Structures of the biological repeating units in the O-chain polysaccharides of Hafnia alvei strains having a typical lipopolysaccharide outer core region

FEMS Immunology and Medical Microbiology 45 (2005) 269–278 www.fems-microbiology.org

Structures of the biological repeating units in the O-chain polysaccharides of Hafnia alvei strains having a typical lipopolysaccharide outer core region Ewa Katzenellenbogen a,*, Nina A. Kocharova b, George V. Zatonsky b, Alexander S. Shashkov b, Maria Bogulska a, Yuriy A. Knirel b a

L. Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wroclaw, Poland b N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospekt 47, Moscow 119991, Russia Received 3 February 2005; received in revised form 15 March 2005; accepted 3 May 2005 First published online 31 May 2005

Abstract Earlier, the structures of the O-chain polysaccharides of the lipopolysaccharides (LPS) of a number of Hafnia alvei strains have been established. However, it remained unknown, which is the first and the last monosaccharide of the O-chain. This is defined by the structure of the so-called biological repeating unit (O-unit), which is pre-assembled and then polymerised in the course of biosynthesis of bacterial polysaccharides by the Wzy-dependent pathway. Now we report on the structures of the O-units in 10 H. alvei strains. The LPS were cleaved by mild acid hydrolysis and oligosaccharide fractions IIIa and IIIb were isolated by gel chromatography subsequently on Sephadex G-50 and BioGel P-2 and studied by methylation analysis and NMR spectroscopy. Fraction IIIb was found to represent the core oligosaccharide containing a terminal upstream a-D-Glc-(1!3)-a-D-Glc or a-DGal-(1!3)-a-D-Glc disaccharide in the outer region that is typical of H. alvei. Fraction IIIa consists of the LPS core with one Ounit linked by a 3-substituted b-D-GalNAc residue (in strains PCM 1189 and PCM 1546) or a 3-substituted b-D-GlcNAc residue (in the other strains studied). In most strains examined the b-configuration of the D-GlcNAc linkage in the first O-unit attached to the core is the same and in some strains is opposite to that found in the interior O-units of the O-chain polysaccharide. Various monosaccharides, including D-Glc, D-Gal, D-GlcA and acyl derivatives of 3-amino-3,6-dideoxy-D-glucose or 4-amino-4,6-dideoxy-Dglucose, occupy the non-reducing end of the O-unit.
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Hafnia alvei; Lipopolysaccharide; O-antigen structure; Biological repeating unit

1. Introduction Strains of Hafnia, a typical member of the Enterobacteriaceae family, represented by only one species Hafnia alvei, are widely distributed in the natural environment. As an opportunistic pathogen, they can be associated with nosocomial infections, especially in hospitalised *

Corresponding author. Tel.: +48 71 3371172; fax: +48 71 3371382. E-mail address: [email protected] (E. Katzenellenbogen).

patients with wounds and urinary and respiratory tract disorders [1,2]. Currently, H. alvei strains are divided into 39 O-serotypes [3]. Numerous serological crossreactions have been observed between strains of H. alvei and other enterobacterial genera, such as Salmonella, Citrobacter, Escherichia and Shigella. Like other Gram-negative bacteria, H. alvei contains a lipopolysaccharide (LPS) as the major component of the outer membrane of the bacterial cell envelope. The LPS plays an important role in the interactions of the bacteria with the host and shows a wide spectrum of

0928-8244/$22.00
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsim.2005.05.003

270

E. Katzenellenbogen et al. / FEMS Immunology and Medical Microbiology 45 (2005) 269–278

biological activities. It consists of three structurally distinct regions: (i) a lipid moiety called lipid A, which is the endotoxic domain and the most conservative part of the LPS; (ii) a structurally variable polysaccharide called O-chain polysaccharide (OPS) or O-antigen, which is built up of the oligosaccharide repeating units and defines the serospecificity of the strain; and (iii) an intervening core oligosaccharide consisting of a heptose-ketodeoxyoctonate (Kdo) inner region proximal to lipid A and a distal hexose outer region. During last decades, the chemical structures of the enterobacterial LPS have been intensively studied in order to elucidate the molecular basis for the antigenic diversity and biological properties of bacterial strains. The LPS of H. alvei was first characterised chemically in 1988 [4], and since that time 28 OPS structures have been determined in various H. alvei strains [5–9]. Typically they are linear or branched heteropolymers with repeating units consisting of two to eight (more often of four to six) monosaccharide residues. Most of them are acidic and contain D-GlcA or D-GalA as well as amino sugars D-GlcNAc or D-GalNAc. In many strains the OPS contain less commonly occurring components of bacterial polysaccharides, such as D-ribose, 6-deoxy-Dtalose, N-acetylneuraminic acid, 2-amino-2,6-dideoxy-, 3-amino-3,6-dideoxy- and 4-amino-4,6-dideoxy-hexoses that are N-acylated with acetyl, 3-hydroxybutyryl or formyl groups, an amide of D-GalA with D-allothreonine. A number of the OPS are phosphorylated, e.g., with a phosphoethanolamine group; some of them have oligosaccharide phosphate repeating units and others are glycerol or arabinitol teichoic acid-like polymers. An LPS core oligosaccharide terminated with an a-DGlc-(1!3)-a-D-Glc disaccharide has been found in most H. alvei strains examined to date [10,11], except for strains PCM 1204 and 1185, in which the terminal Glc is replaced with a terminal Gal [12]. In three other strains, more complex structures of the hexose outer core region have been established, which are identical to those of Salmonella Ra (H. alvei strain 39) and Escherichia coli R4 (H. alvei strains 23 and PCM 1222) [13]. In several H. alvei strains, additional core-like trisaccharides containing galactose, heptose and Kdo have been identified [14–16]. One of the known pathways of bacterial polysaccharides biosynthesis, called the Wzy-dependent or O-antigen polymerase-dependent pathway, includes an assembling of the single oligosaccharide O-unit on an undecaprenyl diphosphate carrier by adding of individual sugar residues under the control of specific glycosyl transferases at the cytoplasmic face of the cytoplasmic membrane [17,18]. Following the translocation to the periplasmic face of the membrane with the help of flippase Wzx, O-units are polymerised with participation of the O-antigen polymerase Wzy and chain length regulator Wzx, and then the polymer is ligated by ligase

WaaL to the core-lipid A moiety. It was proposed [19] that the biosynthesis of heteropolymeric OPS with Ounits larger than disaccharide including in the main chain at least one UDP-activated sugar proceeded by this pathway (‘‘block mechanism’’). The knowledge of the structures of the O-units, called also biological repeating units, is important for elucidation of the LPS biosynthesis pathways, including growth of the O-chain and its ligation to the LPS core, as well as for a better understanding on the molecular level the immunospecificity of bacterial strains defined to a large extent by a saccharide located at the non-reducing end of the O-chain. However, for the majority of enterobacterial LPS examined to date, only the structure of the socalled chemical repeating unit has been elucidated, which may be the same as that of the biological repeating unit (O-unit) or may differ by a cyclic permutation of the monosaccharides in the main chain. Only in some bacteria, including three H. alvei strains (2, 39 and PCM 1209) [20–22], the structures of the O-units have been established. The aim of this work was to determine the structures of the biological repeating units in 10 more H. alvei strains, in which the structures of the chemical repeating unit have been already reported [9,23–31]. For this purpose, the oligosaccharide fraction from each strain, obtained by mild acid hydrolysis of the LPS followed by gel chromatography on Sephadex G-50 in our previous studies on the H. alvei OPS, was further fractionated by gel chromatography on BioGel P-2. Two main fractions, IIIa (core with one O-unit attached) and IIIb (unsubstituted core), were studied by methylation analysis and NMR spectroscopy, and the structures of the O-units thus established were regarded as those of the biological repeating units of the OPS.

2. Materials and methods 2.1. Gel chromatography and GLC–MS Gel chromatography was carried out on columns (2 · 100 cm) of Sephadex G-50 and BioGel P-2 in 0.05 M pyridinium acetate buffer pH 5.6, and monitored by the phenol–H2SO4 method. GLC–MS was carried out on a Hewlett–Packard 5971 instrument (Palo Alto, CA, USA), equipped with an HP-1 glass capillary column (12 m · 0.2 mm), using a temperature program of 150–270 C at 8 C min
1. 2.2. NMR spectroscopy Samples were deuterium-exchanged by freeze-drying twice from D2O and examined as solutions in 99.96% D2O at 30 C on a Bruker DRX-500 spectrometer. The 2D NMR spectra were obtained using standard

E. Katzenellenbogen et al. / FEMS Immunology and Medical Microbiology 45 (2005) 269–278

Bruker software, and the XWINNMR 2.6 program was used to acquire and process the NMR data. Chemical shifts are reported related to internal acetone (dC 31.45). 2.3. Bacterial strains, isolation of LPS and oligosaccharides H. alvei strains PCM 1185, 1188, 1189, 1196, 1199, 1204, 1205, 1211, 1216 and 1546 derived from the collection of the L. Hirszfeld Institute of Immunology and Experimental Therapy (Wrocław, Poland). The growth of the bacteria in liquid medium, extraction and purification of the LPS, isolated in yields 1.6–3.5% of the dry bacterial weight, were performed as described [9,23–31]. An LPS sample from each strain was hydrolysed with aq 1% HOAc (100 C, 40–60 min), and the carbohydrate portion (60–70% of the LPS weight) was fractionated by gel chromatography on Sephadex G-50. From each strain three to four fractions (P1–P4) were obtained in the following yields (given as percentage of the total material eluted from the column): P1 (a long-chain Opolysaccharide, OPS) 12–52%; P2 (a core substituted with a shorter-chain OPS) 3–10%; P3 (core oligosaccharides) 37–50% from strains PCM 1196, 1189, 1199 and 1204 or 30–35% from the other strains; P4 (Kdo-containing fraction) 16–35%. Fraction P3 was further separated by gel chromatography on BioGel P-2 into three to five fractions (IIIa1–IIId), including those of unsubstituted core (IIIb) and core substituted with one O-unit (IIIa). For NMR spectroscopic studies fraction IIIa from each strain having an O-acetylated OPS was O-deacetylated by treatment with aq 12% ammonia (20 C, 16 h) followed by lyophilisation. 2.4. Compositional and methylation analyses Heptose [32], phosphate [33], Kdo [34], O-acetyl groups [35] and free amino groups [36] were quantified directly in oligosaccharides IIIa and IIIb using different colorimetric methods. Fractions IIIa and IIIb were hydrolysed with 2 M CF3CO2H (120 C, 2 h) or 10 M HCl (80 C, 30 min), monosaccharides were converted into the alditol acetates [37] and analysed by GLC– MS. Two different sets of hydrolysis conditions were used to ensure that no sugar constituent is underestimated in sugar analysis. Methylation was performed according to Gunnarsson [38] as described [39], methylated oligosaccharides were hydrolysed as above, reduced with NaBH4 or NaBD4, acetylated, and the partially methylated alditol acetates were identified by GLC–MS.

3. Results LPS from 10 H. alvei strains were subjected to mild acid hydrolysis to release lipid A, and the carbohy-

271

drate-containing material was fractionated on Sephadex G-50 to give three to four fractions (P1–P4). The polysaccharide fraction P1 from each strain was studied by us previously and the OPS structures were determined [9,23–31]. In this work fractions P3 from 10 strains were further fractionated on BioGel P-2 to afford three to five fractions (IIIa1–IIId). Sugar and methylation analyses (for details see below) showed that fraction IIIb from each strain contained only core constituents, including Glc, Gal and Hep, and thus was an unsubstituted core oligosaccharide, whereas fraction IIIa contained both core and OPS constituents, and further studies showed that it corresponded to the core substituted with one O-unit. The content of the unsubstituted core and that substituted with one O-unit was different depending on the strain, the yields of fraction IIIa ranged from 16.5% (from strain PCM 1188) to 51% (from strain PCM 1204) of the fraction P3 and, vice versa, the yields of fraction IIIb varied from 27% (from strain PCM 1204) to 59% (from strain PCM 1188). The content of heptose, phosphate and free amino groups in fraction IIIb (Table 1), together with the methylation analysis data (Table 2), confirmed the structure of the core oligosaccharide reported earlier for some of these and a number of other H. alvei strains [10–12]. The typical H. alvei LPS core is composed of three heptose residues, one glucose and one galactose residue (in strains PCM 1185 and 1204) or two glucose residues in the other strains studied, one phosphate and one ethanolamine diphosphate group. It seems likely that the same core structure is characteristic of H. alvei strains PCM 1199, 1205, 1216, 1188, which have been characterised only serologically [40,41], as well as of strains PCM 1189 and 1546, in which the core structure has not been studied earlier. The free amino groups detected in fraction IIIb may derive not only from ethanolamine but also from glycine, which is present in the H. alvei core [42] but its location remains unknown. Fraction IIIa has essentially the same composition in respect to the core constituents except for the content of Kdo, which is higher than in fraction IIIb for seven of the strains studied (PCM 1188, 1189, 1199, 1205, 1211, 1216 and 1546) (Table 1). This can be due to the presence of an additional Kdo-containing oligosaccharide reported for a number of H. alvei strains [14–16,43], the position of which in the LPS molecule is unclear. O-Acetyl groups found in a nearly stoichiometric amount in fraction IIIa of the most strains belong to the O-unit attached to the core. Comparing the methylation analysis data of fraction IIIa (Table 2) with published OPS data [9,23–31] enabled determination of the terminal monosaccharide at the non-reducing end of the O-unit and, thus, inferring the structures of the biological repeating units in all H. alvei strains studied, which are shown in Table 3. Terminal N-acyl derivatives of Qui3N or Qui4N were

272

E. Katzenellenbogen et al. / FEMS Immunology and Medical Microbiology 45 (2005) 269–278

Table 1 Chemical composition of fractions IIIa and IIIb from H. alvei LPS H. alvei strain PCM

Fraction

1185

Heptose

Phosphorus

Amino groups
1

Kdo

O-Acetyl groups

%

MR

%

MR

lmol mg

MR

%

MR

lmol mg
1

MR

IIIa IIIb

25.0 31.0

3.0 3.0

5.0 5.6

4.0 3.6

0.87 0.60

2.2 1.2

1.7 1.4

0.17 0.12

0.3 n.d.

0.76

1188

IIIa IIIb

21.0 31.0

3.0 3.0

4.2 5.3

3.9 3.0

0.55 0.70

1.7 1.5

3.3 2.2

0.42 0.16

0.3 n.d.

0.9

1189

IIIa IIIb

21.0 37.0

3.0 3.0

3.4 6.0

3.3 3.2

0.55 0.72

1.7 1.2

3.8 1.9

0.50 0.13

0.36 n.d.

1.1

1196

IIIa IIIb

27.0 30.0

3.0 3.0

5.1 6.0

3.7 4.1

0.95 0.80

2.2 1.7

2.1 2.0

0.21 0.17

n.d. n.d.

1199

IIIa IIIb

16.0 28.0

3.0 3.0

3.8 5.5

4.5 4.1

0.45 0.70

1.7 1.6

4.5 2.1

0.70 0.21

0.18a n.d.

0.7a

1204

IIIa IIIb

21.0 28.0

3.0 3.0

4.9 5.2

4.8 3.9

0.85 0.70

2.6 1.6

2.0 1.4

0.24 0.20

0.3 n.d.

0.9

1205

IIIa IIIb

17.0 25.0

3.0 3.0

3.8 5.0

4.4 4.0

0.40 0.55

1.5 1.4

3.9 1.9

0.60 0.12

0.2a n.d.

0.7a

1211

IIIa IIIb

19.0 30.0

3.0 3.0

2.2 4.8

2.4 3.2

0.57 0.90

1.9 1.9

4.8 1.6

0.70 0.14

0.48 n.d.

1.6

1216

IIIa IIIb

18.0 29.0

3.0 3.0

2.5 5.3

2.8 3.7

0.45 0.70

1.5 1.6

4.9 1.6

0.70 0.15

0.4 n.d.

1.4

1546

IIIa IIIb

20.0 30.0

3.0 3.0

2.1 5.8

2.1 4.1

0.50 0.87

1.6 1.9

6.0 1.65

0.80 0.15

0.6 n.d.

1.9

Given is molar ratio (MR) of the components. n.d., not determined. a The content of the O-acetyl groups decreased during the storage of the oligosaccharides at ambient temperature.

identified in fraction IIIa from strains PCM 1185, 1199, 1204, 1205 and 1216, and a terminal Gal residue in that from strain PCM 1196. The corresponding monosaccharide residues were substituted at position 2, 3 or 4 in the OPS, and, hence, in the O-unit they are the last monosaccharides distal from the core. No new terminal monosaccharide was detected among methylation products from fraction IIIa of strains PCM 1211 and 1546 compared to fraction IIIb but the content of 2,3,4,6-tetra-O-methylglucose increased significantly, thus indicating that the last upstream (non-reducing end) monosaccharide in the O-unit is a glucose residue. The same conclusion resulted from the identification of 2,3,6-tri-O-methylglucose among methylation products from fraction IIIa of strain PCM 1189 instead of 2,3di-O-methylglucose from the corresponding OPS [9], which demonstrated a 4-substituted Glc residue in fraction IIIa versus a 4,6-disubstituted Glc residue in the OPS. Finally, the same substitution pattern of Man, Gal, Rha and GlcNAc residues in fraction IIIa and the OPS from strain PCM 1188 showed that the last upstream monosaccharide in the O-unit is a GlcA residue, which was not detected in methylation analysis under the conditions used. Methylation analysis of fraction IIIa from H. alvei PCM 1185, terminated with a Qui3N derivative, revealed 2,3,4-tri-O-methylglucose (from 6-substituted Glc in the O-unit) (Table 2), whereas methylation anal-

ysis of the OPS from this strain resulted in identification of 2,3,4,6-tetra-O-methylglucose (from terminal Glc) and 2,3-di-O-methylglucose (from 4,6-disubstituted Glc) [29]. The difference in the glucosylation patterns between the OPS and the O-unit in fraction IIIa showed that the first repeating unit is devoid of the lateral glucose residue (this is depicted in italics in Table 3), which is present in most, if not all, other repeating units in the O-chain from strain PCM 1185. This finding is in agreement with the concept that glucosylation is a postpolymerisation modification of O-antigens [44], which often occurs non-stoichiometrically (e.g., as in the OPS of H. alvei PCM 1189 [9]). No significant differences were observed between the structures of the first and other repeating units in the remaining H. alvei strains studied. Fraction IIIa from each strain, except for PCM 1199 having an OPS structure closely related to that of PCM 1205 (Table 3), was studied using NMR spectroscopy. Owing to heterogeneity of the samples, the 1H and 13C NMR spectra were too complex to be assigned but important structural information could be extracted by comparison of the 13C NMR chemical shifts of some characteristic signals in fraction IIIa and the corresponding OPS using an H-detected 1H, 13C HSQC experiment. For instance, a higher-field position of the C3 signal of a 2-substituted N-acyl derivative of bQui3N at d 56.7–57.0 in the spectra of the OPS of strains

E. Katzenellenbogen et al. / FEMS Immunology and Medical Microbiology 45 (2005) 269–278

273

Table 2 Methylation analysis data of fractions IIIa and IIIb from H. alvei LPS H. alvei strain PCM

IIIa B

Monosaccharide

B

1185

t-Glc t-Gal !3)Glc !3)Gal !6)Glc t-Hep !3)GlcNAc t-Qui3NAcyl

0.3 0.9 1.0 0.2 0.9 1.5 0.4 0.5

0.2 0.8 1.0 0.1 0.7 1.3 0.2 0.6

t-Gal !3)Glc t-Hep

0.7 1.0 0.5

1188

t-Rha t-Glc !3)Glc !4)Gal !2,3)Man t-Hep !3)GlcNAc

0.4 0.6 0.6 1.2 1.4 1.5 1.0

0.8 0.6 0.6 0.9 1.0 1.3

t-Glc !3)Glc t-Hep

0.8 1.0 0.6

1189

t-Glc !2)Glc !3)Glc !4)Glc !3)Gal !6)Glc t-Hep !4,6)Glc !3)GalNAc !4,6)GalNAc

t-Glc !3)Glc t-Hep

0.8 1.0 1.2

1196

t-Glc t-Gal !3)Glc !6)Glc t-Hep !3)GlcNAc !6)GlcNAc

0.7 1.0 1.0 1.2 t 1.0 0.8

0.8 1.0 0.8 1.0 t

t-Glc !3)Glc t-Hep

0.9 1.0 1.5

1199

t-Glc !3)Glc !2)Gal t-Hep t-Qui4NAc t-GlcNAc !3)GlcNAc

0.2 0.2 0.6 1.5 1.0 0.7 0.7

0.3 0.3 0.6 1.3 1.0 0.5 t

t-Glc !3)Glc t-Hep

1.0 1.0 0.9

1204

t-Gal !2)Man + !3)Glca !3)Man t-Hep t-Qui3NFo !3)GlcNAc !3)GalNAc

1.1 2.5 0.8 >2.0 0.7 1.0 0.45

0.5 1.0 0.3 0.9

t-Gal !3)Glc t-Hep

0.7 1.0 0.9

1205

t-Glc !3)Glc !2)Gal t-Hep t-Qui4NAc t-GlcNAc !3)GlcNAc

0.25 0.15 0.5 1.2 1.0 0.8 1.0

0.5 0.5 0.6 1.3 1.0 0.9 0.6

t-Glc !3)Glc t-Hep

1.0 1.0 0.75

1211

t-Glc !3)Glc t-Hep !2)Fuc3N

1.4 0.3 1.1 0.3

2.7 0.4 1.8 0.8

t-Glc !3)Glc t-Hep

1.0 1.0 1.4

Monosaccharide

IIIb A

3.4 0.6 1.0 0.9 2.0 0.3 >2.0 0.2 1.1 0.2

(continued on next page)

274

E. Katzenellenbogen et al. / FEMS Immunology and Medical Microbiology 45 (2005) 269–278

Table 2 (continued) H. alvei strain PCM

IIIa

IIIb

Monosaccharide

A

B

!3)GlcNAc !6)GlcNAc !3,4)GalNAc !2)Fuc3NAcyl

1.1 1.0 0.7 0.5

0.7 1.0 0.3 0.7

1216

t-Glc !4)Gal + !3)Glca t-Hep !4)GlcNAc !3)GlcNAc t-Qui3NAcyl

0.2 1.1 0.9 1.1 0.7 1.0

0.1 1.0 0.5 0.3

1546

t-Glc !3)Glc !3)Gal t-Hep !4)GalNAc !3)GalNAc

1.3 0.2 1.0 1.0 0.3 0.5

1.0 0.2 1.0 0.8

Monosaccharide

B

t-Glc !3)Glc t-Hep

0.8 1.0 0.7

t-Glc !3)Glc t-Hep

1.4 1.0 1.4

1.0

0.4

Given are molar ratios of partially methylated alditol acetates. t-Glc and t-Hep stand for terminal D-glucose and L-glycero-D-manno-heptose corresponding to identification of 2,3,4,6-tetra-O-methylglucose and 2,3,4,6,7-penta-O-methylheptose, respectively, etc. Hydrolysis conditions in methylation analysis: A, 10 M HCl, 80 C, 30 min; B, 2 M CF3CO2H, 120 C, 2 h. t, present in a trace amount. Italicised is the terminal non-reducing monosaccharide of the O-unit. a The methylated derivatives are not separated in GLC.

PCM 1185 (Fig. 1) and 1204, as compared with that at d 58.3–58.9 in the corresponding fraction IIIa, showed the presence in the former and the absence in the latter of a glycosyl substituent at position 2 of this monosaccharide. A similar difference in the C3 chemical shifts (d 53.6 vs. 55.2) was observed for a 4-substituted N-acyl derivative of a-Qui3N in the OPS and fraction IIIa from strain PCM 1216. In 2-substituted b-Gal and 2-substituted b-Qui3NFo (strains PCM 1196 and 1204), the glycosylation at position 2 caused also an effect on the C1 resonance, which is shifted upfield to d 102.7 and 103.8 in the OPS as compared to its position at d 104.5 and 105.2 in fraction IIIa, respectively. The terminal upstream monosaccharide in the O-unit of strains PCM 1196, 1205 and 1546 was confirmed by comparison of the chemical shifts for C2 of b-Gal, C3 of b-Qui4NAc and C6 of a-Glc. These are the linkage carbons in the OPS, whose signals shifted downfield to d 79.0, 78.6 and 68.8, respectively, owing to glycosylation, but which resonated in fraction IIIa as in the corresponding nonsubstituted monosaccharides. Therefore, the NMR spectroscopic data confirmed the terminal upstream monosaccharide in the O-unit of most strains and thus defined the first downstream (reducing end) monosaccharide, which is either a 3-substituted GlcNAc or a 3-substituted GalNAc residue (Table 3). Furthermore, using the same approach, it could be demonstrated that in all H. alvei strains studied the first monosaccharide is b-linked to the core. Indeed, in fraction IIIa from all strains with 3-substituted GlcNAc, C2 of this monosaccharide resonated at d 55.1–56.3, i.e., in the region typical of the b-linkage [45]. In the OPS of

strains PCM 1188, 1196, 1204, 1211 and 1216, the signal for C2 of the 3-substituted b-GlcNAc residue was observed in the same region, the difference between the C2 chemical shifts in fraction IIIa and OPS of each particular strain being <0.5 ppm. In the spectra of the OPS from strains PCM 1185 (Fig. 1) and 1205, C2 of the 3-substituted a-GlcNAc resonated at a higher field at d 52.5– 52.8, the C2 chemical shift difference between the OPS and fraction IIIa (3.5 ppm) being typical of replacement of a-GlcNAc with b-GlcNAc. In strains PCM 1189 and 1546 the 3-substituted GalNAc residue is b-linked in both fraction IIIa and OPS as was inferred from the same C2 chemical shifts d 51.9–52.0.

4. Discussion The LPS samples from H. alvei strains examined are heterogeneous in respect to the carbohydrate chain length. SDS–PAGE showed that they include molecules of so-called R (rough)-, S (smooth)- and SR (semirough)-forms, which consist of the core, core substituted with an O-chain polysaccharide and core substituted with one O-unit of the O-polysaccharide (for SDS– PAGE profiles see [4,9,31]). The presence of the SR-form LPS molecules enabled isolation by mild acid degradation followed by gel chromatography of core oligosaccharides with one O-unit attached, whose structure elucidation revealed the structures of the biological repeating units in 10 H. alvei strains. The data obtained in this work and published data on two more H. alvei OPS [21,22] show that the first

E. Katzenellenbogen et al. / FEMS Immunology and Medical Microbiology 45 (2005) 269–278

275

Table 3 Biological repeating units of the OPS of H. alvei Strain

Structure of the first O-unit linked to the core

Reference to the reported OPS structure

Data of this work 1185a,b

[29]

1188

[28]

1189

[9]

1196

!2)Gal(b1!6)Glc(a1!6)GlcNAc(a1!4)GalA(a1!3)GlcNAc(b1!

1199b,c

1204

[30] [25]

!2)Qui3NFo(b1!3)GalNAc(a1!4)GlcA(a 1!3)Man(a1!2)Man(a1!3)GlcNAc(b1!

[27]

1205b

[24]

1211

[23]

1216

!4)Qui3NAcyl(a1!4)Gal(b1!4)GlcNAc(b1!4)GlcA(b1!3)GlcNAc(b1!

[26]

1546

!6)Glc(a1!4)GlcA(b1!4)GalNAc(b1!3)Gal(a1!3)GalNAc(b1!

[31]

Published data [21,22] 39

[21]

1209

[22]

All monosaccharides have the D configuration and are present in the pyranose form unless stated otherwise. Acyl and Fo stand for (R)-hydroxybutyryl and formyl, respectively. Position of substitution of the terminal upstream monosaccharide of the O-unit in the OPS is shown in italics. O-Acetyl groups present in many OPS are not shown. a The lateral glucose residue depicted in italics has been reported in the OPS but is absent from the first O-unit linked to the core. b The GlcNAc residue is depicted in bold type when its linkage in the first O-unit has the opposite configuration compared to that in the other O-units of the OPS. c The b configuration of GlcNAc is presumable.

downstream sugar in the O-units is either a D-GlcNAc or a D-GalNAc residue, which is glycosylated at position 3 (Table 3). At least in some LPS molecules of H. alvei 2

(PCM 2386) the first monosaccharide is a 3-substituted D-GalNAc residue too [46,47]. Whether a- or b-linked in the interior repeating units of the O-chain, this sugar

276

E. Katzenellenbogen et al. / FEMS Immunology and Medical Microbiology 45 (2005) 269–278

Fig. 1. Part of 13C NMR spectra of the O-polysaccharide and fraction IIIa from H. alvei PCM 1185 showing the resonance region of nitrogen-bearing carbons C3 of Qui3NAcyl and C2 of GlcNAc.

is b-linked when it forms a bridge between the first O-unit of the O-chain and the core. That the first monosaccharide may have different anomeric configurations in the first and other O-units in the OPS is a result of involvement of two different enzymes, O-antigen polymerase Wzy and ligase WaaL, with polymerisation of the O-unit and ligation of the O-chain to the core in the Wzy-dependent biosynthesis pathway of the LPS [44]. The linkage type between the OPS and the core has been elucidated also for some other bacteria, in which the first sugar of the O-unit is either a 3-substituted b-D-GlcNAc residue too (Salmonella enterica serovars Toucra O48 [48] and Arizonae O62 [49], Plesiomonas shigelloides O54 [50]) or another 3-substituted monosaccharide, including b-D-Gal (S. enterica serovar Typhimurium [51]), 2-acetamido-4-amino2,4,6-trideoxy-b-D-galactose (Shigella sonnei phase I [52]), b-D-QuiNAc (Vibrio cholerae [53–55], Pseudomonas aeruginosa [56–59]) or b-D-FucNAc (P. aeruginosa [60,61]). The substitution of the first monosaccharide at position 3 and the b-linkage between the O-chain and the core seem to be preferable in the Wzy-dependent pathway, which enable prediction of the structures of the biological repeating units. Particularly, most other H. alvei OPS examined to date beyond Table 3 also contain either 3-substituted GlcNAc or 3-substituted GalNAc [5,6,8], which can be considered as a putative first sugar of the O-unit. Repeating units of only two H. alvei strains, 38 and 1223, contain no 3-substituted hexosamine. The O-chain of H. alvei 1223 is a D-mannan identical to that of E. coli O9 and Klebsiella pneumoniae O3

[7]. This and other bacterial mannans and galactans are synthesised by an alternative ABC transporter-dependent mechanism, which includes sequential transfer of monosaccharide residues to the nascent O-chain [44]. The polymer growth occurs on a 3-substituted D-GlcNAc1-(undecaprenol diphosphate) primer, and the O-chain is ligated to the core-lipid A moiety with formation of the b-glycosidic linkage between the GlcNAc primer and the core, e.g., as in K. pneumoniae [62]. The O-chain of H. alvei 38 is a heteropolymer having a disaccharide repeating units of 4-substituted b-D-ManNAc and 4-substituted a-D-GlcNAc [63], and its biosynthesis pathway is difficult to predict. The site of attachment of the O-chain to the H. alvei LPS core also remains obscure. Comparing the results of methylation analysis of the oligosaccharides that correspond to the typical unsubstituted core (fraction IIIb) and core substituted with one O-unit (fraction IIIa) shown in Table 3 indicate that neither the outer Glc/ Gal-(1!3)-Glc disaccharide nor the lateral heptose residue is substituted with the O-chain. On the other hand, in the LPS of H. alvei 39, 23 and 1222, having non-typical core regions [13], the O-chain is linked directly to the hexose outer core region (authorsÕ unpublished data). In at least part of the LPS molecules from H. alvei 2, the O-chain is linked to a Kdo residue of a core-derived Kdo-containing oligosaccharide [46]. Similar Kdo-containing oligosaccharides have been found also in some other H. alvei strains [14–16,43] as well as in K. pneumoniae [62]. In the latter bacterium, such oligosaccharide intervenes between the OPS and the core [62], but in H.alvei its location in the LPS has not been determined since it was cleaved by the acid-labile Kdo linkage in the course of mild acid hydrolysis of the LPS used in our studies of the H. alvei core. The problem of elucidation of the linkage type between the OPS and core can be solved by alkaline degradation of the H. alvei LPS to give an oligosaccharide containing the first O-unit linked to the core-lipid A carbohydrate backbone. This product would be useful also for determination of the full core oligosaccharide structure, which remains to be established in H. alvei strains with both typical and atypical LPS core. Acknowledgement This work was supported by grant for Leading Scientific Schools of the Russian Federation (project NSh.1557.2003.3). References [1] Sakazaki, R. and Tamura, K. (1992) The genus Hafnia In: The Prokaryotes (Balows, A., Tru¨per, H.G., Dworkin, M., Harder, W. and Schleifer, K.-H., Eds.), vol. III, pp. 2816–2821. Springer Verlag, New York.

E. Katzenellenbogen et al. / FEMS Immunology and Medical Microbiology 45 (2005) 269–278 [2] Klapholz, A., Lessnau, K.D., Huang, B., Talavera, W. and Boyle, J.F. (1994) Hafnia alvei. Respiratory tract isolates in a community hospital over a three-year period and a literature review. Chest 105, 1098–1100. [3] Baturo, A.P. and Raginskaya, V.P. (1978) Antigenic scheme for the Hafniae. Int. J. Syst. Bacteriol. 28, 126–127. [4] Romanowska, A., Katzenellenbogen, E., Kułakowska, M., Gamian, A., Witkowska, D., Mulczyk, M. and Romanowska, E. (1988) Hafnia alvei lipopolysaccharides: isolation, sugar composition and SDS–PAGE analysis. FEMS Microbiol. Immunol. 47, 151–155. [5] Romanowska, E. (2000) Immunochemical aspects of Hafnia alvei O-antigens. FEMS Immunol. Med. Microbiol. 27, 219–225. [6] Jachymek, W., Czaja, J., Niedziela, T., Lugowski, C. and Kenne, L. (1999) Structural studies of the O-specific polysaccharide of Hafnia alvei strain PCM 1207 lipopolysaccharide. Eur. J. Biochem. 266, 53–61. [7] Katzenellenbogen, E., Kocharova, N.A., Zatonsky, G.V., Ku¨blerKiełb, J., Gamian, A., Shashkov, A.S., Knirel, Y.A. and Romanowska, E. (2001) Structural and serological studies on Hafnia alvei O-specific polysaccharide of a-D-mannan type isolated from the lipopolysaccharide of strain PCM 1223. FEMS Immunol. Med. Microbiol. 30, 223–227. [8] Katzenellenbogen, E., Kocharova, N.A., Bogulska, M., Shashkov, A.S. and Knirel, Y.A. (2004) Structure of the O-polysaccharide from the lipopolysaccharide of Hafnia alvei strain PCM 1529. Carbohydr. Res. 339, 723–727. [9] Katzenellenbogen, E., Kocharova, N.A., Zatonsky, G.V., Shashkov, A.S., Korzeniowska-Kowal, A., Gamian, A., Bogulska, M. and Knirel, Y.A. (2005) Structure of the O-polysaccharide of Hafnia alvei strain PCM 1189 that has hexa- to octa-saccharide repeating units owing to incomplete glucosylation. Carbohydr. Res. 340, 263–270. [10] Gamian, A., Katzenellenbogen, E., Romanowska, E., Da˛browski, U. and Da˛browski, J. (1995) Lipopolysaccharide core region of Hafnia alvei: structure elucidation using chemical methods, gas chromatography–mass spectrometry, and NMR spectroscopy. Carbohydr. Res. 266, 221–228. [11] Jachymek, W., Petersson, C., Helander, A., Kenne, L., Lugowski, C. and Niedziela, T. (1995) Structural studies of the O-specific chain and a core hexasaccharide of Hafnia alvei strain 1192 lipopolysaccharide. Carbohydr. Res. 269, 125–138. [12] Ku¨bler, J., Katzenellenbogen, E., Gamian, A., Bogulska, M., Ejchart, A. and Romanowska, E. (2000) Structure of the lipopolysaccharide core region of Hafnia alvei strains 1185 and 1204. Carbohydr. Res. 329, 233–238. [13] Romanowska, E., Katzenellenbogen, E., Jachymek, W., Niedziela, T., Bogulska, M. and Ługowski, C. (1999) Non-typical lipopolysaccharide core regions of some Hafnia alvei strains: structural and serological studies. FEMS Immunol. Med. Microbiol. 24, 63–71. [14] Katzenellenbogen, E., Gamian, A., Romanowska, E., Da˛browski, U. and Da˛browski, J. (1993) 3-Deoxy-octulosonic acid-containing trisaccharide fragment of an unusual core type of some Hafnia alvei lipopolysaccharides. Biochem. Biophys. Res. Commun. 194, 1058–1064. [15] Jachymek, W., Ługowski, C., Romanowska, E., Witkowska, D., Petersson, C. and Kenne, L. (1994) The structure of a core oligosaccharide component from Hafnia alvei strain 32 and 1192 lipopolysaccharides. Carbohydr. Res. 251, 327–330. [16] Katzenellenbogen, E., Gamian, A. and Romanowska, E. (1998) Trisaccharide core fragment occuring in lipopolysaccharides of some Hafnia alvei strains. Carbohydr. Lett. 3, 223–230. [17] Heinrichs, D.E., Whitfield, C. and Valvano, M.A. (1999) Biosynthesis and genetics of lipopolysaccharide core In: Endotoxin in Health and Disease (Brade, H., Morrison, D.C., Opal, S. and Vogel, S., Eds.), pp. 305–330. Marcel Dekker, New York.

277

[18] Keenleyside, W.J. and Whitfield, C. (1999) Genetics and biosynthesis of lipopolysaccharide O-antigens In: Endotoxin in Health and Disease (Brade, H., Morrison, D.C., Opal, S. and Vogel, S., Eds.), pp. 331–358. Marcel Dekker, New York. [19] Shibaev, V.N. (1986) Biosynthesis of bacterial polysaccharide chains composed of repeating units. Adv. Carbohydr. Chem. Biochem. 44, 277–339. [20] Gamian, A., Romanowska, E., Dabrowski, U. and Dabrowski, J. (1991) Structure of the O-specific, sialic acid containing polysaccharide chain and its linkage to the core region in lipopolysaccharide in Hafnia alvei strain 2 as elucidated by chemical methods, gas–liquid chromatography/mass spectrometry. Biochemistry 200, 401–407. [21] Katzenellenbogen, E., Romanowska, E., Kocharova, N.A., Shashkov, A.S., Lipkind, G.M., Knirel, Y.A. and Kochetkov, N.K. (1990) The structure of the biological repeating unit of the O-antigen of Hafnia alvei O39. Carbohydr. Res. 203, 219–227. [22] Niedziela, T., Petersson, C., Jachymek, W., Kenne, L. and Lugowski, C. (1996) Structural studies of the O-specific polysaccharide of Hafnia alvei strain 1209 lipopolysaccharide. Eur. J. Biochem. 237, 635–641. [23] Katzenellenbogen, E., Romanowska, E., Da˛browski, U. and Da˛browski, J. (1991) O-specific polysaccharide of Hafnia alvei lipopolysaccharide isolated from strain 1211. Structural study using chemical methods, gas–liquid chromatography/mass spectrometry and NMR spectroscopy. Eur. J. Biochem. 200, 401–407. [24] Katzenellenbogen, E., Romanowska, E., Kocharova, N.A., Knirel, Y.A., Shashkov, A.S. and Kochetkov, N.K. (1992) The structure of a glycerol teichoic acid-like O-specific polysaccharide of Hafnia alvei 1205. Carbohydr. Res. 231, 249–260. [25] Katzenellenbogen, E., Kocharova, N.A., Zatonsky, G.V., Mieszała, M., Gamian, A., Bogulska, M., Shashkov, A.S., Romanowska, E. and Knirel, Y.A. (1998) Immunochemical studies of the lipopolysaccharide O-specific polysaccharide of Hafnia alvei PCM 1199 related to H. alvei PCM 1205. Eur. J. Biochem. 251, 980– 985. [26] Katzenellenbogen, E., Romanowska, E., Shashkov, A.S., Kocharova, N.A., Knirel, Y.A. and Kochetkov, N.K. (1994) The structure of the O-specific polysaccharide of Hafnia alvei strain 1216. Carbohydr. Res. 259, 67–76. [27] Katzenellenbogen, E., Romanowska, E., Kocharova, N.A., Shashkov, A.S., Knirel, Y.A. and Kochetkov, N.K. (1995) Structure of the O-specific polysaccharide of Hafnia alvei 1204 containing 3,6-dideoxy-3-formamido-D-glucose. Carbohydr. Res. 273, 187–195. [28] Gamian, A., Katzenellenbogen, E., Romanowska, E., Garcia Fernandez, J.M., Pedersen, C., Ulrich, J. and Defaye, J. (1995) Structure of the Hafnia alvei strain PCM 1188 O-specific polysaccharide. Carbohydr. Res. 277, 245–255. [29] Katzenellenbogen, E., Ku¨bler, J., Gamian, A., Romanowska, E., Shashkov, A.S., Kocharova, N.A., Knirel, Y.A. and Kochetkov, N.K. (1996) Structural study and serological characterisation of the O-specific polysaccharide of Hafnia alvei PCM 1185, another Hafnia O-antigen that contains 3,6-dideoxy-3-[(R)-3-hydroxybutyramido]-D-glucose. Carbohydr. Res. 293, 61–70. [30] Katzenellenbogen, E., Zatonsky, G.V., Kocharova, N.A., Rowin´ski, S., Gamian, A., Shashkov, A.S., Romanowska, E. and Knirel, Y.A. (2001) Structure of the O-specific polysaccharide of Hafnia alvei PCM 1196. Carbohydr. Res. 330, 523–528. [31] Katzenellenbogen, E., Kocharova, N.A., Zatonsky, G.V., Korzeniowska-Kowal, A., Shashkov, A.S. and Knirel, Y.A. (2003) Structure of the O-polysaccharide from the lipopolysaccharide of Hafnia alvei strain PCM 1546. Carbohydr. Res. 338, 2153–2158. [32] 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, 499–506.

278

E. Katzenellenbogen et al. / FEMS Immunology and Medical Microbiology 45 (2005) 269–278

[33] Chen, P.S., Toribara, T.Y. and Warner, H. (1956) Microdetermination of phosphorus. Anal. Chem. 28, 1756–1758. [34] Karkhanis, Y.A., Zeltner, J.Y., Jackson, J.J. and Carlo, D.J. (1978) A new and improved microassay to determine 2-keto-3deoxyoctonate in lipopolysaccharide of Gram-negative bacteria. Anal. Biochem. 85, 595–601. [35] Hestrin, S. (1949) The reaction of acetylcholine and other carboxylic acid derivatives with hydroxylamine and its analytical applications. J. Biol. Chem. 180, 249–253. [36] Ghuysen, J.M. and Strominger, J.L. (1963) Structure of the cell wall of Staphylococcus aureus strain Copenhagen. I. Preparation of fragments by enzymatic hydrolysis. Biochemistry 338, 1110– 1119. [37] Sawardeker, J.S., Sloneker, J.H. and Jeans, A. (1965) Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography. Anal. Chem. 37, 1602–1604. [38] Gunnarsson, A. (1987) N- and O-Alkylation of glycoconjugates and polysaccharides by solid base in dimethyl sulphoxide/alkyl iodide. Glycoconj. J. 4, 239–245. [39] Katzenellenbogen, E., Kocharova, N.A., Zatonsky, G.V., Witkowska, D., Bogulska, M., Shashkov, A.S., Gamian, A. and Knirel, Y.A. (2003) Structural and serological studies on a new 4deoxy-D-arabino-hexose-containing O-specific polysaccharide from the lipopolysaccharide of Citrobacter braakii PCM 1531 (serogroup O6). Eur. J. Biochem. 270, 2732–2738. [40] Lugowski, C., Niedziela, T., Jachymek, W., Klonowska, A., Czarny, A., Rowinski, S., Petersson, C. and Kenne, L. (1995) Structural and serological characterization of Hafnia alvei lipopolysaccharide core region. Acta Biochim. Pol. 42, 51–54. [41] Lugowski, C., Jachymek, W., Niedziela, T., Romanowska, A., Witkowska, D. and Romanowska, E. (1995) Lipopolysaccharide core region of Hafnia alvei: serological characterization. FEMS Immunol. Med. Microbiol. 10, 119–124. _ [42] Gamian, A., Mieszała, M., Katzenellenbogen, E., Czarny, A., Zal, T. and Romanowska, E. (1996) The occurence of glycine in bacterial lipopolysaccharides. FEMS Immunol. Med. Microbiol. 13, 261–268. [43] Jachymek, W., Niedziela, T., Lukasiewicz, J., Dzieciatkowska, M., Kenne, L. and Lugowski, C. (2002) Core oligosaccharides of Hafnia alvei 32. Structural and serological analysis of the endotoxin core region, the O-antigen and the linkage between them. Abstract. In: XXI International Carbohydrate Symposium, Cairns, Australia, 7–12 July 2002, p. 281. [44] Raetz, C.R.H. and Whitfield, C. (2002) Lipopolysaccharide endotoxins. Annu. Rev. Biochem. 71, 635–700. [45] Lipkind, G.M., Shashkov, A.S., Knirel, Y.A., Vinogradov, E.V. and Kochetkov, N.K. (1988) A computer-assisted structural analysis of regular polysaccharides on the basis of 13C n.m.r. data. Carbohydr. Res. 175, 59–75. [46] Gamian, A., Ulrich, J., Defaye, J., Mieszała, M., Witkowska, D. and Romanowska, E. (1998) Structural heterogeneity of the sialicacid-containing oligosaccharides from the lipopolysaccharide of Hafnia alvei strain 2 as detected by FABMS studies. Carbohydr. Res. 314, 201–209. [47] Gamian, A. and Kenne, L. (1993) Analysis of 7-substituted sialic acid in some enterobacterial lipopolysaccharides. J. Bacteriol. 175, 1508–1513. [48] Gamian, A., Jones, C., Lipinski, T., Korzeniowska-Kowal, A. and Ravenscroft, N. (2000) Structure of the sialic acid-containing O-specific polysaccharide from Salmonella enterica serovar Toucra O48 lipopolysaccharide. Eur. J. Biochem. 267, 3160–3166. [49] Olsthoorn, M.M.A., Petersen, B.O., Schlecht, S., Haverkamp, J., Bock, K., Thomas-Oates, J.E. and Holst, O. (1998) Identification of a novel core type in Salmonella lipopolysaccharide. Complete structural analysis of the core region of the lipopolysaccharide from Salmonella enterica sv. Arizonae 062. J. Biol. Chem. 273, 3817–3829.

[50] Niedziela, T., Lukasiewicz, J., Jachymek, W., Dzieciatkowska, M., Lugowski, C. and Kenne, L. (2002) Core oligosaccharides of Plesiomonas shigelloides O54: H2 (strain CNCTC 113/92). Structural and serological analysis of the lipopolysaccharide core region, the O-antigen biological repeating unit, and the linkage between them. J. Biol. Chem. 277, 11653–11663. [51] Olsthoorn, M.M., Petersen, B.O., Duus, J., Haverkamp, J., Thomas-Oates, J.E., Bock, K. and Holst, O. (2000) The structure of the linkage between the O-specific polysaccharide and the core region of the lipopolysaccharide from Salmonella enterica serovar Typhimurium revisited. Eur. J. Biochem. 267, 2014–2027. [52] Gamian, A. and Romanowska, E. (1982) The core structure of Shigella sonnei lipopolysaccharide and the linkage between Ospecific polysaccharide and the core region. Eur. J. Biochem. 129, 105–109. [53] Vinogradov, E.V., Holst, O., Thomas-Oates, J.E., Broady, K.W. and Brade, H. (1992) The structure of the O-antigenic polysaccharide from lipopolysaccharide of Vibrio cholerae strain H11 (non-O1). Eur. J. Biochem. 210, 491–498. [54] Knirel, Y.A., Widmalm, G., Senchenkova, S.N., Jansson, P.-E. and Weintraub, A. (1997) Structural studies on the short-chain lipopolysaccharide of Vibrio cholerae O139 Bengal. Eur. J. Biochem. 247, 402–410. [55] Cox, A.D., Brisson, J.-R., Thibault, P. and Perry, M.B. (1997) Structural analysis of the lipopolysaccharide from Vibrio cholerae serotype O22. Carbohydr. Res. 304, 191–208. [56] Bystrova, O.V., Shashkov, A.S., Kocharova, N.A., Knirel, Y.A., Za¨hringer, U. and Pier, G.B. (2003) Elucidation of the structure of the lipopolysaccharide core and the linkage between the core and the O-antigen in Pseudomonas aeruginosa immunotype 5 using strong alkaline degradation of the lipopolysaccharide. Biochemistry (Moscow) 68, 918–925. [57] Bystrova, O.V., Lindner, B., Moll, H., Kocharova, N.A., Knirel, Y.A., Za¨hringer, U. and Pier, G.B. (2003) Structure of the biological repeating unit of the O-antigen of Pseudomonas aeruginosa immunotype 4 containing both 2-acetamido-2,6-dideoxy-D-glucose and 2-acetamido-2,6-dideoxy-D-galactose. Carbohydr. Res. 338, 1801–1806. [58] Bystrova, O.V., Lindner, B., Moll, H., Kocharova, N.A., Knirel, Y.A., Za¨hringer, U. and Pier, G.B. (2003) Structure of the lipopolysaccharide of Pseudomonas aeruginosa O-12 with a randomly O-acetylated core region. Carbohydr. Res. 338, 1895– 1905. [59] Bystrova, O.V., Shashkov, A.S., Kocharova, N.A., Knirel, Y.A., Lindner, B., Za¨hringer, U. and Pier, G.B. (2002) Structural studies on the core and the O-polysaccharide repeating unit of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide. Eur. J. Biochem. 269, 2194–2203. [60] Sadovskaya, I., Brisson, J.-R., Thibault, P., Richards, J.C., Lam, J.S. and Altman, E. (2000) Structural characterization of the outer core and the O-chain linkage region of lipopolysaccharide from Pseudomonas aeruginosa serotype O5. Eur. J. Biochem. 267, 1640– 1650. [61] Dean, C.R., Datta, A., Carlson, R.W. and Goldberg, J.B. (2002) WbjA adds glucose to complete the O-antigen trisaccharide repeating unit of the lipopolysaccharide of Pseudomonas aeruginosa serogroup O11. J. Bacteriol. 184, 323–326. [62] Vinogradov, E., Frirdich, E., Mac Lean, L.L., Perry, M.B., Petersen, B.O., Duus, J.O. and Whitfield, C. (2002) Structures of lipopolysaccharides from Klebsiella pneumoniae. Elucidation of the structure of the linkage region between core and polysaccharide O chain and identification of the residues at the non-reducing termini of the O chains. J. Biol. Chem. 277, 25070–25081. [63] Katzenellenbogen, E., Romanowska, E., Witkowska, D. and Shashkov, A.S. (1992) The structure of the O-specific polysaccharide from Hafnia alvei strain 38 lipopolysaccharide. Carbohydr. Res. 231, 51–54.