Phosphorylation and metabolism of sucrose and its five linkage-isomeric alpha-D-glucosyl-D-fructoses by Klebsiella pneumoniae

Not only sucrose but the five isomeric alpha-D-glucosyl-D-fructoses trehalulose, turanose, maltulose, leucrose, and palatinose are utilized by Klebsiella pneumoniae as energy sources for growth, thereby undergoing phosphorylation by a phosphoenolpyruvate-dependent phosphotransferase system uniformly at 0-6 of the glucosyl moiety. Similarly, maltose, isomaltose, and maltitol, when exposed to these conditions, are phosphorylated regiospecifically at O-6 of their non-reducing glucose portion. The structures of these novel compounds have been established unequivocally by enzymatic analysis, acid hydrolysis, FAB negative-ion spectrometry, and 1H and 13C NMR spectroscopy. In cells of K. pneumoniae, hydrolysis of sucrose 6-phosphate is catalyzed by sucrose 6-phosphate hydrolase from Family 32 of the glycosylhydrolase superfamily. The five 6'-O-phosphorylated alpha-D-glucosyl-fructoses are hydrolyzed by an inducible (approximately 49-50 Kda) phospho-alpha-glucosidase from Family 4 of the glycosylhydrolase superfamily.     

25-Hydroxyvitamin D3 3-sulphate is a major circulating form of vitamin D in man

25-Hydroxyvitamin D3 3 beta-sulphate has been identified in human plasma. The compound was isolated by anion-exchange chromatography and following hydrolysis it was characterized by high-performance liquid chromatography and gas chromatography-mass spectrometry. The mean concentration of sulphated 25-hydroxyvitamin D3 in plasma from 60 patients was 16.7 +/- 7.1 ng/ml and the levels often exceeded those of the corresponding free compound. The study also shows that unconjugated 25-hydroxyvitamin D3 is not readily sulphated by man in vivo.     

The cholecalciferol sulphate system in mammals

7-Dehydrocholesterol sulphate has been identified in human and rat skin. The compound was isolated by anion exchange chromatography and following hydrolysis it was characterized by high-performance liquid chromatography and gas chromatography-mass spectrometry. Experiments with rats showed that 7-dehydrocholesterol sulphate can serve as a precursor of cholecalciferol sulphate and 25-hydroxy-cholecalciferol 3-sulphate, the latter compound being present in significant amounts in human blood. The sulphated sterols identified represent a previously unknown secosteroid system in mammals.     

Benzene from bacterial cleavage of the carbon-phosphorus bond of phenylphosphonates.

An organism, identified as a strain of Klebsiella pneumoniae, was proved by direct assay to utilize ionic methyl phenylphosphonate as sole phosphorus source. One product from C-P-bond cleavage was identified as benzene by combined g.l.c.-mass spectrometry. The molar yield of benzene from the phosphonate was 89%.     

Bile acids. XLIX. Allocholic acid, the major bile acid of Uromastix hardwickii.

Tauroallocholate is the major bile salt of the lizard, Uromastix hardwickii. Alkaline hydrolysis of bile from 25 gallbladders provided 1.21 g of acidic material, about 90% of which was allocholic acid. Analyses by gas-liquid chromatography, and mass spectrometry verified the presence of almost 10% of deoxycholic acid and smaller amounts of other 5alpha and 5beta-bile acids.     

Structural studies of the O-antigen polysaccharides of Klebsiella O5 and Escherichia coli O8

The O-antigen polysaccharides of Klebsiella serotype O5 and Escherichia coli serotype O8 are serologically very similar or identical. The structures of these two polysaccharides have now been re-investigated. N.m.r. spectroscopy, chromium trioxide oxidation, hydrolysis with a specific phage enzyme, and f.a.b. mass spectrometry were the principal methods used. It is concluded that the O-antigen has the following structure, in which D-Man3Me is 3-O-methyl-D-mannose and n is approximately 10. (Formula: see text) Biosynthetic studies indicate that these antigens are synthesised by addition of D-mannopyranosyl groups to the "non-reducing" end of the mannan chain, and it seems possible that addition of a 3-O-methyl-D-mannopyranosyl group involves termination.     

Klebsiella serotype-13 capsular polysaccharide: primary structure and depolymerization by a bacteriophage-borne glycanase

Periodate oxidation and Smith degradation, methylation analysis including uronic acid degradation, partial hydrolysis with acid, bacteriophage degradation, and p.m.r. spectroscopy have been used to elucidate the primary structure of the Klebsiella serotype-13 capsular polysaccharide. The polymer consists of pentasaccharide repeating-units comprising a 4)-beta-D-Manp-(1 leads to 4)-alpha-D-Glcp-(1 leads to 3)-beta-D-Glcp-(1 leads to chain with a 3,4-O-(1-carboxyethylidene)-beta-D-Galp-(1 leads to 4)-alpha-D-GlcAp-(1 leads to branch at position 3 of the mannose. It is shown that there is a glycanase activity associated with particles of Klebsiella bacteriophage No. 13, which catalyses hydrolysis of chain beta-D-Glcp-(1 leads to 4)-beta-D-Manp linkages in the type-13 polysaccharide. The chemical basis of some serological cross-reactions of the Klebsiella K13 antigen is discussed.     

Occurrence of 3-O-( -D-glucopyranosyluronic acid)-D-galactose in the capsular polysaccharide of Klebsiella K-type 20

Partial hydrolysis and methylation studies have been used to show that the capsular polysaccharide of Klebsiella K-type 20 is composed of a main chain of d-mannose and d-galactose units with the aldobiouronic acid attached as a side chain to d-mannose     

Structural analysis of the alpha-D-glucan (EPS35-5) produced by the Lactobacillus reuteri strain 35-5 glucansucrase GTFA enzyme

The neutral exopolysaccharide EPS35-5 (reuteran) produced from sucrose by the glucansucrase GTFA enzyme from Lactobacillus reuteri 35-5 was found to be a (1-->4,1-->6)-alpha-D-glucan, with no repeating units present. Based on linkage analysis and 1D/2D 1H and 13C NMR spectroscopy of intact EPS35-5, as well as MS and NMR analysis of oligosaccharides obtained by partial acid hydrolysis and enzymatic hydrolysis, using pullulanase M1 (Klebsiella planticola), of EPS35-5, a composite model, that includes all identified structural elements, was formulated as follows: [Formula: see text].     

Structure of a phosphoethanolamine-containing O-polysaccharide of Citrobacter freundii strain PCM 1443 from serogroup O39 and its relatedness to the Klebsiella pneumoniae O1 polysaccharide

Lipopolysaccharide was extracted from cells of Citrobacter freundii PCM 1443 from serogroup O39 and degraded by mild acid hydrolysis to give an O-polysaccharide. Based on enzymatic and methylation analyses, along with 1H and 13C nuclear magnetic resonance spectroscopy, it was found that the lipopolysaccharide studied has two different linear polysaccharide chains of d-galactan type containing 3-substituted galactose residues. One of the galactans has the disaccharide repeating units of alpha-D-galactopyranose and beta-D-galactofuranose and the other is comprised of alpha-D-galactopyranose and beta-D-galactopyranose, the latter being substituted in 25% repeats with PEtN at O-6. An immunoblotting assay demonstrated that the lipopolysaccharide of C. freundii PCM 1443 is serologically related to that of Klebsiella pneumoniae O1, which contains the same galactan chains but is devoid of phosphoethanolamine.     

Optimized glucuronide hydrolysis for the detection of psilocin in human urine samples

In order to develop a sensitive and reliable analytical method for psilocin (PC) in urine samples, the hydrolysis conditions including the acid, alkaline and enzymatic hydrolyses have been investigated by monitoring not only PC but also psilocin glucuronide (PCG) by liquid chromatography tandem mass spectrometry (LC-MS-MS); PCG was initially identified in a "magic mushroom (MM)" user's urine by liquid chromatography mass spectrometry (LC-MS) and LC-MS-MS. The proposed conditions optimized for the hydrolysis are as follows: hydrolysis, enzymatic hydrolysis; enzyme, Escherichia coli beta-glucuronidase (5000 units/ml urine); incubation, pH 6 at 37 degrees C for 2h. The complete hydrolysis of PCG in urine was obtained under these conditions, while the enzymatic hydrolyses with three types of beta-glucuronidases originated from bovine liver (Type B-1), Helix pomatia (Type H-1) and Ampullaria provided uncompleted hydrolysis of PCG. Also, neither the acid nor alkaline hydrolysis was found to be applicable. According to the present method, 3.55 microg/ml of psilocin was detected in the "magic mushroom" user's urine after the enzymatic hydrolysis, though psilocin was not detected without hydrolysis.     

Investigating microwave hydrolysis for the traceable quantification of peptide standards using gas chromatography-mass spectrometry

Over the past decade, a number of endogenous peptides and endogenous peptide analogs have been employed in therapeutics and as diagnostic markers. The use of peptides as standards for the absolute quantification of proteins has become commonly accepted. Consequently, the requirement for standard peptides traceable to the International System of Units with low associated measurement uncertainty, and for accurate methods of peptide quantification, has increased. Here we describe a method of peptide quantification involving microwave-assisted acid hydrolysis followed by gas chromatography–mass spectrometry that enables traceable quantification of a peptide by exact matching isotope dilution mass spectrometry where the total hydrolysis time required is only 3 h. A solution of angiotensin I was quantified using this method, and the results were in agreement with those obtained previously using an oven hydrolysis liquid chromatography–tandem mass spectrometry method.     

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)     

Reduction of fensulfothion to fensulfothion sulfide by Klebsiella pneumoniae.

A cell suspension of Klebsiella pneumoniae converted the organophosphorus pesticide fensulfothion to a product that was shown by chemical oxidation, gas-liquid chromatography, infrared spectrophotometry, and mass spectrometry to be fensulfothion sulfide. Further alteration of this metabolite was not noted.     

Reduction of fensulfothion to fensulfothion sulfide by Klebsiella pneumoniae.

A cell suspension of Klebsiella pneumoniae converted the organophosphorus pesticide fensulfothion to a product that was shown by chemical oxidation, gas-liquid chromatography, infrared spectrophotometry, and mass spectrometry to be fensulfothion sulfide. Further alteration of this metabolite was not noted.     

Ammonium hydroxide hydrolysis: a valuable support in the MALDI-TOF mass spectrometry analysis of Lipid A fatty acid distribution

Lipid A is the lipophilic moiety of lipopolysaccharides (LPSs), the major components of the external membrane of almost all gram-negative bacteria. It is responsible for the toxicity of LPS and has a heterogeneous structure composed of a bis-phosphorylated glucosamine disaccharide backbone that is acylated at the positions 2, 3 of the GlcN I (proximal) and GlcN II (distal) residue with O- and N-linked 3-hydroxy fatty acids (primary substitution). These fatty acids are further acylated by means of their 3-hydroxy groups (secondary substitution). The toxicity of Lipid A is dependent on its primary structure; the number, the length, and the distribution of the fatty acids on the disaccharide backbone strongly influence the endotoxic activity. In this paper a general and easy methodology to obtain secondary fatty acid distribution, which is one of the most difficult issues in the structural determination of Lipid A, is proposed. The method combines ammonium hydroxide hydrolysis and matrix assisted laser desorption ionization (MALDI)-mass spectrometry analysis and has been successfully proven with five different Lipid A species. The procedure exploits the lower stability under mild alkaline conditions of acyl and acyloxyacyl esters with respect to that of the acyl and acyloxyacyl amides. The partially degraded Lipid A species obtained are analyzed by MALDI-MS. The generality of this approach was tested on five Lipid As, namely those arising from Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas reactans, and Burkholderia caryophylli.     

Molecular analysis of an aesculinase activity from Klebsiella oxytoca

A DNA fragment containing a Klebsiella oxytoca gene for aesculinase activity was cloned on the multicopy plasmid pBR322. This β-glucosidase activity was confined to aesculin hydrolysis only. It was expressed equally well in Escherichia coli, Salmonella typhimurium and Aeromonas hydrophila. Two polypeptides were found to be encoded within the 2·6 kb of the cloned DNA encoding aesculinase activity.     

Chromosome-encoded beta-lactamases of Citrobacter diversus. Interaction with beta-iodopenicillanate and labelling of the active site.

Both forms of the chromosome-encoded beta-lactamase of Citrobacter diversus react with beta-iodopenicillanate at a rate characteristic of class A beta-lactamases. The active site of form I was labelled with the same reagent. The sequence of the peptide obtained after trypsin hydrolysis is identical with that of a peptide obtained in a similar manner from the chromosome-encoded beta-lactamase of Klebsiella pneumoniae.     

Structure of Saccharomyces cerevisiae mating hormone a-factor. Identification of S-farnesyl cysteine as a structural component

Mating type a cells of the yeast Saccharomyces cerevisiae produce a mating hormone, the a-factor, that we have previously characterized as a very hydrophobic, modified dodecapeptide (Betz, R., Crabb, J. W., Meyer, H. E., Wittig, R., and Duntze, W. (1987) J. Biol. Chem. 262, 546-548). We have investigated the molecular structure in detail using mass spectrometry and proton NMR spectrometry of the intact hormone and authentic component molecules. Tandem mass spectrometry confirms the previously determined peptide sequence of the hormone and shows that it contains additional structural components with masses of 205 and 15 daltons. These were identified by proton NMR and mass spectrometry as a farnesyl (C15H25) residue and a terminal methyl ester group. The farnesyl moiety is attached to the sulfur atom of the carboxyl-terminal cysteine residue, as revealed by NMR of synthetic S-farnesyl cysteine methyl ester. The stereochemical configuration of the farnesyl moiety was determined to be trans,trans by comparison of gas chromatography retention times, mass spectra, and NMR spectra with those of standards. These results define the structure of a-factor as: (Sequence: see text). Replacement of the farnesyl by a methyl group leads to a partial reduction in specific biological activity of the a-factor, whereas hydrolysis of the carboxyl-terminal methyl ester causes a complete loss of activity.     

Structural investigation of the capsular polysaccharide of Klebsiella serotype K35

The structure of the capsular polysaccharide (K antigen) of Klebsiella K35 has been established as having the pentasaccharide repeating unit shown ("four plus one" type). The structural investigation utilized the techniques of methylation, beta-elimination, Smith degradation, and partial hydrolysis. N.m.r. spectroscopy (1H and 13C) was used extensively to establish the configurations of the anomeric linkages and to delineate the sequence of the sugars in the structure of the polysaccharide. (Formula: see text).