Characterization of extended-host-range pseudo-T-even bacteriophage Kpp95 isolated on Klebsiella pneumoniae

Abstract

Kpp95, isolated on Klebsiella pneumoniae, is a bacteriophage with the morphology of T4-type phages and is capable of rapid lysis of host cells. Its double-stranded genomic DNA (ca. 175 kb, estimated by pulsed-field gel electrophoresis) can be cut only by restriction endonucleases with a cleavage site flanked either by A and T or by T, as tested, suggesting that it contains the modified derivative(s) of G and/or C. Over 26 protein bands were visualized upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the virion proteins. N-terminal sequencing indicated that the most abundant band (46 kDa) is the major coat protein (gp23) which has been cleaved from a signal peptide likely with a length similar to that of T4. Phylogenetic analyses based on the sequences of the central region (263 amino acid residues) of gp23 and the full length of gp18 and gp19 placed Kpp95 among the pseudo-T-even subgroup, most closely related to the coliphage JS98. In addition to being able to lyse many extended-spectrum beta-lactamase strains of K. pneumoniae, Kpp95 can lyse Klebsiella oxytoca, Enterobacter agglomerans, and Serratia marcescens cells. Thus, Kpp95 deserves further studies for development as a component of a therapeutic cocktail, owing to its high efficiencies of host lysis plus extended host range.

FIG. 1.

Electron micrograph of bacteriophage Kpp95. The sample was stained with 2% uranyl acetate.

FIG. 2.

One-step growth curve of bacteriophage Kpp95 on K. pneumoniae strain 10693 in chloroform-treated culture (○) and in untreated cultures (•).

FIG. 3.

PFGE of the Kpp95 genome. Electrophoresis was performed with 1% agarose gel at 14°C and 200 V, with an initial time of 1 s, a final time of 40 s, and a running time of 20 h. Lane M contains molecular size markers (Lambda DNA ladder).

FIG. 4.

SDS-PAGE of Kpp95 coat proteins. Lane M contains broad-range protein molecular mass markers.

FIG. 5.

Phylogenetic trees based on the aa sequence of gp23 (A), gp18 (B), and gp19 (C) from T4-type phages. The numbers at branching points are bootstrap values based on 1,000 replicates. Parentheses contain the GenBank accession numbers, followed by the lengths (aa) of the protein/protein segment (except that each of the gp23 segments was 263 aa in length) and then identity/similarity. The sequences include (i) T-even phages T4, T6, RB32, and RB69 infecting E. coli; (ii) pseudo-T-even phages 25 and 31 infecting Aeromonas salmonicida, phage 42 of Burkholderia cepacia, and coliphages AR1, JS98, RB42, RB43, RB49, and SV14; and (iii) schizo-T-even phage Aeh1 of Aeromonas hydrophila, KVP40 of Vibrio cholerae, and nt1 of Vibrio natriegens.