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)-␤GlcA-(1,4)-␣GlcNAc-(1-] is a receptor for the capsulespecific 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 capsulespecific 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.At least 80 distinct capsular polysaccharides (K antigens) have been described previously for Escherichia coli (26). Expression of a polysaccharide capsule on the cell surface confers resistance to host immune defenses and other adverse environmental conditions (3,29). In addition, many of these structurally distinct capsules act as receptors for bacteriophages, and variation of capsular structure may be a mechanism for evasion of bacteriophage infection. Due to their specificity, capsule-specific bacteriophages can be used for identification of numerous E. coli K serotypes. Integral to the tail structure of many capsule-specific bacteriophages are enzymes which degrade the bacterial polysaccharide and provide the bacteriophages access to receptors on the cell surface (5, 32-35).The E. coli K5 capsular polysaccharide is a polymer of -4)-␤GlcA-(1,4)-␣GlcNAc-(1-(40). This structure is identical to N-acetylheparosan, the precursor polymer of heparin and heparan sulfate (17). Coliphage K5A, a bacteriophage specific for E. coli K5 capsule-expressing strains, has be...

The K5 Lyase KflA Combines a Viral Tail Spike Structure with a Bacterial Polysaccharide Lyase Mechanism

“…KflA does not require metal ions for function (28), and no such cofactor was observed in the crystal structure, leading us to conclude the catalytic role of Glu 206 is not the coordination of a metal cofactor. Rather, we propose Glu 206 is involved in direct hydrogen bond formation with the carboxylic group of GlcA substrate components in a role analogous to that of Glu 205 in the syn-␤-elimination catalytic site of heparinase II.…”

“…Strains and Plasmids-The arabinose-inducible plasmid pLYA100 was used for expression, purification, and mutagenesis, as described previously (28), encoding an N-terminally His 6 -tagged variant of full-length K5 lyase (Swiss-Prot accession no. O09496) from coliphage K5A (His-KflA).…”

“…Expression and Purification-His-KflA was expressed as described previously (28). Cells were lysed as described previously (28), but in a modified buffer (50 mM Tris-HCl, pH 8.5, 500 mM NaCl, 10% glycerol, 12.5 mM imidazole) supplemented with 1 mg/ml lysozyme and 0.1% (v/v) protease inhibitor mixture (Sigma, P8849).…”

“…Cells were lysed as described previously (28), but in a modified buffer (50 mM Tris-HCl, pH 8.5, 500 mM NaCl, 10% glycerol, 12.5 mM imidazole) supplemented with 1 mg/ml lysozyme and 0.1% (v/v) protease inhibitor mixture (Sigma, P8849). The lysate was centrifuged (40,000 ϫ g, 10 min, 10°C), and the supernatant was loaded onto a HisTrap column (GE Healthcare).…”

Proteolytic Processing and Oligomerization of Bacteriophage-derived Endosialidases

“…The alignment includes the C-terminal domains of the neck appendage protein gp12 of the Bacillus subtilis phage GA-1 (EMBL accession no. X96987.2), the L-shaped tail fiber protein of coliphage T5 (27), the K5-specific lyases of coliphages K1-5 (20) and K5 (28), and the corresponding K5 lyase of E. coli K5 (29). The protein alignment reveals that the cleavage site identified for endoNE and endoNF (marked with an arrowhead in Fig.…”