Protein-sharing network of KBNP1315.

<p>A network for KBNP1315 was produced using Cytoscape 3.1.2. Nodes represent phages, while edges indicate significant relationships between nodes. Each node is colored in accordance with its ICTV classification.</p

Complete Genomic and Lysis-Cassette Characterization of the Novel Phage, KBNP1315, which Infects Avian Pathogenic <i>Escherichia coli</i> (APEC)

<div><p>Avian pathogenic <i>Escherichia coli</i> (APEC) is a major pathogen that causes avian colibacillosis and is associated with severe economic losses in the chicken-farming industry. Here, bacteriophage KBNP1315, infecting APEC strain KBP1315, was genomically and functionally characterized. The evolutionary relationships of KBNP1315 were analyzed at the genomic level using gene (protein)-sharing networks, the Markov clustering (MCL) algorithm, and comparative genomics. Our network analysis showed that KBNP1315 was connected to 30 members of the <i>Autographivirinae</i> subfamily, which comprises the SP6-, T7-, P60-, phiKMV-, GAP227- and KP34-related groups. Network decomposition suggested that KBNP1315 belongs to the SP6-like phages, but our comparison of putative encoded proteins revealed that key proteins of KBNP1315, including the tail spike protein and endolysin, had relative low levels of amino acid sequence similarity with other members of the SP6-like phages. Thus KBNP1315 may only be distantly related to the SP6-like phages, and (based on the difference in endolysin) its lysis mechanism may differ from theirs. To characterize the lytic functions of the holin and endolysin proteins from KBNP1315, we expressed these proteins individually or simultaneously in <i>E</i>. <i>coli</i> BL21 (DE3) competent cell. Interestingly, the expressed endolysin was secreted into the periplasm and caused a high degree of host cell lysis that was dose-dependently delayed/blocked by NaN₃-mediated inhibition of the SecA pathway. The expressed holin triggered only a moderate inhibition of cell growth, whereas coexpression of holin and endolysin enhanced the lytic effect of endolysin. Together, these results revealed that KBNP1315 appears to use a pin-holin/signal-arrest-release (SAR) endolysin pathway to trigger host cell lysis.</p></div

<i>Autographivirinae</i> subfamily phages are represented as an independent network.

<p>Each node is illustrated as a pie chart, with the wedges representing the proportion of edges connected with nodes belonging to each cluster. The left panel shows the clusters in different colors. The lower panel represents the membership matrix. The rows indicate phages related to <i>Autographivirinae</i> subfamily and the columns represent the clusters that share wedges with the nodes more than 0.01. Shared boxes indicate the clusters into which the phages were assigned by the MCL algorithm.</p

Morphology of bacteriophage KBNP1315.

<p>Transmission electron micrograph (TEM) of purified phage particles negatively stained with 2% uranyl acetate.</p

Comparative genome analysis among KBNP1315 and select relatives (KBNP1315, SP6, K1E, K1-5, and ACG-c91).

<p>Each ORF is represented by a color assigned according to the protein function. The degree of amino acid similarity between paired ORFs was analyzed by BLASTp search, and the percentage of similarity is represented in color from pale gray to dark gray.</p

Growth curves of <i>E</i>.<i>coli</i> BL21 cells expressing two putative lysis proteins of KBNP1315.

<p>(A) ORF 51 (putative endolysin) was expressed in <i>E</i>.<i>coli</i> BL21 in the presence of different concentrations of NaN₃. IPTG (1 mM) was added at 90 min (arrow) in the induction group (open symbols). The non-induction group is represented with closed symbols. NaN₃ was simultaneously applied at concentrations of 0 mM (circle), 0.1 mM (triangle), 0.5 mM (square), and 1 mM (diamond). (B) Growth curves of induction (open symbols) and non-induction (closed symbols) groups expressing ORF46/pDEST42 (circle), ORF51/pDEST42 (triangle), and ORF46/ORF51/pDEST42 (square) are shown. Induction was performed by the addition of 0.3 mM IPTG (arrow).</p