Phage K gp102 Drives Temperature-Sensitive Antibacterial Activity on USA300 MRSA.

There is widespread interest in using obligately lytic bacteriophages ("phages") to treat human bacterial infections. Among Staphylococcus aureus infections, the USA300 lineage is a frequent cause of invasive disease. We observed that phage K, a model S. aureus myophage, exhibits temperature-sensitive growth on USA300 strains, with the wild-type phage providing poorer growth suppression in broth and forming smaller and fainter plaques at 37 °C vs. 30 °C. We isolated 65 mutants of phage K that had improved plaquing characteristics at 37 °C when compared to the parental phage. In all 65 mutants, this phenotype was attributable to loss-of-function (LoF) mutations in gp102, which encodes a protein of unknown function that has homologs only among the Herelleviridae (SPO1-like myophages infecting gram-positive bacteria). Additional experiments with representative mutants consistently showed that the temperature-sensitive plaque phenotype was specific to USA300 MRSA strains and that Gp102 disruption was correlated with improved suppression of bacterial growth in broth and improved antibacterial activity in a mouse model of upper respiratory tract infection. The same genotype and in vitro phenotypes could be replicated in close relatives of phage K. Gp102 disruption did not have a detectable effect on adsorption but did delay cell culture lysis relative to wild-type under permissive infection conditions, suggesting that gp102 conservation might be maintained by selective pressure for more rapid replication. Expression of gp102 on a plasmid was toxic to both an MSSA and a USA300 MRSA strain. Molecular modeling predicts a protein with two helix-turn-helix domains that displays some similarity to DNA-binding proteins such as transcription factors. While its function remains unclear, gp102 is a conserved gene that is important to the infection process of Kayvirus phages, and it appears that the manner in which USA300 strains defend against them at 37 °C can be overcome by gp102 LoF mutations.

Complete Genome Sequence of Citrobacter freundii Myophage Maleficent.

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.

Complete Genome Sequence of Citrobacter freundii Myophage Maroon.

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.

Complete Genome Sequence of Salmonella enterica Serovar Typhimurium Siphophage Seabear.

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.

Complete Genome Sequence of Salmonella enterica Serovar Enteritidis Siphophage Seafire.

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.

Complete Genome Sequence of Proteus mirabilis Siphophage Stubb.

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.

Complete Genome Sequence of Enterotoxigenic Escherichia coli Podophage LL11.

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.

Complete Genome Sequence of Salmonella enterica Serovar Typhimurium Siphophage Skate.

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.

Complete Genome Sequence of Staphylococcus aureus Siphophage Lorac.

Staphylococcus aureus is a leading cause of a wide range of clinical infections. Here, we announce the complete genome sequence of S. aureus siphophage Lorac, a phiETA-like temperate phage that is similar at the nucleotide level to the previously described S. aureus prophage phiNM2.

Complete Genome Sequence of Salmonella enterica Serovar Heidelberg Siphophage Sepoy.

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.

Complete Genome Sequence of Salmonella enterica Serovar Typhimurium Siphophage Siskin.

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.

Complete Genome Sequence of Klebsiella pneumoniae Podophage Patroon.

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.

Complete Genome Sequence of Serratia marcescens Siphophage Scapp.

Serratia marcescens is an opportunistic pathogen that typically infects the respiratory and urinary tract, with the majority of cases being hospital acquired. The study of S. marcescens phages may help control drug-resistant S. marcescens strains. In this study, we announce the complete genome sequence and the features of S. marcescens siphophage Scapp.

Complete Genome Sequence of Serratia marcescens Siphophage Serbin.

Serratia marcescens is an opportunistic human pathogen that is known to cause hospital-acquired respiratory and urinary tract infections. Here, we announce the complete genome sequence and the features of S. marcescens phage Serbin.

Phage spanins: diversity, topological dynamics and gene convergence.

BACKGROUND: Spanins are phage lysis proteins required to disrupt the outer membrane. Phages employ either two-component spanins or unimolecular spanins in this final step of Gram-negative host lysis. Two-component spanins like Rz-Rz1 from phage lambda consist of an integral inner membrane protein: i-spanin, and an outer membrane lipoprotein: o-spanin, that form a complex spanning the periplasm. Two-component spanins exist in three different genetic architectures; embedded, overlapped and separated. In contrast, the unimolecular spanins, like gp11 from phage T1, have an N-terminal lipoylation signal sequence and a C-terminal transmembrane domain to account for the topology requirements. Our proposed model for spanin function, for both spanin types, follows a common theme of the outer membrane getting fused with the inner membrane, effecting the release of progeny virions. RESULTS: Here we present a SpaninDataBase which consists of 528 two-component spanins and 58 unimolecular spanins identified in this analysis. Primary analysis revealed significant differences in the secondary structure predictions for the periplasmic domains of the two-component and unimolecular spanin types, as well as within the three different genetic architectures of the two-component spanins. Using a threshold of 40% sequence identity over 40% sequence length, we were able to group the spanins into 143 i-spanin, 125 o-spanin and 13 u-spanin families. More than 40% of these families from each type were singletons, underlining the extreme diversity of this class of lysis proteins. Multiple sequence alignments of periplasmic domains demonstrated conserved secondary structure patterns and domain organization within family members. Furthermore, analysis of families with members from different architecture allowed us to interpret the evolutionary dynamics of spanin gene arrangement. Also, the potential universal role of intermolecular disulfide bonds in two-component spanin function was substantiated through bioinformatic and genetic approaches. Additionally, a novel lipobox motif, AWAC, was identified and experimentally verified. CONCLUSIONS: The findings from this bioinformatic approach gave us instructive insights into spanin function, evolution, domain organization and provide a platform for future spanin annotation, as well as biochemical and genetic experiments. They also establish that spanins, like viral membrane fusion proteins, adopt different strategies to achieve fusion of the inner and outer membranes.

Localization and Regulation of the T1 Unimolecular Spanin.

Spanins are bacteriophage lysis proteins responsible for disruption of the outer membrane, the final step of Gram-negative host lysis. The absence of spanins results in a terminal phenotype of fragile spherical cells. The phage T1 employs a unimolecular spanin gp11 that has an N-terminal lipoylation signal and a C-terminal transmembrane domain. Upon maturation and localization, gp11 ends up as an outer membrane lipoprotein with a C-terminal transmembrane domain embedded in the inner membrane, thus connecting both membranes as a covalent polypeptide chain. Unlike the two-component spanins encoded by most of the other phages, including lambda, the unimolecular spanins have not been studied extensively. In this work, we show that the gp11 mutants lacking either membrane localization signal were nonfunctional and conferred a partially dominant phenotype. Translation from internal start sites within the gp11 coding sequence generated a shorter product which exhibited a negative regulatory effect on gp11 function. Fluorescence spectroscopy time-lapse videos of gp11-GFP expression showed gp11 accumulated in distinct punctate foci, suggesting localized clusters assembled within the peptidoglycan meshwork. In addition, gp11 was shown to mediate lysis in the absence of holin and endolysin function when peptidoglycan density was depleted by starvation for murein precursors. This result indicates that the peptidoglycan is a negative regulator of gp11 function. This supports a model in which gp11 acts by fusing the inner and outer membranes, a mode of action analogous to but mechanistically distinct from that proposed for the two-component spanin systems.IMPORTANCE Spanins have been proposed to fuse the cytoplasmic and outer membranes during phage lysis. Recent work with the lambda spanins Rz-Rz1, which are similar to class I viral fusion proteins, has shed light on the functional domains and requirements for two-component spanin function. Here we report, for the first time, a genetic and biochemical approach to characterize unimolecular spanins, which are structurally and mechanistically different from two-component spanins. Considering similar predicted secondary structures within the ectodomains, unimolecular spanins can be regarded as a prokaryotic version of type II viral membrane fusion proteins. This study not only adds to our understanding of regulation of phage lysis at various levels but also provides a prokaryotic genetically tractable platform for interrogating class II-like membrane fusion proteins.

A New Class of Phantom Materials for Poroelastography Imaging Techniques.

Poroelastography is an elastographic technique used to image the temporal mechanical behavior of tissues. One of the major challenges in determining experimental potentials and limitations of this technique has been the lack of complex and realistic controlled phantoms that could be used to corroborate the limited number of theoretical and simulation studies available in the literature as well as to predict its performance in complex experimental situations and in a variety of conditions. In the study described here, we propose and analyze a new class of phantom materials for temporal elastography imaging. The results indicate that, by using polyacrylamide, we can generate inhomogeneous elastographic phantoms with controlled fluid content and fluid flow properties, while maintaining mechanical and ultrasonic properties similar to those of soft tissues.

Complete Genome Sequence of Bacillus megaterium Siphophage Stills.

Bacillus megaterium is a soil-dwelling bacterium frequently used in research as a model organism and in industry in protein production applications. Bacteriophages may be used to enhance the use of this bacterium. Here, we describe the complete genome of B. megaterium siphophage Stills and its core features.

Membrane fusion during phage lysis.

In general, phages cause lysis of the bacterial host to effect release of the progeny virions. Until recently, it was thought that degradation of the peptidoglycan (PG) was necessary and sufficient for osmotic bursting of the cell. Recently, we have shown that in Gram-negative hosts, phage lysis also requires the disruption of the outer membrane (OM). This is accomplished by spanins, which are phage-encoded proteins that connect the cytoplasmic membrane (inner membrane, IM) and the OM. The mechanism by which the spanins destroy the OM is unknown. Here we show that the spanins of the paradigm coliphage lambda mediate efficient membrane fusion. This supports the notion that the last step of lysis is the fusion of the IM and OM. Moreover, data are provided indicating that spanin-mediated fusion is regulated by the meshwork of the PG, thus coupling fusion to murein degradation by the phage endolysin. Because endolysin function requires the formation of µm-scale holes by the phage holin, the lysis pathway is seen to require dramatic dynamics on the part of the OM and IM, as well as destruction of the PG.

Complete Genome of Clavibacter michiganensis subsp. sepedonicusis Siphophage CN1A.

Clavibacter michiganensis subsp. sepedonicusis is a Gram-positive actinomycete that is the causative agent of the potato disease ring rot. Here, we announce the complete genome sequence of the Clavibacter michiganensis subsp. sepedonicusis siphophage CN1A. CN1A is only the second fully sequenced Clavibacter michiganensis subsp. sepedonicusis phage reported to date. Core and unique features of its genome are described.