, J
C-1,H-1
164 Hz, and
J
1,2
8.8 Hz. Thecorrelation of H2C tothe C2C at55.4 p.p.m.
is consistent with the presence of a C2 acetamido substituent,
and the lack of a proton at C6, considered in conjunction
with the long-range correlation of H5C to the carbonyl shift
at 175.4 p.p.m., seen in a HMBC experiment, is consistent
with the presence of a C6 carboxylic acid function and allows
C to be identified as a b-GlcpANAc residue.
Residue B was identified as an a-linked 2-acetamido-2,6-
dideoxyhexos-4-ulose residue from further NMR data. The
observed correlation from H2B to the corresponding C2B in
an HSQC experiment, the anomeric coupling constants J
1,2
of 3 Hz and J
C-1,H-1
of 180 Hz, considered in conjunction
with the fact that connectivities could only be followed from
H1B to H2B and H3B, and from the methyl resonance of
H6B to H5B with no evidence of connectivities to any
proton signals at C4B. The presence of a C4 keto group
function and the absence of a proton at C4B was further
supported from an observed long-range correlation between

H-
NMR spectrum with that of an authentic sample, and the
fact that its reduced (NaBD
4
) and acetylated product on
GLC/MS gave a single peak corresponding in retention
time (T
G
0.93) and mass spectrum to an authentic sample
of 1,3,4,5,-tetra-O-acetyl-2-acetamido-2,6-dideoxy-
D
-galact-
itol-[1-
2
H].
The hexuronic acid component C was identified as
2-acetamido-2-deoxy-
D
-glucuronic acid from the isolation
Fig. 2.
1
H-
13
CHSQCshiftcorrelationmap
of the spectral regions
1
H (1.0–5.5 p.p.m.)
and
13
C (10–104 p.p.m.) of the F. columnare

D
-configuration was confirmed from
the specific optical rotation of its hydrochloride derivative
{[a]
D
+67° (c0.3,water).Lit.[a]
D
+72°} and by GLC
analysis of its derived acetylated 2-(S)-butyl glycosides [11].
Residue B was identified as 2-acetamido-2,6-dideoxy-
D
-
xylo-hexos-4-ulose (D-Sug). The above preparative paper
chromatography of the hydrolysed reduced (NaBD
4
)
carbodiimide-activated O-PS also yielded two separated
aminoglycose fractions identified as a mixture of 2-amino-
2,6-dideoxy-
D
-(and
L
)galactose {R
GN
1.48; [a]
D
)4 ° (c 0.2,
water) [13]} and 2-amino-2,6-dideoxy-
D
-glucose {R

-forms of the
aminoglycose, as evidenced from its optical rotation, and
from GLC analysis of its acetylated 2-(S)-butyl glycoside
derivatives. This finding is consistent with this fraction being
composed of a
L
-FucN component originating from the
O-PS residue A and the
D
-FucN from the reduced residue B.
The isolation of optically pure 2-amino-2,6-dideoxy-
D
-
glucose (D-QuiN), the major reduction product of residue
B, further confirms the
D
-configuration assigned to residue
B. Preparative paper chromatographic separation of the
hydrolysis products of NaBH
4
-reduced carbodiimide-
activated O-PS afforded the hydrochloride derivatives of
2-amino-2-deoxyglucose, 2-amino-2,6-dideoxyglucose, and
2-amino-2,6-dideoxygalactose, the
1
H-NMR spectra of
which were identical with those of authentic reference
glycoses, and further confirms their characterization. The
combined MS data and the isolation of the two aminoglyc-
oses with the respective

repeating unit with the structure:
Consistent with the above conclusion, the NMR analysis
of the native O-PS showed that the chemical shifts of the
linkage position carbon atoms C4A,C3B,andC4C
experience significant deshielding, further confirming the
linkage position assignments. As NMR data indicated the
presence of two O-acetyl substituents in the native O-PS,
they can only be located at the available O3 positions of
residues A and C. Partial de-O-acetylation of the O-PS with
dilute ammonium hydroxide (50 °C,1h)resultedinthe
hydrolytic removal of the acetyl substituent on residue A (a-
L-FucpNAm) and partial ( 20%) removal from residue C
(b-D-GlcpNAcA). The de-O-acetylation of A effected
deshielding of C3A (71.5–68.2 p.p.m.) and H3A (5.16–
4.04 p.p.m.), thus establishing the acetyl substituent loca-
tion at C3A in the native O-PS. The O-acetyl substitution on
residue C (b-D-GlcpNAcA) was indicated to be at position
C3C as these
1
Hand
13
C resonances experience similar
downfield shifts on de-O-acetylation. A consideration of the
experimental evidence thus leads to the full structure of the
F. columnare ATCC 43622 LPS native O-chain being an
unbranched polymer of a repeating trisaccharide having the
structure:
½C½A½B
½! 4Þ-b-d-GlcpNAcA-ð1!4Þ-a-l-FucpNAm-ð1!3Þ-a-d-Sugp-ð1!
½C½A½B

agreement with previous studies, we also found that the
presence of this 4-ketoglycose in the polymeric structure
rendered the O-PS unstable under alkaline conditions
and even prolonged storage in aqueous solutions at
pH 7. A similar result was found in a study of forbeside
C, a saponin of Asterias forbesi [18], which also has a
component D-Sug residue.
After the precipitation of the LPS from the phenol phase
extract of F. columnare cells by ultracentrifugation, a low
yield of CPS material was obtained from the ultracentri-
fugate by acetone precipitation followed by Sephadex G-50
gel-filtration chromatography, yielding a lipid-free high-
molecular-mass void-volume fraction. On analysis, the
material proved to have the same structure as the homo-
logous LPS O-PS. This material could be considered to be a
putative capsule or simply free O-PS. The significance of the
O-PS and putative CPS in pathogenesis requires further
investigation. In the fish pathogens, Vibrio ordalii O:2 [15]
and Vibrio anguillarum O:2 [17], their respective LPS O-PS
components and CPSs shared the same respective homo-
logous structures, and the same constitution may pertain in
F. columnare.
Pathogenesis studies have shown a correlation between the
capacity of F. columnare to adhere to fish gill epithelium and
virulence [6,19,20]. However, the nature of the adhesins
involved have not been identified, but possible candidates are
LPS, capsule, fimbriae or other appendages of the bacterium,
a hypothesis requiring further investigation.
It is of note that the structure of the LPS O-antigen of
F. columnare differs structurally from the LPS O-antigen

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3446 L. L. MacLean et al.(Eur. J. Biochem. 270) Ó FEBS 2003