Molecular structure for microbial polysaccharides: conformation of the Klebsiella serotype K9 capsular polysaccharide

Fibre type X-ray diffraction patterns have been obtained from oriented, semicrystalline films prepared from the sodium salt form of the bacterial capsular polysaccharide of Klebsiella serotype K9. The molecule has a pentasaccharide repeating sequence, with four neutral residues in the backbone and a glucoronic acid side chain. A novel feature of the molecule is the incorporation of α-l-rhamnose residues, one 1,2 linked and two 1,3 linked in the backbone. Analysis of the X-ray diffraction results indicate an extended three-fold helical conformation with an axially projected chemical repeat of 1.377 nm. Both left and right handed helices have been examined using linked atom least squares techniques to optimize the stereochemistry while simultaneously meeting the observed helical parameters.     

Structure of the serine-containing capsular poly-saccharide K40 antigen from Escherichia coli O8:K40:H9

The structure of the K40 antigenic capsular polysaccharide (K40 antigen) of E. coli O8:K40:H9 was elucidated by determination of the composition, ¹H- and ¹³C-n.m.r. spectroscopy, periodate oxidation and Smith degradation, and methylation analysis. The K40 polysaccharide consists of [(O-β- -glucopyranosyluronic acid)-(1→4)-O-(2-acetamido-2-deoxy-- -glucopyranosyl)-(1→6)-O-(2-acetamido-2-deoxy-- -glucopyranosyl)-(1→4)] repeating units. All of the glucuronic acid residues are substituted amidically with -serine.     

The structure of Klebsiella serotype 11 capsular polysaccharide

Using periodate oxidation, methylation analysis, the characterization of oligosaccharides obtained by partial acid hydrolysis, p.m.r. spectroscopy, and analytical ultracentrifugation, the structure of the (mildly alkali-treated) Klebsiella serotype 11 capsular polysaccharide has been elucidated. The tetrasaccharide repeating-unit comprises the sequence ?3)-β-D-Glcp-(1?3)-β-D-GlcUAp-(1?3)-α-D-Galp-(1→ with a 4,6-O-(1-car?yethylidene)-α-D-galactosyl residue linked to O-4 of the glucuronic acid residue. The structural basis for some serological cross-reactions of the Klebsiella K11 antigen is discussed, and it is shown that rabbit antisera against the Klebsiella K11 test-strain predominantly contain K agglutinins specific for branch-terminal 4,6-O-(1-car?yethylidene)-D-galactose.     

Primary structure of the Escherichia coli serotype K30 capsular polysaccharide.

Methylation, 1H nuclear magnetic resonance, and bacteriophage degradation results indicate that the Escherichia coli serotype K30 capsular polysaccharide consists of leads to 2)-alpha-D-Manp-(1 leads to 3)-beta-D-Galp-(1 leads to chains carrying beta-D-GlcUAp-(1 leads to 3)-alpha-D-Galp-(1 leads to branches at position 3 of the mannoses.     

Primary structure of the Klebsiella serotype 16 capsular polysaccharide

The primary structure of the Klebsiella serotype 16 capsular polysaccharide consists of tetrasaccharide repeating-units comprising a/ar3)-α-D-Glcp-(1/ar4)β-D-GlcAp-(1/ar4)-α-L-Fucp-(1/ar chain with a β-D-Galp-(1→ branch at position 4 of the D-glucosyl residue.     

Studies of the primary structure of the capsular polysaccharide from Klebsiella serotype K15

The capsular polysaccharide of Klebsiella serotype K15 has been investigated mainly by methylation analysis, characterisation of the oligosaccharides obtained by partial acid hydrolysis, periodate oxidation, enzymic degradation, and 1H- and 13C-n.m.r. spectroscopy, and shown to have the hexasaccharide repeating-unit 1. The glycan does not contain any pyruvic acetal or O-acetyl substituents. (formula; see text)     

Structural determination of the O-antigenic polysaccharide from Escherichia coli O74

The structure of the O-antigen polysaccharide (PS) from Escherichia coli O74 has been determined. Component analysis, together with ¹H and ¹³C NMR spectroscopy as well as ¹H,¹⁵N-HSQC experiments were employed to elucidate the structure. Inter-residue correlations were determined by ¹H,¹H-NOESY and ¹H,¹³C-heteronuclear multiple-bond correlation experiments. The PS is composed of tetrasaccharide repeating units with the following structure:

Full-size image (5K)

Cross-peaks of low intensity from an α-linked N-acetylglucosamine residue were present in the NMR spectra, and spectral analysis indicates that they originate from the penultimate residue in the polysaccharide. Consequently, the biological repeating unit has a 3-substituted N-acetyl-d-glucosamine residue at its reducing end. The ¹H, ¹³C and ¹⁵N NMR chemical shifts of the α- and β-anomeric forms of d-Fucp3NAc are also reported. The repeating unit of the E. coli O74 O-antigen is identical to that of the capsular polysaccharide from E. coli K45.     

Structure of the capsular polysaccharide of Klebsiella serotype K79

The structure of the capsular polysaccharide from Klebsiella K79 was determined by the techniques of methylation, periodate oxidation, beta-elimination, chromic acid oxidation, and partial hydrolysis. N.m.r. spectroscopy (1H and 13C) was used extensively to establish the nature of the anomeric linkages of the polysaccharide and of oligosaccharides derived through degradative procedures. The polysaccharide was found to have the heptasaccharide, "5 + 2" repeating unit: (Formula: see text).     

Structure of the capsular polysaccharide of Klebsiella serotype K40

The capsular polysaccharide from Klebsiella Serotype K40 contains D-galactose, D-mannose, L-rhamnose, and D-glucuronic acid in the ratios of 4:1:1:1. Methylation analysis of the native and carboxyl-reduced polysaccharide provided information about the glycosidic linkages in the repeating unit. Degradation of the permethylated polymer with base established the identity of the sugar unit preceding the glycosyluronic acid residue. The modes of linkages of different sugar residues were further confirmed by Smith degradation and partial hydrolysis of the K40 polysaccharide. The anomeric configurations of the different sugar residues were determined by oxidation of the peracetylated native and carboxyl-reduced polysaccharide with chromium trioxide. Based on all of these results, the heptasaccharide structure 1 was assigned to the repeating unit of the K40 polysaccharide. (Formula: see text)     

Structural investigation of the capsular polysaccharide of klebsiella serotype K12

Klebsiella K12 capsular polysaccharide has been investigated by the techniques of methylation, Smith degradation—periodate oxidation, uronic acid degradation, and partial hydrolysis, in conjunction with ¹H-n.m.r. spectroscopy at 100 and 220 MHz, and ¹³C-n.m.r. spectroscopy at 20 MHz. The structure has been found to consist of the hexasaccharide repeating-unit shown, having a d-galactofuranosyl residue at the branch point. In this series, a d-galactofuranosyl residue has previously only been found in the polysaccharide from Klebsiella K41.

     

Molecular cloning of the fnr gene of Escherichia coli K12

Summary Mutations in the fnr gene of Escherichia coli have pleiotropic effects leading to deficiencies in the reduction of fumarate and nitrate, hydrogen production and the ability to grow anaerobically with fumarate or nitrate as terminal electron acceptors. Transducing phages (fnr) carrying the wild-type fnr gene were isolated from populations of artificially-constructed recombinant lambda phages by their ability to complement the lesions of fnr mutants. The fnr phages restored anaerobic growth with fumarate and nitrate as electron acceptors and, as prophages, they promoted normal synthesis of fumarate reductase, nitrate reductase and hydrogenase in fnr mutants. Five independently-isolated fnr phages each contained a R.HindIII fragment (11.5 kilobases) that possessed three internal R.EcoRI targets and had inserted with the same orientation relative to the phage. A physical map of the fnr region was constructed by restriction analysis and flanking fragments were identified by DNA:DNA hybridization.     

Structural investigation of the capsular polysaccharide of Klebsiella serotype K80

The use of methylation, periodate oxidation, β-elimination, and selective hydrolysis enabled the structure of the K80 polysaccharide to be determined. The nature of the anomeric linkages was established by ¹H-n.m.r. spectroscopy, and confirmed by the results of oxidation of the fully acetylated polysaccharide with chromic acid. The K80 polysaccharide is comprised of repeating units of the pentasaccharide shown, and contains a pyruvic acetal on each repeating unit. This pattern constitutes the first instance, in this series of polysaccharides, of a pyruvic acetal attached to a side-chain rhamnosyl group.

     

Structural investigation of the capsular polysaccharide of klebsiella serotype K26

The structure of the capsular polysaccharide from Klebsiella K26 has been determined by using the techniques of methylation, periodate oxidation, partial hydrolysis, and β-elimination. N.m.r. spectroscopy (¹H and ¹³C) was used to establish the nature of the anomeric linkages and to identify oligosaccharides obtained by the different degradative techniques employed.The polysaccharide is comprised of repeating units of the heptasaccharide shown.

     

Structural investigation of Klebsiella serotype K10 capsular polysaccharide

The primary structure of Klebsiella serotype K10 capsular polysaccharide has been investigated using mainly the techniques of methylation, partial hydrolysis, and 1H and 13C NMR spectroscopy. The polysaccharide was found to consist of hexasaccharide repeating units having the following structure: (formula; see text)     

Morphogenesis of Escherichia coli

Morphogenesis of the rod-shaped Escherichia coli is determined by controlled growth of an exoskeleton made of murein (peptidoglycan). Recent insights in the growth strategy of the stress-bearing murein sacculus has contributed to our understanding of how the required concerted action of murein polymerizing and hydrolyzing enzymes is achieved. The proteins involved are coordinated by the formation of multienzyme complexes. In this review, we summarize the recent results on murein structure and metabolism. On the basis of these findings, we present a model that explains maintenance of the specific rod shape of E. coli.     

Curing of Escherichia coli K12 plasmids by coumermycin

Summary Low concentrations of the antibiotic coumermycin A₁, the inhibitor of bacterial DNA gyrase, effectively eliminate pBR322, pMB9 and other ColE1 related plasmids from E. coli K12 strains. The curing action of antibiotic seems to result from the plasmid degradation and not just from the inhibition of replication.     

On the mechanism of conjugation in Escherichia coli K 12

Summary The phenomenon of conjugation consists of many stages. The most important are: the formation of contacts between mating cells, the transfer of DNA from the donor to the recipient, and the integration of the transfered DNA fragments into the chromosome of the recipient. Only after completion of all these stages are recombinants formed. With the aid of specific inhibitiors (nalidixic acid, FUDR), thymine starvation, and use of special thermosensitive mutants it is possible to study the role of DNA synthesis during every stage of conjugation. It was demonstrated that the genetic transfer is due to semiconservative DNA-replication in the donor cell. The fragments of DNA transfered are synthesized in the period of mating by a special replication system (F-replicon). In case of T _DNA ^S mutants unable to grow at 41°, the ability to synthesize DNA during conjugation is preserved.The inhibition of the DNA synthesis in the donor cell by poisons leads to complete inhibition of genetic transfer. The third stage — formation of recombinants requires DNA synthesis in the recipient cell and is inhibited by poisoning, thymine starvation or T _DNA ^S mutations in the recipient. In cases where recombination is not involved (i.e. sexduction) the inhibition of DNA synthesis in the recipient has no significant effect.     

Temperature dependent survival of UV-irradiated Escherichia coli K12

Summary We have found that several excision deficient derivatives of Escherichia coli K12 survive better after UV irradiation if incubated at 42°C than if incubated at 30°C. The highest survival was observed when incubation at 42°C followed UV irradiation and was maintained for at least 16 h. Our results indicate that this temperature dependent resistance (TDR) requires a functional recA gene, but not uvr A, uvrB, recF, or recB genes, or the recA441 (tif-1) mutation which allows thermoinduction of the recA-lexA regulon. Our data are consistent with the idea that the increase in survival observed at 42°C reflects enhanced daughterstrand gap repair by DNA strand exchange. Although the conditions used to elicit TDR can induce heat shock proteins and thermotolerance in E. coli, the relationship between the two responses remains to be elucidated.