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Hyaluronic acid was obtained from filtrates of heat-killed cultures of Streptococcus pyogenes group A, strain K56, by simple ethanol precipitation and treatment with an adsorbent. The hyaluronic acid is pure as judged from chemical and sedimentation analyses. Particles of streptococcal bacteriophage 12/12 were isolated from phage-lysed group A streptococci by polyethylene glycol precipitation and isopyenic centrifugation. Electron micrographs of negatively stained preparations showed a typical Bradley group B virus with a long, flexible, cross-striated tail and a knob- or star-like structure at the distal tip of the tail. The hyaluronic acid is depolymerized upon incubation with the phage 12/12 virions. After extensive digestion, a mixture of at least four oligosaccharides is formed, the two smallest of which are a tetra- and octasaccharide terminating in reducing N-acetyl-D-glucosamine. The tetrasaccharide shows an absorption maximum at 231.5 nm with a molar extinction coefficient epsilon = 4820 litres X mole-1 X cm-1, and it is therefore concluded that the bacteriophage-borne hyaluronidase catalyses a beta-elimination. Accordingly it is classified as a hyaluronate lyase (EC 4.2.99.1).

The thrombin-like serine protease ancrod from the Malayan pit viper Agkistrodon rhodostoma was expressed in mouse epithelial cells (C127). Oligosaccharide constituents were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F. Neutral oligosaccharide alditols obtained after reduction and enzymic desialylation were separated by two-dimensional HPLC and characterized by methylation analysis, liquid secondary-ion mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sequential degradation with exoglycosidases. In contrast to natural ancrod, the recombinant glycoprotein carries exclusively diantennary, triantennary and tetraantennary N-glycans with Gal beta 4 GlcNAc beta (type-2) antennae which were, in part, further substituted by host-cell-specific structural elements such as Gal alpha 3 residues or N-acetyllactosamine repeats. As a characteristic feature, a substantial proportion of the oligosaccharides bears a GalNAc beta 4Glc-NAc antenna. Studies at the level of individual N-glycosylation sites demonstrated that glycans with N, N'-diacetyllactosediamine units are not specifically attached but occur at all sites in varying amounts. Hence, the putative recognition signal (Pro70-Lys-Lys) for glycoprotein hormone N-acetylgalactosaminyltransferase, present in this glycoprotein in close proximity to Asn79, does not convey site-specific transfer of GalNAc residues in these cells.

The disulfide-bonding pattern of glycoprotein 70 (gp70), the surface glycoprotein (SU) encoded by the envelope gene of polytropic Friend milk cell focus-inducing virus, was elucidated and compared with that of glycoprotein 71 (gp71), the corresponding glycoprotein of the ecotropic Friend murine leukemia virus, which had previously been determined (M. Linder, D. Linder, J. Hahnen, H.-H. Schott, and Stirm, Eur. J. Biochem. 203:65-73, 1992). In the carboxy-terminal constant domain, in which these glycoproteins have about 97% sequence homology, the location of the four disulfide bonds was found to be analogous. In the amino-terminal differential domain, with about 37% sequence homology, 8 of the 12 cysteine residues of the ecotropic SU are conserved in the polytropic SU. In this domain, a similar clustering of disulfide bonds was detected, which led to the identification of three distinct disulfide-bonded regions in both glycoproteins. However, because of deletions and sequence deviations, the glycoproteins must have significantly different three-dimensional structures in these regions. Since the receptor-binding functions of both glycoproteins have been attributed to their amino-terminal domains and since each binds to a different receptor, these disulfide-bonded structures are likely candidates for receptor-binding functions. Limited proteolysis of both glycoproteins with various endoproteinases led to the identification of preferential proteolytic sites between disulfide-bonded regions, at the beginning of the hypervariable proline-rich region, and between differential and constant domains, further confirming the structural organization of the folded glycoproteins.

Sulphated N-linked carbohydrate chains isolated from recombinant human tissue plasminogen activator expressed in mouse epithelial (C127) cells were analysed as oligosaccharide alditols by methylation analysis, liquid secondary ion mass spectrometry, and one- and two-dimensional 1H-NMR spectroscopy. The results demonstrate that the major component has the following novel structure: NeuAc-alpha 2-6Gal beta 1-4GlcNAc beta 1-2[NeuAc alpha 2-3Gal beta 1- 4GlcNAc beta 1-4]-Man alpha 1-3[NeuAc alpha 2-3(SO4-6)Gal beta 1- 4-GlcNAc beta 1-2Man alpha 1-6]-Man beta 1-4GlcNAc beta 1- 4[Fuc alpha 1-6]GlcNAc-o1.

Mutants of Escherichia coli C were selected for resistance towards a set of cell wall LPS core specific bacteriophages, including oX174. Increasingly deficient LPS's from wt and mutant E. coli C were tested for inactivation of oX174, and the core oligosaccharides were subjected to structural analysis by methylation/g.l.c./m.s. Loss of the terminal galactose in the following basic structure of the E. coli C wt core was found to lead to adsorption resistance towards oX174

Different interactions between particles of Escherichia coli capsule bacteriophage 29 and its receptor, the E. coli serotype 29 capsular polysaccharide have been studied. The inactivation of phage 29 (8 x 10(3) PFU/ml) by isolated host capsular glycan was found to be physiologically insignificant (50% inactivation dose equals 100 mug after 1 h at 37 C). No adsorption (less than 2 x 10(4) PFU/mug) of the viruses to K29 polysaccharide-coated erythroyctes (at 0 or 37 C) was observed either. The phage particles were, however, found to catalyze the hydrolysis of beta-D-glucosido-(1leads to 3)-D-glucuronic acid bonds (arrow) in the receptor polymer, leading, ultimately, to the formation of a mixture of K29 hexasaccharide (one repeating unit), dodecasaccharide, and octadecasaccharide: (see article). Testing derivatives of K29 polysaccharide, as well as 82 heterologous bacterial (mainly Enteriobactericeae) capsular glycans, the viral glycanase was found to be highly specific; in accordance with the host range of phage 29, only one enzymatic cross-reaction (with the Klebsiella K31 polysaccharide) was observed. These and previous results, as well as the electron optical findings of M. E. Bayer and H. Thurow (submitted for publication), are discussed in terms of a unifying mechanism of phage 29-host capsule interaction. We propose that the viruses penetrate the capsules by means of their spike-associated glycanase activity, which leads them along capsular polysaccharide strands to membrane-cell wall adhesions where ejection of the viral genomes occurs.

From Escherichia coli R4, and from some deeper rough mutants of it, the cell wall lipopolysaccharides were isolated and subjected to mild acid hydrolysis. By methylation/g.l.c./m.s. and other analyses of the core oligosaccharides thus obtained, the primary structure of the E. coli R4 core (hexose and heptose region) was elucidated

Two bacteriophages degrading 3-deoxy-D-manno-2-octulosonic acid-(KDO)-containing capsules of Escherichia coli strains were identified. Using modifications of the thiobarbituric acid assay, it was shown that each phage contains a glycan hydrolase activity cleaving one type of ketosidic linkage of KDO. Thus, the enzyme from phage phi 95 catalyzes the hydrolysis of beta-octulofuranosidonic linkages of the K95 glycan; and phi 1092, the alpha-octulopyranosidonic linkages of the K? antigen of E. coli LP1092. No cross-reactivity of the phage enzymes with other KDO-containing capsular polysaccharides was observed.

The lipopolysaccharides of ampicillin-resistant cell-wall-defective mutants of Escherichia coli K-12 were analyzed. From their lipopolysaccharides the respective core oligosaccharides were obtained. Following dephosphorylation, the core oligosaccharides were methylated and analyzed by gas chromatography/mass spectrometry. From core-defective mutants substructures of the K-12 core were obtained. Analysis of the lipopolysaccharide preparations from wild-type K-12 indicated the presence of several core structures with different degrees of completion. The lipopolysaccharide preparation was degraded and the oligosaccharide mixture was partially resolved by gel filtration chromatography. Methylation, gas chromatography and mass spectrometry of the oligosaccharides permitted the tentative formulation of the K-12 core structure. Alternative interpretations for this heterogeneity are discussed.

Using periodate oxidation, methylation analysis, characterization of oligosaccharides by Smith degradation or partial acid hydrolysis, as well as proton magnetic resonance, the primary structure of the Escherichia coli serotype 29 capsular polysaccharide (the receptor of E. coli K phage 29) was reinvestigated. The polymer was found to consist of hexasaccharide repeating units of the following structure: (see article).

The surface glycoprotein (mixture of isoglycoproteins with Mr 69 000 and 71 000) was isolated from the particles of Friend murine leukemia virus, and was successively digested with protease and with endo-beta-N-acetylglucosaminidase from Streptomyces griseus. Roughly 20% (w/w) of the carbohydrates in this glycoprotein were thus released, and they were fractionated by high-performance liquid chromatography after reduction with KB3H4/NaBH4. The radioactive oligosaccharitol fractions obtained were analyzed by exoglycosidase digestion, by acetolysis, and, after permethylation, by fast atom-bombardment mass spectrometry, as well as by capillary gas-liquid chromatography/mass spectrometry following hydrolysis, reduction and peracetylation. Around 85% (mol/mol) of the endo-H-sensitive viral glycans were thus found to be oligomannosidic oligosaccharitols of size classes Man5GlcNAcOH, Man6GlcNAcOH, Man8GlcNAcOH, Man7GlcNAcOH, and Man9GlcNAcOH (in order of prevalence), and the major structural isomers of each size class were identified. About another 15% (mol/mol) of the oligosaccharitols were shown to be of the 'mixed type', comprising mainly four species in which the Man(alpha 1----6)-branch of the Man alpha 1----6 (Man alpha 1----3) Man beta 1----4GlcNAcOH core is substituted by one or two additional alpha-mannoses, while the Man(alpha 1----3)-branch carries an N-acetyllactosamine unit substituted by sialic acid, or by Gal(alpha 1----3).

The Escherichia coli capsule bacteriophages (K phages) described herein are specific for certain capsular strains of E. coli , all of them test strains for different E. coli K antigens. The phages are not adsorbed to the acapsular mutants of their host organisms nor to similar strains with serologically and chemically different capsular polysaccharides. Thirteen E. coli (and one Klebsiella ) K phages were visualized in the electron microscope. Most viruses are similar to P22 and thus belong to Bradley group C; however, one each of group A (long, contractile tail) and group B (long, noncontractile tail) was also found. All K phages were seen to carry spikes but no tail fibers were detected. These results suggest that the structures responsible for the recognition of the thick (about 400 nm or more) capsular polysaccharide gels are located in these spikes.

A capsule depolymerase induced by bacteriophage No. 11 on Klebsiella 390 (03:K11), has been isolated. The material was essentially homogeneous in analytical ultracentrifugation and immunoelectrophoresis. It catalyzes the hydrolysis mainly of β- d -glucopyranosido-(1 → 3)- d -glucuronic acid, but also that of a few (1 in about 20) d -galactopyranosido-(1 → 3)- d -glucose bonds in K11 capsular polysaccharide. The depolymerase consists of dimers of club- or drop-shaped particles of a molecular weight of 155,000 ± 10,000 containing one polypeptide chain of about 62,500 and one of 94,000 dalton. Klebsiella bacteriophage No. 11 is an isometric virus; its head has a diameter of about 450 A. It bears a base plate of about 265 A diameter which has the shape of a six-pointed star with a central hole or prop, carrying two spikes for each of its six points. The following observations support the view that the depolymerase consists of pairs of free spikes of phage 11: (I) The depolymerase monomers and phage spikes in situ have the same dimensions. (II) Polypeptide chains of the same size as the two in the depolymerase also occur in whole virus. (III) The free depolymerase shows approximately the same enzymatic activity as do phage spikes in situ. Purified depolymerase and bacteriophage particles catalyze the hydrolysis of the same bonds in Klebsiella K11 capsular polysaccharide, ultimately forming oligosaccharide fragments of one, two, and three repeating units of the (alkali-treated) polymer. (IV) Phage 11 and the depolymerase strongly cross-react serologically, and anti-depolymerase IgG antibodies specifically adhere to the spike region of whole virus.

Some Escherichia coli K bacteriophage particles, capable of interacting specifically with bacterial polysaccharide capsules, carry an endo-glycosidase activity, probably located in the spikes.

The glycoprotein from Friend murine leukemia virus was digested with protease from Staphylococcus aureus V8. A glycopeptide comprising the N-terminal glycosylation site (Asn-12) was isolated from the mixture of fragments and analyzed by amino acid sequencing and methylation-capillary gas chromatography—mass spectrometry before and after treatment with sialidase from Vibrio cholerae. Asn-12 was thus found to be substituted by a family of partially sialylated, fucosylated, and intersected glycoprotein N-glycans of the hybrid type.

The structures of the oligosaccharides of the hemagglutinin of fowl plague virus [influenza A/FPV/Rostock/34 (H7N1)] have been elucidated by one‐ and two‐dimensional 1H n.m.r. spectroscopy at 500 MHz and by microscale methylation analysis. N‐Glycosidic oligosaccharides of the oligomannosidic (OM) and of the N‐acetyllactosaminic type have been found, the latter type comprising biantennary structures, without (A) or with (E) bisecting N‐acetylglucosamine, and triantennary (C) structures. Analysis of the tryptic and thermolytic glycopeptides of the hemagglutinin allowed the allocation of these oligosaccharides to the individual glycosylation sites. Each attachment site contained a unique set of oligosaccharides. Asn12 contains predominantly structures C and E which are highly fucosylated. Asn28 contains OM and A structures that lack fucose and sulfate. Asn123 shows A that has incomplete antennae but is highly fucosylated and sulfated. Asn149 has fucosylated A and E. Asn231 shows fucosylated A and E with incomplete antennae. Asn406 has OM oligosaccharides. Asn478 has A and E with little fucose. Localization of the oligosaccharides on the three‐dimensional structure of the hemagglutinin revealed that the oligomannosidic glycans are attached to glycosylation sites at which the enzymes responsible for carbohydrate processing do not have proper access. These observations demonstrate that an important structural determinant for the oligosaccharide side chains is the structure of the glycoprotein itself. In addition, evidence was obtained that the rate of glycoprotein synthesis also has an influence on carbohydrate structure.

The lipopolysaccharide of Escherichia coli BB and a number of R-phage selected (e.g. T3, T4) cell-wall-defective mutants were analyzed. From their lipopolysaccharides the respective core oligosaccharides were obtained. Following dephosphorylation, the oligosaccharides were methylated and analyzed by gas chromatography/mass spectrometry. This revealed the sugar sequence in the hexose-heptose region of the core. The linkage of heptose (Hep) to 2-keto-3-deoxyoctonate (KDO) was established as ... Hep 1,5 leads to KDO ... by methylation analysis. The substituted derivative of KDO was identified by gas chromatography and mass spectrometry. The KDO region contains three KDO units. Its structure was elaborated by (a) selective removal and identification of 7-phosphoryl ethanolamine-KDO (KDO-PN), (b) periodate oxidation and thiobarbituric acid reaction in conjunction with mild hydrolysis, (c) a modified methylation analysis. Phosphate substitution of E. coli BB core was studied by beta-elimination and using the information obtained with KDO-PN. The structures of the cell wall lipopolysaccharides from E. coli BB and cell-wall-defective mutants are given.

† Mutants of Escherichia coli B with increasing structural defects in the cell wall lipopolysaccharide (above) were tested for adsorption resistance to bacteriophages T3 and T4, and the lipopolysaccharide extracted from these mutants was assayed for loss of ability to inactivate the viruses. Absence of terminal glucose, or of any of the substituents (branch heptose, phosphate and pyrophosphorylethanolamine) on the two chain heptoses was found to have little effect on T3 and T4 susceptibility. However, all mutants lacking both glucoses were resistant to both phages.

Publisher Summary Glycan glycanohydrolase activities catalyzing the depolymerization of bacterial surface polysaccharides are associated with the tail parts of many bacteriophages. During the infective process, they help the viruses to penetrate the outer layers of their host cells. These bacterioviral hydrolases are highly specific for one or a few structurally related heteroglycans; therefore, they contribute to the restriction of bacteriophage host range. This chapter describes the propagation and purification of Escherichia coli bacteriophage ϕ92,7 the depolymerization of colominic acid, [-α-NeuAc-(2 → 8)-]n, by the virus particles, and the isolation of the oligosialic acid products. The chapter discusses the process of propagation and purification of bacteriophage. There is a discussion on the materials used, bacteriophage stock, large-scale propagation of bacteriophage, and purification of bacteriophage particles. The chapter describes the procedures of depolymerization of colominic acid and isolation of sialic acid oligomers and their principles, substrates, and reagents. The chapter examines how colominic oligosaccharides may also be separated by gel filtrations and how larger sialic acid oligomers are obtained after short periods of incubation with the virus.

The free host capsule depolymerase, induced by Escherichia coli capsule bacteriophage no. 29, and causing the formation of haloes around its plaques, has been purified to homogeneity. As judged from the following facts, this "enzyme" consists of free phage 29 spikes. (i) Detached phage organelles and depolymerase 29 particles exhibit the same molecular weight (about 245,000, as determined from the sedimentation equilibrium), contain polypeptide chains of the same two sizes (57,000 plus or minus 3,000 and 29,500 plus or minus 2,000, as determined by SDS-PAA gel electrophoresis), and have (within experimental error) the same sedimentation coefficient, isoelectric point, and amino acid composition. (ii) Isolated depolymerase and phage spikes in situ both catalyze the hydrolysis of glucosidic bonds in host capsular polysaccharide, leading ultimately to the formation of oligosaccharide fragments of one, two, and three hexasaccharide repeating units. (iii) Depolymerase 29 and phage 29 spikes have roughly the same electron optical dimensions. As tentatively estimated from the total and the virus-associated capsule depolymerase activity in the lysates, phage 29 infection seems to produce eight to seventeen times more free than incorporated spikes.

The 30 O-methylated hexitol and 2-deoxy-2-(N-methyl)acetamidohexitol acetates which commonly may be obtained during methylation analysis of N-glycosidically linked glycoprotein oligosaccharides or their biosynthetic precursors, were subjected to chromatography through glass capillary columns, wall-coated with either Silar 9CP, Dexsil 410, SE-30, or OV 101. All 22 methylhexitol acetates (e.g., 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol and -mannitol) were optimally resolved on the (most polar) Silar column, whereas the 8 aminohexitol derivatives (as well as the corresponding unmethylated hexitol and aminohexitol acetates) were best separated on either Dexsil 410 or OV 101.

A bacteriophage growing on Escherichia coli K13, K20, and K23 strains carries a glycanase that catalyzes the hydrolytic cleavage of the beta-ketopyranosidic linkages of 3-deoxy-D-manno-2-octulosonic acid (KDO) in the respective capsular polysaccharides. The main cleavage product of the K23 polysaccharide has been identified by 1H- and 13C-n.m.r. spectroscopy as beta beta Ribfl----7 beta KDOp2----3-beta Ribfl----7KDO. Cleavage of polysaccharides containing alpha-pyranosidic, or 5-substituted beta-pyranosidic KDO is not catalyzed by the enzyme.

RECENTLY, I. Orskov, F. Orskov, W. J. Sojka and J. M. Leach1 described a new genetical donor strain of Escherichia coli. This strain, G7 (O8 : K87(B) : K88ab(L) : H19), was at first isolated from a case of pig enteritis. Investigations in different countries have shown that this serotype is frequently found in enteric diseases in pigs. A characteristic feature was the simultaneous existence of two K antigens in the same strain. The K87(B) antigen is a polysaccharide as are all hitherto examined B (and A) antigens2. The K88 antigen is thermolabile, that is, its ability to agglutinate and to bind antibody and its capacity to induce antibody formation in rabbits are destroyed by heating to 100° C for 1 h and thus it must be grouped as an L antigen according to established definitions3. Since the first detection of the K88 antigen it has been found in many E. coli strains, mainly from young pigs with intestinal diseases, and in different serological combinations4–6. Furthermore, it has been shown that K88 exists in at least two varieties, one of which is symbolized as K88ab, and the other K88ac (ref. 4). Strain G7 is a genetical donor and can, in conjugation experiments, transfer chromosomal markers to suitable recipients. In addition the genetic determinant of the K88 antigen can be passed to other E. coli serotypes by episomal transfer7.

The K88 antigen was carried by episomal transfer to D282, a nonmotile Escherichia coli strain without K antigen. D520, obtained by this episomal transfer, was used for the extraction of K88 antigen. It was shown by the agar gel precipitation technique that some K88 antigen was released from D520 into suspending aqueous medium. The amount of liberated material was increased by gentle heating (60 C) or treatment in a Waring Blendor. The antigen was obtained from the extracts in a purified form by making use of its insolubility between p H 3.5 and 5.5 and of its high sedimentation rate ( S 0 20,w = 36.7 S ). The homogeneity of the material was demonstrated by agar gel precipitation with D520 antiserum, by analytical ultracentrifugation, and by moving-boundary electrophoresis. Chemical analysis revealed that K88 is a pure protein containing all the common amino acids with the exception of cysteine-cystine. Purified K88 selectively precipitated the K88 antibodies from D520 antiserum and was shown to be immunogenic in rabbits.

A set of five Escherichia coli K phages has been isolated. These phages are adsorbed to and lyse the capsular forms of the host bacteria, whereas their spontaneous, acapsular mutants are not affected. All host strains are heavily encapsulated test strains for E. coli K antigens of the thermostable A type and they readily segregate acapsular mutants. In four of the phage-host systems, all secondary growth obtained was found to be acapsular. When tested for host-range mutants on 38 strains of E. coli and Klebsiella , less than one mutant per 10 5 plaque-forming units was found. No cross-reacting neutralizing antibodies were obtained when rabbits were immunized with the K phages. The latent periods (between 16 and 30 min) and average burst sizes (between 145 and 580) were determined by one-step growth experiments.

Two nonfimbriate strains of Escherichia coli and their K88 + counterparts, obtained by episomal transfer of this antigen, were studied with the electron microscope. Specimens were prepared with the spray drop method and were studied after shadow-casting. Under these conditions, the alteration in surface structure, due to the acquisition of K88 antigen, appeared as a fur of fine filaments, distinctly more flexible than fimbriae. Purified K88 antigen was also studied and found to have the same structure.