Figure 3

Capsule depolymerase expression of phages K5-2 and K5-4 and enzymatic activity of their purified capsule depolymerases. (A) Purity of recombinant K5-2 ORF37, K5-4 ORF37 and K5-4 ORF38 proteins. The purified K5-2 ORF37, K5-4 ORF37 and K5-4 ORF38 proteins were separated on SDS-PAGE with Coomassie blue staining, and the molecular weights were indicated beside the protein markers. Immunoblots showing the purified recombinant proteins were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and detected using a mouse anti-His antibody (1:5000) and a rabbit anti-mouse IgG-HRP (1:10,000). (B) Activity of purified K5-2 ORF37, K5-4 ORF37 and K5-4 ORF38 proteins among different capsular types in K30, K69, K8 and K5 Klebsiella strains. Five K30 strains (ref K30, DM3, DM23, Co39 and Co45 and two K69 strains (ref K69 and Co94) were spotted with K5-2 ORF37 protein (10–700 ng). Three K8 strains (ref K8, C0413 and C0523) and five K5 strains (ref K5, K44, K9534, C0522 and C0525) were spotted with K5-4 ORF37 protein (10–125 ng) and K5-4 ORF38 protein (10–1500 ng). (C) Alcian blue staining of CPS treated with K5, K8 and K30/K69 capsule depolymerases. Polysaccharide extracts from the reference K5, K8, K30 and K69 strains were treated with 5 μg K5dep, K8dep and K30/K69dep, respectively, and stained with Alcian blue.