Abstract
Pasteurella multocida Type F, the minor fowl cholera pathogen, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported that the capsule was removed by treating microbes with chondroitin AC lyase. We found by acid hydrolysis that the polysaccharide contained galactosamine and glucuronic acid. We molecularly cloned a Type F polysaccharide synthase and characterized its enzymatic activity. The 965-residue enzyme, called P. multocida chondroitin synthase (pmCS), is 87% identical at the nucleotide and the amino acid level to the hyaluronan synthase, pmHAS, from P. multocida Type A. A recombinant Escherichia coli-derived truncated, soluble version of pmCS (residues 1-704) was shown to catalyze the repetitive addition of sugars from UDP-GalNAc and UDP-GlcUA to chondroitin oligosaccharide acceptors in vitro. Other structurally related sugar nucleotide precursors did not substitute in the elongation reaction. Polymer molecules composed of approximately 10(3) sugar residues were produced, as measured by gel filtration chromatography. The polysaccharide synthesized in vitro was sensitive to the action of chondroitin AC lyase but resistant to the action of hyaluronan lyase. This is the first report identifying a glycosyltransferase that forms a polysaccharide composed of chondroitin disaccharide repeats, [beta(1,4)GlcUA-beta(1,3)GalNAc](n). In analogy to known hyaluronan synthases, a single polypeptide species, pmCS, possesses both transferase activities.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF195517
Expression of Recombinant P. multocida Chondroitin Synthase—In our previous studies with pmHAS [33], we found that a functional, soluble enzyme would be created if a portion of the carboxyl terminus was truncated by molecular genetic means
We verify that P. multocida Type F produces a chondroitin or chondroitin-like capsule
Summary
Materials and Pasteurella Strains—Unless otherwise noted, all chemicals were from Sigma or Fisher, and all molecular biology reagents were from Promega. After 40 cycles of PCR (94 °C 30 s; 42 °C 30 s; 72 °C 4 min) with Taq DNA polymerase in the supplied buffer (Fisher), the samples were separated by agarose gel electrophoresis. A portion of the pmCS open reading frame (residues 1–704) in the insert of one of the excised clones, pPmF4A, was amplified by 20 cycles of PCR [16] with Taq polymerase. The resulting PCR product was purified and concentrated using GeneClean This insert was cloned using the pETBlue-1 Acceptor system (Novagen) according to the manufacturer’s instructions. To identify the radiolabeled polymers, portions of some reactions were dialyzed into water (3-kDa cutoff), and the high molecular weight product was digested with various glycolytic enzymes for 7 h at 37 °C. The enzyme concentrations and digestion buffers were: Flavobacterium chondroitin AC lyase, 1 milliunit/l, 50 mM Tris acetate, pH 7.5; Proteus chondroitin AC lyase, 1 milliunit/l, 50 mM Tris acetate, pH 8; Streptomyces HA lyase, 266 milliunits/l, 50 mM sodium acetate, pH 5.4
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