Revised Structure of the Capsular Polysaccharide of Klebsiella Serotype 15

System. Appl. Microbiol. 15, 505-512 (1992) © Gustav Fischer Verlag, StuttgartfNew York

Revised Structure of the Capsular Polysaccharide of Klebsiella Serotype 15 ATREYEE MUKHERJEE and NIRMOLENDU ROY'f Department of Biological Chemistry, Indian Association for the Cultivation of Scienc, Calcutta-700 032, India

Received April 21, 1992

Summary The capsular polysaccharide from Klebsiella Type 15 was found to contain D-galactose, D-glucose, and Dglucuronic acid in the ratios 4: 1 : 1. Methylation analysis of the native and carboxyl-reduced polysaccharide provided informaton about the linkages of different sugar residues in the repeating unit. Partial acid hydrolysis of the polysaccharides gave three acidic and one neutral oligomers whose structures were established. The sequence of different sugar moieties were established from the structures of oligomers and from the Smith degradation of the native antigen. The anomeric configurations of different sugar residues were established with the help of IH and 13C NMR spectral analysis of the native polysaccharides, the aldobliouronic acid and the glyceryl glycoside of the trisaccharide from Smith degradation together with the findings of enzymic hydrolysis. Based on all these results, the hexasaccharide repeating unit 1 was assigned to the K-15 polysaccharide. The results of the inhibition study of several oligomers related to K15 antigen on the homologous immune system support this structure. a-D-Glcp

1

!

6

(1)

-3)-a-D-Galp-(I~)-~-D-Galp-(1~3)-~-D-Galp-(1~6)-~-D-Galp(l~

3

t

1 ~-D-GlcpA

Key words: Klebsiella serotype 15 - Capsular polysaccharide - Structure

Introduction The genus Klebsiella was classified by Orskov (1956) into approximately 80 serotypes based on their antigenic capsular polysaccharides. Qualitative sugar analysis of all the polysacchrides were reported (Nimmich, 1968). The structure of the polysaccharide from Klebsiella Type 15, as reported recently (Nath, and Chakraborty, 1987) had a hexasaccharide repeating unit 2. * Corresponding author

~-D-Glcp-(l ~4 )-a-D-GlcpA

1 ~ 2

-4 )-~-D-Galp-( 1~3 )-~-D-Galp-(l ~3 )-~-D-Galp-(l ~6) -~-D-Galp-(l~

(2)

As a part of our program to study the relation between the structure of the antigen and its immunological property we isolated the aldobio- and aldotriouronic acids from

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A. Mukherjee and N. Roy

K15 polysaccharide which were analyzed and found not to be in conformity with the published structure of K15 polysaccharide. This necessitated the reexamination of structure and immunochemical behaviour of the K15 polysaccharide. We now report our completed investigation.

Materials and Methods Isolation and purification of polysaccharide. Klebsiella type 15 bacteria was grown on 3 % sucrose-yeast extract-agar medium and the polysaccharide was isolated as described earlier (Sarkar and Roy, 1986). The polysaccharide was purified by passing through a column of Sephadex G-I00 and column was eluted with 4: 10: 1000 pyridine-acetic acid-water. The homogeneity of the polysaccharide was established by high voltage electrophoresis in borate buffer. Miscellaneous methods. Uronic acid content of the native polysaccharide was determined by Carbazole method (Galambos, 1967). The native polysaccharide was carboxyl reduced by standard method (Taylor and Conrad, 1972). Acid hydrolysis of oligo- and polysaccharides with 2M trifluoroacetic acid and subsequent conversion of the products to their alditol acetates were conducted as described before (Sarkar and Ray, 1986). Oxida..tion of K15 polysaccharide with chromium trioxide was conducted in the usual way (Lindberg et al., 1972). Inhibition of oligomers (Lonn, 1987 and Johansson et aI., 1963) on the homologous immune system was carried out in the usual way (Sarkar et al., 1989). Chromatographic methods. Paper chromatograp'hy was performed on Whatman No. 1 and No.3 MM papers. Solvent systems (v/v) used were (A) 9: 2 : 2 ethyl acetate-acetic acid-water and (B) 4: 1 : 5 I-butanol-acetic acid-water (upper layer). Gas liquid chromatography (GC) was performed by using a Hewlett Packward Model 5730A instrument having a flame ionization detector and a Hewlett-Packard Model 3380A electronic integrator. The columns used were glass (1.85 m x 6 mm) Packed with (1) 3% ECNSS-M on Gas Chrom Q (100-120 mesh) and (2) 3% OV-225 on Gas Chrom Q (100-200 mesh). The chromatography was performed at 180° for neutral sugars and at 170° for methylated sugars by converting the sugars into their corresponding alditol acetates (Sarkar et aI., 1986). Retention times of partially methylated alditol acetates were measured with respect to that of 1.5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol as unity. GC-MS analyses of the partially methylated alditol acetates were carried out on a Hewlett-Packard Model 5988A Mass spectrometry System using Supelco SP-2330 fused silica capillary column (15 m x 0.25 mm, 0.20 ~m film). Colorimetric estimations were performed by using a Hitachi Model 100-60 spectrophotometer. NMR spectroscopy and polarimetry. Proton NMR spectra were recorded with a Bruker WM 300 instrument in D2 0 at 70° with acetone as internal standard. 13C NMR spectra were taken with the same instrument at 75 MHz with sodium trimethylsilyltetradeutero-propionate as external standard or dioxane as internal standard. Optical rotations were measured with a PerkinElmer Model 241 MC Spectropolarimeter. Methylation analysis. The methylation of native and Carboxyl-reduced polysaccharides were carried out first by Hakomori (1964) and then by modified Kuhn procedure (Walker et al., 1962). The oligosaccharides were methylated according to modified Kuhn procedure. The permethylated polysaccharide and oligo saccharides were reduced with lithium aluminium hydride (Lindberg et al., 1972) and the products were purified over a LH20 column, hydrolysed with 2M trifluoracetic acid, converted to alditol acetates and analysed by GC or GC-MS or both.

Periodate oxidation of K15 Polysaccharide. The method used was basically that of Goldstein et al. (1965). To an aqueous 0.05% solution of the polysaccharide (200 mg in 400 mL) was added to 0.2 M sodium metaperiodate (100 mL) and the mixture was kept in the dark for 46 h at 4°. The excess of periodate was decomposed by adding ethylene glycol (10 mL) and the mixture was kept at room temperature for 3 h. The solution was dialyzed against distilled water for 2 days and then concentrated to 5 mL. Sodium borohydride (250 mg) was added and the solution was kept for 4 h at room temperature, decationised with Dowex 50W-X8 (H+) resin and concentrated to dryness. Boric acid was removed from the residue by repeated addition and evaporation of methanol. Alditol acetates prepared from a portion of the residue showed only galactose. The rest of the polyol was hydrolyzed with 0.5 M trifluoroacetic acid for 17 h at room temperature. The acid was evaporated off by codistillation with water and the product was lyophilized. The product (35 mg) gave a single spot in PC (solvent A) and was purified by preparative paper chromatography. A portion of the product (2 mg) was subjected to acid hydrolysis and methylation analysis. Proton and Carbon-13 NMR analysis of the product was carried out. Partial hydrolysis of K15 polysaccharide. The polysaccharide (100 mg) was hydrolyzed with 0.5 M trifluoroacetic acid (50 mL) for .1.5 h at 100°C. The acid was removed by evaporation. The solution was passed through a column of Dowex 1 x 4 (OAc) anion exchange resin. The neutral oligomers were eluted with water and freeze dried. The acidic oligomers were recovered by eluting with 30% acetic acid. The eluate was freeze dried and resolved into different components by preparative paper chromatography (solvent A). Hydrolysis with enzymes. Native K15 polysaccharide (5 mg each) in acetate buffer (pH 5.0, 3 mL) was incubated separately with a-D-glucopyranosidase (Bakers yeast, Sigma) and ~-D­ glucopranosidase (Almonds, Sigma) at 37°C for 36 h. The enzyme was deactivated, the mixture was filtered and dialysed. The dialysate was concentrated, freeze dried and analysed by GC as alditol acetate. In similar experiments, the trisaccharide from Smith degradation was subjected to hydrolysis with a-D-galactopyranosidase (Aspergillus niger, Sigma) and ~-D-galactopy­ ranosidase (E. coli, Sigma) and the dialysate analysed as above.

Results Isolation and composition of K15 polysaccharide Klebsiella K15 bacteria were grown on an agar medium and the purified capsular polysaccharide was isolated from the bacteria. Hydrolysis of the polysaccharide followed by PC, showed the presence of glucose, galactose and glucuronic acid together with some slower moving components. GC of the derived alditol acetates confirmed the identities of galactose and glucose in the approximate ratio 4: 1. The sugar components were isolated by preparative paper chromatography and they were identified as D-galactose, D-glucose and D-glucuronic acid from their specific rotations. The proportion of uronic acid was estimated (Galambos, 1967) to be 15.8%. GC analysis of the alditol acetates of carboxyl reduced polysaccharide showed D-galactose and D-glucose in the ratio 2 : 1. The results of acid hydrolysis are shown in Table 1. NMR data ,of K15 polysaccharide is given in Table 2. Carbon-13 NMR spectrum is shown in Fig. 1.

Revised Structure of the Capsular Polysaccharide of Klebsiella Serotype 15 Table 1. Acid hydrolysis of Klebsiella K15 polysaccharide and physical data of the oligomers

Mole percents of sugars obtained in K15 polysaccharide

Sugars

D-Galactose G-Glucose D-Glucuronic acid Aldobiuronic acid Aldotriouronic acid Aldotetraouronic acid Neutral disaccharide Glyceryl trisaccharide from Smith degradation

Native

Carboxyl reduced

67.2 17.0 15.8

67.1 32.9

Oligomers from partial hydrolysis Yield (mg)

7.0 2.7 6.6 5.0 35.0

Methylation analysis Results of the methylation analysis of K15 polysaccharides and the oligosaccharides were compiled in Table 2. Alditol acetates obtained from methylated polysaccharide revealed (GC) derivatives of 2,3,4,6-tetra-0methylglucose, 2,4,6-tri-0-methylgalactose, 2,3,4-tri-0methyl-galactose and 2-0-methylgalactose in the approximate ratio 1 : 2 : 1 : 1. Reduction (Lindberg et aI., 1972) of the methylated polysaccharide with lithium aluminium hydride followed by conversion of the product into alditol acetates gave 2,3,4,6-tetra -0-methy19lticose, 2,4, 6-tri -0-meth ylgalactose, 2,3,4-tri-0-methylgalactose, 2,3,4,6-tetra-0-methylglucose and 2-0-methylgalactose in the approximate ratio 1 : 2 : 1 : 1 : 1. The peaks were identified by comparing with authentic samples and also by GC-MS analysis (Table 3). Methylation analysis of the carboxyl reduced K15 poly-

507

RLact

[alD

0.93 0.65 0.48 0.90 0.71

+79° +52° +25° +2° -29.6° +6° -5.8° +30°

saccharide gave 2,3,4,6-tetramethylglucose, 2,4,6-tri-0methylgalactose, 2,3,4-tri-0-methylgalactose and 2-0methyl-galactose in the approximate ratio 2: 2 : 1 : 1.

Oxidation of the polysaccharide wth periodate Smith degradation of K15 polysaccharide gave the glyceryl glycoside of a trisaccharide (35 mg) having Rlactose 0.71 and [afoD + 30°, as the only sugar moiety. Acid hydrolysis of a portion of the trisaccharide gave galactose and glycerol in the ratio 3 : 1. Methylation analysis of the trisaccharide showed the presence of 2,3,4,6-tetra-0-methylgalactose, 2,4,6-tri-0-methylgalactose and 2,3,6-triO-methylgalactose in approximately equimolar amounts. The peaks were confirmed by comparing with authentic samples and also by GC-MS analysis (Tables 3). Proton and Carbon-13 NMR spectra of the glyceryl trisaccharide is shown in Fig.2a and 2b while major signals are compiled in Table 2.

Table 2. NMR data for Klebsiella K15 polysaccharide, aldobiouronic acid and glyceryl trisaccharide IHNMR

Compounds

Aldobiouronic acid

BC NMR

Chemical shift PPM (b)

J

Hz

Ratio of integral

Proton assignment

Chemical shift PPM (b)

Anomeric carbon assignment

5.2 4.6 4.5

3 8 8

0.3 1.2 1.1

a-Gal-OH p-GlcA p-Gal-OH

185.4 104.2 96.7 92.7

C-6 of GlcA p-GlcA a-Gal

~-Gal

Glyceryl trisaccharide

4.95 4.60 4.56

2.4 7.5 7.8

3.8 4.0 4.0

a-Gal p-Gal p-Gal

105.2 105.1 99.2

p-Gal p-Gal a-Gal

K15 polysaccharide

5.07 4.95 4.7 4.60 4.47 4.40

1.5 3.0 8.0 8.0 8.0 8.0

0.8 0.7 1.1 1.2 1.1 0.8

a-Gal a-Gal p-Gal p-GlcA p-Gal p-Gal

174.4 104.93 103.77 103.55 102.72 99.46 98.56

C-6 of GlcA p-GlcA p-Gal p-Gal p-Gal a-Gal a-Gal

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A. Mukherjee and N. Roy

J

180

140

160

12~

60

80

Hl~

20

Fig. 1. Carbon-13 NMR spectrm of the native polysaccharide from Klebsiella serotype 15.

Partial hydrolysis of the polysaccharide Partial hydrolysis of K15 polysaccharide gave one aldobio- (A 2 ), one aldotrio- (A 3 ) and one aldotetraouronic acid (A4) together with D-~lucuronic acid. The glucuronic acid thus obtained had [al °D + 30°, corresponding to the specific rotation of an authentic D-glucuronic acid. Preparative paper chromatography of the neutral fraction gave a disaccharide having [alD - 5.8° together with galactose and glucose. Physical data of the oligosaccharides are shown in Table 1. Proton and Carbon-13 NMR spectra of

the aldobiouronic acid are shown in Fig. 3a and 3b while major signals are compiled in Table 2. Acid hydrolysis of A2 , A3 , and A4 with 2M trifluoroacetic acid gave galactose as the only neutral sugar. In each case PC of the hydrolysates gave spots corresponding to galactose and glucuronic acid. Acid hydrolysis of the neutral disaccharide also gave galactose only. The results of the methylation analysis of the oligomers are summarised in Table 3. It was noted that 2,3,4-tri-O-methylglucose appeared only after reduction with LiAIH4 •

Table 3. Relative retention times and mole percents of partially methylated sugars identified as their alditol acetates obtained from permethylated poly- or oligosaccharide Methylated sugars

2,3,4,6,-GIc 2,3,4,6-Gal 2,4,6-Gal 2,3,6-Gal 2,3,4-Gal 2,3,4-Gal 2,6-Gal 2-Gal

Ta

1.00 1.17 2.02 2.19 2.20 2.87 3.14 6.35

Mole percent of sugars obtained in GC

Tb

1.00 1.22 2.26 2.40 2.45 3.46 3.65 8.17

II

III

20.6

33.6

17.8

41.2

35.3

35.6

17.5

14.3

14.3 14.3

20.7

16.8

18.0

IV

V

VI

VII

VIII

35.9 33.5 30.6

53.1 46.9

52.6

69.0

26.3 25.7

47.4

31.0

25.8 22.2

2,3,4,6-Glc etc. indicates 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-D-glucitol etc. Ta and Tb indicates retention time relative to 2,3,4,6Glc on a column of OV-225 and ECNSS-M respectively. I, K15 polysaccharide; II, carboxal reduced polysaccharide; III, LiAIH4 reduced permethylated polysaccharide; IV, trisaccharide obtained by Smith degradation of the polysaccharide; V, Neutral disaccharide; VI, VII, and VIII, aldobio-, aldotrio- and aldotetraouronic acid after LiAIH4 reduction respectively.

Revised Structure of the Capsular Polysaccharide of Klebsiella Serotype 15

S.I

PPM

509

4.5

Fig. 2a. Proton NMR spectrum of aldobiouronic acid.

Fig. 2b. Carbon-13 NMR spectrum of aldobiouronic acid.

Discussion The native K15 polysaccharide was found to contain Dglucose, D-galactose and D-glucuronic acid in the ratio 1 : 4 : 1. The result suggested the polysaccharide to be having a hexasaccharide repeating unit in conformity with the results published earlier (Nath and Chakraborty, 1987). The 3C-NMR spectrum (Fig. 1) of the polysaccharide indicated the presence of six anomeric carbons (Table 2) of which two were a-glycosidic appearing at 6 99.46 and 99.56 the other four being ~-glycosidic appearing at 6

102.75, 103.55, 103.77 and 104.9. The signals obtained in the proton NMR spectrum (Table 2) corroborated these findings. The earlier results (Nath and Chakraborty, 1987) showing only one a-glycosidic linkage does not seem to be correct. Methylation analysis of the native K15 polysaccharide and carboxyl reduced K15 polysaccharide (Table 3) showed that both Dcglucose arid D-glucuronic acid were present as non-reducing units, and attached to one galactose moiety which thus appeared as 2-0-methylgalactose. Of the remaining galactose units, two were present as 0-3

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A. Mukherjee and N. Roy

5 lJ

4. 5

4. 0

I

I

3 5

-3 .lT

Fig. 3a. Proton NMR spectrum of glyceryl trisaccharide from Smith degradation.

,

?

Fig. 3b. Carbon-13 NMR spectrum of glyceryl trisaccharide from Smith degradation.

i --

I

2 5 PPM

Revised Structure of the Capsular Polysaccharide of Klebsiella Serotype 15 linked and one as 0-6 linked sugars. The peaks obtained in GC were identified by comparing with authentic samples and also by GC-MS analysis. During methylation analysis of native K15 polysaccharide, since 2,3,4-trimethylglucose was not formed before reduction with lithium aluminium hydride, it must have originated from D-glucuronic acid moieties present as non-reducing units. As expected, during methylation analysis of carboxyl reduced K15 polysaccharide, one unit of tetramethyl glucose have appeared from the D-glucuronic acid moieties. The results of methylation analysis in the earlier communication on K15 structure indicated two 2,3,6-tri-0-methylgalactose and one 4,6-di-0-methylgalactose in place of two 2,4,6-0-methylgalactose and one 2-0-methylgalactose respectively as confirmed in the present study. Something must therefore went wrong in the earlier study because our results are confirmed by periodate oxidation. As stated above, the results of the periodate oxidation and Smith degradation of the antigen agreed well with the results of methylation analysis. Methylation analysis of the resulting glyceryl trisaccharide showed one terminal, one 0-3 linked and one 0-4 linked galactose. The presence of glycerol during acid hydrolysis was expected. Proton NMR spectrum of the glyceryl trisaccharide (Fig.2a) showed one anomeric proton signal for a-galactoside at <:> 4.95 (d, J = 2.4Hz) and two anomeric proton signals for ~-galactoside at <:> 4.60 (d, J = 7.5Hz) and <:> 4.56 (d, J = 7.8Hz) respectively while total proton count turned out to be very close to 26. Carbon-13 NMR spectrum of the trisaccharide (Fig. 2b) also provided an anomeric carbon signal for a-galactoside at <:> 99.2 and two anomeric carbon signals for ~-galactoside at () 105.1 and 105.2 respectively while total count of carbon was 21. Hydrolysis of the trisaccharide with a-D-galactopyranosidase cleaved one galactose unit while ~-D-galactopyranosidase cleaved none. This proved that the terminal non-reducing galactose was a-linked. The structure of the trisaccharide can therefor be written as

511

corresponded to the a and ~-forms of the reducing galac· tose unit. The structure of the aldobiouronic acid was therefore confirmed to be ~-d-GlcpA-(1 ~3 )-D-Galp

(4)

While the previous investigators did not report the NMR spectra of aldobiouronic acid but arbitrarily reported the anomeric configuration of D-glucuronic acid to be a, the present study confirmed the linkage to be ~-from its NMR spectra. Acid hydrolysis and methylation results of aldotriouronic acid together with its more negative and rotation compared to the aldobiouronic acid definitely points its structure to be ~-D-GlcpA(l ~3 )-~-D-Galp-(l ~3 )-D-Galp

(5)

Since the two galactose units in the aldotriouronic acid are also the same two non-terminal galactose units of the glyceryl trisaccharide from Smith degradation, the structure 3 must be the correct one for the glyceryl trisaccharide. Enzymic hydrolysis of K15 polysaccharide revealed that D-glucose was easily cleaved by a-D-glucopyranosidase whereas ~-D-glucopyranosidase had no cleaving effect. These results together with what we gathered from above discussion proved that D-glucose moiety in K15 antigen was a-linked. Moreover chromium trioxide oxidation of peracetylated K15 polysaccharide showed no appreciable change in the amount of glucose with time indicating glucose to be a-linked. Accordingly the structure of the repeating unit of the K15 polysaccharide must be 1. a-D-Glcp 1 ~ 6 -3)-a-D-Galp-(1~)-~-D-Galp-(1~3)-~-D-Galp-(1~6)

a-D-Galp-(l ~ )-~-D-Galp-(l ~3 )-~-D-Galp-O-glcerol (3)

3

while another structure with interchanging of two nontermin a galactose moieties is also a possibility. Previous investigators have not conducted the periodate oxidation. Acid hydrolysis and methylation analysis suggested the aldobiouronic acid (A 2) to be D-GlcpA- (1~3)-D-Galp. The low specific rotation (+2°) of A2 indicated that the glucuronic acid moiety was ~-linked to D-galactose. This assumption was confirmed from its IH and 13C-NMR spectral results. The IH-NMR spectrum (Fig. 3a, Table 2) of the aldobiouronic acid showed three signals in the anomeric region. The signal of () 4.6 (d, J 8Hz) for 1.2 integral of proton represented the ~-anomeric linkage of D-glucuronic acid residue. The other two signals at () 5.2 (d, J 3Hz) and () 4.5 (d, J 8Hz) for 0.3 and 1.1 integrals respectively of proton were assigned to the a and ~-forms of the reducing galactose moiety. In 13C-NMR spectrum (Fig.3b, Table 2), the signal for C-l of glucuronic acid appeared at () 104.25 confirming its ~-configuration. Other anomeric signals appearing at () 92.77 and 96.74

1

i

-~-D-Galp(l~

(1)

~-D-GlcpA

The aldotetraouronic acid and the neutral disaccharide whose structure could be written from acid hydrolysis and methylation analysis as 6 and 7, fit well in structure 1. a-D-Galp-(1~4)-~-D-Galp-(1~3)-D-Galp

3

(6)

i

1 ~-D-GlcpA ~-D-Galp-(1~3)~D-Galp

(7)

While Klebsiella serotypes are specific antigens, it is not possible for any strain to contain two different capsular polysaccharides. That we were dealing with the right strain was confirmed by serological identification of the

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Inhibitor

None Methyl a-D-glucopyranoside Methyl P-D-glucopyranoside Methyl a-D-galactopyranoside Methyl p-D-galactopyranoside D-glucuronic acid Disaccharide (7) Glyceryl trisaccharide (3) Aldobiouronic acid (4) Aldotriouronic acid (5) Aldotetraouronic acid (6) Pseudocellobiouronic acid (8) Disaccharide (9)

Inhibitor added

IgGa precepitated

(~mole)

(~g)

0.0 3.9 4.1 3.5 4.1 3.7 2.5 2.0 2.1 1.9 2.5 2.1 2.25

610 372 403 504 473 369 304 293 220 179 153 445 250

% Inhibitionb

39.0 34.0 17.4 22.5 39.5 50.2 52.0 64.0 70.7 75.0 27.0 59.0

Inhibitor needed for 50% inhibition (~ mole)

Table 4. Inhibition of homologous precipitin reaction in the Klebsiella Type 15 immune system

2.40 1.95 1.65 1.05 1.30 1.40

Key: a Results are calculated to 1.0 ml of undiluted serum. b Average of duplicate runs.

strain by Dr. 1. 0rskov after the investigation was completed. Inhibition of several mono- and oligo saccharides on the homologous immune system (Table 4) showed that the aldobiouronic acid (4), aldotriouronic acid (5) and aldotetraouronic acid (6) inhibited the quantitative precipitin reaction to a significant extent at a very low concentration whereas the pseudocellobiouronic acid (8) was found not to be an effective inhibitor at moderately high concentration. It has been suggested that (Sutherland, 1977) the serological specificity resides with oligosaccharide side chain specially if it contained an uronic acid moiety (Heidelberger et aI., 1969). Thus the fact that pseudocellobiouronic acid (8) which is an oligosaccharide side chain of 2 does not inhibit the precipitin reaction to a significant extent but 6-0-(a-D-glucopyranosyl)-D-galactose (9) and also the oligomers 3-7 which are parts of structure 1 inhibit the reaction significantly confirm structure I to be correct for Klebsiella K15 repeating unit. Acknowledgements. The authors are indebted to Dr. I. 0rskov, Statens Serum Institute, Copenhagen, for providing the bacterial strain and also for establishing its authenticity once again after the investigation was completed. The authors are thankful to Drs. H. Mayer and S. Basu of Max-Planck-Institut fur Immunbiologie, Freiburg, Germany, for GC-MS and NMR spectral analysis, to Drs. G. Magnusson and A. K. Ray of Lund Institute of Technology, Sweden also for NMR spectral analysis and to Dr. A. K. Guha of this department for growing the bacteria and for helpful discussions.

References 1. Dutton, G. G. S., Paulin, M.: The capsular polysaccharide of Klebsiella serotype K60j A novel structural pattern. Carbohydr. Res. 87, 107-117 (1980) 2. Galambos, J. T.: The reaction of carbozole with carbohydrates. 1. Effect of borate and sulfamate on the carbazole color of sugars. Analyt. Biochem. 19, 119-132 (1967) 3. Goldstein, I. j., Hay, G. W., Lewis, B. A., Smith, F.: Controlled degradation of polysaccharides by periodate oxida-

4. 5. 6.

7. 8. 9.

10. 11. 12. 13. 14. 15.

16.

tion, reduction and hydrolysis. pp. 361-366. In: Methods. Carbohydr. Chern. (R. L. Whistler, ed.), Vol. 5. New York, Academic Press 1965 Hakomori, S.: A rapid permethylation of glycolipid and polysaccharide catalyzed by methylsulfinyl carbanion in dimethyl sulfoxide. J. Biochem. 55, 205-208 (1964) Heidelberger, M., Roy, N., Glaudemans, C. P. j.: Inhibition by Aldobiouronates in the precipitation of pneumococcal type II and III systems. Biochemistry 8, 4822-4824 (1969) Hoffman, j., Lindberg, B., Svensson, S.: Determination of anomeric configuration of sugar residues in acetylated oligoand polysaccharides by oxidation with chromium trioxide in acetic acid. Acta Chern. Scand. 26, 661-666 (1972) Johannson, I., Lindberg, B., Theander, 0.,: Pseudocellobiouronic acid, synthesis and Acid hydrolysis. Acta Chern. Scand. 17,2019-2024 (1963) Lindberg, B., Lonngren, j., Nimmich, W.: Structural studies of the Klebsiella O-group 9 Lipopolysaccharide. Carbohydr. Res. 23, 47-55 (1972) Lonn, H.: Glycosylation using a thioglycoside and methyl trifluoromethanesulfonate. A new and efficient method for cis and trans glycoside formation J. Carbohydr. Chern. 6, 301-306 (1987) Nath, K., Chakraborty, A. K.: Studies of the primary structure of the capsular polysaccharide from Klebsiella serotype K15. Carbohydr. Res. 161,91-96 (1987) Nimmich, W.: Zur Isolierung und qualitativen Bausteinanalyse der K-Antigene von Klebsiellen. Z. med. Mikrobiol. Immunol. 154, 117-131 (1968) 0rskov, I.: Serological Investigations in the Klebsiella group. Acta Path. Microbiol. Scand. 38, 375-384 (1956) Sarkar, A. K., Roy, N.: Structure of the Klebsiella type 10 capsular polysaccharide. Carbohydr. Res. 152, 205-216 (1986) Sutherland, I. W.: pp. 399-443. In: Immunochemistry, An Advanced Text Book (L. E. Glynn, and M. W. Steward, eds.). New York, Wiley (1977) Taylor, R. L., Conrad, H. E.: Stoichiometric depolymerization of polyuronides and glucosaminoglucurans to monosaccharides following reduction of their carbodiimide activated carboxyl groups. Biochemistry 11, 1383-1388 (1972) Walker jr., H. G., Gee, M., McCready, R. M.: Complete methylation of reducing carbohydrates. J. Org. Chern. 27, 2100-2102 (1962)

Dr. Nirmolendu Roy, Dept. of Biological Chemistry, Indian Association for the Cultivation of Science, Calcutta-700 032, India