Eur. J. Biochem. 185,645- 650 (1 989)
C FEBS 1989
Structure of Proteus mirabilis 0 2 7 0-specific polysaccharide
containing amino acids and phosphoethanolamine
-
Evgeny V. VINOGRADOV', Danuta KRAJEWSKA-PIETRASIK'. Wiestaw KACA3, Alexander S. SHASHKOV', Yuriy A. KNIREL'
and Nikolay K. KOCHETKOV'
' N. D. Zelinsky Institute of Organic Chemistry, Academy of Sciences of the USSR, Moscow. USSR
Institute of Microbiology, University ol' t b d i . Poland
' Centre of Microbiology and Virology, Polish Academy of Scicnccs, Lodi, Poland
(Received April lO/July 17, 1989) - EJB 89 0441
6
I
80% P!EtN
The main methods used for structural analysis of the polysaccharide were selective solvolysis with anhydrous
hydrogen fluoride and Smith degradation, as well as N M R spectroscopy and mass spectrometry.
The immunodominant role of the lateral N-acetyl-D-glucosamine and phosphoethanolamine in manifesting
the serological specificity of P. miruhilis 027 has been established.
Proteus mirabilis is an important human facultative pathogen which frequently causes urinary tract infections [l].The
composition of lipopolysaccharides of different P. mirabilis
serogroups was studied and many of them were found to
contain the amino acids alanine and lysine [2]. The latter
was found to be the immunodominant component of the 0specific part of lipopolysaccharide from P.inirahilis S1959 [ 3 ,
41. Lysine is also present in the 0-region of lipopolysaccharide
from P. mirahilis 027 which additionally includes other noncarbohydrate components, such as L-alanine, ethanolamine
and phosphate [5, 61.
In this paper we describe the structural determination of
P. miruhilis 0 2 7 0-specific polysaccharide and the investigation of the role of its non-carbohydrate components in
manifesting serological specificity.
MATERIALS AND METHODS
Miscellaneous m e l h d s
NMK spectra were recorded on a Uruker AM-300 instrument in D 2 0(in CDC13 for compound 3; Fig. 2) with acetone
as an internal standard (6, = 2.23 ppm; 6, = 31.45 ppm), at
30' C for oligosaccharides and 80°C for polysaccharides. GasC'orrcspondence fo D. Krajewska-Pietrasik, Institute of Microbiology, University of t6&, ul. Banacha 12/16, PL-90-237 Cbdi,
Poland
Ahhreviution. GLC/MS, gas-liquid chromatography/mass spectrometry.
liquid chroinatography/mass spectrometry (GLC/MS) and
direct-probe inlet MS were performed on a Varian MAT 31 1
instrument, ionisation potential 70 eV, equipped with a glasscapillary column, coated with OV-1 stationary phase. Monosaccharide analysis was performed using a Technicon sugar
analyzer and a Biotronik amino acid analyzer; analysis of
sugar alditol acetates by GLC was carried out as described in
[7]. All oligosaccharide separation and desalting procedures
were done by gel chromatography on a Fractogel TSK HW
40(S) column (1.6 cm x 80 cm) in 1% acetic acid monitored
with a Knauer differential rcfractometer.
N-Acelylation of free amino groups in the 0-specific
polysaccharide was performed according to the procedure [El.
Determination of the absolute configurations ofthe amino
components and hexoses has been performed as described
previously [5, 91.
Bucteriul struins arid culture conditions
Proteus mirubilis 0 2 7 (50157) was derived from Czechoslovak National Collection of Type Cultures of the Institute
OF Epidemiology and Microbiology, Prague, Czechoslovakia;
P. mirahilis S1959 strain came from the strains collection of
the Institute of Microbiology, University of t 6 d i , Poland.
Cultivation of bacteria was carried out in nutrient broth
(Warsaw Serum and Vaccine Laboratory) with 1 % glucose
added. 'The bacteria were harvested at the end of the logarithmic growth phase, centrifuged, washed with saline and finally
freeze-dried.
646
Lipopolysaccharide was isolated from killed cells by the
Wcstphal procedure [lo]. Thc lipid moiety was split off by
mild acid hydrolysis and the 0-specific polysaccharide was
isolated by gel chromatography on Sephadex (3-50 as described previously [4].
(10O"C, 2.5 h) bacterial suspensions (10" cell-forming units/
ml) in doses of 0.25 ml, 0.5 ml and 1 ml over a three-week
period [ 131.
A quantitativc microprecipitation method and its inhibition were used as essentially described in [14].
The double immunodiffusion tests were performed according to 1131.
Preparcition qr the oligosaccharides 1 - 4,6 and 7 (Fig.2 )
RESULTS
Two samples of the P. rnirabilis 0 2 7 polysaccharide (80 mg
each) were treated with anhydrous hydrogen fluoride
(approximately 5 ml) for 2 h at 20°C and 0°C. The excess
reagent was removed under diminished pressure and the residue purified by gel chromatography. Two oligosaccharide
fractions containing mainly trisaccharide 1 and tetrasaccharide 2 were obtained. These were treated with sodium
borohydride in water (20 "C, 2 h) and desalted to give reduced
oligosaccharides 3 and 6, which were treated with acetic anhydride in saturated aqueous NaHC03 solution (20°C, 2 h) and
pure oligosaccharides 4 and 7 were isolated by gel
chromatography.
Structural studies
Isolation of Iipopolysacrharides
Preparation qf oligosaccharide 5 fFig. 2 )
Oligosaccharide 4 (20 mg) was dissolved in one drop of
water, diluted with methanol (2 ml) and ether solution or
diazomethane was added until precipitation of the oligosaccharide began. The mixture was evaporated and the procedure repeated until a yellow transparent solution was
obtained. This was kcpt for 20 min, evaporated and the product acetylated with a 1:1 mixture of acetic anhydride/pyridine
(20 h a t 20 "C), evaporated with toluene; acetate 5 was isolated
by preparative TLC on a Merck precoated SiOz plate in a
chloroform/methanol (9: 1 ) mixture. 'H-NMR data for
oligosaccharide 5 (A, glucuronic acid residue; B, galacturonic
acid residue), 6/ppm: 5.96 (H-4B, dd, J4,? = 1.8 Hz), 5.24
(H-3A, t , J3,4= 9 Hz), 5.18 (2H, H-lB, 2B, m), 5.14 (H-4A,
dd, J4,5 = 9.5 Hz), 4.90 (H-2A, dd, J 2 . 3 = 9.0 Hz), 4.81 (HlA, d, J I , =
~ 7.5Hz), 4.60 (H-5B, d), 4.23 (H-3B, J3.4 =
3.5 Hz), 4.05 (H-5A, d).
Methylation analysis
Methylation analysis was performed according to
Hakomori procedure [I 11, methylated substances were isolated by absorbtion on Sep-Pak CI8cartridges [12]. To obtain
partially methylated alditol acetates samples were hydrolysed
with 2 M CF3COOH (120-C, 1 h), reduced with NaBH, and
acetylated with acetic anhydride in pyridine.
Mass spectrum data for oligosaccharide 8 ( m / z ; relative
intensities in parenthescs): 1027 (0.22), 1026 (0.28), 1009
(0.31), 996 (0.56), 995 (1.05), 994 (0.95), 981 (0.39), 979 (0.33),
967 (0.56), 963 (0.69), 938 (0.56), 937 (1.22), 748 (2.28), 735
(16.7), 734 (42.5), 720 (5.3), 702 (14.4), 674 (2.6), 575 (2.0),
536(1.4),476(2.6),432(10.6),431 (40.1),417(4.5),400(12.1),
399 (45.5), 387 (22.0), 385 (2.6), 371 (2.3), 370 (3.1), 286 (26.4),
277 (16.7), 276 (loo), 260 (15.1), 258 (18.9), 257 (11.4), 254
(12.9), 244 (12.9), 243 (9.9), 231 (7.6), 230 (17.4), 229 (16.0),
214 (8.3), 202 (7.7), 200 (10.6), 169 (69.6), 141 (55.3).
Serological techniques
Anti-0 sera were obtained by immunization of New
Zealand rabbits (approximately 2.5 - 3.8 kg) with heat-killed
Analysis of the polysaccharide hydrolysate (4 M HCl,
10O"C, 3 h) by conventional methods revealed the presence
of D-glucosamine, L-lysine, L-alanine, D-glucuronic and Dgalacturonic acids, and ethanolamine. Absolute configurations of D-glucuronic and o-galacturonic acids were determined by enzymatic oxidation of the corresponding hcxoses,
as obtained from the two uronic acids by carboxyl reduction.
Phosphatc content in the polysaccharide was estimated as
2.5% by colourimetric analysis [1 51.
13C-NMK spectrum of the polysaccharide (Fig. 1)
contained the signals for four anomeric carbons at 98.7105.1 ppm, 14 carbons linked to oxygen in the region 68.282.5 ppm, C2 atoms of two amino sugars at 55.2 pprn and
56.8 pprn and their C6 at 61.9 ppm and 65.4 ppm, C6 atoms
of two hexuronamides at 170.3 ppm and 170.5 ppm, and the
signals for residues of lysine, alanine, ethanolamine and two
N-acetyl groups (Table 1). The signals Ihr ethanolamine residue at 63.2 ppm and 41.5 ppm and for C5 and C6 of one of
the hexosamine residues at 75.4 ppm and 65.4 ppm were split
into doublcts due to coupling wit.h phosphorus. These data
suggested that the polysaccharide is built up of tetrasaccharide
repeating units containing two N-acetylglucosamine residues,
and residues of glucuronic acid, galacturonic acid, lysine,
alanine, ethanolamine and phosphate. Ethanolaminc is evidently linked through phosphate to C6 of one of the glucosamine residues. The "P-NMR spectrum of the polysaccharide
contained the only signal at 1.33 pprn belonging to a
phosphodiester group. Lysine and alanine are linked to the
uronic acids through a-amino groups, as was previously
shown [5].
The polysaccharide was cleaved selectively by treatment
with anhydrous H F at 0°C and 20°C. Solvolysis at 20°C led
to a single oligosaccharide product, identified as trisaccharide
1. Reaction under the milder conditions (at 0°C) gave mainly
tetrasaccharide 2 along with non-separable higher oligomers.
Both oligosaccharides were isolated by gel chromatography
on TSK HW-40(S) gel, reduced with NaBH4 into
oligosaccharides 3 and 6 and N-acetylated at the 6-amino
group of lysine to yield oligosaccharides 4 and 7 (Fig. 2).
The I3C-NMR spectra showed that trisaccharide 4 contains
residues of N-acetylglucosaminitol, two uronic acids, lysine
and alanine. Tetrasaccharide 7 contains, additionally, a residue of Iv--acetylglucosamine. Phosphoethanolamine was absent from both oligosaccharides.
Methylation analysis of trisaccharide 4 indicated that the
residues of N-acetylglucosaminitol and one of the uronic acids
arc substituted at position 3, whereas the second uronic acid
residue is terminal. The mass spectrum of the methylated
trisaccharide 8 contained the peaks of primary ions with mjz
1027 ( M + H ) , 1026 (M), 734 (biosyl cation), 579, 431
(glycosyl cation) and 276 (Fig. 2). The formation of the ions
with mjz 579 and 431 is indicative for the connection of lysine
to the terminal uronic acid residue and alanine to the central
647
I
I
100
90
80
?O
60
50
PPN
I
40
30
Fig. 1. “ C - N M R spectrum of polysaccharide from P. mirabilis 027
Table 1. Chemical shijts in I3C-NMRspectra
Assignments of signals lying closer to one another than 0.5 ppm may be interchanged. Chemical shifts (S/ppm) for alanine: 177.0- 177.5 (CI),
49.6-51.0 (C2), 17.7-18.7 (C3); for ethanolamine: 41.4-41.6 (CH,NH,), 63.1 -63.2 (CH,O); for N-acetyl groups: 23.3-23.9 (CH,),
175.1- 175.7 (CO). PS, intact polysaccharide; PS*, Smith-degraded polysaccharide; OS, oligosaccharide. The data given i s for the rcpeating
units containing phosphoethanolarnine of the polysaccharide. Unit A, glucuronic acid; unit B, galacturonic acid; unit C , N-acctylglucosamine
or N-acetylglucosaminitol; unit D, N-acetylglucosarnine. The last four compounds are with lysine
Compound
Unit
S
c1
c2
C3
c4
c5
C6
PS* 10
PS* 9
0s 7
0s 4
105.1
105.0
105.0
105.0
105.2
74.3
74.1
74.0
73.8
74.1
79.0
85.0
85.3
75.2
76.2
75.3
71.I
71.1
78.2
72.5
75.4
76.1
76.1
74.9
76.2
170.6
170.7
170.7
?
171.5
PS
PS* 10
PS* 9
0s 7
0s 4
101.2
101.1
101.1
101.5
101.7
68.2
68.3
68.3
68.3
68.3
80.6
80.4
80.4
80.2
80.2
70.7
70.7
70.7
70.6
70.7
72.4
72.5
72.5
72.4
72.5
170.4
170.5
170.5
PS
PS* I0
l’S* 9
0s 7
0s 4
101.2
102.6
102.6
61.9
61.9
55.2
55.6
55.6
55.1
54.8
82.5
82.6
82.2
78.9
78.7
71.5
72.1
71.7
71.5
71.4
75.4
76.7
75.5
72.8
71.7
65.4
62.0
65.6
64.1
64.1
PS
98.7
100.8
56.8
56.7
74.7
74.5
71.1
71 .O
77.2
77.2
61.9
61.8
PS
178.9
177.3
55.5
54.5
54.5
53.9
31.9
31.8
32.0
31.4
23.2
23.1
23.1
23.1
27.4
27.4
28.9
28.9
40.6
40.7
40.2
40.3
PS
0s 7
PS *
0s 7
0s 4
A
?
177.9
?
171.2
648
COOH
COOH
HO
1
OH
A
OH
CNHAC
COOH
COOR'
COOH
COOR'
OR
A
MOH
COOMe
COOH
3
4
5
R=R'.R'~H
DNHAC
F! mirabilis 027
R - R z h , R"=Ac
R = R' =Ac, R'=Me
C
COOMe
P mirabilis S 1959
Fig. 3 . Structures qf Smith-degruded and intact P. mirabilis 0 2 7
polysuccharide und 1'. mirabilis S195Y polysaccharide
the initial polysaccharide. Its 13C-NMR spectrum comprised
two sets of signals, which were assigned to structure 9 containing phosphoethanolamine, and structure 10 lacking this
component (Fig. 3). The main difference between the two
subspectra was the respectivc shift or the signals of C4, C5,
residue; the corresponding trisaccharide with the reversed C6 or the remaining N-acetylglucosamine residue (unit C)
position of the amino acids would give fragments with mjz from 71.7 ppni, 75.5 ppm and 65.6 ppm in structure 9 to
692 and 328, which were absent from the spectrum.
72.1 ppm, 76.7 ppm and 62.0 ppm in structure 10. From the
Trisaccharide 4 was converted into the acetylated deriva- integral intensity ratios of these signals it was concluded that
tive of dimethyl cster 5 by sequential treatment with the linear polymer contains approximately 1 non-phosdiazomethane and acctic anhydride in pyridine. 'H-NMR phorylated repeating unitj4 phosphorylated units. This data
spectrum of this derivative was interpreted by using selective allowed location of the phosphoethanolamine substituent at
segmental spin-decoupling expcrirnents. The coupling con- C6 of unit C. The phosphodiester linkage may not be split
stant ( J l , z = 8.0 Hz) for the residue of glucuronic acid proved during Smith degradation and, hence, the initial polyits j?-configuration. Appearance of the nuclear Overhauser saccharide also contains nonphosphorylated and phosphoryeffect at C3H of galacturonic acid on irradiation of C1H of lated repeating units in the same proportion. Its I3C-NMR
glucuronic acid showed that the latter is linked to C3 of the spectrum was not so well-resolved as that of the Smith-deformer. The anomeric configuration of the galacturonic acid gradcd polysaccharide. but the minor signals at 71.9 ppm
residue could not be dctermined in this way due to overlapping and 76.4 ppm, which belong to the C4 and C5 of the nonC I H and C2H signals. The a-configuration of this sugar was phosphorylated unit C, were also observed.
deduced from the results of the interpretation of thc 13CThe position of substitution of the glucuronic acid residue
NMR spectrum of trisaccharide 4 (Table 1). This was (unit A) and the anomeric confipration of the glucosamine
performed using glycosylation effects data [I 61 and confirmed residue in the backbone (unit C) were determined as a result
finally the structure of the trisaccharide.
of the interpretation of the I3C-KMR spectrum of the SmithAnalysis of the "C-NMR spectrum of tetrasaccharide 7 degraded polysaccharide (Table 1). Anomeric configurations
showed that this compound differs from trisaccharide 4 by of all sugar residues werc further confirmed by thc coupling
the presence of the N-acetyl-/Y-o-glucosamineresidue (unit D) constants ' J c l , determined from the gated-decoupling speclinked to C4 of the glucuronic acid residue.
trum of the Smith-degraded product. The values of 173 Hz,
Smith degradation of the polysaccharide resulted in re- 162 Hz and 159 Hz correspond to an a-configuration for thc
moval of the lateral glucosamine residue (unit D) and forma- galacturonic acid residue (Cl signal at 101.1 ppm) and a 1tion of a linear polymer containing all other components of configuration for the glucosamirie and glucuronic acid resiFig. 2. Structures of oligosacchclride F derived from polyvarrharidr 0 2 7
649
Fig. 4. Double irnmunoelectrophoresis of' P. mirabilis 0 2 7 antiserum with lipopolysaccharide 0 2 7 ( A ) and its 0-specific polysaccharide [ R )
hillyen lm!
Fig. 5 . Precipitation qf 0 2 7 antiserum by various untigms. ( X ) lntact
P. inirabilis 0 2 7 polysaccharide; ( 0 ) Smith-degraded 0 2 7
polysaccharide devoid of the lateral GlcNAc; (0)
N-acetylated 0 2 7
polysaccharide; ( A ) intact P. mirabilis S1959 polysdcchande; (m)
tetrasaccharide 6; ( 0 )tetrasaccharide 7. Precipitation of anti-027
antiserum by intact P. mirahilis 0 2 7 polysaccharide, is inhibited by
tetrasaccharides 6 and 7
dues (C1 signals at 102.6 ppm and 105.0 ppm, respectively)
[17]. Comparison of the structures of tetrasaccharide 7 and
the Smith-degraded polysaccharide allowed to establish the
complete structure of the P. miruhilis 0 2 7 0-specific polysaccharide shown in Fig. 3.
This branched structure is consistent with the data of
Inethylation analysis of this polysaccharide [S] resulted in
identification of thc terminal glucosamine residue.
Stvolog ical studies
In an inimunoelectrophoresis test, lipopolysaccharidc
from P. mira1~ili.s027 as well as its 0-specific part migrated
towards the anode, indicating their negative charge (Fig. 4).
In the quantitative precipitation test 1 1 pg of the
polysaccharide precipitated 92 pg of its specific antibody. Periodate oxidation of the lateral GlcNAc in the polysaccharide
resulted in a significant decrease of serological activity of this
antigen. This confirms the previous data showing that the
release of the lateral GlcNAc by acid hydrolysis led to a strong
decrease in serological activity [5].
N-Acetylation of free amino groups of L-lysinc and
phosphoethanolamine also significantly decreased precipitation activity of the polysaccharide (Fig. 5). This indicates
that L-lysine and/or phosphoethanolamine also play some
serological role.
Two further tetrasaccharides 6 and 7 were used as inhibitors of the precipitation test with anti-027 serum (Fig. 5).
These tetrasaccharides were devoid of phosphoethanolamine
and possessed the lateral residue of GlcNAc. Both were able,
in amounts up to 60 pg, to inhibit 30-40% of the precipitation of anti-027 serum by polysaccharide. Thus, serological
activity of-tetrasaccharide 6 is not affected by blocking of the
6-amino group of L-lysine.
In the polysaccharidc from thc strain P. mirabilis S1959
(Fig. 3 ) studied by us earlier the immunodominant L-lysine is
attached to the carboxyl group of galacturonic acid via its
1-amino group [3, 41.
In precipitation test with anti-027 serum, polysaccharide
S1959 showed only a very weak cross-reaction. Similar results
were obtained when anti-S1959 serum was precipitated by
polysaccharide 027. These results were confirmed by a passive
hcmolysis test in which only a weak cross-reaction between
P. miruhilis 027 antiserum and lipopolysaccharide S1959 was
observed.
DISCUSSION
An unusual feature of P. mirubilis 0 2 7 0-polysaccharide
antigcn is the presence of several different non-carbohydrate
substituents: two amino acids, t-lysine and r-alanine, linked
through an amide group to the carboxyl groups of uronic
acids and ethanolamine attached at position 6 of the N acetylglucosamine residue by a phosphodiester linkage.
Amino acids occur rather rarely in bacterial 0-antigens; however, alanine and lysine are characteristic for many of Proteus
lipopolysaccharides [2]. Ethanolamine phosphate is well
known as a component of the core region of lipopolysaccharides of the majority of Gram-negative bacteria [I 81, but,
to the test of our knowledge. it has never been identified in
the 0-specific polysaccharide chain except of those for P.
mirubilis 0 2 7 [6] and P. mirubilis strain D52 [19].
Due to the presence of the above-mentioned non-carbohydrate constituents the 027 polysaccharide contains both
acidic (carboxyl groups of two amino acids and a phosphate
group) and basic functions (free amino groups of lysinc and
ethanolamine). This endows the polysaccharide with amphoteric properties that may be important for adaptation of the
microorganism to grow under different pH conditions.
Non-sugar components seem also to play an important
role in manifesting the serological specificity of Proteus 0-
650
antigens. The role of lysine in the P . rnirabilis strain S1959 0antigen has been established prcviously 131. In P . miruhilis
0 2 7 a decrease of serological activity in a precipitation test
(up to 40%) is observed as a result of acetylation of the free
amino groups in the 0-specific polysaccharide. This decrease
is most likely related to the substitution of the amino group
of ethanolamine, since no difference was observed in the inhibition of thc precipitation by the aid of tetrasaccharide fragments 6 and 7 both of which are devoid of ethanolamine
phosphate. and in one of which 6-amino group of lysine is
free whereas in the other it is N-acetylated. It could be pointed
that the role of ethanolamine phosphate is not so crucial as
the role of the immunodominant lateral N-acetylglucosamine
residue, removal of which resulted in the stronger decrease of
the serological activity [5].
That lysine is not very important for the manifesting the
serological specificity of the 027 antigen is confirmed by the
very weak cross-reaction between the anti-027 serum and
0-polysaccharide of S1 9 59 lipopolysaccharide, in which this
amino acid is known to play the immunodominant role [3,
141.
The weak serological relationship between these O-antigens may result from the different spatial arrangement of
other structural constituents around lysine, since this common
amino acid component is attached to different sugar
components, to the lateral residue of galacturonic acid in the
strain S19.59 antigen [3, 41 and to the residue of glucuronic
acid occurring in the main chain of 027 antigen. Evaluation
of the confirmation of both polysaccharides would be helpful
in supporting this suggestion.
We are indebted to Prof. Krystyna Kotelko for her constructive
criticism and valuable discussion. We thank mgr Jolanta Makowska
and Janusz Wlodarczyk for excellent technical assistance,
REFERENCES
1. Kotelko. K . (1986) Curr. Top. ktierohiol. Immunol. 129, 181 21 5.
2. Sidorczyk, Z., Kaca, W. & Kotelko, K. (1975) Bull. Acad. Pol.
Sci. Ser. Sci. Biol. 23, 603 -609.
3. Gromska, W. & Mayer, H. (1976) Eur. J . Biochem. 62,391 -399.
4. Kaca, W., Knirel, Y. A , ,Vinogradov, E. V. &Kotekko, K. (1987)
Arch. Immunol. Ther. Exp. 35,431 -431.
5. Gromska, W. & Krajewska, D. (1981) Arch. Imraunol. Ther. Exp.
29, 595 -600.
6. Krdjewska, D. & Gromska, W. (1982) Arch. Zmmunol. Ther. Exp.
29, 581 -587.
7. Dmitriev, B. A., Knirel, Y. A., Kochetkov. N . K. & Hofman, I.
L. (1976) Eur. J . Biochem. 66, 559-566.
8. Roseman, S. & Ludowieg, J. (1954) J . Am. Chem. SOC.76, 302.
9 Vinogradov, E. V., Shashkov, A. S . , Knirel, Y. A., Kochetkov,
N. K., Kholodkova, E. V. & Stanislavsky, A. S. (1987) Bioorg.
Khim. 13, 660 - 669.
10 Weslphal, 0. & Jann, K. (1965) Methods Carbohydr. Chem. 5,
83-91.
11 Hakomori: S. (1964) J . Biochem. (Tokyo) 55, 205-208.
12 Waeghe, T. J., Darvill, A. G., McNeil, M. & Albersheim, P. (1983)
Carbohydr. Res. 123, 281 -304.
13. Vinogradov, E. V., Kaca, W., Knirel, Y. A., R6ialski, A. &
Kochetkov, N. K. (1989) Eur. J . Biochem. 180,95-99.
14. Gromska, W., Kaca, W. & Kotelko, K. (1978) Bull. Arad. Pol.
Sci. Seu. Sci. Biol. 26, 7 13.
15. Danilov, L,. L. & Chojnacki, T. (1981) FEBS Letf. 131, 310-312.
16. Lipkind, G. M., Shdshkov, A. S., Knirel, Y. A,, Vinogradov, E.
V. & Kochctkov, N. K. (1988) Carbolzvdr. Res. 175, 59-75.
17. Bock, K. & Pedersen, C. (1974) J . Chem. Soc. Perkin Trans. [I,
293-297.
18. Liideritz, O., Freutienberg, M. A,, Galanos, Ch., Lehmann, V.,
Rictschel, E. Th. & Shaw, D. H. (1982) Current Top. Membr.
Trans. 17, 79-151.
19. Gmeiner, J. (1977) Eur. J . Biochem. 74, 171 -180.
-