0021-9193/78/0133-0390$02.00/0 JOURNAL OF BACTFROLOGY, Jan. 1978, p. 390-391 Copyright a 1978 American Society for Microbiology
Vol. 133, No. 1 Printed in U.S.A.
Primary Structure of the Escherichia coli Serotype K42 Capsular Polysaccharide and Its Serological Identity with the Klebsiella K63 Polysaccharide HEINER N1EMANN,1 AJIT K.
CHAKRABORTY,' HORST FRIEBOLIN,2 AND STEPHAN STIRMI*
Max-Planck-Institut fiur Immunbiologie, Freiburg,I and Institut fur Organische Chemie der Universitat,
Heidelberg,2 Germcan Federal Republic Received for publication 6 September 1977
The Escherichia coli K42 capsular polysaccharide consists of --3)-a-D-Galp(1--'3)-a-D-GalUAp-(1--'3)-a-L-Fucp-(l-. repeating units. The E. coli K42 and Klebsiella K63 antigens are serologically identical. Recent advances in the conformation analysis of bacterial polysaccharides (2, 5, 6) have led to a new interest in complete primary structures of these polymers. For this reason, we have reinvestigated the Escherichia coli K42 capsular polysaccharide, a partial primary structure of which has previously been published (3). The K42 glycan, consisting of D-galactose (Gal), D-galacturonic acid (GalUA), fucose (Fuc), and 0-acetyl in a molar ratio of 1:1:1:0.5 (3), was isolated by the phenol-water-Cetavlon technique from E. coli A295b [08:K42(A):H-] (3). From its optical rotation, the fucose, prepared by hydrolysis and paper chromatography, was assigned to the L series. After partial acid hydrolysis (20 min at 100°C in 0.5 N H2S04), the aldobiouronic acid GalUA Fuc and the aldotriouronic acid Gal GalUA Fuc were isolated by paper electrophoresis, as described by Jann et al. (3). Using the procedures previously cited and described (7, 9), the glycan and the trisaccharide (repeating unit) were methylated and analyzed by gas-liquid chromatography-mass spectrome-
try of the alditol acetates (1) before and after reduction of the carboxyl groups. The polymer and the aldobiouronic and aldotriouronic acids were also subjected to proton magnetic resonance spectroscopy. The results are summarized in Tables 1 and 2. The 0-acetyl residues on every second repeating unit were not localized. Because there were some chemical indications (8; J. P. Joseleau, personal communication) that the E. coli K42 and Klebsiella K63 capsular polysaccharides might be similar, E. coli A295b was serologically compared with the test strain for the Klebsiella K63 antigen (4, 8) as follows: both strains were subjected to tube agglutinations in an E. coli 08:K42 rabbit serum, prepared with E. coli A295b (4) (homologous K titer, 1:80), as well as in a Difco Klebsiella K63 serum (homologous K titer, 1:160). The strains crossreacted to the same titers, and all homologous K agglutinins could be absorbed (4) from both sera with heterologous bacteria.
-.
--
-.
We express our gratitude to D. Borowiak for his expert handling of the gas-liquid chromatography-mass spectrome-
TABLE 1. Identification and ratios of O-acetyl-O-methylalditols obtained from E. coli serotype 42 capsular polysaccharide and the repeating unit triouronic acid Peracetyl derivative of:
2,4-FucOH 2,3,4,6-GalOH 2,4,6-GalOH 2,4-GalOH
'
_
Literal
Found
45
1.12 1.25 2.28 6.35
1.14 1.24 2.29 6.28
+ +
P4r;asn, froav".v,+ 1-axl{ /.I rmary 1ragmetLs LUUroU 1twe/
117
131
+ + + + + 2,4-di-O-methyl-L-fucitol, etc.
161
189
+ +
D.f;- -f
ratoUo1UU1 peaK itegas
205 233 247
If
II
III
IV
+
0.8
0.8
0.7 1.0
0.7 1.0
+
+ +
1.0
1.0
(191)d
0.8d
0.8d
2,4-FucOH b Retention time, relative to peracetylated 2,3,4,6-GlcOH (T - 1.00) and 2,3-GlcOH (T 5.39) in gas-liquid =
on an ECNSS-M column (1). chromatography c I, K42 polysaccharide, methylated (the GlcUA derivative was not detected with the methods used); II, K42 polysaccharide, methylated, and then carboxyl reduced-dideuterated; III, K42 aldotriouronic acid, methylated (9); IV, K42 aldotriouronic acid, methylated (9), and then carboxyl reduced-dideuterated. d Dideuterated fragment found instead.
390
391
NOTES
VOiL. 133, 1978
TABLE 2. Proton magnetic resonance signals of anomeric and fucose methyl protons in E. coli serotype 42 oligosaccharides and capsular polysaccharide": summary of structures Oligo- or polysaccharide
8
Aldobiouronic acid
a-D-Ga1UAp(41 -* 3)-L-Fuc
J [Hz]
Approx ratio of integrals
3)-L-Fuc
Type-42 polysaccharide 3)-a-D-Galp-(1-. 3)-a-D-GalUAP(1-. 3)-aL-Fucp-(l a
a-D-GalUAp
5.36 5.30 4.72 1.30
3.01 2.8
1.6
6.5
0.4
a-L-Fuc f-L-Fuc
7.0
3.0
CH3 of L-Fuc
5.40
4.0 2.5
5.32 4.60
>' 3.57 7.0
ca-D-GaIp a-L-Fuc
0.5
1.32
7.0
3.0
8-L-FuC CH3 of L-Fuc
5.45 5.40 5.35 1.30
3.5 3.0 3.5 7.0
1.0 1.0 1.0 3.0
Aldotriouronic acid a-D-Gap-(1--. 3)-a-D-GalUAp-(1
Proton assignment
c (a-D-GaIUAp
)
(a-D-GalUAp
ca-D-GalpJ
a-L-Fucp CH3 of L-Fucp
Solutions in absolute deuterium oxide were run at 700C and 90 MHz.
try instrument, and to H. Thoma for excellent technical assistance. This project was supported by Fonds der Chemischen Industrie (S.S.) and by the Deutsche Forschungsgemeinschaft (H.F.).
LITERATURE CITED 1. Bjorndal, H., C. G. Helierqvist, B. Lindberg, and S. Svenson. 1970. Gas-Flhssigkeits-Chromatographie und Massenspektrometrie bei der Methylierungsanalyse von Polysacchariden. Angew. Chem. 82:643-674. 2. Holzwarth, G. 1976. Confonnation of the extracellular polysaccharide of Xanthomonas campestris. Biochem-
istry 15:4333-4339. 3. Jann, K., B. Jann, F. 0rskov, I. 0rskov, and 0. Westphal. 1965. Immunchemische Untersuchungen an K-Antigen von Escherichia coli. I. Das K-Antigen von E. coli 08:K42(A):H-. Biochem. Z. 342:1-22. 4. Kauffmann, F. 1966. The bacteriology of Enterobacteriaceae. Munksgaard, Copenhagen.
5. Moorhouse, R., W. T. Winter, S. Arnott, and M. E. Bayer. 1977. Conformation and molecular organization in fibers of the capsular polysaccharide from Escherichia coli M41 mutant. J. Mol. Biol. 109:373-391. 6. Morris, E. R., D. A. Rees, G. Young, M. D. Walkinshaw, and A. Darke. 1977. Order-disorder transition for a bacterial polysaccharide in solution. A role for polysaccharide conformation in recognition between Xanthomonas pathogen and its plant host. J. Mol. Biol. 110:1-16. 7. Niemann, H., B. Kwiatkowski, U. Westphal, and S. Stirm. 1977. Klebsiella serotype 25 capsular polysaccharide: primary structure and depolymerization by a bacteriophage-borne glycanase. J. Bacteriol. 130:366-374. 8. Nimmich, W. 1968. Zur Isolierung und qualitativen Bausteinanalyse der K-Antigene von Klebsillen. Z. Med.
Mikrobiol. Immunol. 154:117-131.
9. Thurow, IL, Y.-ML Choy, N. Frank, H. Niemann, and S. Stirm. 1975. The structure of the Klebsiella serotype 11 capsularpolysaccharide. Carbohydr. Res. 41:241-255.
Vol. 133, No. 1 Printed in U.S.A.
Primary Structure of the Escherichia coli Serotype K42 Capsular Polysaccharide and Its Serological Identity with the Klebsiella K63 Polysaccharide HEINER N1EMANN,1 AJIT K.
CHAKRABORTY,' HORST FRIEBOLIN,2 AND STEPHAN STIRMI*
Max-Planck-Institut fiur Immunbiologie, Freiburg,I and Institut fur Organische Chemie der Universitat,
Heidelberg,2 Germcan Federal Republic Received for publication 6 September 1977
The Escherichia coli K42 capsular polysaccharide consists of --3)-a-D-Galp(1--'3)-a-D-GalUAp-(1--'3)-a-L-Fucp-(l-. repeating units. The E. coli K42 and Klebsiella K63 antigens are serologically identical. Recent advances in the conformation analysis of bacterial polysaccharides (2, 5, 6) have led to a new interest in complete primary structures of these polymers. For this reason, we have reinvestigated the Escherichia coli K42 capsular polysaccharide, a partial primary structure of which has previously been published (3). The K42 glycan, consisting of D-galactose (Gal), D-galacturonic acid (GalUA), fucose (Fuc), and 0-acetyl in a molar ratio of 1:1:1:0.5 (3), was isolated by the phenol-water-Cetavlon technique from E. coli A295b [08:K42(A):H-] (3). From its optical rotation, the fucose, prepared by hydrolysis and paper chromatography, was assigned to the L series. After partial acid hydrolysis (20 min at 100°C in 0.5 N H2S04), the aldobiouronic acid GalUA Fuc and the aldotriouronic acid Gal GalUA Fuc were isolated by paper electrophoresis, as described by Jann et al. (3). Using the procedures previously cited and described (7, 9), the glycan and the trisaccharide (repeating unit) were methylated and analyzed by gas-liquid chromatography-mass spectrome-
try of the alditol acetates (1) before and after reduction of the carboxyl groups. The polymer and the aldobiouronic and aldotriouronic acids were also subjected to proton magnetic resonance spectroscopy. The results are summarized in Tables 1 and 2. The 0-acetyl residues on every second repeating unit were not localized. Because there were some chemical indications (8; J. P. Joseleau, personal communication) that the E. coli K42 and Klebsiella K63 capsular polysaccharides might be similar, E. coli A295b was serologically compared with the test strain for the Klebsiella K63 antigen (4, 8) as follows: both strains were subjected to tube agglutinations in an E. coli 08:K42 rabbit serum, prepared with E. coli A295b (4) (homologous K titer, 1:80), as well as in a Difco Klebsiella K63 serum (homologous K titer, 1:160). The strains crossreacted to the same titers, and all homologous K agglutinins could be absorbed (4) from both sera with heterologous bacteria.
-.
--
-.
We express our gratitude to D. Borowiak for his expert handling of the gas-liquid chromatography-mass spectrome-
TABLE 1. Identification and ratios of O-acetyl-O-methylalditols obtained from E. coli serotype 42 capsular polysaccharide and the repeating unit triouronic acid Peracetyl derivative of:
2,4-FucOH 2,3,4,6-GalOH 2,4,6-GalOH 2,4-GalOH
'
_
Literal
Found
45
1.12 1.25 2.28 6.35
1.14 1.24 2.29 6.28
+ +
P4r;asn, froav".v,+ 1-axl{ /.I rmary 1ragmetLs LUUroU 1twe/
117
131
+ + + + + 2,4-di-O-methyl-L-fucitol, etc.
161
189
+ +
D.f;- -f
ratoUo1UU1 peaK itegas
205 233 247
If
II
III
IV
+
0.8
0.8
0.7 1.0
0.7 1.0
+
+ +
1.0
1.0
(191)d
0.8d
0.8d
2,4-FucOH b Retention time, relative to peracetylated 2,3,4,6-GlcOH (T - 1.00) and 2,3-GlcOH (T 5.39) in gas-liquid =
on an ECNSS-M column (1). chromatography c I, K42 polysaccharide, methylated (the GlcUA derivative was not detected with the methods used); II, K42 polysaccharide, methylated, and then carboxyl reduced-dideuterated; III, K42 aldotriouronic acid, methylated (9); IV, K42 aldotriouronic acid, methylated (9), and then carboxyl reduced-dideuterated. d Dideuterated fragment found instead.
390
391
NOTES
VOiL. 133, 1978
TABLE 2. Proton magnetic resonance signals of anomeric and fucose methyl protons in E. coli serotype 42 oligosaccharides and capsular polysaccharide": summary of structures Oligo- or polysaccharide
8
Aldobiouronic acid
a-D-Ga1UAp(41 -* 3)-L-Fuc
J [Hz]
Approx ratio of integrals
3)-L-Fuc
Type-42 polysaccharide 3)-a-D-Galp-(1-. 3)-a-D-GalUAP(1-. 3)-aL-Fucp-(l a
a-D-GalUAp
5.36 5.30 4.72 1.30
3.01 2.8
1.6
6.5
0.4
a-L-Fuc f-L-Fuc
7.0
3.0
CH3 of L-Fuc
5.40
4.0 2.5
5.32 4.60
>' 3.57 7.0
ca-D-GaIp a-L-Fuc
0.5
1.32
7.0
3.0
8-L-FuC CH3 of L-Fuc
5.45 5.40 5.35 1.30
3.5 3.0 3.5 7.0
1.0 1.0 1.0 3.0
Aldotriouronic acid a-D-Gap-(1--. 3)-a-D-GalUAp-(1
Proton assignment
c (a-D-GaIUAp
)
(a-D-GalUAp
ca-D-GalpJ
a-L-Fucp CH3 of L-Fucp
Solutions in absolute deuterium oxide were run at 700C and 90 MHz.
try instrument, and to H. Thoma for excellent technical assistance. This project was supported by Fonds der Chemischen Industrie (S.S.) and by the Deutsche Forschungsgemeinschaft (H.F.).
LITERATURE CITED 1. Bjorndal, H., C. G. Helierqvist, B. Lindberg, and S. Svenson. 1970. Gas-Flhssigkeits-Chromatographie und Massenspektrometrie bei der Methylierungsanalyse von Polysacchariden. Angew. Chem. 82:643-674. 2. Holzwarth, G. 1976. Confonnation of the extracellular polysaccharide of Xanthomonas campestris. Biochem-
istry 15:4333-4339. 3. Jann, K., B. Jann, F. 0rskov, I. 0rskov, and 0. Westphal. 1965. Immunchemische Untersuchungen an K-Antigen von Escherichia coli. I. Das K-Antigen von E. coli 08:K42(A):H-. Biochem. Z. 342:1-22. 4. Kauffmann, F. 1966. The bacteriology of Enterobacteriaceae. Munksgaard, Copenhagen.
5. Moorhouse, R., W. T. Winter, S. Arnott, and M. E. Bayer. 1977. Conformation and molecular organization in fibers of the capsular polysaccharide from Escherichia coli M41 mutant. J. Mol. Biol. 109:373-391. 6. Morris, E. R., D. A. Rees, G. Young, M. D. Walkinshaw, and A. Darke. 1977. Order-disorder transition for a bacterial polysaccharide in solution. A role for polysaccharide conformation in recognition between Xanthomonas pathogen and its plant host. J. Mol. Biol. 110:1-16. 7. Niemann, H., B. Kwiatkowski, U. Westphal, and S. Stirm. 1977. Klebsiella serotype 25 capsular polysaccharide: primary structure and depolymerization by a bacteriophage-borne glycanase. J. Bacteriol. 130:366-374. 8. Nimmich, W. 1968. Zur Isolierung und qualitativen Bausteinanalyse der K-Antigene von Klebsillen. Z. Med.
Mikrobiol. Immunol. 154:117-131.
9. Thurow, IL, Y.-ML Choy, N. Frank, H. Niemann, and S. Stirm. 1975. The structure of the Klebsiella serotype 11 capsularpolysaccharide. Carbohydr. Res. 41:241-255.
