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Although the F-specific ssRNA phage MS2 has long had paradigm status, little is known about penetration of the genomic RNA (gRNA) into the cell. The phage initially binds to the F-pilus using its maturation protein (Mat), and then the Mat-bound gRNA is released from the viral capsid and somehow crosses the bacterial envelope into the cytoplasm. To address the mechanics of this process, we fluorescently labeled the ssRNA phage MS2 to track F-pilus dynamics during infection. We discovered that ssRNA phage infection triggers the release of F-pili from host cells, and that higher multiplicity of infection (MOI) correlates with detachment of longer F-pili. We also report that entry of gRNA into the host cytoplasm requires the F-plasmid–encoded coupling protein, TraD, which is located at the cytoplasmic entrance of the F-encoded type IV secretion system (T4SS). However, TraD is not essential for pilus detachment, indicating that detachment is triggered by an early step of MS2 engagement with the F-pilus or T4SS. We propose a multistep model in which the ssRNA phage binds to the F-pilus and through pilus retraction engages with the distal end of the T4SS channel at the cell surface. Continued pilus retraction pulls the Mat-gRNA complex out of the virion into the T4SS channel, causing a torsional stress that breaks the mature F-pilus at the cell surface. We propose that phage-induced disruptions of F-pilus dynamics provides a selective advantage for infecting phages and thus may be prevalent among the phages specific for retractile pili.

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.

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 Qβ, 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.

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.

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 ( i nsensitivity to L l ysis) 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 dnaJ P330Q 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 dnaJ P330Q mutant was recessive and could be suppressed by L o vercomes d na J ( L odj ) alleles selected for restoration of lysis. All three L odj 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 L odj 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.

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 Qβ 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.

Mammals do not regenerate axons in their central nervous system (CNS) spontaneously. This phenomenon is the cause of numerous medical conditions after damage to nerve fibers in the CNS of humans. The study of the mechanisms of nerve regeneration in other vertebrate animals able to spontaneously regenerate axons in their CNS is essential for understanding nerve regeneration from a scientific point of view, and for developing therapeutic approaches to enhance nerve regeneration in the CNS of humans. RICH proteins are a novel group of proteins implicated in nerve regeneration in the CNS of teleost fish, yet their mechanisms of action are not well understood. A number of mutant versions of the zebrafish RICH (zRICH) protein were generated and characterized at biochemical and cellular levels in our laboratory. With the aim of understanding the effects of RICH proteins in neuronal axon outgrowth, stable transfectants derived from the neuronal model PC12 cell line expressing zRICH Wild-Type or mutant versions of zRICH were studied. Results from differentiation experiments suggest that RICH proteins enhance neuronal plasticity by facilitating neurite branching. Biochemical co-purification results have demonstrated that zRICH binds to the cytoskeletal protein tubulin. The central domain of the protein is sufficient for tubulin binding, but a mutant version of the protein lacking the terminal domains, which cannot bind to the plasma membrane, was not able to enhance neurite branching. RICH proteins may facilitate axon regeneration by regulating the axonal cytoskeleton and facilitating the formation of new neurite branches.

Engineered nanomaterials display significant advantages due to their unique nanostructure, along with their tuneable properties for the designed application. Silver nanoparticles (Ag-NPs) have drawn attention due to their use as potent bactericidal agents and were characterized in this research to provide an understanding of the interaction between nanomaterials and bacteria. This work presents the bactericidal performance of Ag-NPs using Escherichia coli (E. coli) as a model microorganism. Several state-of-the-art techniques, such as high-angle annular dark-field detector in scanning transmission electron microscopy, and energy filtered imaging in electron energy loss spectroscopy, were employed to obtain nanostructural and elemental information. The bactericidal activities of Ag-NPs were then compared with two commonly used disinfectants, sodium hypochlorite (NaClO) and phenol (C(6)H(5)OH). These two chemical disinfectants exhibited rapid bactericidal activity, showing a minimal bactericidal concentration (MBC) of 16 parts-per-million (ppm) and 16 part-per-thousand (ppth), respectively for NaClO and C(6)H(5)OH within about 10 min. In contrast, Ag-NPs exhibit slow but long-term bactericidal effect demonstrating MBCs of 0.6 parts-per-million (ppm) within 6h when used as disinfectant. An advantage using Ag-NPs to inactivate E coli at low dosages is negligible environmental waste or hazardous by-products. The results showed that Ag-NPs caused bacterial inactivation by a mechanism involving several steps, including cell wall and cytoplasmic membrane damage.

Using environmentally friendly synthesis techniques, monodispersive silver nanoparticles (AgNPs) were engineered. These particles exhibited bactericidal activity against Escherichia coli under both light and dark conditions. Unlike sodium hypochlorite, which demonstrated almost immediate disinfection, AgNPs required 30 min. In contrast to hypochlorite, however, the minimum dose of AgNPs decreased as the incubation time increased to less than 1 part per million. The mechanism whereby the nanoparticles inactivate the microbe is speculated to incorporate three distinct pathways. Collectively, the advantages of using AgNPs are (1) long-term efficacy; (2) effectiveness at ultralow doses; and (3) generation of little or no waste during the synthesis.

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.

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 .

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.

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.

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.

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 ϕKMV-like A. baumannii podophage Petty.

Phage DNA wassequencedusing454pyrosequencingattheEmoryGRAGenomeCenter(EmoryUniversity,Atlanta,GA).TrimmedFLXTitaniumreadswereassembledtoasinglecontigat121-foldcoverageusingtheNewblerassembler,version2.5.3(454LifeSciences),atdefaultsettings.PCRconfirmedcompletedcontigs.Geneswerepredictedusing GeneMarkS (5), and gene predictions were corrected usingsoftware tools available on the Center for Phage Technology(CPT) Portal (https://cpt.tamu.edu/cpt-software/portal/). Elec-tron microscopy was performed at the Texas A&M Microscopyand Imaging Center.CN1A is a siphophage with a unique genome of 55,837 bp. IthasaG C content of 62.1%, which is noticeably lower than the72.6%G Ccontentofitshost(3).Theunitgenomehasa90.1%codingdensity,with78predictedcodingsequencesandonetRNAgene. Based on BLASTp and InterProScan results, 18 genes werepredictedtoencodeproteinswithaknownfunction(6,7).Oftherest, 15 genes were hypothetical conserved genes and 45 were hy-pothetical novel genes. An examination of the raw sequence datausing the PAUSE (https://cpt.tamu.edu/cpt-software/releases/pause/)methodrevealedthatCN1Ahasalongterminalrepeatof952 bp.Theannotatedgenescorrespondtocorefunctionssuchasrep-lication,nucleotidemetabolism,morphogenesis,andlysis.Genesencoding replication proteins include those for helicase, DNApolymerase exonuclease subunit epsilon, and a single-strandedDNA (ssDNA) binding protein. A T4 PseT-like polynucleotide5 =-kinase/3 -phosphatase gene and a T7 gp

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.

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.

Salmonella enterica serovar Enteritidis is a food-borne pathogen that causes salmonellosis in the United States. Bacteriophages are emerging as viable biocontrol agents against this pathogen. Here, we present the complete annotated genome sequence of Salmonella Enteritidis T4-like myophage Marshall, which has potential as a phage therapy agent.

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.

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.

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.

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.

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.

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 .

hasbeenshowntobeofgreat interest to the pharmaceutical industry, and it is used inproduction of penicillin, antifungals, and antivirals. The bacte-rium has important use as a model organism, as it is exploited instudiesofmembraneformation,thesporulationprocess,andpro-tein localization (2, 3).BacteriophageSlashwasobtainedfromasoilsamplecollectedin College Station, TX. Phage DNA was sequenced using 454 py-rosequencingattheEmoryGRAGenomeCenter(EmoryUniver-sity,Atlanta,GA).TrimmedFLXTitaniumreadswereassembledto a single contig at 111-fold coverage using the Newbler assem-bler version 2.5.3 (454 Life Sciences), with the default settings.PCR confirmed the completed contig. The genes were initiallypredicted using GeneMarkS (4), and gene predictions were cor-rected using software tools available on the Center for PhageTechnology (CPT) portal (https://cpt.tamu.edu/cpt-software/portal/).TransmissionelectronmicroscopywasperformedattheMicroscopy and Imaging Center at Texas A&M University.Phage Slash is a siphophage containing a 79,815-bp double-stranded DNA (dsDNA) genome with a G C content of 35.6%and a coding density of 90.1%. Thirty-two genes were assignedfunctionsbyBLASTpandInterProanalyses(5,6).Inaddition,16hypothetical conserved genes and 64 hypothetical novel geneswere annotated.Several morphogenesis genes were identified, encoding tapemeasure protein, head-to-tail joining protein, tailspike protein,and the terminase large subunit. Although the large terminaselacks homology to the terminases of known packaging strategies,an analysis of the raw sequencing data using the Pause method(https://cpt.tamu.edu/cpt-software/releases/pause/)revealedthatSlash has a direct terminal repeat of 567 bp. The genes encodingDNA metabolism enzymes, including recombinase, DnaB-likehelicase, DNA polymerase III (epsilon subunit), DNA primase,Hollidayjunctionresolvaseprotein,andaRecB-likeexonuclease,wereidentified.SlashalsocarriesDNAbiosynthesisgenes(encod-ing dUTPase, thymidylate synthase, guanylate kinase, and the al-phaandbetasubunitsofribonucleotidereductase).Fourputativesigma/transcriptionfactorswerealsoidentified.Fourhomingen-donucleaseswithunknownsequencespecificitieswereidentified,including two with an AP2 domain. The AP2 domain is a DNAbinding domain that was originally identified in plant transcrip-tion factors (7). Recent structural analysis suggests that the plantAP2 domain is likely an evolutionary ancestor of homing endo-nucleases(8).AlysiscassetteencodingaclassIIholin(twotrans-membrane domains in an N-in C-in topology) and an endolysin(