ABSTRACT
The infection cycle of phage λ terminates in lysis mediated by three types of lysis proteins, each disrupting a layer in the bacterial envelope: the S105 holin, the R endolysin, and the Rz/Rz1 spanin complex targeting the inner membrane, cell wall or peptidoglycan, and the outer membrane, respectively. Video microscopy has shown that in most infections, lysis occurs as a sudden, explosive event at a cell pole, such that the initial product is a less refractile ghost that retains rod-shaped morphology. Here, we investigate the molecular basis of polar lysis using time-lapse fluorescence microscopy. The results indicate that the holin determines the morphology of lysis by suddenly forming two-dimensional rafts at the poles about 100 s prior to lysis. Given the physiological and biochemical similarities between the lambda holin and other class I holins, dynamic redistribution and sudden concentration may be common features of holins, probably reflecting the fitness advantage of all-or-nothing lysis regulation.IMPORTANCEIn this study, we use fluorescent video microscopy to track -green fluorescent protein (GFP)-labeled holin in the minutes prior to phage lysis. Our work contextualizes prior genetic and biochemical data, showing when hole formation starts and where holin oligomers form in relation to the site of lytic rupture. Furthermore, prior work showed that the morphology of lambda-infected cells is characterized by an explosive event starting at the cell pole; however, the basis for this was not clear. This study shows that holin most often oligomerizes at cell poles and that the site of the oligomerization is spatially correlated with the site of lytic blowout. Therefore, the holin is the key contributor to polar lysis morphology for phage lambda.
Subject(s)
Bacteriophage lambda , Viral Proteins , Viral Proteins/metabolism , Bacteriophage lambda/genetics , Cell Death , Cell Wall/metabolism , BacteriolysisABSTRACT
Most phages of Gram-negative hosts encode spanins for disruption of the outer membrane, the last step in host lysis. However, bioinformatic analysis indicates that â¼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the OM. Here, we show that the T7-like coliphage phiKT causes the 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 E. 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 prior to lysis. Gp28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. Synthesized gp28 behaved similar 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. Significance We provide evidence that phiKT produces an antimicrobial peptide for outer membrane disruption during lysis. This protein, designated as 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.
ABSTRACT
Asymptomatic Salmonella carriage in beef cattle is a food safety concern and the beef feedlot environment and cattle hides are reservoirs of this pathogen. Bacteriophages present an attractive non-antibiotic strategy for control of Salmonella in beef. In this study, four diverse and genetically unrelated Salmonella phages, Sergei, Season12, Sw2, and Munch, were characterized and tested alone and in combination for their ability to control Salmonella in cattle hide and soil systems, which are relevant models for Salmonella control in beef production. Phage Sergei is a member of the genus Sashavirus, phage Season12 was identified as a member of the Chivirus genus, Sw2 was identified as a member of the T5-like Epseptimavirus genus, and Munch was found to be a novel "jumbo" myovirus. Observed pathogen reductions in the model systems ranged from 0.50 to 1.75 log10 CFU/cm2 in hides and from 0.53 to 1.38 log10 CFU/g in soil, with phages Sergei and Sw2 producing greater reductions (â¼1 log10 CFU/cm2 or CFU/g) than Season12 and Munch. These findings are in accordance with previous observations of phage virulence, suggesting the simple ability of a phage to form plaques on a bacterial strain is not a strong indicator of antimicrobial activity, but performance in liquid culture assays provides a better predictor. The antimicrobial efficacies of phage treatments were found to be phage-specific across model systems, implying that a phage capable of achieving bacterial reduction in one model is more likely to perform well in another. Phage combinations did not produce significantly greater efficacy than single phages even after 24 h in the soil model, and phage-insensitive colonies were not isolated from treated samples, suggesting that the emergence of phage resistance was not a major factor limiting efficacy in this system.
ABSTRACT
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.
Subject(s)
Bacteriolysis/genetics , Evolution, Molecular , Genes, Viral/genetics , Levivirus/genetics , Viral Proteins/metabolism , Escherichia coli/virology , Levivirus/pathogenicity , Mutagenesis, Site-Directed , Mutation , RNA, Viral/genetics , Viral Proteins/geneticsABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Salmonella enterica serovar Typhimurium is a Gram-negative pathogen and a primary cause of foodborne illnesses worldwide. Here, we present the complete 47,393-bp genome sequence of the siphophage Skate, which was isolated against S. Typhimurium strain LT2.
ABSTRACT
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.
