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Hafnia alvei lipopolysaccharides: Isolation, sugar composition and SDS-PAGE analysis
FEMS Microbiology Immunology 47 (1988) 151-156 Published by Elsevier
151
F1M00024
Hafnia alvei lipopolysaccharides: Isolation, sugar composition and SDS-PAGE analysis A n n a R o m a n o w s k a , Ewa K a t z e n e l l e n b o g e n , M a l g o r z a t a K u I a k o w s k a , Andrzej G a m i a n , D a n u t a W i t k o w s k a , M a r i a n M u l c z y k a n d E12bieta R o m a n o w s k a Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wroctaw, Poland
Received 22 April 1988 Revision received and accepted 24 June 1988
Key words: Lipopolysaccharide; Hafnei ah, ei; SDS-PAGE analysis
1. SUMMARY Lipopolysaccharides (LPS) of 33 strains of Hafnia alvei were isolated and purified. LPS content of the dry bacterial mass ranged from 1.2 to 4.5%. All examined lipopolysaccharides contained glucose, glucosamine, heptose, 3-deoxy-octulosonic acid and often galactose. Rhamnose, mannose, galactosamine, mannosamine and unidentified amino sugars were found in some H. alvei strains. Sialic acid was present in LPS of one strain. D-3-Hydroxybutyryl groups also were identified in lipopolysaccharides of 5 strains of this genus. SDS-PAGE of the lipopolysaccharides was presented in the paper. According to these results two core types exist in H. alvei.
Correspondence to: A. Romanowska, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, 12 Czerska, 53-114 Wroc/aw, Poland.
2. I N T R O D U C T I O N The name Hafnia alvei was proposed by Moller in 1954 for a new group of Enterobacteriaceae [1]. The numerical taxonomy study by Johnson et al. and D N A reassociation studies by Steigerwalt et al. confirmed the status of Hafnia as a separate genus [2]. According to the serological classification of Sakazaki [2] among H. alvei strains 68 O-antigens and 34 H-antigens exist. Seven O-subgroups have been recognized in this genus. H. alvei strains show numerous serological cross-reactions with Escherichia coli, Enterobacter cloaceae, Citrobacter freundii and Shigella [2]. In general H. alvei occur in stool specimens of healthy persons, however many incidences of nosocomial infections with Hafnia have also been reported [2]. So far no immunochemical data have been obtained for this enterobacterial group. We now report the preliminary chemical characteristics of H. alvei lipopolysaccharides (O-antigens) isolated from 33 strains.
0920-8534/88/$03.50 © 1988 Federation of European Microbiological Societies
152
39
3. MATERIALS A N D M E T H O D S
38
3.1 H. alvei organisms." standard strain A TCC 13337
1
I I
31
1191
20 strains derived from the collection of Pasteur Institute (Paris) kindly provided by Dr. LeMinor, 3 strains from National Institute of Hygiene (Warsaw) kindly given by Dr. Z. Tyc and 9 strains from the collection of Institute of Immunology and Experimental Therapy (Wrodaw) were used in the experiments. All the strains were found to possess characteristics typical for H. alvei based on biochemical criteria. They gave positive lysine and ornithine decarboxylase reactions and negative result for the arginine dihydrolase test. All the strains did not ferment raffinose, sorbitol, adonitol and inositol. Growing the bacteria in liquid medium, isolation and purification of the lipopolysaccharides were carried out as described previously [3,4].
1209
3.2 Sugar analysis
32 37 17 114160 1M 1190 .......
1192 1188 1187
m
1224 1204 1211
I
1214. 13337
1216 451-L 1203 1221 23
Ill
1196 2 1199 1200 1205
For neutral sugars determination the lipopolysaccharide samples were hydrolyzed with 0.1 M HCI at 100°C for 48 h and monosaccharides identified as alditol acetates [5] by gas-liquid chromatography (Varian 2100 instrument with flame ionization detector and Gas chrom Q column 3% OV-225 at 200 ° C). For amino sugars determination the lipopolysaccharide samples were hydrolyzed with 4 M HCI at 1 0 0 ° C for 12 h. Amino sugars were analyzed according to modified Ludowieg-Benmaman method [6,7] or as alditol acetates by gas-liquid chromatography (see above) at 220 °C. Heptose was determined according to Osborn [8], 3-deoxyoctulosonic acid by the method of Karkhanis et al. [9], 6-deoxyhexose according to Gibbons [10] and sialic acid by the resorcinol method [11]. Neutral glycoses were estimated according to Dubois et al. [12]. Paper chromatography was performed on Whatman no. 1 paper by using 1-butanol/
1220 1213
Fig. 1. Silver-stained SDS-PAGE of purified lipopolysaccharides from Hafnia alvei strains.
153 Table 1 S u g a r c o m p o s i t i o n of the l i p o p o l y s a c c h a r i d e s i s o l a t e d f r o m Source
Strain
Neutral glycoses
Hafnia alvei
strains
S u g a r c o m p o n e n t s in % Glc
Gal
Man
Hep
KDO
6-deoxy
GlcN
GalN
ManN
Hex ATCC d 13337
18.5
8.9
-
7.8
3,1
-
3.5
5.0
Pasteur Institute
1187 d
23.5
9.6
-
8.4
3.6
-
4.5
5.7
(Paris)
1188
29.0
6.8
4.7
2.7
9.4
3,2
2.5 ~
4.5
-
-
6.3
2.7
6.8 ~
5.4
-
6.8
3.8
-
6.0
4.5
1190
28.0
8.1
2.9
1191
31.5
12.1
3.l
1192
28.0
6.2
1.2
-
8.3
2.5
5.2 "
7.0
-
1196 b
22.0
7.3
3.3
-
6.4
5.8
-
9.7
-
1199
27.0
7.7
0.7
-
7.2
3.0
-
7.8
1200
23.0
nd
nd
8.0
3.1
-
8.6
1203
24.0
10.6
4.8
-
8.4
3.8
-
10.0
1204 8
25.5
5.0
4.3
7.4
8.7
1.6
-
3.8
1205
24.0
7.7
3.5
-
9.8
1.6
1209
29.0
11.7
2.8
-
10.2
3.0
5.3 ~
2.5
3.0
1211 b'a
26.0
17.8
1.2
-
6.2
2.2
-
6.3
5.2
1213
33.0
21.9
1.7
-
8.0
2.2
-
1.5
7.8
1214
29.0
8.9
12.5
-
8.4
1.7
-
7.7
1 2 1 6 8'd
23.0
9.0
5.6
-
9.0
3.2
-
11.6
1220 b
43.0
nd
nd
nd
7.2
2.9
-
8.2
1221 d
31.0
nd
nd
nd
8.8
3.8
3.8
8.0
1222
42.0
nd
nd
nd
7.5
nd
7.8
8.0
1.5
1224
29.0
11.4
-
7.0
2.7
-
5.5
6.0
481-L
18.5
6.8
3.7
-
8.0
3.0
-
2.3
7.7
1M
18.5
5.2
0.5
-
10.2
3.6
-
4.8
114-60
22.0
7.6
-
12.6
3.0
-
5.2
I n s t i t u t e of
1
29.0
12.6
3.0
-
8.8
2.0
-
6.8
-
4.2
Immunology and
2c
31.0
14.9
7.7
-
6.0
3.0
-
3.2
3.8
-
-
-
14.5
-
6.0
-
National Institute of Hygiene (Warsaw)
Experimental
17
20.0
T h e r a p y (Wroclaw).
23
27.5
-
7.5 nd
nd
2.6
-
6.7
1.1
nd
nd
5.5
2.0
-
2.2
8.6
10.0
6.0
-
12.0
31
18.0
nd
nd
nd
4.5
1.4
12.0
32
18.5
nd
nd
nd
5.2
1.1
6.5
3.0
7.0
37
15.5
nd
nd
nd
5.2
1.0
9.0
-
11.0
38
21.0
nd
nd
nd
6.0
1.7
4.5
10.0
-
1 1 . 5
39
27.5
nd
nd
nd
5.5
2.2
10.0
6.0
3.0
7.5
= a b s e n t c o m p o n e n t ; nd = n o t d e t e r m i n e d ; ~ 6 - d e o x y h e x o s e w a s i d e n t i f i e d as r h a m n o s e ; b u n k n o w n a m i n o s u g a r p r e s e n t ; ~ sialic
acid present; d D - 3 - h y d r o x y b u t y r i c a c i d p r e s e n t ; G a l , g a l a c t o s e ; Glc, g l u c o s e ; H e p , h e p t o s e ; M a n , m a n n o s e ; 6 - d e o H e x , 6 - d e o x y h e xose; G l c N , g l u c o s a m i n e ; GAIN, g a l a c t o s a m i n e ; M a n N , m a n n o s a m i n e ; K D O , 3 - d e o x y - o c t u l o s o n i c acid.
154
pyridine/water ( 6 : 4 : 3, v / v / v ) as the irrigant and Trevelyan spray or ninhydrin (0.25% in acetone) reagent as the detectors. Determination of D-3-hydroxybutyric acid was performed using D-3-hydroxybutyrate dehydrogenase [13]. Lipopolysaccharide samples (1 mg) were hydrolyzed with 4 M HC1 at 100 ° C for 2.5 h prior to the analysis. SDS-polyacrylamide gel electrophoresis (SDSPAGE) was carried out by the method of Laemmli [14] with the following modifications: (a) slab gels (10 × 15 × 0.1 cm) contained the linear 10-20% acrylamide gel gradient and 5% acrylamide stacking gel; (b) LPS suspension (0.5 mg/ml) in 50 mM Tris-HCl-10 mM EDTA buffer, pH 6.8 containing 4% SDS, 10% glycerol and 0.005% bromophenol blue was boiled for 20 min and then 2 /~1 portion was applied to the gel. Electrophoresis was run with a constant current of 30 mA per slab gel. LPS in the gel was visualized by silver stain procedure of Tsai and Frash [15].
RESULTS AND DISCUSSION Lipopolysaccharides of H. aluei after phenolwater extraction of bacterial cells were isolated from aqueous phase and purified by gel filtration on Sepharose 2B. LPS containing fractions, free of RNA contamination showed A 2 6 0 / A 2 4 0 lower than 1.0 (see reference 4). LPS yield ranged from 1.2 to 4.5% of the dry bacterial mass. The sugar compositions of lipopolysaccharides obtained from 33 strains are shown in Table 1. All lipopolysaccharides contained glucose, glucosamine, heptose and 3-deoxy-octulosonic acid; galactose was a frequent component of Hafnia lipopolysaccharides. Furthermore, the following sugars were identified in Hafnia genus: galactosamine (13 strains), 6-deoxyhexose (9 strains), mannosamine (7 strains), mannose (2 strains), sialic acid (1 strain) and unknown amino sugars (5 strains). In general, amino sugars of Hafnia lipopolysaccharides were N-acetylated, but in 5 strains N-3-D-hydroxybutyryl residues also were found. The LPS content of D-3-hydroxybutyric acid ranged from 1.05 to 2.5% in these strains. Lipopolysaccharides from Hafnia strains were
analyzed by SDS-PAGE and silver staining for molecular heterogeneity. The majority of them showed high molecular weight ladderlike pattern of bands up the gel (Fig. 1). These bands are characteristic for S-type lipopolysaccharides. There is a group of the lipopolysaccharides (strains: 2, 23, 1199, 1203, 1205, 1213, 1221) whose O-specific polysaccharide chains are rather short and occur in markedly lower amount by comparison with the core oligosaccharide region. Finally, the LPSs of some strains: 1 M, 114-60, 1191, 1196 and 1200, showed R-type character by the presence of fast migrating bands only. These bands were formed by low molecular weight core lipopolysaccharide. It should be emphasized however, that R-type character of a lipopolysaccharide observed in SDS-PAGE because of the limited sensitivity of the method, could not exclude the presence of minute O-specific polysaccharide portion in this LPS preparation. The fractionation on BioGel P-4 of the carbohydrate material isolated from the lipopolysaccharides of strains 1191, 1196 and 11460 proved that besides the main core fraction small amounts of O-specific material also were present (unpublished results).
REFERENCES [1] Bergan, T. (1984) Classification of Enterobacteriaceae. Methods Microbiol. 14, 1. [2] Sakazaki, R. (1984) Serology of Enterobaeter and Hafnia. Methods Microbiol. 14, 165. [31 Romanowska, E. and Mulczyk, M. (1968) Chemical studies on the specific fragment of Shigella sonnei phase II. Eur. J. Biochem. 5, 109. [41 Romanowska, E. (1970) Sepharose gel filtration method of purification of lipopolysaccharides. Anal. Biochem. 33, 383. [5] Sawardeker, J.S., Sloneker, J.N. and Jeanes, A. (1965) Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography. Anal. Chem. 37, 1602. [6] Ludowieg, J. and Benmaman, D. (1967) Colorimetric differentiation of hexosamines. Anal. Biochem. 19, 80. [7] Romanowska, E. and Reinhold, V. (1973) 2-Amino-2-deoxyhexuronic acid: a constituent of Shigella sonnei phase I lipopolysaccharide. Eur. J. Biochem. 36, 160. [8] Osborn, M.J. (1963) Studies on the gram-negative cell wall. I. Evidence for the role of 2-keto-3-deoxyoctonate in the lipopolysaccharide of Salmonella typhimurium. Proc. Natl. Acad. Sci. USA 50, 499.
155 [9] Karkhanis, Y.D., Zeltner, J.Y., Jackson, J.J. and Carlo, D.J. (1978) A new and improved microassay to determine 2-keto-3-deoxyoctonate in lipopolysaccharide of Gramnegative bacteria. Anal. Biochem. 85, 595. [10] Gibbons, M.N. (1962) Reaction of 6-deoxyhexoses with thioglycolic acid and sulfuric acid. Methods Carbohydr. Res. 1. 502. [11] Svennerholm, L. (1957) Quantitative estimation of sialic acids lI. A colorimetric resorcinol-hydrochloric acid method. Biochem. Biophys. Acta 24, 604. [12] Dubois, H., Gilles, K.A., Hamilton, J.K., Rebers, P. and
Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350. [13] Williamson, D.H. and Mellanby, J. (1974) D-(-)-3-Hydroxybutyrat in H.U. Bergmeyer (ed.), Methoden der Enzymatische Analyse, Verlag Chemie, Weinheim. F.R.G. [14] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680. [15] Tsai, C.M. and Frash, C.E. (1982) A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119, 115.
151
F1M00024
Hafnia alvei lipopolysaccharides: Isolation, sugar composition and SDS-PAGE analysis A n n a R o m a n o w s k a , Ewa K a t z e n e l l e n b o g e n , M a l g o r z a t a K u I a k o w s k a , Andrzej G a m i a n , D a n u t a W i t k o w s k a , M a r i a n M u l c z y k a n d E12bieta R o m a n o w s k a Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Wroctaw, Poland
Received 22 April 1988 Revision received and accepted 24 June 1988
Key words: Lipopolysaccharide; Hafnei ah, ei; SDS-PAGE analysis
1. SUMMARY Lipopolysaccharides (LPS) of 33 strains of Hafnia alvei were isolated and purified. LPS content of the dry bacterial mass ranged from 1.2 to 4.5%. All examined lipopolysaccharides contained glucose, glucosamine, heptose, 3-deoxy-octulosonic acid and often galactose. Rhamnose, mannose, galactosamine, mannosamine and unidentified amino sugars were found in some H. alvei strains. Sialic acid was present in LPS of one strain. D-3-Hydroxybutyryl groups also were identified in lipopolysaccharides of 5 strains of this genus. SDS-PAGE of the lipopolysaccharides was presented in the paper. According to these results two core types exist in H. alvei.
Correspondence to: A. Romanowska, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, 12 Czerska, 53-114 Wroc/aw, Poland.
2. I N T R O D U C T I O N The name Hafnia alvei was proposed by Moller in 1954 for a new group of Enterobacteriaceae [1]. The numerical taxonomy study by Johnson et al. and D N A reassociation studies by Steigerwalt et al. confirmed the status of Hafnia as a separate genus [2]. According to the serological classification of Sakazaki [2] among H. alvei strains 68 O-antigens and 34 H-antigens exist. Seven O-subgroups have been recognized in this genus. H. alvei strains show numerous serological cross-reactions with Escherichia coli, Enterobacter cloaceae, Citrobacter freundii and Shigella [2]. In general H. alvei occur in stool specimens of healthy persons, however many incidences of nosocomial infections with Hafnia have also been reported [2]. So far no immunochemical data have been obtained for this enterobacterial group. We now report the preliminary chemical characteristics of H. alvei lipopolysaccharides (O-antigens) isolated from 33 strains.
0920-8534/88/$03.50 © 1988 Federation of European Microbiological Societies
152
39
3. MATERIALS A N D M E T H O D S
38
3.1 H. alvei organisms." standard strain A TCC 13337
1
I I
31
1191
20 strains derived from the collection of Pasteur Institute (Paris) kindly provided by Dr. LeMinor, 3 strains from National Institute of Hygiene (Warsaw) kindly given by Dr. Z. Tyc and 9 strains from the collection of Institute of Immunology and Experimental Therapy (Wrodaw) were used in the experiments. All the strains were found to possess characteristics typical for H. alvei based on biochemical criteria. They gave positive lysine and ornithine decarboxylase reactions and negative result for the arginine dihydrolase test. All the strains did not ferment raffinose, sorbitol, adonitol and inositol. Growing the bacteria in liquid medium, isolation and purification of the lipopolysaccharides were carried out as described previously [3,4].
1209
3.2 Sugar analysis
32 37 17 114160 1M 1190 .......
1192 1188 1187
m
1224 1204 1211
I
1214. 13337
1216 451-L 1203 1221 23
Ill
1196 2 1199 1200 1205
For neutral sugars determination the lipopolysaccharide samples were hydrolyzed with 0.1 M HCI at 100°C for 48 h and monosaccharides identified as alditol acetates [5] by gas-liquid chromatography (Varian 2100 instrument with flame ionization detector and Gas chrom Q column 3% OV-225 at 200 ° C). For amino sugars determination the lipopolysaccharide samples were hydrolyzed with 4 M HCI at 1 0 0 ° C for 12 h. Amino sugars were analyzed according to modified Ludowieg-Benmaman method [6,7] or as alditol acetates by gas-liquid chromatography (see above) at 220 °C. Heptose was determined according to Osborn [8], 3-deoxyoctulosonic acid by the method of Karkhanis et al. [9], 6-deoxyhexose according to Gibbons [10] and sialic acid by the resorcinol method [11]. Neutral glycoses were estimated according to Dubois et al. [12]. Paper chromatography was performed on Whatman no. 1 paper by using 1-butanol/
1220 1213
Fig. 1. Silver-stained SDS-PAGE of purified lipopolysaccharides from Hafnia alvei strains.
153 Table 1 S u g a r c o m p o s i t i o n of the l i p o p o l y s a c c h a r i d e s i s o l a t e d f r o m Source
Strain
Neutral glycoses
Hafnia alvei
strains
S u g a r c o m p o n e n t s in % Glc
Gal
Man
Hep
KDO
6-deoxy
GlcN
GalN
ManN
Hex ATCC d 13337
18.5
8.9
-
7.8
3,1
-
3.5
5.0
Pasteur Institute
1187 d
23.5
9.6
-
8.4
3.6
-
4.5
5.7
(Paris)
1188
29.0
6.8
4.7
2.7
9.4
3,2
2.5 ~
4.5
-
-
6.3
2.7
6.8 ~
5.4
-
6.8
3.8
-
6.0
4.5
1190
28.0
8.1
2.9
1191
31.5
12.1
3.l
1192
28.0
6.2
1.2
-
8.3
2.5
5.2 "
7.0
-
1196 b
22.0
7.3
3.3
-
6.4
5.8
-
9.7
-
1199
27.0
7.7
0.7
-
7.2
3.0
-
7.8
1200
23.0
nd
nd
8.0
3.1
-
8.6
1203
24.0
10.6
4.8
-
8.4
3.8
-
10.0
1204 8
25.5
5.0
4.3
7.4
8.7
1.6
-
3.8
1205
24.0
7.7
3.5
-
9.8
1.6
1209
29.0
11.7
2.8
-
10.2
3.0
5.3 ~
2.5
3.0
1211 b'a
26.0
17.8
1.2
-
6.2
2.2
-
6.3
5.2
1213
33.0
21.9
1.7
-
8.0
2.2
-
1.5
7.8
1214
29.0
8.9
12.5
-
8.4
1.7
-
7.7
1 2 1 6 8'd
23.0
9.0
5.6
-
9.0
3.2
-
11.6
1220 b
43.0
nd
nd
nd
7.2
2.9
-
8.2
1221 d
31.0
nd
nd
nd
8.8
3.8
3.8
8.0
1222
42.0
nd
nd
nd
7.5
nd
7.8
8.0
1.5
1224
29.0
11.4
-
7.0
2.7
-
5.5
6.0
481-L
18.5
6.8
3.7
-
8.0
3.0
-
2.3
7.7
1M
18.5
5.2
0.5
-
10.2
3.6
-
4.8
114-60
22.0
7.6
-
12.6
3.0
-
5.2
I n s t i t u t e of
1
29.0
12.6
3.0
-
8.8
2.0
-
6.8
-
4.2
Immunology and
2c
31.0
14.9
7.7
-
6.0
3.0
-
3.2
3.8
-
-
-
14.5
-
6.0
-
National Institute of Hygiene (Warsaw)
Experimental
17
20.0
T h e r a p y (Wroclaw).
23
27.5
-
7.5 nd
nd
2.6
-
6.7
1.1
nd
nd
5.5
2.0
-
2.2
8.6
10.0
6.0
-
12.0
31
18.0
nd
nd
nd
4.5
1.4
12.0
32
18.5
nd
nd
nd
5.2
1.1
6.5
3.0
7.0
37
15.5
nd
nd
nd
5.2
1.0
9.0
-
11.0
38
21.0
nd
nd
nd
6.0
1.7
4.5
10.0
-
1 1 . 5
39
27.5
nd
nd
nd
5.5
2.2
10.0
6.0
3.0
7.5
= a b s e n t c o m p o n e n t ; nd = n o t d e t e r m i n e d ; ~ 6 - d e o x y h e x o s e w a s i d e n t i f i e d as r h a m n o s e ; b u n k n o w n a m i n o s u g a r p r e s e n t ; ~ sialic
acid present; d D - 3 - h y d r o x y b u t y r i c a c i d p r e s e n t ; G a l , g a l a c t o s e ; Glc, g l u c o s e ; H e p , h e p t o s e ; M a n , m a n n o s e ; 6 - d e o H e x , 6 - d e o x y h e xose; G l c N , g l u c o s a m i n e ; GAIN, g a l a c t o s a m i n e ; M a n N , m a n n o s a m i n e ; K D O , 3 - d e o x y - o c t u l o s o n i c acid.
154
pyridine/water ( 6 : 4 : 3, v / v / v ) as the irrigant and Trevelyan spray or ninhydrin (0.25% in acetone) reagent as the detectors. Determination of D-3-hydroxybutyric acid was performed using D-3-hydroxybutyrate dehydrogenase [13]. Lipopolysaccharide samples (1 mg) were hydrolyzed with 4 M HC1 at 100 ° C for 2.5 h prior to the analysis. SDS-polyacrylamide gel electrophoresis (SDSPAGE) was carried out by the method of Laemmli [14] with the following modifications: (a) slab gels (10 × 15 × 0.1 cm) contained the linear 10-20% acrylamide gel gradient and 5% acrylamide stacking gel; (b) LPS suspension (0.5 mg/ml) in 50 mM Tris-HCl-10 mM EDTA buffer, pH 6.8 containing 4% SDS, 10% glycerol and 0.005% bromophenol blue was boiled for 20 min and then 2 /~1 portion was applied to the gel. Electrophoresis was run with a constant current of 30 mA per slab gel. LPS in the gel was visualized by silver stain procedure of Tsai and Frash [15].
RESULTS AND DISCUSSION Lipopolysaccharides of H. aluei after phenolwater extraction of bacterial cells were isolated from aqueous phase and purified by gel filtration on Sepharose 2B. LPS containing fractions, free of RNA contamination showed A 2 6 0 / A 2 4 0 lower than 1.0 (see reference 4). LPS yield ranged from 1.2 to 4.5% of the dry bacterial mass. The sugar compositions of lipopolysaccharides obtained from 33 strains are shown in Table 1. All lipopolysaccharides contained glucose, glucosamine, heptose and 3-deoxy-octulosonic acid; galactose was a frequent component of Hafnia lipopolysaccharides. Furthermore, the following sugars were identified in Hafnia genus: galactosamine (13 strains), 6-deoxyhexose (9 strains), mannosamine (7 strains), mannose (2 strains), sialic acid (1 strain) and unknown amino sugars (5 strains). In general, amino sugars of Hafnia lipopolysaccharides were N-acetylated, but in 5 strains N-3-D-hydroxybutyryl residues also were found. The LPS content of D-3-hydroxybutyric acid ranged from 1.05 to 2.5% in these strains. Lipopolysaccharides from Hafnia strains were
analyzed by SDS-PAGE and silver staining for molecular heterogeneity. The majority of them showed high molecular weight ladderlike pattern of bands up the gel (Fig. 1). These bands are characteristic for S-type lipopolysaccharides. There is a group of the lipopolysaccharides (strains: 2, 23, 1199, 1203, 1205, 1213, 1221) whose O-specific polysaccharide chains are rather short and occur in markedly lower amount by comparison with the core oligosaccharide region. Finally, the LPSs of some strains: 1 M, 114-60, 1191, 1196 and 1200, showed R-type character by the presence of fast migrating bands only. These bands were formed by low molecular weight core lipopolysaccharide. It should be emphasized however, that R-type character of a lipopolysaccharide observed in SDS-PAGE because of the limited sensitivity of the method, could not exclude the presence of minute O-specific polysaccharide portion in this LPS preparation. The fractionation on BioGel P-4 of the carbohydrate material isolated from the lipopolysaccharides of strains 1191, 1196 and 11460 proved that besides the main core fraction small amounts of O-specific material also were present (unpublished results).
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Smith, F. (1956) Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350. [13] Williamson, D.H. and Mellanby, J. (1974) D-(-)-3-Hydroxybutyrat in H.U. Bergmeyer (ed.), Methoden der Enzymatische Analyse, Verlag Chemie, Weinheim. F.R.G. [14] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680. [15] Tsai, C.M. and Frash, C.E. (1982) A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal. Biochem. 119, 115.