Rapid de novo evolution of lysis genes in single-stranded RNA phages.

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.

Single-gene lysis in the metagenomic era.

The small lytic phages (Microviridae and Leviviridae), effect bacterial lysis with the product of a single gene. The three well-studied single-gene lysis (Sgl) proteins (E of φX174, A2 of Qß, and LysM of phage M) lack direct muralytic activity, and have been shown to function as 'protein antibiotics' by acting as noncompetitive inhibitors of conserved peptidoglycan (PG) biosynthesis enzymes, MurA, MraY, and MurJ respectively. The fourth, protein L of MS2, does not inhibit PG biosynthesis but instead is hypothesized to trigger host autolytic response through an unknown mechanism. Recent advances in meta-omics approaches have led to an explosion in the available genomes of small lytic phages. Of the thousands of new genomes, only one annotated Sgl shared some sequence similarity with a known Sgl (L of MS2), highlighting the diversity in Sgls. The newly available genomic space serves as an untapped resource for discovering novel Sgls.

A viral protein antibiotic inhibits lipid II flippase activity.

For bacteriophage infections, the cell walls of bacteria, consisting of a single highly polymeric molecule of peptidoglycan (PG), pose a major problem for the release of progeny virions. Phage lysis proteins that overcome this barrier can point the way to new antibacterial strategies 1 , especially small lytic single-stranded DNA (the microviruses) and RNA phages (the leviviruses) that effect host lysis using a single non-enzymatic protein 2 . Previously, the A2 protein of levivirus Qß and the E protein of the microvirus ϕX174 were shown to be 'protein antibiotics' that inhibit the MurA and MraY steps of the PG synthesis pathway 2-4 . Here, we investigated the mechanism of action of an unrelated lysis protein, LysM, of the Escherichia coli levivirus M 5 . We show that LysM inhibits the translocation of the final lipid-linked PG precursor called lipid II across the cytoplasmic membrane by interfering with the activity of MurJ. The finding that LysM inhibits a distinct step in the PG synthesis pathway from the A2 and E proteins indicates that small phages, particularly the single-stranded RNA (ssRNA) leviviruses, have a previously unappreciated capacity for evolving novel inhibitors of PG biogenesis despite their limited coding potential.

Mutational analysis of the MS2 lysis protein L.

Small single-stranded nucleic acid phages effect lysis by expressing a single protein, the amurin, lacking muralytic enzymatic activity. Three amurins have been shown to act like 'protein antibiotics' by inhibiting cell-wall biosynthesis. However, the L lysis protein of the canonical ssRNA phage MS2, a 75 aa polypeptide, causes lysis by an unknown mechanism without affecting net peptidoglycan synthesis. To identify residues important for lytic function, randomly mutagenized alleles of L were generated, cloned into an inducible plasmid and the transformants were selected on agar containing the inducer. From a total of 396 clones, 67 were unique single base-pair changes that rendered L non-functional, of which 44 were missense mutants and 23 were nonsense mutants. Most of the non-functional missense alleles that accumulated in levels comparable to the wild-type allele are localized in the C-terminal half of L, clustered in and around an LS dipeptide sequence. The LS motif was used to align L genes from ssRNA phages lacking any sequence similarity to MS2 or to each other. This alignment revealed a conserved domain structure, in terms of charge, hydrophobic character and predicted helical content. None of the missense mutants affected membrane-association of L. Several of the L mutations in the central domains were highly conservative and recessive, suggesting a defect in a heterotypic protein-protein interaction, rather than in direct disruption of the bilayer structure, as had been previously proposed for L.

MS2 Lysis of Escherichia coli Depends on Host Chaperone DnaJ.

The L protein of the single-stranded RNA phage MS2 causes lysis of Escherichia coli without inducing bacteriolytic activity or inhibiting net peptidoglycan (PG) synthesis. To find host genes required for L-mediated lysis, spontaneous Ill (insensitivity to Llysis) mutants were selected as survivors of L expression and shown to have a missense change of the highly conserved proline (P330Q) in the C-terminal domain of DnaJ. In the dnaJP330Q mutant host, L-mediated lysis is completely blocked at 30°C without affecting the intracellular levels of L. At higher temperatures (37°C and 42°C), both lysis and L accumulation are delayed. The lysis block at 30°C in the dnaJP330Q mutant was recessive and could be suppressed by Lovercomes dnaJ (Lodj ) alleles selected for restoration of lysis. All three Lodj alleles lack the highly basic N-terminal half of the lysis protein and cause lysis ∼20 min earlier than full-length L. DnaJ was found to form a complex with full-length L. This complex was abrogated by the P330Q mutation and was absent with the Lodj truncations. These results suggest that, in the absence of interaction with DnaJ, the N-terminal domain of L interferes with its ability to bind to its unknown target. The lysis retardation and DnaJ chaperone dependency conferred by the nonessential, highly basic N-terminal domain of L resembles the SlyD chaperone dependency conferred by the highly basic C-terminal domain of the E lysis protein of ϕX174, suggesting a common theme where single-gene lysis can be modulated by host factors influenced by physiological conditions.IMPORTANCE Small single-stranded nucleic acid lytic phages (Microviridae and Leviviridae) lyse their host by expressing a single "protein antibiotic." The protein antibiotics from two out of three prototypic small lytic viruses have been shown to inhibit two different steps in the conserved PG biosynthesis pathway. However, the molecular basis of lysis caused by L, the lysis protein of the third prototypic virus, MS2, is unknown. The significance of our research lies in the identification of DnaJ as a chaperone in the MS2 L lysis pathway and the identification of the minimal lytic domain of MS2 L. Additionally, our research highlights the importance of the highly conserved P330 residue in the C-terminal domain of DnaJ for specific protein interactions.

Complete Genome of Salmonella enterica Serovar Typhimurium T5-Like Siphophage Stitch.

Salmonellosis, caused by Salmonella, is a leading cause of food poisoning worldwide. With the continuing rise of bacterial antibiotic resistance, efforts are focused on seeking new approaches for treatment of bacterial infections, namely, bacteriophage therapy. Here, we report the complete genome of S. Typhimurium siphophage Stitch.

Complete Genome of Bacillus megaterium Podophage Page.

Bacillus megaterium is a Gram-positive, spore-forming saprophytic inhabitant of diverse environments. It is a reservoir for industrial chemical production and is emerging as a model organism for studying sporulation and protein localization. Here, we introduce the complete genome of Page, a novel podophage infecting B. megaterium.

Complete Genome of Salmonella enterica Serovar Typhimurium Myophage Maynard.

Salmonella enterica serovar Typhimurium is a pathogenic bacterium that has been a major concern for food and public safety. Phages infecting S. Typhimurium may prove to be useful therapeutics against this harmful bacterium. Here, we announce the complete genome of S. Typhimurium T4-like myophage Maynard and describe its features.

Complete Genome of Bacillus subtilis Myophage CampHawk.

The study of bacteriophages infecting the model organism Bacillus subtilis has provided an abundance of general knowledge and a platform for advances in biotechnology. Here, we announce the annotated genome of CampHawk, a B. subtilis phage. CampHawk was found to be an SPO1-like phage with similar gene content and arrangement.

Complete Genome of Acinetobacter baumannii Podophage Petty.

Acinetobacter baumannii is an emerging pathogen that was isolated from wounded soldiers in military treatment facilities in Iraq but has since become a problem in civilian hospitals. Here, we announce and describe the complete genome of the KMV-like A. baumannii podophage Petty.

Complete Genome of Acinetobacter baumannii N4-Like Podophage Presley.

Acinetobacter baumannii is an emerging multidrug-resistant nosocomial pathogen. Bacteriophages may be useful as an alternative method of treatment against this and other multidrug-resistant bacteria. Here, we present the complete genome sequence of A. baumannii phage Presley, an N4-like podophage.

Complete Genome of Bacillus thuringiensis Myophage BigBertha.

BigBertha is a myophage of Bacillus thuringiensis, a widely used biocontrol agent that is active against many insect pests of plants. Here, we present the complete annotated genome of BigBertha. The genome shares 85.9% sequence identity with Bacillus cereus phage B4.

Complete Genome of Bacillus pumilus Siphophage Blastoid.

Phage Blastoid is a siphophage that infects Bacillus pumilus. B. pumilus is widely used in agriculture but has recently been linked to cases of food poisoning. Here, we report the complete genome of Blastoid and discuss unique genomic characteristics.

Complete Genome of Bacillus pumilus Siphophage Glittering.

Bacillus pumilus is a Gram-positive bacterium widely used in agriculture both as an antifungal and as a growth-promoting symbiont. B. pumilus is rarely infectious but has recently been shown to infect humans. Here, we present the complete genome of B. pumilus phage Glittering, a potential biocontrol agent for B. pumilus.

Complete Genome of Bacillus subtilis Myophage Grass.

Bacillus subtilis is a ubiquitous Gram-positive model organism. Here, we describe the complete genome of B. subtilus myophage Grass. Aside from genes encoding core proteins pertinent to the life cycle of the phage, Grass has several interesting features, including an FtsK/SpoIIIE protein.

Complete Genome of Bacillus megaterium Podophage Pony.

Bacillus megaterium podophage Pony was isolated from a soil sample collected in College Station, TX. Here, we report the sequencing and annotation of the 39,844-bp genome of phage Pony and describe the major features identified.

Complete Genome of Bacillus pumilus Siphophage Riggi.

Bacillus pumilus is primarily used in the agricultural industry to promote plant growth and provide resistance to bacterial and fungal plant diseases. It has recently, however, been shown to cause disease in humans. Here, we announce the complete genome of B. pumilus phage Riggi.

Complete Genome of Bacillus thuringiensis Myophage Spock.

Bacillus thuringiensis is a Gram-positive, sporulating soil microbe with valuable pesticide-producing properties. The study of bacteriophages of B. thuringiensis could provide new biotechnological tools for the use of this bacterium. Here, we present the complete annotated genome of Spock, a myophage of B. thuringiensis, and describe its features.

Complete Genome of Bacillus megaterium Siphophage Staley.

Siphophage Staley was isolated because of its ability to grow on Bacillus megaterium. Here we report the complete genome and annotation of phage Staley and describe core features. Among its interesting genes is one encoding an SleB germination protein.

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.