Cloning, Expression, and Purification of the K5 Capsular Polysaccharide Lyase (KflA) from Coliphage K5A: Evidence for Two Distinct K5 Lyase Enzymes

<jats:title>ABSTRACT</jats:title> <jats:p> The <jats:italic>Escherichia coli</jats:italic> K5 capsular polysaccharide [-4)-βGlcA-(1,4)-αGlcNAc-(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 ( <jats:italic>kflA</jats:italic> ) was cloned and sequenced. The <jats:italic>kflA</jats:italic> 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 <jats:italic>E. coli</jats:italic> SEBR 3282. There was only limited nucleotide homology between the <jats:italic>kflA</jats:italic> and <jats:italic>elmA</jats:italic> 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 <jats:italic>kflA</jats:italic> was not present on the chromosome of the <jats:italic>E. coli</jats:italic> strains examined. In contrast, <jats:italic>elmA</jats:italic> was present in a subset of <jats:italic>E. coli</jats:italic> strains. Homology was observed between DNA flanking the <jats:italic>kflA</jats:italic> gene of bacteriophage K5A and DNA flanking a small open reading frame (ORF <jats:sub>L</jats:sub> ) located 5′ of the endosialidase gene of the <jats:italic>E. coli</jats:italic> 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 <jats:sub>L</jats:sub> in bacteriophage K1E exhibited homology to the N terminus of KflA from bacteriophage K5A, suggesting that ORF <jats:sub>L</jats:sub> is a truncated remnant of KflA. The presence of this truncated <jats:italic>kflA</jats:italic> gene implies that bacteriophage K1E has evolved from bacteriophage K5A by acquisition of the endosialidase gene and subsequent loss of functional <jats:italic>kflA</jats:italic> . A (His) <jats:sub>6</jats:sub> -KflA fusion protein was overexpressed in <jats:italic>E. coli</jats:italic> 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. </jats:p>