Klebsiella phages representing a novel clade of viruses with an unknown DNA modification and biotechnologically interesting enzymes

Abstract

Lytic bacteriophages and phage-encoded endolysins (peptidoglycan hydrolases) provide a source for the development of novel antimicrobial strategies. In the present study, we focus on the closely related (96 % DNA sequence identity) environmental myoviruses vB_KpnM_KP15 (KP15) and vB_KpnM_KP27 (KP27) infecting multidrug-resistant Klebsiella pneumoniae and Klebsiella oxytoca strains. Their genome organisation and evolutionary relationship are compared to Enterobacter phage phiEap-3 and Klebsiella phages Matisse and Miro. Due to the shared and distinct evolutionary history of these phages, we propose to create a new phage genus "Kp15virus" within the Tevenvirinae subfamily. In silico genome analysis reveals two unique putative homing endonucleases of KP27 phage, probably involved in unrevealed mechanism of DNA modification and resistance to restriction digestion, resulting in a broader host spectrum. Additionally, we identified in KP15 and KP27 a complete set of lysis genes, containing holin, antiholin, spanin and endolysin. By turbidimetric assays on permeabilized Gram-negative strains, we verified the ability of the KP27 endolysin to destroy the bacterial peptidoglycan. We confirmed high stability, absence of toxicity on a human epithelial cell line and the enzymatic specificity of endolysin, which was found to possess endopeptidase activity, cleaving the peptide stem between L-alanine and D-glutamic acid.

Keywords: Bacteriophage; DNA modification; Klebsiella spp.; Kp15virus; Thermostable endolysin.

Fig. 1

The graphical comparison of KP15 and KP27 genomes. The outer ring of the ideogram represents phage KP15 genome, while the inner ring shows phage KP27 genome. Differences were marked inside the inner circle, indicating proper name for particular genes. Lines that connect two rings are linking proteins that are homologues or fulfil similar function for both phages. Genes have been grouped according to their predicted function: DNA replication, morphogenesis genes, auxiliary metabolism, structural and additional genes with known function, as well as homing endonuclease genes, present only in KP27 genome (coloured black)

Fig. 2

The similarities of “Kp15virus” and related species of bacteriophages. a Phylogenetic analysis based on large subunit terminase proteins. b Phylogenomic tree calculated using Gegenees 2.2.1 based on pairwise comparisons

Fig. 3

The similarity of YP_007348875 (a) and YP_007348891 (b) homing endonucleases region among the “Kp15virus” representatives

Fig. 4

Specific activity of KP27 endolysin. a Schematic representation of the solubilized muropeptides after muramidase treatment of Escherichia coli murein sacculi. The KP27 endolysin treatment hydrolyzed the peptide bound between l-alanyl-d-glutamate (red arrows). NAM N-acetyl-muramic acid, NAG N-acetyl-glucosamine, l -Ala l-alanine, d -Glu d-glutamic acid, m-DAP mesodiaminopimelic acid, d -Ala d-alanine. b In vitro endopeptidase assay of KP27 endolysin on E. coli sacculi. The numbers represent the order in which the reaction was performed. Prior to the reaction with a second enzyme, the individual enzymatic reaction was heat inactivated. M4 GlcNac-β-(1 → 4)-MurNac-l-Ala-d-Glu-γ-meso-DAP-d-Ala, D44 cross-linked M4. c Mass muropeptide analysis. Experimental MS corresponds to m + z data acquired, while best match corresponds to the theoretical MS value given for each muropeptide. M1 GlcNac-β-(1 → 4)-MurNac-l-Ala (colour figure online)