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Most phages of Gram-negative bacteria hosts encode spanins for disruption of the outer membrane, which is the last step in host lysis. However, bioinformatic analysis indicates that ∼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the outer membrane (OM). Here, we show that the T7-like coliphage phiKT causes an explosive cell lysis associated with spanin activity despite not encoding spanins. A putative lysis cassette cloned from the phiKT late gene region includes the hypothetical novel gene 28 located between the holin and endolysin genes and supports inducible lysis in Escherichia coli K-12. Moreover, induction of an isogenic construct lacking gene 28 resulted in divalent cation-stabilized spherical cells rather than lysis, implicating gp28 in OM disruption. Additionally, gp28 was shown to complement the lysis defect of a spanin-null λ lysogen. Gene 28 encodes a 56-amino acid cationic protein with predicted amphipathic helical structure and is membrane-associated after lysis. Urea and KCl washes did not release gp28 from the particulate, suggesting a strong hydrophobic membrane interaction. Fluorescence microscopy supports membrane localization of the gp28 protein before lysis. The protein gp28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. Synthesized gp28 behaved similarly to LL-37 in standard assays mixing peptide and cells to measure bactericidal and inhibitory effects. Taken together, these results indicate that phiKT gp28 is a phage-encoded cationic antimicrobial peptide that disrupts bacterial outer membranes during host lysis and, thus, establishes a new class of phage lysis proteins, the disruptins. IMPORTANCE We provide evidence that phiKT produces an antimicrobial peptide for outer membrane disruption during lysis. This protein, designated a disruptin, is a new paradigm for phage lysis and has no similarities to other known lysis genes. Although many mechanisms have been proposed for the function of antimicrobial peptides, there is no consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic system to support such studies. Therefore, the phiKT disruptin may represent the first genetically tractable antimicrobial peptide, facilitating mechanistic analyses.

Leviviruses are bacteriophages with small single-stranded RNA genomes consisting of 3-4 genes, one of which (sgl) encodes a protein that induces the host to undergo autolysis and liberate progeny virions. Recent meta-transcriptomic studies have uncovered thousands of leviviral genomes, but most of these lack an annotated sgl, mainly due to the small size, lack of sequence similarity, and embedded nature of these genes. Here, we identify sgl genes in 244 leviviral genomes and functionally characterize them in Escherichia coli. We show that leviviruses readily evolve sgl genes and sometimes have more than one per genome. Moreover, these genes share little to no similarity with each other or to previously known sgl genes, thus representing a rich source for potential protein antibiotics., Leviviruses are phages with ssRNA genomes that encode a protein (Sgl) that induces host autolysis by interfering with bacterial cell wall synthesis. Identification of sgl genes is complicated by their small size and lack of sequence similarity. Here, Chamakura et al. use bioinformatic and experimental approaches to identify sgl genes in 244 leviviral genomes.

As an opportunistic pathogen, Citrobacter freundii is involved in a wide spectrum of nosocomial infections. C. freundii phages may prove useful as therapeutics for treating infections caused by multidrug-resistant C. freundii strains. Here, we report the complete genome sequence of C. freundii siphophage Sazh, which is closely related to Enterobacteria phages T1 and TLS.

Proteus mirabilis is a pathogen that has been linked to nosocomial infections. Studies on phages infecting P. mirabilis may provide therapeutics for infections caused by antibiotic-resistant strains of this pathogen. Here, we announce the complete genome sequence of a P. mirabilis myophage, Mydo, which is distantly related to Escherichia coli phage rv5.

Staphylococcus aureus bacteria, especially the multidrug resistance strains, are responsible for a wide range of clinical infections. Here, we announce the genome sequence of S. aureus podophage Portland, which is closely related to a group of phi29‐like S. aureus podophages, including phages phi44AHJD and phiP68. The exact genome sequence ends of phage Portland were not determined and may be obscured by terminal proteins.

Proteus mirabilis as a nosocomial pathogen is often the cause of urinary tract infections. This announcement describes the complete genome sequence of a P. mirabilis myophage named Myduc. Phage Myduc is related to Enterobacteria phage phiEcoM-GJ1, which belongs to a group of myophages with small genome sizes (52,000 to 56,000 bp) possessing a T7-like RNA polymerase.

Carbapenemase-producing Klebsiella pneumoniae is an important opportunistic pathogen due to its drug resistance. This study reports on the isolation and characterization of a podophage, named Pylas, infecting this bacterium. The complete genome of phage Pylas is described, and it is distantly related to the well-studied phage N4.

Klebsiella pneumoniae is an opportunistic pathogen associated with hospital-acquired infections. This report describes the complete genome of the K. pneumoniae myophage Mulock, which appears to be a temperate myophage distantly related to other Klebsiella myophages in morphogenesis genes and is partially syntenic with the canonical Escherichia phage lambda in genes encoding lambda-like functions.

Carbapenemase-producing Klebsiella pneumoniae poses a significant public health threat due to its resistance to antibiotics. Siphophage Seifer was isolated and characterized as part of an effort to develop phage therapeutics to control this pathogen. This report describes the complete genome sequence of phage Seifer, which is a distant member of the χ-like siphovirus phage cluster.

Klebsiella pneumoniae is an opportunistic pathogen that is the cause of several hospital-acquired infections. Bacteriophages that target this bacterium could be used therapeutically as novel antimicrobial agents. Here, we present the complete genome sequence of the T1-like K. pneumoniae phage Sanco.

Salmonella enterica serovar Typhimurium is a foodborne pathogen that causes gastroenteritis. Due to increases in antibiotic resistance, bacteriophage therapy may be an alternative method for preventing Salmonella foodborne infections. We report here the complete genome sequence of a T5-like phage, Seabear, which was isolated against S. Typhimurium.

Citrobacter freundii, a member of the Enterobacteriaceae family, has been linked to opportunistic infections in neonates and immunocompromised adults. Here, we report the complete genome sequence of a T4-like myophage, Maleficent, which infects C. freundii.

Bacteriophages infecting Salmonella enterica subsp. enterica serovar Enteritidis may be used as biocontrol agents in food products or animals for preventing foodborne diseases caused by this pathogen. The complete genome sequence of phage Seafire, a T5-like siphophage infecting S. Enteritidis, is described in this report.

Citrobacter freundii is a nosocomial opportunistic pathogen that can cause urinary and bloodstream infections. Phage therapies against C. freundii may prove useful in treating infections caused by this ubiquitous bacterium. Here, we report the complete genome of a T4-like myophage, Maroon, that infects C. freundii.

Proteus mirabilis, a Gram-negative bacterium belonging to the family Enterobacteriaceae, is a common cause of urinary tract infections. Phages infecting Proteus mirabilis could be used as therapeutics to treat infections caused by this bacterium. This announcement describes the complete genome sequence of the T5-like P. mirabilis phage Stubb.

Enterotoxigenic Escherichia coli (ETEC) is an opportunistic pathogen that commonly causes foodborne illness. Study of bacteriophages against this pathogen could be useful to develop alternative treatment approaches. Here, we present the complete genome sequence of LL11, a T7-like podophage that infects ETEC.

Phage Sepoy infects Salmonella enterica serovar Heidelberg, a Gram-negative bacterium that causes severe foodborne illnesses. Bacteriophages infecting this pathogen may be used as biocontrol agents for preventing Salmonella foodborne diseases. Here, we present the complete genome sequence of Sepoy, a T5-like siphophage.

Staphylococcus epidermidis is an opportunistic pathogen that commonly colonizes human skin and mucous membranes. We report here the complete genome sequences of three S. epidermidis phages, Quidividi, Terranova, and Twillingate, which are members of the Twort-like group of large myophages infecting Gram-positive hosts.

Klebsiella pneumoniae infection is a serious concern in hospital settings due to the continuing emergence of multidrug-resistant strains. The study of K. pneumoniae phages may help the development of new treatment strategies., Klebsiella pneumoniae infection is a serious concern in hospital settings due to the continuing emergence of multidrug-resistant strains. The study of K. pneumoniae phages may help the development of new treatment strategies. Here, the complete genome sequence of K. pneumoniae phage Patroon, a T3/T7-like phage, is presented.

Bacteriophage Siskin is a member of the χ-like siphovirus phage cluster that infects Salmonella enterica serovar Typhimurium strain LT2. Here, we report the complete 58,476-bp sequence of the Siskin genome, provide confirmation of its genomic termini, and describe a potentially new class of holins and endolysins found in the lysis cassette.

Multiple antimicrobial-resistant strains of Salmonella enterica serovar Newport have been recorded. Study on phages infecting S. Newport may provide new therapeutics or diagnostics for this pathogen. Here, we describe the complete genome sequence of the T4-like phage Melville that uses S. Newport as one of its hosts.

Salmonella enterica serovar Enteritidis is a Gram-negative bacterium and one of the most common foodborne pathogens. Biocontrol using bacteriophage in food products or animals is one possible means by which pathogenic salmonellosis infection could be inhibited. Here, we report the complete genome sequence of the T4-like Salmonella Enteritidis myophage Mooltan.

Spanins are required for the last step in bacteriophage lysis: the disruption of the outer membrane. Bioinformatic analysis has shown that ~15% of phages lack a spanin gene, which suggests an alternate mechanism of outer membrane disruption. To address this, we selected virulent podophage ϕKT as a spaninless exemplar and tested ϕKT genes for outer membrane disruption during lysis. Hypothetical novel gene 28 causes outer membrane disruption when co-expressed with ϕKT lysis genes and complements the lysis defect of a λ spanin mutant. Gp28 is a 56 aa cationic peptide with predicted amphipathic helical structure and is associated with the particulate fraction after lysis. Urea and KCl washes did not release gp28 from the particulate, suggesting a strong hydrophobic interaction with the membrane. Super high-resolution microscopy supports a primarily outer membrane localization for the peptide. Additionally, holin function is not required for gp28-mediated lysis. Gp28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. In standard assays to measure bactericidal and inhibitory effects of antimicrobial peptides on bacterial cells, synthesized gp28 performed equivalently to LL-37. The studies presented here suggest that ϕKT Gp28 disrupts bacterial outer membranes during lysis in a manner akin to antimicrobial peptides.SignificanceHere we provide evidence that ϕKT produces an antimicrobial peptide for outer membrane disruption during lysis. The disruptin is a new paradigm for phage lysis, and has no similarities to other known lysis genes. Many mechanisms have been proposed for the function of antimicrobial peptides, however there is not a consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic selection system to support such studies. Therefore, the ϕKT disruptin may represent the first genetically tractable antimicrobial peptide.

The final step of lysis in phage λ infections of Escherichia coli is mediated by the spanins Rz and Rz1. These proteins form a complex that bridges the cell envelope and that has been proposed to cause fusion of the inner and outer membranes. Accordingly, mutations that block spanin function are found within coiled-coil domains and the proline-rich region, motifs essential in other fusion systems. To gain insight into spanin function, pseudorevertant alleles that restored plaque formation for lysis-defective mutants of Rz and Rz1 were selected. Most second-site suppressors clustered within a coiled-coil domain of Rz near the outer leaflet of the cytoplasmic membrane and were not allele specific. Suppressors largely encoded polar insertions within the hydrophobic core of the coiled-coil interface. Such suppressor changes resulted in decreased proteolytic stability of the Rz double mutants in vivo . Unlike the wild type, in which lysis occurs while the cells retain a rod shape, revertant alleles with second-site suppressor mutations supported lysis events that were preceded by spherical cell formation. This suggests that destabilization of the membrane-proximal coiled coil restores function for defective spanin alleles by increasing the conformational freedom of the complex at the cost of its normal, all-or-nothing functionality. IMPORTANCE Caudovirales encode cell envelope-spanning proteins called spanins, which are thought to fuse the inner and outer membranes during phage lysis. Recent genetic analysis identified the functional domains of the lambda spanins, which are similar to class I viral fusion proteins. While the pre- and postfusion structures of model fusion systems have been well characterized, the intermediate structure(s) formed during the fusion reaction remains elusive. Genetic analysis would be expected to identify functional connections between intermediates. Since most membrane fusion systems are not genetically tractable, only few such investigations have been reported. Here, we report a suppressor analysis of lambda spanin function. To our knowledge this is the first suppression analysis of a class I-like complex and also the first such analysis of a prokaryote membrane fusion system.