Construction and characterization of DNA libraries from cultured phages and environmental viromes.

Despite many efforts to understand and leverage the functional potential of environmental viromes, most bacteriophage genes are largely uncharacterized. To explore novel biology from uncultivated microbes like phages, metagenomics has emerged as a powerful tool to directly mine new genes without the need to culture the diverse microbiota and the viruses within. When a pure computational approach cannot infer gene function, it may be necessary to create a DNA library from environmental genomic DNA, followed by the screening of that library for a particular function. However, these screens are often initiated without a metagenomic analysis of the completed DNA library being reported. Here, we describe the construction and characterization of DNA libraries from a single cultured phage (ΦT4), five cultured Escherichia coli phages, and three metagenomic viral sets built from freshwater, seawater, and wastewater samples. Through next-generation sequencing of five independent samplings of the libraries, we found a consistent number of recovered genes per replicate for each library, with many genes classifiable via the KEGG and Pharokka databases. By characterizing the size of the genes and inserts, we found that our libraries contain a median of one to two genes per contig with a median gene length of 303-381 bp for all libraries, reflective of the small genomes of viruses. The environmental libraries were genetically diverse compared to the single phage and multi-phage libraries. Additionally, we found reduced coverage of individual genomes when five phages were used as opposed to one. Taken together, this work provides a comprehensive analysis of the DNA libraries from phage genomes that can be used for metagenomic exploration and functional screens to infer and identify new biology.IMPORTANCEFunctional metagenomics is an approach that aims to characterize the putative biological function of genes in the microbial world. This includes an examination of the sequencing data collected from a pooled source of diverse microbes and inference of gene function by comparison to annotated and studied genes from public databases. At times, DNA libraries are made from these genes, and the library is screened for a specific function. Hits are validated using a combination of biological, computational, and structural analysis. Left unresolved is a detailed characterization of the library, both its diversity and content, for the purposes of imputing function entirely by computational means, a process that may yield findings that aid in designing useful screens to identify novel gene functions. In this study, we constructed libraries from cultured phages and uncultured viromes from the environment and characterized some important parameters, such as gene number, genes per contig, ratio of hypothetical to known proteins, total genomic coverage and recovery, and the effect of pooling genetic information from multiple sources, to provide a better understanding of the nature of these libraries. This work will aid the design and implementation of future screens of pooled DNA libraries to discover and isolate viral genes with novel biology across various biomes.

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.

Phage-Antibiotic Synergy Is Driven by a Unique Combination of Antibacterial Mechanism of Action and Stoichiometry.

The continued rise in antibiotic resistance is precipitating a medical crisis. Bacteriophage (phage) has been hailed as one possible therapeutic option to augment the efficacy of antibiotics. However, only a few studies have addressed the synergistic relationship between phage and antibiotics. Here, we report a comprehensive analysis of phage-antibiotic interaction that evaluates synergism, additivism, and antagonism for all classes of antibiotics across clinically achievable stoichiometries. We combined an optically based real-time microtiter plate readout with a matrix-like heat map of treatment potencies to measure phage and antibiotic synergy (PAS), a process we term synography. Phage-antibiotic synography was performed against a pandemic drug-resistant clonal group of extraintestinal pathogenic Escherichia coli (ExPEC) with antibiotic levels blanketing the MIC across seven orders of viral titers. Our results suggest that, under certain conditions, phages provide an adjuvating effect by lowering the MIC for drug-resistant strains. Furthermore, synergistic and antagonistic interactions are highly dependent on the mechanism of bacterial inhibition by the class of antibiotic paired to the phage, and when synergism is observed, it suppresses the emergence of resistant cells. Host conditions that simulate the infection environment, including serum and urine, suppress PAS in a bacterial growth-dependent manner. Lastly, two different related phages that differed in their burst sizes produced drastically different synograms. Collectively, these data suggest lytic phages can resuscitate an ineffective antibiotic for previously resistant bacteria while also synergizing with antibiotics in a class-dependent manner, processes that may be dampened by lower bacterial growth rates found in host environments.IMPORTANCE Bacteriophage (phage) therapy is a promising approach to combat the rise of multidrug-resistant bacteria. Currently, the preferred clinical modality is to pair phage with an antibiotic, a practice thought to improve efficacy. However, antagonism between phage and antibiotics has been reported, the choice of phage and antibiotic is not often empirically determined, and the effect of the host factors on the effectiveness is unknown. Here, we interrogate phage-antibiotic interactions across antibiotics with different mechanisms of action. Our results suggest that phage can lower the working MIC for bacterial strains already resistant to the antibiotic, is dependent on the antibiotic class and stoichiometry of the pairing, and is dramatically influenced by the host microenvironment.

Complete Genome Sequence of Klebsiella pneumoniae Myophage Mulock.

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.

Complete Genome Sequence of Escherichia coli Myophage Minorna.

The Gram-negative bacterium Escherichia coli causes many diseases, and antibiotic resistance has become a problem for their treatment. Bacteriophages may present a viable treatment alternative. Here, the complete genome sequence of E. coli-infecting myophage Minorna is presented. Proteins needed for replication, morphogenesis, and lysis were identified in the Minorna coding sequence.

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.