Lipopolysaccharides Possessing Twol-Glycero-d-manno-heptopyranosyl-α-(1→5)-3-deoxy-d-manno-oct-2-ulopyranosonic Acid Moieties in the Core Region
2002; Elsevier BV; Volume: 277; Issue: 12 Linguagem: Inglês
10.1074/jbc.m110283200
ISSN1083-351X
AutoresAntonio Molinaro, Cristina De Castro, Rosa Lanzetta, Antonio Evidente, Michelangelo Parrilli, Otto Holst,
Tópico(s)Legume Nitrogen Fixing Symbiosis
ResumoThe carbohydrate backbone of the core-lipid A region was characterized from the lipopolysaccharides (LPSs) of the plant-pathogenic bacterium Burkholderia caryophylli. For the first time, the presence of two moieties ofl-glycero-d-manno-heptopyranosyl-α-(1→5)-3-deoxy-d-manno-oct-2-ulopyranosonic acid was identified in a core region, which is of particular interest with regard to the biosynthesis of this and of LPSs in general. The LPSs of B. caryophylli were degraded by mild hydrazinolysis (de-O-acylation), treatment with 48% aqueous HF at 4 °C (cleavage of phosphate groups and destruction of the O-specific polysaccharides), reduction with NaBH4, and de-N-acylation utilizing hot KOH. The major oligosaccharide representing the carbohydrate backbone of the core region and lipid A was isolated by high-performance anion-exchange chromatography. Its analysis employing compositional and methylation analyses, matrix-assisted laser desorption/ionization mass spectrometry, and1H and 13C NMR spectroscopy applying various one-dimensional and two-dimensional experiments identified the following structure.STRUCTURE I All sugars are pyranoses and α-linked, if not stated otherwise. Hep isl-glycero-d-manno-heptose, Kdo is 3-deoxy-d-manno-oct-2-ulosonic acid. The carbohydrate backbone of the core-lipid A region was characterized from the lipopolysaccharides (LPSs) of the plant-pathogenic bacterium Burkholderia caryophylli. For the first time, the presence of two moieties ofl-glycero-d-manno-heptopyranosyl-α-(1→5)-3-deoxy-d-manno-oct-2-ulopyranosonic acid was identified in a core region, which is of particular interest with regard to the biosynthesis of this and of LPSs in general. The LPSs of B. caryophylli were degraded by mild hydrazinolysis (de-O-acylation), treatment with 48% aqueous HF at 4 °C (cleavage of phosphate groups and destruction of the O-specific polysaccharides), reduction with NaBH4, and de-N-acylation utilizing hot KOH. The major oligosaccharide representing the carbohydrate backbone of the core region and lipid A was isolated by high-performance anion-exchange chromatography. Its analysis employing compositional and methylation analyses, matrix-assisted laser desorption/ionization mass spectrometry, and1H and 13C NMR spectroscopy applying various one-dimensional and two-dimensional experiments identified the following structure.STRUCTURE I All sugars are pyranoses and α-linked, if not stated otherwise. Hep isl-glycero-d-manno-heptose, Kdo is 3-deoxy-d-manno-oct-2-ulosonic acid. lipopolysaccharide 3-deoxy-d-manno-oct-2-ulosonic acid l-glycero-d-manno-heptose high-performance anion-exchange chromatography gas-liquid chromatography-mass spectrometry heteronuclear multiple quantum coherence heteronuclear multiple bond correlation matrix-assisted laser desorption ionization-time-of-flight Burkholderia caryophylli is a phytopathogenic Gram-negative bacterium that had earlier been included in the genusPseudomonas (1Palleroni N.J. Antonie Leeuwenhoek. 1993; 64: 231-251Crossref PubMed Scopus (116) Google Scholar). However, application of ribosomal RNA (rRNA) similarity studies showed that this original genusPseudomonas is diverse and contains five distantly related groups. Of these, RNA group I contains the members of the true genusPseudomonas. The new genus Burkholderia (RNA group II) contains species that are either plant or animal pathogens.B. caryophylli is responsible for the wilting of carnation (2Jones L.K. Phytopathology. 1941; 31: 199Google Scholar), and it shares with other Gram-negative species the presence of lipopolysaccharides (LPSs)1in its cell wall. One characteristic feature of LPSs from the genusBurkholderia is the occurrence of two differentO-specific polysaccharides. In the case of LPSs fromB. caryophylli, two linear homo-polysaccharides were identified as O-specific polysaccharides, one of which is furnished from 3,6,10-trideoxy-4-C-(d-glycero-1-hydroxyethyl)-d-erythro-d-gulo-decose (caryophyllose, α-1→7-linked, caryophyllan) and the other from 4,8-cyclo-3,9-dideoxy-l-erythro-d-ido-nonose (caryose, β-1→7-linked, caryan) (3Adinolfi M. De Corsaro M.M. Castro C. Lanzetta R. Parrilli M. Evidente A. Lavermicocca P. Carbohydr. Res. 1995; 267: 307-311Crossref Scopus (27) Google Scholar, 4Adinolfi M. De Corsaro M.M. Castro C. Evidente A. Lanzetta R. Mangoni L. Parrilli M. Carbohydr. Res. 1995; 274: 223-232Crossref Scopus (26) Google Scholar, 5De Castro C. Evidente A. Lanzetta R. Lavermicocca P. Manzo E. Molinaro A. Parrilli M. Carbohydr. Res. 1996; 284: 119-133Crossref Scopus (32) Google Scholar, 6Adinolfi M. De Corsaro M.M. Castro C. Evidente A. Lanzetta R. Molinaro A. Parrilli M. Carbohydr. Res. 1996; 284: 111-118Crossref Scopus (35) Google Scholar). The caryan is acetylated in nonstoichiometric amounts, leading to a block pattern and, thus, to the establishment of repeating units in a homopolymer (7De Molinaro A. Castro C. Petersen B.O. Duus J.Ø. Parrilli M. Holst O. Angew. Chem. Int. Ed. 2000; 39: 156-160Crossref PubMed Scopus (18) Google Scholar), while only the side chain the caryophyllan is randomly acetylated, and no chemical repeating unit was possible to define (8De Castro C. Lanzetta R. Molinaro A. Parrilli M. Piscopo V. Carbohydr. Res. 2001; 335: 205-211Crossref PubMed Scopus (12) Google Scholar).In LPSs, the O-specific polysaccharide is linked to the core region, which in turn is bound to the lipid A (9Mamat U. Seydel U. Grimmecke D. Holst O. Rietschel E.Th. Barton D. Nakanishi K. Meth-Cohn O. Pinto B.M. Comprehensive Natural Products Chemistry. 3. Elsevier Science Ltd., Oxford1998: 179-239Google Scholar). All core regions identified so far (10Holst O. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 115-154Google Scholar) possess at least one residue of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) that links this region to the lipid A (Kdo I). A second characteristic molecule of the core region isl-glycero-d-manno-heptose (Hep); however, there are heptose-free LPSs (e.g. of the genera Chlamydia and Acinetobacter). In those cases Hep is present, Kdo I is usually substituted by a Hep residue at O-5, regardless to the number of Kdo residues in the structure, and elongation of the core occurs from this Hep residue. Thus, the presence of one Hep-α-(1→5)-Kdo moiety is a characteristic feature of heptose-containing core regions of LPSs. Kdo I may further be substituted at O-4 by a second Kdo residue (Kdo II, e.g. inSalmonella enterica and Escherichia coli). In a few cases, Kdo II is further substituted by neutral sugar residues,e.g. l-rhamnose or d-galactose, in particular E. coli strains. As elucidated best for LPSs ofE. coli (11Rick P.D. Raetz C.R.H. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 283-304Google Scholar, 12Heinrichs D.E. Valvano M.A. Whitfield C. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 305-330Google Scholar, 13Gronow S. Brade H. J. Endotoxin Res. 2001; 7: 3-23PubMed Google Scholar), biosynthesis of the initial parts of the core region begins with the attachment of the Kdo-α-(2→4)-Kdo disaccharide to tetraacyl-lipid A (precursor IVA). After completion of the acylation of lipid A, the first Hep is attached to O-5 of Kdo I, followed by further elongation steps.LPSs of plant-pathogenic bacteria have been shown to play a role in phytopathogenicity (14Dow M. Newman M.-A. von Roepenack E. Annu. Rev. Phytopathol. 2000; 38: 241-261Crossref PubMed Scopus (198) Google Scholar). Since there is only little information on the structure-function relationship of LPSs from B. caryophylli, we have begun with the characterization of its LPSs. Here, we report the structure of the core region which possesses as a novel feature two Hep-α-(1→5)-Kdo moieties.DISCUSSIONB. caryophylli had been named Pseudomonas caryophylli before 1973 and, thus, was taxonomically included in the genus Pseudomonas, which, because of the diversity of functions found in its members, harbored a large number of species (1Palleroni N.J. Antonie Leeuwenhoek. 1993; 64: 231-251Crossref PubMed Scopus (116) Google Scholar). Many attempts to develop systems of classification ofPseudomonas species had failed during the first half of the 20th century, and it was then the research on rRNA sequence similarities among Pseudomonas species that resulted in an internal subdivision of the genus into five RNA homology groups. This subdivision was largely confirmed by investigations on e.g.fatty acid compositions, the appearance of the outer membrane protein OprP, and genome structure and organization. The first of the RNA homology groups (RNA group 1) contains authentic Pseudomonasspecies, and RNA group 2 consists of species of a new genus namedBurkholderia. Quite a number of structures of LPSs fromPseudomonas and some from Burkholderia species have been investigated so far. With regard to the core region of LPSs, which is structurally more conserved than the O-specific polysaccharide, published structures indicate that the core regions ofPseudomonas LPSs differ from those ofBurkholderia LPSs (10Holst O. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 115-154Google Scholar, 33Isshiki, Y., Kawahara, K., and Zähringer, U. (1998) in The Fifth Conference of the International Endotoxin Society September 12–15, Santa Fe, NM, Poster Abstract 70, International Endotoxin SocietyGoogle Scholar), which is confirmed by the data presented in this paper. In particular, one residue ofd-glycero-α-d-talo-oct-2-ulopyranosonic acid (Ko) was identified in the LPSs of Burkholderia cepaciaand Burkholderia pseudomallei, replacing the branching Kdo (Kdo II). This has not been identified in any LPS fromPseudomonas and, thus, might be of chemotaxonomical importance for the differentiation of both genera.The linkages of the sugars in the O-specific polymers of the LPSs of B. caryophylli are acid-labile, and in preliminary experiments it could be shown that treatment of the LPSs with 48% aqueous HF (4 °C, 48 h) not only removed the phosphate groups but also cleaved the O-specific polysaccharides. Thus, we applied this method and succeeded, after additional reduction and deacylation of the LPS, in the isolation of the complete carbohydrate backbone of the core-lipid A region. Its structure (Fig. 1) could be established from chemical and methylation analyses and from NMR spectroscopic and mass spectrometric investigations. The core region contains a structural element that commonly occurs in theSalmonella type core regions of enterobacterial LPS,e.g. from S. enterica or E. coli, namely α-d-Glcp-(1→3)-[l-α-d-Hepp-(1→7)-]-l-α-d-Hepp-(1→3)-l-α-d-Hepp-(1→5)-[α-Kdo-2→4)]-α-Kdo-(2→ (10Holst O. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 115-154Google Scholar). The Glcp residue of this moiety is substituted by two hexoses, i.e. Glcp and Galp, which is similar to several enterobacterial core structures. In dissimilarity to the Salmonella type core regions, the core region from LPSs of B. caryophylli is free of phosphate and contains a Glcp residue that is β-(1→4)-linked to Hep E. The last structural element represents a characteristic feature of phosphate-deficient (e.g. Yersinia enterocolitica, Proteus mirabilis) or phosphate-free (e.g. Klebsiella pneumoniae) core regions. In the core region of LPSs from B. caryophylli it is substituted at O-6 by another α-Glcp residue. The same disaccharidic substituent occurs also in the core region of LPS from P. mirabilis strain R110/1959 (34Radziejewska-Lebrecht J. Mayer H. Eur. J. Biochem. 1989; 183: 573-581Crossref PubMed Scopus (41) Google Scholar, 35Vinogradov E. Radziejewska-Lebrecht J. Kaca W. Eur. J. Biochem. 2000; 267: 262-268Crossref PubMed Scopus (35) Google Scholar). Most strikingly and identified for the first time, the core region of B. caryophylli LPSs possesses twol-α-d-Hepp-(1→5)-α-Kdo-2→ moieties (N-D andE-C), one of which is linked to lipid A and the other to Kdo C. In several cases, a (nonstoichiometric) substitution of the branching Kdo residue with other sugars has been identified. Despite the fact that in several LPSs this residue is substituted at O-4 (S. enterica, E. coli,Chlamydia (9Mamat U. Seydel U. Grimmecke D. Holst O. Rietschel E.Th. Barton D. Nakanishi K. Meth-Cohn O. Pinto B.M. Comprehensive Natural Products Chemistry. 3. Elsevier Science Ltd., Oxford1998: 179-239Google Scholar, 36Brade H. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 229-242Google Scholar)) or O-8 (Chlamydia (36Brade H. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 229-242Google Scholar)) by a third Kdo residue, it may carry a substituent at O-4 (d-GalpA in Rhizobium etli CE3 (37Kannenberg E.L. Reuhs B. Forsberg L.S. Carlson R.W. Spaink H.H. Kondrosi A. Hooykaas P.J.J. The Rhizobiaceae. Kluwer, Amsterdam, The Netherlands1998: 119-154Crossref Google Scholar)), at O-5 (α-l-Rhap in E. coli K-12 (38Holst O. Zähringer U. Brade H. Zamojski A. Carbohydr. Res. 1991; 215: 323-335Crossref PubMed Scopus (39) Google Scholar); d-GalpA in R. etli CE3 (37Kannenberg E.L. Reuhs B. Forsberg L.S. Carlson R.W. Spaink H.H. Kondrosi A. Hooykaas P.J.J. The Rhizobiaceae. Kluwer, Amsterdam, The Netherlands1998: 119-154Crossref Google Scholar);d-Glcp-(1→4)-d-GalpA disaccharide in Ochrobacterium anthropi (21Velasco J. Moll H. Knirel Y.A. Sinnwell V. Moriyón I. Zähringer U. Carbohydr. Res. 1998; 306: 283-290Crossref PubMed Scopus (19) Google Scholar)), at O-7 (α-d-Galp in E. coli R2 strain EH100 (39Holst O. Röhrscheidt-Andrzejewski E. Cordes H.-P. Brade H. Carbohydr. Res. 1989; 188: 212-218Crossref PubMed Scopus (19) Google Scholar)) and at O-8 (β-l-Arap4N inLegionella pneumophila (40Moll H. Knirel Y.A. Helbig J.H. Zähringer U. Carbohydr. Res. 1997; 304: 91-94Crossref PubMed Scopus (27) Google Scholar)).With regard to these structures, a substitution of Kdo D at O-5 by Hep may be considered as just another variant. However, with regard to biosynthesis of LPS, this substitution is of higher bearing. Biosynthesis of the core region is best established for the LPSs ofE. coli (11Rick P.D. Raetz C.R.H. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 283-304Google Scholar, 12Heinrichs D.E. Valvano M.A. Whitfield C. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 305-330Google Scholar, 13Gronow S. Brade H. J. Endotoxin Res. 2001; 7: 3-23PubMed Google Scholar). It begins with the attachment of two Kdo residues to precursor IVA, the tetraacylated and bisphosphorylated GlcN-disaccharide, which is performed by one Kdo-transferase WaaA (KdtA). This step occurs differently in LPS biosynthesis of Pseudomonas aeruginosa, where both Kdo residues are transferred to the completed (fully acylated) lipid A (41Goldman R.C. Doran C.C. Kadam S.K. Capobianco J.Q. J. Biol. Chem. 1988; 263: 5217-5223Abstract Full Text PDF PubMed Google Scholar,42Mohan S. Raetz C.R.H. J. Bacteriol. 1994; 176: 6944-6951Crossref PubMed Google Scholar). However, here and in E. coli, after completion of lipid A employing two additional acylation steps, the Kdo that is attached to lipid A (Kdo I, residue C in Fig. 3) is substituted at O-5 by Hepp, a step that is brought about by heptosyltransferase I, which is encoded by the gene waaC. In a next step ofE. coli LPS biosynthesis, this Hepp residue is then substituted at O-3 by another Hepp through the action of heptosyltransferase II, which is encoded by the genewaaF. Then further steps of core biosynthesis follow,i.e. attachment of the first Glcp (bywaaG) and the third Hepp to Hep II (bywaaQ), and completion of the outer core region. Possibly, decorations of the core, like the attachment of branching sugars or outer core heptose residues (e.g. Hepp inE. coli K-12 (38Holst O. Zähringer U. Brade H. Zamojski A. Carbohydr. Res. 1991; 215: 323-335Crossref PubMed Scopus (39) Google Scholar), dd-Hepp inK. pneumoniae (19Süsskind M. Brade L. Brade H. Holst O. J. Biol. Chem. 1998; 273: 7006-7017Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) or P. mirabilis R110/1959 (34Radziejewska-Lebrecht J. Mayer H. Eur. J. Biochem. 1989; 183: 573-581Crossref PubMed Scopus (41) Google Scholar,35Vinogradov E. Radziejewska-Lebrecht J. Kaca W. Eur. J. Biochem. 2000; 267: 262-268Crossref PubMed Scopus (35) Google Scholar)), occur at later stages of the biosynthesis. With regard to the introduction of outer core heptose residues in LPS core biosynthesis, it is unknown whether the same heptosyltransferases that furnish the inner core region are utilized again or whether other, specific heptosyltransferases are activated. This is also true for the biosynthesis of the twol-α-d-Hepp-(1→5)-α-Kdo-2→ moieties in the LPSs of B. caryophylli, and current data do not favor one possibility over the other.Several core structures of LPSs from Ps. aeruginosa andPs. fluorescens have been published (10Holst O. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 115-154Google Scholar); however, with the core structure of LPSs from B. caryophylli these only share the structural elementl-α-d-Hepp-(1→3)-l-α-d-Hepp-(1→5)-[α-Kdo-2→4)]-α-Kdo-(2→. One of the distinguishing features of B. caryophylli LPSs is their low phosphate content. Of LPSs from the genusBurkholderia, two core structures are known, i.e.from LPSs of B. cepacia GIFU 645 (43Kawahara K. Isshiki Y. Dejsirilert S. Ezaki T. Zähringer U. The Fifth Conference of the International Endotoxin Society.1998Google Scholar) and from B. pseudomallei GIFU 12046 (33Isshiki, Y., Kawahara, K., and Zähringer, U. (1998) in The Fifth Conference of the International Endotoxin Society September 12–15, Santa Fe, NM, Poster Abstract 70, International Endotoxin SocietyGoogle Scholar). Both structures are similar to that of the core region from B. caryophylli, since they are free of phosphate and possess the structural elementl-α-d-Hepp-(1→7)-l-α- d-Hepp-(1→3)-[β-d-Glcp-(1→4)]-l-α-d-Hepp-(1→5)-α-Kdo-(2→. However, in these cases Kdo I is substituted at O-4 byd-glycero-α-d-talo-oct-2-ulopyranosonic acid (Ko) rather than Kdo. In the LPSs of B. caryophylli, only small amounts of Ko could be detected, and no core oligosaccharide possessing this sugar could be isolated. 2A. Molinaro, C. De Castro, R. Lanzetta, M. Parrilli, and O. Holst, unpublished data. It is thus unclear whether the same α-Ko-(2→4)-α-Kdo disaccharide is present in this core region. Whereas in the core region of B. pseudomalleiHep II is substituted at O-3 by α-d-Glcp, this particular Hep carries in the core region of LPS from B. cepacia an l-Rhap residue at O-2, which represents another unusual structural feature. Burkholderia caryophylli is a phytopathogenic Gram-negative bacterium that had earlier been included in the genusPseudomonas (1Palleroni N.J. Antonie Leeuwenhoek. 1993; 64: 231-251Crossref PubMed Scopus (116) Google Scholar). However, application of ribosomal RNA (rRNA) similarity studies showed that this original genusPseudomonas is diverse and contains five distantly related groups. Of these, RNA group I contains the members of the true genusPseudomonas. The new genus Burkholderia (RNA group II) contains species that are either plant or animal pathogens.B. caryophylli is responsible for the wilting of carnation (2Jones L.K. Phytopathology. 1941; 31: 199Google Scholar), and it shares with other Gram-negative species the presence of lipopolysaccharides (LPSs)1in its cell wall. One characteristic feature of LPSs from the genusBurkholderia is the occurrence of two differentO-specific polysaccharides. In the case of LPSs fromB. caryophylli, two linear homo-polysaccharides were identified as O-specific polysaccharides, one of which is furnished from 3,6,10-trideoxy-4-C-(d-glycero-1-hydroxyethyl)-d-erythro-d-gulo-decose (caryophyllose, α-1→7-linked, caryophyllan) and the other from 4,8-cyclo-3,9-dideoxy-l-erythro-d-ido-nonose (caryose, β-1→7-linked, caryan) (3Adinolfi M. De Corsaro M.M. Castro C. Lanzetta R. Parrilli M. Evidente A. Lavermicocca P. Carbohydr. Res. 1995; 267: 307-311Crossref Scopus (27) Google Scholar, 4Adinolfi M. De Corsaro M.M. Castro C. Evidente A. Lanzetta R. Mangoni L. Parrilli M. Carbohydr. Res. 1995; 274: 223-232Crossref Scopus (26) Google Scholar, 5De Castro C. Evidente A. Lanzetta R. Lavermicocca P. Manzo E. Molinaro A. Parrilli M. Carbohydr. Res. 1996; 284: 119-133Crossref Scopus (32) Google Scholar, 6Adinolfi M. De Corsaro M.M. Castro C. Evidente A. Lanzetta R. Molinaro A. Parrilli M. Carbohydr. Res. 1996; 284: 111-118Crossref Scopus (35) Google Scholar). The caryan is acetylated in nonstoichiometric amounts, leading to a block pattern and, thus, to the establishment of repeating units in a homopolymer (7De Molinaro A. Castro C. Petersen B.O. Duus J.Ø. Parrilli M. Holst O. Angew. Chem. Int. Ed. 2000; 39: 156-160Crossref PubMed Scopus (18) Google Scholar), while only the side chain the caryophyllan is randomly acetylated, and no chemical repeating unit was possible to define (8De Castro C. Lanzetta R. Molinaro A. Parrilli M. Piscopo V. Carbohydr. Res. 2001; 335: 205-211Crossref PubMed Scopus (12) Google Scholar). In LPSs, the O-specific polysaccharide is linked to the core region, which in turn is bound to the lipid A (9Mamat U. Seydel U. Grimmecke D. Holst O. Rietschel E.Th. Barton D. Nakanishi K. Meth-Cohn O. Pinto B.M. Comprehensive Natural Products Chemistry. 3. Elsevier Science Ltd., Oxford1998: 179-239Google Scholar). All core regions identified so far (10Holst O. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 115-154Google Scholar) possess at least one residue of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) that links this region to the lipid A (Kdo I). A second characteristic molecule of the core region isl-glycero-d-manno-heptose (Hep); however, there are heptose-free LPSs (e.g. of the genera Chlamydia and Acinetobacter). In those cases Hep is present, Kdo I is usually substituted by a Hep residue at O-5, regardless to the number of Kdo residues in the structure, and elongation of the core occurs from this Hep residue. Thus, the presence of one Hep-α-(1→5)-Kdo moiety is a characteristic feature of heptose-containing core regions of LPSs. Kdo I may further be substituted at O-4 by a second Kdo residue (Kdo II, e.g. inSalmonella enterica and Escherichia coli). In a few cases, Kdo II is further substituted by neutral sugar residues,e.g. l-rhamnose or d-galactose, in particular E. coli strains. As elucidated best for LPSs ofE. coli (11Rick P.D. Raetz C.R.H. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 283-304Google Scholar, 12Heinrichs D.E. Valvano M.A. Whitfield C. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 305-330Google Scholar, 13Gronow S. Brade H. J. Endotoxin Res. 2001; 7: 3-23PubMed Google Scholar), biosynthesis of the initial parts of the core region begins with the attachment of the Kdo-α-(2→4)-Kdo disaccharide to tetraacyl-lipid A (precursor IVA). After completion of the acylation of lipid A, the first Hep is attached to O-5 of Kdo I, followed by further elongation steps. LPSs of plant-pathogenic bacteria have been shown to play a role in phytopathogenicity (14Dow M. Newman M.-A. von Roepenack E. Annu. Rev. Phytopathol. 2000; 38: 241-261Crossref PubMed Scopus (198) Google Scholar). Since there is only little information on the structure-function relationship of LPSs from B. caryophylli, we have begun with the characterization of its LPSs. Here, we report the structure of the core region which possesses as a novel feature two Hep-α-(1→5)-Kdo moieties. DISCUSSIONB. caryophylli had been named Pseudomonas caryophylli before 1973 and, thus, was taxonomically included in the genus Pseudomonas, which, because of the diversity of functions found in its members, harbored a large number of species (1Palleroni N.J. Antonie Leeuwenhoek. 1993; 64: 231-251Crossref PubMed Scopus (116) Google Scholar). Many attempts to develop systems of classification ofPseudomonas species had failed during the first half of the 20th century, and it was then the research on rRNA sequence similarities among Pseudomonas species that resulted in an internal subdivision of the genus into five RNA homology groups. This subdivision was largely confirmed by investigations on e.g.fatty acid compositions, the appearance of the outer membrane protein OprP, and genome structure and organization. The first of the RNA homology groups (RNA group 1) contains authentic Pseudomonasspecies, and RNA group 2 consists of species of a new genus namedBurkholderia. Quite a number of structures of LPSs fromPseudomonas and some from Burkholderia species have been investigated so far. With regard to the core region of LPSs, which is structurally more conserved than the O-specific polysaccharide, published structures indicate that the core regions ofPseudomonas LPSs differ from those ofBurkholderia LPSs (10Holst O. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 115-154Google Scholar, 33Isshiki, Y., Kawahara, K., and Zähringer, U. (1998) in The Fifth Conference of the International Endotoxin Society September 12–15, Santa Fe, NM, Poster Abstract 70, International Endotoxin SocietyGoogle Scholar), which is confirmed by the data presented in this paper. In particular, one residue ofd-glycero-α-d-talo-oct-2-ulopyranosonic acid (Ko) was identified in the LPSs of Burkholderia cepaciaand Burkholderia pseudomallei, replacing the branching Kdo (Kdo II). This has not been identified in any LPS fromPseudomonas and, thus, might be of chemotaxonomical importance for the differentiation of both genera.The linkages of the sugars in the O-specific polymers of the LPSs of B. caryophylli are acid-labile, and in preliminary experiments it could be shown that treatment of the LPSs with 48% aqueous HF (4 °C, 48 h) not only removed the phosphate groups but also cleaved the O-specific polysaccharides. Thus, we applied this method and succeeded, after additional reduction and deacylation of the LPS, in the isolation of the complete carbohydrate backbone of the core-lipid A region. Its structure (Fig. 1) could be established from chemical and methylation analyses and from NMR spectroscopic and mass spectrometric investigations. The core region contains a structural element that commonly occurs in theSalmonella type core regions of enterobacterial LPS,e.g. from S. enterica or E. coli, namely α-d-Glcp-(1→3)-[l-α-d-Hepp-(1→7)-]-l-α-d-Hepp-(1→3)-l-α-d-Hepp-(1→5)-[α-Kdo-2→4)]-α-Kdo-(2→ (10Holst O. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 115-154Google Scholar). The Glcp residue of this moiety is substituted by two hexoses, i.e. Glcp and Galp, which is similar to several enterobacterial core structures. In dissimilarity to the Salmonella type core regions, the core region from LPSs of B. caryophylli is free of phosphate and contains a Glcp residue that is β-(1→4)-linked to Hep E. The last structural element represents a characteristic feature of phosphate-deficient (e.g. Yersinia enterocolitica, Proteus mirabilis) or phosphate-free (e.g. Klebsiella pneumoniae) core regions. In the core region of LPSs from B. caryophylli it is substituted at O-6 by another α-Glcp residue. The same disaccharidic substituent occurs also in the core region of LPS from P. mirabilis strain R110/1959 (34Radziejewska-Lebrecht J. Mayer H. Eur. J. Biochem. 1989; 183: 573-581Crossref PubMed Scopus (41) Google Scholar, 35Vinogradov E. Radziejewska-Lebrecht J. Kaca W. Eur. J. Biochem. 2000; 267: 262-268Crossref PubMed Scopus (35) Google Scholar). Most strikingly and identified for the first time, the core region of B. caryophylli LPSs possesses twol-α-d-Hepp-(1→5)-α-Kdo-2→ moieties (N-D andE-C), one of which is linked to lipid A and the other to Kdo C. In several cases, a (nonstoichiometric) substitution of the branching Kdo residue with other sugars has been identified. Despite the fact that in several LPSs this residue is substituted at O-4 (S. enterica, E. coli,Chlamydia (9Mamat U. Seydel U. Grimmecke D. Holst O. Rietschel E.Th. Barton D. Nakanishi K. Meth-Cohn O. Pinto B.M. Comprehensive Natural Products Chemistry. 3. Elsevier Science Ltd., Oxford1998: 179-239Google Scholar, 36Brade H. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 229-242Google Scholar)) or O-8 (Chlamydia (36Brade H. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 229-242Google Scholar)) by a third Kdo residue, it may carry a substituent at O-4 (d-GalpA in Rhizobium etli CE3 (37Kannenberg E.L. Reuhs B. Forsberg L.S. Carlson R.W. Spaink H.H. Kondrosi A. Hooykaas P.J.J. The Rhizobiaceae. Kluwer, Amsterdam, The Netherlands1998: 119-154Crossref Google Scholar)), at O-5 (α-l-Rhap in E. coli K-12 (38Holst O. Zähringer U. Brade H. Zamojski A. Carbohydr. Res. 1991; 215: 323-335Crossref PubMed Scopus (39) Google Scholar); d-GalpA in R. etli CE3 (37Kannenberg E.L. Reuhs B. Forsberg L.S. Carlson R.W. Spaink H.H. Kondrosi A. Hooykaas P.J.J. The Rhizobiaceae. Kluwer, Amsterdam, The Netherlands1998: 119-154Crossref Google Scholar);d-Glcp-(1→4)-d-GalpA disaccharide in Ochrobacterium anthropi (21Velasco J. Moll H. Knirel Y.A. Sinnwell V. Moriyón I. Zähringer U. Carbohydr. Res. 1998; 306: 283-290Crossref PubMed Scopus (19) Google Scholar)), at O-7 (α-d-Galp in E. coli R2 strain EH100 (39Holst O. Röhrscheidt-Andrzejewski E. Cordes H.-P. Brade H. Carbohydr. Res. 1989; 188: 212-218Crossref PubMed Scopus (19) Google Scholar)) and at O-8 (β-l-Arap4N inLegionella pneumophila (40Moll H. Knirel Y.A. Helbig J.H. Zähringer U. Carbohydr. Res. 1997; 304: 91-94Crossref PubMed Scopus (27) Google Scholar)).With regard to these structures, a substitution of Kdo D at O-5 by Hep may be considered as just another variant. However, with regard to biosynthesis of LPS, this substitution is of higher bearing. Biosynthesis of the core region is best established for the LPSs ofE. coli (11Rick P.D. Raetz C.R.H. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 283-304Google Scholar, 12Heinrichs D.E. Valvano M.A. Whitfield C. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 305-330Google Scholar, 13Gronow S. Brade H. J. Endotoxin Res. 2001; 7: 3-23PubMed Google Scholar). It begins with the attachment of two Kdo residues to precursor IVA, the tetraacylated and bisphosphorylated GlcN-disaccharide, which is performed by one Kdo-transferase WaaA (KdtA). This step occurs differently in LPS biosynthesis of Pseudomonas aeruginosa, where both Kdo residues are transferred to the completed (fully acylated) lipid A (41Goldman R.C. Doran C.C. Kadam S.K. Capobianco J.Q. J. Biol. Chem. 1988; 263: 5217-5223Abstract Full Text PDF PubMed Google Scholar,42Mohan S. Raetz C.R.H. J. Bacteriol. 1994; 176: 6944-6951Crossref PubMed Google Scholar). However, here and in E. coli, after completion of lipid A employing two additional acylation steps, the Kdo that is attached to lipid A (Kdo I, residue C in Fig. 3) is substituted at O-5 by Hepp, a step that is brought about by heptosyltransferase I, which is encoded by the gene waaC. In a next step ofE. coli LPS biosynthesis, this Hepp residue is then substituted at O-3 by another Hepp through the action of heptosyltransferase II, which is encoded by the genewaaF. Then further steps of core biosynthesis follow,i.e. attachment of the first Glcp (bywaaG) and the third Hepp to Hep II (bywaaQ), and completion of the outer core region. Possibly, decorations of the core, like the attachment of branching sugars or outer core heptose residues (e.g. Hepp inE. coli K-12 (38Holst O. Zähringer U. Brade H. Zamojski A. Carbohydr. Res. 1991; 215: 323-335Crossref PubMed Scopus (39) Google Scholar), dd-Hepp inK. pneumoniae (19Süsskind M. Brade L. Brade H. Holst O. J. Biol. Chem. 1998; 273: 7006-7017Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) or P. mirabilis R110/1959 (34Radziejewska-Lebrecht J. Mayer H. Eur. J. Biochem. 1989; 183: 573-581Crossref PubMed Scopus (41) Google Scholar,35Vinogradov E. Radziejewska-Lebrecht J. Kaca W. Eur. J. Biochem. 2000; 267: 262-268Crossref PubMed Scopus (35) Google Scholar)), occur at later stages of the biosynthesis. With regard to the introduction of outer core heptose residues in LPS core biosynthesis, it is unknown whether the same heptosyltransferases that furnish the inner core region are utilized again or whether other, specific heptosyltransferases are activated. This is also true for the biosynthesis of the twol-α-d-Hepp-(1→5)-α-Kdo-2→ moieties in the LPSs of B. caryophylli, and current data do not favor one possibility over the other.Several core structures of LPSs from Ps. aeruginosa andPs. fluorescens have been published (10Holst O. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 115-154Google Scholar); however, with the core structure of LPSs from B. caryophylli these only share the structural elementl-α-d-Hepp-(1→3)-l-α-d-Hepp-(1→5)-[α-Kdo-2→4)]-α-Kdo-(2→. One of the distinguishing features of B. caryophylli LPSs is their low phosphate content. Of LPSs from the genusBurkholderia, two core structures are known, i.e.from LPSs of B. cepacia GIFU 645 (43Kawahara K. Isshiki Y. Dejsirilert S. Ezaki T. Zähringer U. The Fifth Conference of the International Endotoxin Society.1998Google Scholar) and from B. pseudomallei GIFU 12046 (33Isshiki, Y., Kawahara, K., and Zähringer, U. (1998) in The Fifth Conference of the International Endotoxin Society September 12–15, Santa Fe, NM, Poster Abstract 70, International Endotoxin SocietyGoogle Scholar). Both structures are similar to that of the core region from B. caryophylli, since they are free of phosphate and possess the structural elementl-α-d-Hepp-(1→7)-l-α- d-Hepp-(1→3)-[β-d-Glcp-(1→4)]-l-α-d-Hepp-(1→5)-α-Kdo-(2→. However, in these cases Kdo I is substituted at O-4 byd-glycero-α-d-talo-oct-2-ulopyranosonic acid (Ko) rather than Kdo. In the LPSs of B. caryophylli, only small amounts of Ko could be detected, and no core oligosaccharide possessing this sugar could be isolated. 2A. Molinaro, C. De Castro, R. Lanzetta, M. Parrilli, and O. Holst, unpublished data. It is thus unclear whether the same α-Ko-(2→4)-α-Kdo disaccharide is present in this core region. Whereas in the core region of B. pseudomalleiHep II is substituted at O-3 by α-d-Glcp, this particular Hep carries in the core region of LPS from B. cepacia an l-Rhap residue at O-2, which represents another unusual structural feature. B. caryophylli had been named Pseudomonas caryophylli before 1973 and, thus, was taxonomically included in the genus Pseudomonas, which, because of the diversity of functions found in its members, harbored a large number of species (1Palleroni N.J. Antonie Leeuwenhoek. 1993; 64: 231-251Crossref PubMed Scopus (116) Google Scholar). Many attempts to develop systems of classification ofPseudomonas species had failed during the first half of the 20th century, and it was then the research on rRNA sequence similarities among Pseudomonas species that resulted in an internal subdivision of the genus into five RNA homology groups. This subdivision was largely confirmed by investigations on e.g.fatty acid compositions, the appearance of the outer membrane protein OprP, and genome structure and organization. The first of the RNA homology groups (RNA group 1) contains authentic Pseudomonasspecies, and RNA group 2 consists of species of a new genus namedBurkholderia. Quite a number of structures of LPSs fromPseudomonas and some from Burkholderia species have been investigated so far. With regard to the core region of LPSs, which is structurally more conserved than the O-specific polysaccharide, published structures indicate that the core regions ofPseudomonas LPSs differ from those ofBurkholderia LPSs (10Holst O. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 115-154Google Scholar, 33Isshiki, Y., Kawahara, K., and Zähringer, U. (1998) in The Fifth Conference of the International Endotoxin Society September 12–15, Santa Fe, NM, Poster Abstract 70, International Endotoxin SocietyGoogle Scholar), which is confirmed by the data presented in this paper. In particular, one residue ofd-glycero-α-d-talo-oct-2-ulopyranosonic acid (Ko) was identified in the LPSs of Burkholderia cepaciaand Burkholderia pseudomallei, replacing the branching Kdo (Kdo II). This has not been identified in any LPS fromPseudomonas and, thus, might be of chemotaxonomical importance for the differentiation of both genera. The linkages of the sugars in the O-specific polymers of the LPSs of B. caryophylli are acid-labile, and in preliminary experiments it could be shown that treatment of the LPSs with 48% aqueous HF (4 °C, 48 h) not only removed the phosphate groups but also cleaved the O-specific polysaccharides. Thus, we applied this method and succeeded, after additional reduction and deacylation of the LPS, in the isolation of the complete carbohydrate backbone of the core-lipid A region. Its structure (Fig. 1) could be established from chemical and methylation analyses and from NMR spectroscopic and mass spectrometric investigations. The core region contains a structural element that commonly occurs in theSalmonella type core regions of enterobacterial LPS,e.g. from S. enterica or E. coli, namely α-d-Glcp-(1→3)-[l-α-d-Hepp-(1→7)-]-l-α-d-Hepp-(1→3)-l-α-d-Hepp-(1→5)-[α-Kdo-2→4)]-α-Kdo-(2→ (10Holst O. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 115-154Google Scholar). The Glcp residue of this moiety is substituted by two hexoses, i.e. Glcp and Galp, which is similar to several enterobacterial core structures. In dissimilarity to the Salmonella type core regions, the core region from LPSs of B. caryophylli is free of phosphate and contains a Glcp residue that is β-(1→4)-linked to Hep E. The last structural element represents a characteristic feature of phosphate-deficient (e.g. Yersinia enterocolitica, Proteus mirabilis) or phosphate-free (e.g. Klebsiella pneumoniae) core regions. In the core region of LPSs from B. caryophylli it is substituted at O-6 by another α-Glcp residue. The same disaccharidic substituent occurs also in the core region of LPS from P. mirabilis strain R110/1959 (34Radziejewska-Lebrecht J. Mayer H. Eur. J. Biochem. 1989; 183: 573-581Crossref PubMed Scopus (41) Google Scholar, 35Vinogradov E. Radziejewska-Lebrecht J. Kaca W. Eur. J. Biochem. 2000; 267: 262-268Crossref PubMed Scopus (35) Google Scholar). Most strikingly and identified for the first time, the core region of B. caryophylli LPSs possesses twol-α-d-Hepp-(1→5)-α-Kdo-2→ moieties (N-D andE-C), one of which is linked to lipid A and the other to Kdo C. In several cases, a (nonstoichiometric) substitution of the branching Kdo residue with other sugars has been identified. Despite the fact that in several LPSs this residue is substituted at O-4 (S. enterica, E. coli,Chlamydia (9Mamat U. Seydel U. Grimmecke D. Holst O. Rietschel E.Th. Barton D. Nakanishi K. Meth-Cohn O. Pinto B.M. Comprehensive Natural Products Chemistry. 3. Elsevier Science Ltd., Oxford1998: 179-239Google Scholar, 36Brade H. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 229-242Google Scholar)) or O-8 (Chlamydia (36Brade H. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 229-242Google Scholar)) by a third Kdo residue, it may carry a substituent at O-4 (d-GalpA in Rhizobium etli CE3 (37Kannenberg E.L. Reuhs B. Forsberg L.S. Carlson R.W. Spaink H.H. Kondrosi A. Hooykaas P.J.J. The Rhizobiaceae. Kluwer, Amsterdam, The Netherlands1998: 119-154Crossref Google Scholar)), at O-5 (α-l-Rhap in E. coli K-12 (38Holst O. Zähringer U. Brade H. Zamojski A. Carbohydr. Res. 1991; 215: 323-335Crossref PubMed Scopus (39) Google Scholar); d-GalpA in R. etli CE3 (37Kannenberg E.L. Reuhs B. Forsberg L.S. Carlson R.W. Spaink H.H. Kondrosi A. Hooykaas P.J.J. The Rhizobiaceae. Kluwer, Amsterdam, The Netherlands1998: 119-154Crossref Google Scholar);d-Glcp-(1→4)-d-GalpA disaccharide in Ochrobacterium anthropi (21Velasco J. Moll H. Knirel Y.A. Sinnwell V. Moriyón I. Zähringer U. Carbohydr. Res. 1998; 306: 283-290Crossref PubMed Scopus (19) Google Scholar)), at O-7 (α-d-Galp in E. coli R2 strain EH100 (39Holst O. Röhrscheidt-Andrzejewski E. Cordes H.-P. Brade H. Carbohydr. Res. 1989; 188: 212-218Crossref PubMed Scopus (19) Google Scholar)) and at O-8 (β-l-Arap4N inLegionella pneumophila (40Moll H. Knirel Y.A. Helbig J.H. Zähringer U. Carbohydr. Res. 1997; 304: 91-94Crossref PubMed Scopus (27) Google Scholar)). With regard to these structures, a substitution of Kdo D at O-5 by Hep may be considered as just another variant. However, with regard to biosynthesis of LPS, this substitution is of higher bearing. Biosynthesis of the core region is best established for the LPSs ofE. coli (11Rick P.D. Raetz C.R.H. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 283-304Google Scholar, 12Heinrichs D.E. Valvano M.A. Whitfield C. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 305-330Google Scholar, 13Gronow S. Brade H. J. Endotoxin Res. 2001; 7: 3-23PubMed Google Scholar). It begins with the attachment of two Kdo residues to precursor IVA, the tetraacylated and bisphosphorylated GlcN-disaccharide, which is performed by one Kdo-transferase WaaA (KdtA). This step occurs differently in LPS biosynthesis of Pseudomonas aeruginosa, where both Kdo residues are transferred to the completed (fully acylated) lipid A (41Goldman R.C. Doran C.C. Kadam S.K. Capobianco J.Q. J. Biol. Chem. 1988; 263: 5217-5223Abstract Full Text PDF PubMed Google Scholar,42Mohan S. Raetz C.R.H. J. Bacteriol. 1994; 176: 6944-6951Crossref PubMed Google Scholar). However, here and in E. coli, after completion of lipid A employing two additional acylation steps, the Kdo that is attached to lipid A (Kdo I, residue C in Fig. 3) is substituted at O-5 by Hepp, a step that is brought about by heptosyltransferase I, which is encoded by the gene waaC. In a next step ofE. coli LPS biosynthesis, this Hepp residue is then substituted at O-3 by another Hepp through the action of heptosyltransferase II, which is encoded by the genewaaF. Then further steps of core biosynthesis follow,i.e. attachment of the first Glcp (bywaaG) and the third Hepp to Hep II (bywaaQ), and completion of the outer core region. Possibly, decorations of the core, like the attachment of branching sugars or outer core heptose residues (e.g. Hepp inE. coli K-12 (38Holst O. Zähringer U. Brade H. Zamojski A. Carbohydr. Res. 1991; 215: 323-335Crossref PubMed Scopus (39) Google Scholar), dd-Hepp inK. pneumoniae (19Süsskind M. Brade L. Brade H. Holst O. J. Biol. Chem. 1998; 273: 7006-7017Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) or P. mirabilis R110/1959 (34Radziejewska-Lebrecht J. Mayer H. Eur. J. Biochem. 1989; 183: 573-581Crossref PubMed Scopus (41) Google Scholar,35Vinogradov E. Radziejewska-Lebrecht J. Kaca W. Eur. J. Biochem. 2000; 267: 262-268Crossref PubMed Scopus (35) Google Scholar)), occur at later stages of the biosynthesis. With regard to the introduction of outer core heptose residues in LPS core biosynthesis, it is unknown whether the same heptosyltransferases that furnish the inner core region are utilized again or whether other, specific heptosyltransferases are activated. This is also true for the biosynthesis of the twol-α-d-Hepp-(1→5)-α-Kdo-2→ moieties in the LPSs of B. caryophylli, and current data do not favor one possibility over the other. Several core structures of LPSs from Ps. aeruginosa andPs. fluorescens have been published (10Holst O. Brade H. Morrsion D.C. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker Inc., New York1999: 115-154Google Scholar); however, with the core structure of LPSs from B. caryophylli these only share the structural elementl-α-d-Hepp-(1→3)-l-α-d-Hepp-(1→5)-[α-Kdo-2→4)]-α-Kdo-(2→. One of the distinguishing features of B. caryophylli LPSs is their low phosphate content. Of LPSs from the genusBurkholderia, two core structures are known, i.e.from LPSs of B. cepacia GIFU 645 (43Kawahara K. Isshiki Y. Dejsirilert S. Ezaki T. Zähringer U. The Fifth Conference of the International Endotoxin Society.1998Google Scholar) and from B. pseudomallei GIFU 12046 (33Isshiki, Y., Kawahara, K., and Zähringer, U. (1998) in The Fifth Conference of the International Endotoxin Society September 12–15, Santa Fe, NM, Poster Abstract 70, International Endotoxin SocietyGoogle Scholar). Both structures are similar to that of the core region from B. caryophylli, since they are free of phosphate and possess the structural elementl-α-d-Hepp-(1→7)-l-α- d-Hepp-(1→3)-[β-d-Glcp-(1→4)]-l-α-d-Hepp-(1→5)-α-Kdo-(2→. However, in these cases Kdo I is substituted at O-4 byd-glycero-α-d-talo-oct-2-ulopyranosonic acid (Ko) rather than Kdo. In the LPSs of B. caryophylli, only small amounts of Ko could be detected, and no core oligosaccharide possessing this sugar could be isolated. 2A. Molinaro, C. De Castro, R. Lanzetta, M. Parrilli, and O. Holst, unpublished data. It is thus unclear whether the same α-Ko-(2→4)-α-Kdo disaccharide is present in this core region. Whereas in the core region of B. pseudomalleiHep II is substituted at O-3 by α-d-Glcp, this particular Hep carries in the core region of LPS from B. cepacia an l-Rhap residue at O-2, which represents another unusual structural feature. We thank Regina Engel for technical assistance, Hans-Peter Cordes for recording the NMR spectra, Angela Amoresano for recording the MALDI-TOF mass spectrum, Hermann Moll for help with GC-MS, and Yasunori Isshiky for valuable discussions.
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