title: Cloning, expression, and purification of the K5 capsular polysaccharide lyase (KflA) from coliphage K5A: evidence for two distinct K5 lyase enzymes.
abstract: The Escherichia coli K5 capsular polysaccharide [-4)-betaGlcA-(1, 4)-alphaGlcNAc-(1-] is a receptor for the capsule-specific bacteriophage K5A. Associated with the structure of bacteriophage K5A is a polysaccharide lyase which degrades the K5 capsule to expose the underlying bacterial cell surface. The bacteriophage K5A lyase gene (kflA) was cloned and sequenced. The kflA gene encodes a polypeptide with a predicted molecular mass of 66.9 kDa and which exhibits amino acid homology with ElmA, a K5 polysaccharide lyase encoded on the chromosome of E. coli SEBR 3282. There was only limited nucleotide homology between the kflA and elmA genes, suggesting that these two genes are distinct and either have been derived from separate progenitors or have diverged from a common progenitor for a considerable length of time. Southern blot analysis revealed that kflA was not present on the chromosome of the E. coli strains examined. In contrast, elmA was present in a subset of E. coli strains. Homology was observed between DNA flanking the kflA gene of bacteriophage K5A and DNA flanking a small open reading frame (ORF(L)) located 5' of the endosialidase gene of the E. coli K1 capsule-specific bacteriophage K1E. The DNA homology between these noncoding sequences indicated that bacteriophages K5A and K1E were related. The deduced polypeptide sequence of ORF(L) in bacteriophage K1E exhibited homology to the N terminus of KflA from bacteriophage K5A, suggesting that ORF(L) is a truncated remnant of KflA. The presence of this truncated kflA gene implies that bacteriophage K1E has evolved from bacteriophage K5A by acquisition of the endosialidase gene and subsequent loss of functional kflA. A (His)(6)-KflA fusion protein was overexpressed in E. coli and purified to homogeneity with a yield of 4.8 mg per liter of bacterial culture. The recombinant enzyme was active over a broad pH range and NaCl concentration and was capable of degrading K5 polysaccharide into a low-molecular-weight product.
authors.affiliation: School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.
authors.forename: B R
authors.initials: BR
authors.lastname: Clarke
authors.forename: F
authors.initials: F
authors.lastname: Esumeh
authors.forename: I S
authors.initials: IS
authors.lastname: Roberts
mesh_terms: D000595:Amino Acid Sequence; D016667:Bacterial Capsules; D001483:Base Sequence; D003001:Cloning, Molecular; D003090:Coliphages / Q000201:enzymology* / Q000235:genetics; D004279:DNA, Viral; D004792:Enzyme Precursors / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D006639:Histidine; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000235:genetics; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D011991:Receptors, Virus / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D004279:DNA, Viral; D004792:Enzyme Precursors; D029968:Escherichia coli Proteins; D011135:Polysaccharides, Bacterial; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; C050673:capsular polysaccharide K5; D006639:Histidine; C035355:endo-N-acetylneuraminidase; D009439:Neuraminidase; C411971:KflA protein, E coli; D011133:Polysaccharide-Lyases