Assay of N-acetylheparosan deacetylase with a capsular polysaccharide from Escherichia coli K5 as substrate

A new substrate for the deacetylase which catalyzes the removal of the N-acetyl groups from N-acetylheparosan in the course of heparin biosynthesis has been prepared. The capsular polysaccharide from Escherichia coli 010:K5:H4, which is structurally identical to N-acetylheparosan, was partially N-deacetylated by hydrazinolysis and was then radioactively labeled by N-acetylation with [3H]acetic anhydride. Upon incubation of the labeled polysaccharide with microsomes from the Furth mastocytoma, [3H]acetyl groups were released, demonstrating that the bacterial polysaccharide was a substrate for the N-deacetylase. Reaction conditions were established which permitted the quantitative assay of N-deacetylase activity; a Km of 74 mg polysaccharide/liter was determined, which corresponds to 2.1 X 10(-4) M, expressed as concentration of uronic acid; Vmax was 3.4 nmol/mg protein/liter. In confirmation of previous results, it was observed (a) that the reaction was stimulated by 3'-phosphoadenylylsulfate (up to a maximum of 45% at a concentration of 0.5 mM), suggesting that N-sulfation occurred which facilitated continued action of the N-deacetylase, and (b) that NaCl and KCl inhibited the enzyme, with 50% reduction of activity at a concentration of 25 mM. In the course of this work, a simple, single-vial assay procedure was used. Released [3H]acetate was extracted from the acidified reaction mixture with a toluene- or xylene-based scintillation fluid containing 10% isoamyl alcohol and measured directly by scintillation spectrometry.     

Neuraminidase associated with coliphage E that specifically depolymerizes the Escherichia coli K1 capsular polysaccharide.

Plaque morphology indicated that the five Escherichia coli K1-specific bacteriophages (A to E) described by Gross et al. (R. J. Gross, T. Cheasty, and B. Rowe, J. Clin. Microbiol. 6:548-550, 1977) encode K1 depolymerase activity that is present in both the bound and free forms. The free form of the enzyme from bacteriophage E was purified 238-fold to apparent homogeneity and in a high yield from ammonium sulfate precipitates of cell lysates by a combination of CsCl density gradient ultracentrifugation, gel filtration, and anion-exchange chromatography. The enzyme complex had an apparent molecular weight of 208,000, as judged from its behavior on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was dissociated by sodium dodecyl sulfate at 100 degrees C to yield two polypeptides with apparent molecular weights of 74,000 and 38,500. Optimum hydrolytic activity was observed at pH 5.5, and activity was strongly inhibited by Ca2+; the Km was 7.41 X 10(-3) M. Rapid hydrolysis of both the O-acetylated and non-O-acetylated forms of the K1 antigen, an alpha 2----8-linked homopolymer of N-acetylneuraminic acid, and of the meningococcus B antigen was observed. Limited hydrolysis of the E. coli K92 antigen, an N-acetylneuraminic acid homopolymer containing alternating alpha 2----8 and alpha 2----9 linkages, occurred, but the enzyme failed to release alpha 2----3-, alpha 2----6-, or alpha 2----9-linked sialic residues from a variety of other substrates.     

Production of capsular polysaccharide from Escherichia coli K4 for biotechnological applications

The production of industrially relevant microbial polysaccharides has recently gained much interest. The capsular polysaccharide of Escherichia coli K4 is almost identical to chondroitin, a commercially valuable biopolymer that is so far obtained from animal tissues entailing complex and expensive extraction procedures. In the present study, the production of capsular polysaccharide by E. coli K4 was investigated taking into consideration a potential industrial application. Strain physiology was first characterized in shake flask experiments to determine the optimal culture conditions for the growth of the microorganism and correlate it to polysaccharide production. Results show that the concentration of carbon source greatly affects polysaccharide production, while the complex nitrogen source is mainly responsible for the build up of biomass. Small-scale batch processes were performed to further evaluate the effect of the initial carbon source concentration and of growth temperatures on polysaccharide production, finally leading to the establishment of the medium to use in following fermentation experiments on a bigger scale. The fed-batch strategy next developed on a 2-L reactor resulted in a maximum cell density of 56 g_cww/L and a titre of capsular polysaccharide equal to 1.4 g/L, approximately ten- and fivefold higher than results obtained in shake flask and 2-L batch experiments, respectively. The release kinetics of K4 polysaccharide into the medium were also explored to gain insight into the mechanisms underlying a complex aspect of the strain physiology.     

IS2-mediated overexpression of kfoC in E. coli K4 increases chondroitin-like capsular polysaccharide production

Transposons are developing molecular tools commonly used for several applications: one of these is the delivery of genes into microorganisms. These mobile genetic elements are characterised by two repeated insertion sequences that flank a sequence encoding one or more orfs for a specific transposase that moves these sequences to other DNA sites. In the present paper, the IS2 transposon of Escherichia coli K4 was modified in vitro by replacing the sequence coding for the transposase with that of the kfoC gene that codes for chondroitin polymerase. KfoC is responsible for the polymerisation of the bacterial capsular polysaccharide whose structure is analogous to that of chondroitin sulphate, a glycosaminoglycan with established and emerging biomedical applications. The recombinant construct was stably integrated into the genome of E. coli K4 by exploiting the transposase from endogenous copies of IS2 in the E. coli chromosome. A significant improvement of the polysaccharide production was observed, resulting in 80 % higher titres in 2.5-L fed-batch cultivations and up to 3.5 g/L in 22-L fed-batch cultures.     

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.     

Activity of CMP-2-keto-3-deoxyoctulosonic acid synthetase in Escherichia coli strains expressing the capsular K5 polysaccharide implication for K5 polysaccharide biosynthesis.

The activity of the cytoplasmic CMP-2-keto-3-deoxyoctulosonic acid synthetase (CMP-KDO synthetase), which is low in Escherichia coli rough strains such as E. coli K-12 and in uncapsulated strains such as E. coli O111, was significantly elevated in encapsulated E. coli O10:K5 and O18:K5. This enzyme activity was even higher in an E. coli clone expressing the K5 capsule. This and the following findings suggest a correlation between elevated CMP-KDO synthetase activity and the biosynthesis of the capsular K5 polysaccharide. (i) Expression of the K5 polysaccharide and elevated CMP-KDO synthetase activity were observed with bacteria grown at 37 degrees C but not with cells grown at 20 degrees C or below. (ii) The recovery kinetics of capsule expression of intact bacteria, in vitro K5 polysaccharide-synthesizing activity of bacteria, and CMP-KDO synthetase activity of bacteria after temperature upshift from 18 to 37 degrees C were the same. (iii) Chemicals which inhibit capsule (polysaccharide) expression also inhibited the elevation of CMP-KDO synthetase activity. The chromosomal location of the gene responsible for the elevation of this enzyme activity was narrowed down to the distal segment of the transport region of the K5 expression genes.     

Analysis of the enzymatic cleavage (beta elimination) of the capsular K5 polysaccharide of Escherichia coli by the K5-specific coliphage: reexamination.

The capsular K5 polysaccharide of Escherichia coli is the receptor of the capsule-specific coliphage K5, which harbors an enzyme that degrades the capsular K5 polysaccharide to a number of oligosaccharides. Analysis of the degradation products using gel permeation chromatography, the periodate-thiobarbituric acid and bicinchoninic acid reactions, and nuclear magnetic resonance spectroscopy showed that the major reaction products are hexa-, octa-, and decasaccharides with 4,5-unsaturated glucuronic acid (delta4,5GlcA) at their nonreducing end. Thus, the bacteriophage enzyme is a K5 polysaccharide lyase and not, as we had reported previously, an endo-N-acetylglucosaminidase.     

Synthesis of the K5 (group II) capsular polysaccharide in transport-deficient recombinant Escherichia coli

Abstract The genes directing the expression of group II capsules in Escherichia coli are organized into three regions. The central region 2 is type specific and thought to determine the synthesis of the respective polysaccharide, whilst the flanking regions 1 and 3 are common to all group II gene clusters and direct the surface expression of the capsular polysaccharide. In this communication we analyze the involvement of region 1 and 3 genes in the synthesis of the capsular KS polysaccharide. Recombinant E. coli strains harboring all KS specific region 2 genes and having various combinations of region 1 and 3 gene were studied using immunoelectron microscopy. Membranes from these bacteria were incubated with UDP[¹⁴C]GlcA and UDPG1cNAc in the absence or presence of KS polysaccharide as an exogenous acceptor. It was found that recombinant strains with only gene region 2 did not produce the K5 polysaccharide. Membranes of such strains did not synthesize the polymer and did not elongate K5 polysaccharide added as an exogenous acceptor. An involvement of genes from region 1 (notably kps C and kps S) and from region 3 (notably kps T) in the K5 polysaccharide synthesis was apparent and is discussed.     

Expression of the Escherichia coli K5 capsular antigen: immunoelectron microscopic and biochemical studies with recombinant E. coli.

The capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three gene regions responsible for polymerization and surface expression have been defined (I. S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1310, 1988). In this report, we describe the immunoelectron microscopic analysis of recombinant bacteria expressing the K5 antigen and of mutants defective in either region 1 or region 3 gene functions, as well as the biochemical analysis of the K5 capsular polysaccharide. Whereas the K5 clone expressed the K5 polysaccharide as a well-developed capsule in about 25% of its population, no capsule was observed in whole mount preparations and ultrathin sections of the expression mutants. Immunogold labeling of sections from the region 3 mutant revealed the capsular K5 polysaccharide in the cytoplasm. With the region 1 mutant, the capsular polysaccharide appeared associated with the cell membrane, and, unlike the region 3 mutant polysaccharide, the capsular polysaccharide could be detected in the periplasm after plasmolysis of the bacteria. Polysaccharides were isolated from the homogenized mutants with cetyltrimethylammonium bromide. The polysaccharide from the region 1 mutant had the same size as that isolated from the capsule of the original K5 clone, and both polysaccharides were substituted with phosphatidic acid. The polysaccharide from the region 3 mutant was smaller and was not substituted with phosphatidic acid. These results prompt us to postulate that gene region 3 products are involved in the translocation of the capsular polysaccharide across the cytoplasmic membrane and that region 1 directs the transport of the lipid-substituted capsular polysaccharide through the periplasm and across the outer membrane.     

Functional homology between the 30S ribosomal protein S7 from E. coli K12 and S7 from E. coli MRE600

Summary It is known that the 30S protein S7 from E. coli strain MRE600 is chemically different from the S7 from E. coli strain K12 (Q13). We have reconstituted 30S subunits using S7 from MRE600 and all other molecular components from K12 and compared the functional activity of the reconstituted particles with those of the particles reconstituted using the S7 from K12. Both reconstituted particles showed the same activity in several functions tested. Since the presence of S7 is essential for the reconstitution of active 30S subunits, we conclude that the S7 from strain K12 is functionally equivalent to the S7 from strain MRE600.This is paper No. 1612 of the Laboratory of Genetics and paper XVIII in the series, Structure and Function of Bacterial Ribosomes. Papers XVI and XVII in this series are Fahnestock, Held, and Nomura (1972) and Fahnestock, Erdmann, and Nomura (1973), respectively.     

Primary structure of the Escherichia coli serotype K42 capsular polysaccharide and its serological identity with the Klebsiella K63 polysaccharide.

The Escherichia coli K42 capsular polysaccharide consists of leads to 3)-alpha-D-Galp-(1 leads to 3)-alpha-D-GalUAp-(1 leads to 3)-alpha-L-Fucp-(1 leads to repeating units. The E. coli K42 and Klebsiella K63 antigens are serologically identical.     

Structure of the Escherichia coli 0104 polysaccharide and its identity with the capsular K9 polysaccharide

Spontaneous mutants of Rhizobium leguminosarum biovar viciae strain C1204b were selected for their ability to tolerate 0.2 M NaCl, a growth-inhibiting level of salt for the parental strain. Transposon-mediated salt-sensitive mutants of strain C1204b were screened for their inability to grow in 0.08 M NaCl. Quantitation of the free-amino acid pools in the mutants grown in NaCl revealed a dramatic increase in glutamine, serine, glutamate and proline, and to a lesser extent alanine and glycine in the salt-tolerant mutants in comparison with the parental strain exposed to NaCl; but only glutamate and proline increased in the salt-sensitive mutants under NaCl stress. Extracellular polysaccharide levels were quantitated for the salt-tolerant mutants and determined to be approximately two-fold higher than for the parental strain. Although the mutations that occurred in the NaCl-tolerant and NaCl-sensitive strains did not interfere with nodule formation, no nitrogenase activity could be observed in the NaCl tolerant mutants as evaluated by acetylene reduction.     

Structure of the K95 antigen from Escherichia coli O75:K95:H5, a capsular polysaccharide containing furanosidic KDO-residues

The structure of the K95 antigenic capsular polysaccharide (K95 antigen) of Escherichia coli O75:K95:H5 was elucidated by determination of the composition, 1H- and 13C-n.m.r. spectroscopy, periodate oxidation, and methylation analysis. The K95 polysaccharide, which contains furanosidic 3-deoxy-D-manno-2-octulosonic acid (KDOf) residues, consists of----3)-beta-D-Rib-(1----8)-KDOf-(2----repeating units, has a molecular weight of approximately 25,000 (approximately 65 repeating units), and is randomly O-acetylated (1 acetyl group per repeating unit at unknown positions).     

Semisynthetic fluorohydrolases prepared by chemical modification of ribonuclease.

A process for conformational modification of protein, which we have previously reported, was investigated as a means of generating fluorohydrolase activity in bovine ribonuclease (RNase). The resulting modified RNase had catalytic activity that depended upon the chosen modifier. Bovine pancreatic ribonuclease, modified by addition of hexamethylphosphoramide (HMPA) at pH 3, was derivatized with diimidates of chain lengths from C1 to C8. The derivative with the highest activity was obtained when RNase was crosslinked with dimethyl pimelimidate (C5). This derivative, which was active over a pH range of 6.5 to 8.0 with an optimum pH of 7.4, hydrolyzed phenylmethylsulfonylfluoride (PMSF) and the potent acetylcholinesterase inhibitor, diisopropyl phosphorofluoridate (DFP). The mean fluorohydrolase activity for four preparations using dimethyl pimelimidate was 0.8 +/- 0.2 U mg-1. Gel filtration on G-75 Sephadex and SDS-polyacrylamide gel electrophoresis showed components having a molecular weight of 13,000 and 27,000, with activity restricted to the 27,000 molecular weight fraction. After gel filtration, the specific activity was 9.1 +/- 2.4 U mg-1, resulting in a molecular activity of 125 min-1. The mechanism of this unique transformation of RNase into a fluorohydrolase is not known, nor has the location of the active site been determined.