Shutoff of host transcription triggers a toxin-antitoxin system to cleave phage RNA and abort infection.

Toxin-antitoxin (TA) systems are widespread in bacteria, but their activation mechanisms and bona fide targets remain largely unknown. Here, we characterize a type III TA system, toxIN, that protects E. coli against multiple bacteriophages, including T4. Using RNA sequencing, we find that the endoribonuclease ToxN is activated following T4 infection and blocks phage development primarily by cleaving viral mRNAs and inhibiting their translation. ToxN activation arises from T4-induced shutoff of host transcription, specifically of toxIN, leading to loss of the intrinsically unstable toxI antitoxin. Transcriptional shutoff is necessary and sufficient for ToxN activation. Notably, toxIN does not strongly protect against another phage, T7, which incompletely blocks host transcription. Thus, our results reveal a critical trade-off in blocking host transcription: it helps phage commandeer host resources but can activate potent defense systems. More generally, our results now reveal the native targets of an RNase toxin and activation mechanism of a phage-defensive TA system.

Xenogeneic Regulation of the Bacterial Transcription Machinery.

The parasitic life cycle of viruses involves the obligatory subversion of the host's macromolecular processes for efficient viral progeny production. Viruses that infect bacteria, bacteriophages (phages), are no exception and have evolved sophisticated ways to control essential biosynthetic machineries of their bacterial prey to benefit phage development. The xenogeneic regulation of bacterial cell function is a poorly understood area of bacteriology. The activity of the bacterial transcription machinery, the RNA polymerase (RNAP), is often regulated by a variety of mechanisms involving small phage-encoded proteins. In this review, we provide a brief overview of known phage proteins that interact with the bacterial RNAP and compare how two prototypical phages of Escherichia coli, T4 and T7, use small proteins to "puppeteer" the bacterial RNAP to ensure a successful infection.

Transposon Insertion Sequencing Elucidates Novel Gene Involvement in Susceptibility and Resistance to Phages T4 and T7 in Escherichia coli O157.

Experiments using bacteriophage (phage) to infect bacterial strains have helped define some basic genetic concepts in microbiology, but our understanding of the complexity of bacterium-phage interactions is still limited. As the global threat of antibiotic resistance continues to increase, phage therapy has reemerged as an attractive alternative or supplement to treating antibiotic-resistant bacterial infections. Further, the long-used method of phage typing to classify bacterial strains is being replaced by molecular genetic techniques. Thus, there is a growing need for a complete understanding of the precise molecular mechanisms underpinning phage-bacterium interactions to optimize phage therapy for the clinic as well as for retrospectively interpreting phage typing data on the molecular level. In this study, a genomics-based fitness assay (TraDIS) was used to identify all host genes involved in phage susceptibility and resistance for a T4 phage infecting Shiga-toxigenic Escherichia coli O157. The TraDIS results identified both established and previously unidentified genes involved in phage infection, and a subset were confirmed by site-directed mutagenesis and phenotypic testing of 14 T4 and 2 T7 phages. For the first time, the entire sap operon was implicated in phage susceptibility and, conversely, the stringent starvation protein A gene (sspA) was shown to provide phage resistance. Identifying genes involved in phage infection and replication should facilitate the selection of bespoke phage combinations to target specific bacterial pathogens.IMPORTANCE Antibiotic resistance has diminished treatment options for many common bacterial infections. Phage therapy is an alternative option that was once popularly used across Europe to kill bacteria within humans. Phage therapy acts by using highly specific viruses (called phages) that infect and lyse certain bacterial species to treat the infection. Whole-genome sequencing has allowed modernization of the investigations into phage-bacterium interactions. Here, using E. coli O157 and T4 bacteriophage as a model, we have exploited a genome-wide fitness assay to investigate all genes involved in defining phage resistance or susceptibility. This knowledge of the genetic determinants of phage resistance and susceptibility can be used to design bespoke phage combinations targeted to specific bacterial infections for successful infection eradication.

Genetically manipulated phages with improved pH resistance for oral administration in veterinary medicine.

Orally administered phages to control zoonotic pathogens face important challenges, mainly related to the hostile conditions found in the gastrointestinal tract (GIT). These include temperature, salinity and primarily pH, which is exceptionally low in certain compartments. Phage survival under these conditions can be jeopardized and undermine treatment. Strategies like encapsulation have been attempted with relative success, but are typically complex and require several optimization steps. Here we report a simple and efficient alternative, consisting in the genetic engineering of phages to display lipids on their surfaces. Escherichia coli phage T7 was used as a model and the E. coli PhoE signal peptide was genetically fused to its major capsid protein (10 A), enabling phospholipid attachment to the phage capsid. The presence of phospholipids on the mutant phages was confirmed by High Performance Thin Layer Chromatography, Dynamic Light Scattering and phospholipase assays. The stability of phages was analysed in simulated GIT conditions, demonstrating improved stability of the mutant phages with survival rates 102-107 pfu.mL-1 higher than wild-type phages. Our work demonstrates that phage engineering can be a good strategy to improve phage tolerance to GIT conditions, having promising application for oral administration in veterinary medicine.

Natural selection underlies apparent stress-induced mutagenesis in a bacteriophage infection model.

The emergence of mutations following growth-limiting conditions underlies bacterial drug resistance, viral escape from the immune system and fundamental evolution-driven events. Intriguingly, whether mutations are induced by growth limitation conditions or are randomly generated during growth and then selected by growth limitation conditions remains an open question(1). Here, we show that bacteriophage T7 undergoes apparent stress-induced mutagenesis when selected for improved recognition of its host's receptor. In our unique experimental set-up, the growth limitation condition is physically and temporally separated from mutagenesis: growth limitation occurs while phage DNA is outside the host, and spontaneous mutations occur during phage DNA replication inside the host. We show that the selected beneficial mutations are not pre-existing and that the initial slow phage growth is enabled by the phage particle's low-efficiency DNA injection into the host. Thus, the phage particle allows phage populations to initially extend their host range without mutagenesis by virtue of residual recognition of the host receptor. Mutations appear during non-selective intracellular replication, and the frequency of mutant phages increases by natural selection acting on free phages, which are not capable of mutagenesis.

Genetic requirements for sensitivity of bacteriophage t7 to dideoxythymidine.

We previously reported that the presence of dideoxythymidine (ddT) in the growth medium selectively inhibits the ability of bacteriophage T7 to infect Escherichia coli by inhibiting phage DNA synthese (N. Q. Tran, L. F. Rezende, U. Qimron, C. C. Richardson, and S. Tabor, Proc. Natl. Acad. Sci. U. S. A. 105:9373-9378, 2008, doi:10.1073/pnas.0804164105). In the presence of T7 gene 1.7 protein, ddT is taken up into the E. coli cell and converted to ddTTP. ddTTP is incorporated into DNA as ddTMP by the T7 DNA polymerase, resulting in chain termination. We have identified the pathway by which exogenous ddT is converted to ddTTP. The pathway consists of ddT transport by host nucleoside permeases and phosphorylation to ddTMP by the host thymidine kinase. T7 gene 1.7 protein phosphorylates ddTMP and ddTDP, resulting in ddTTP. A 74-residue peptide of the gene 1.7 protein confers ddT sensitivity to the same extent as the 196-residue wild-type gene 1.7 protein. We also show that cleavage of thymidine to thymine and deoxyribose-1-phosphate by the host thymidine phosphorylase greatly increases the sensitivity of phage T7 to ddT. Finally, a mutation in T7 DNA polymerase that leads to discrimination against the incorporation of ddTMP eliminates ddT sensitivity.

Empirical complexities in the genetic foundations of lethal mutagenesis.

From population genetics theory, elevating the mutation rate of a large population should progressively reduce average fitness. If the fitness decline is large enough, the population will go extinct in a process known as lethal mutagenesis. Lethal mutagenesis has been endorsed in the virology literature as a promising approach to viral treatment, and several in vitro studies have forced viral extinction with high doses of mutagenic drugs. Yet only one empirical study has tested the genetic models underlying lethal mutagenesis, and the theory failed on even a qualitative level. Here we provide a new level of analysis of lethal mutagenesis by developing and evaluating models specifically tailored to empirical systems that may be used to test the theory. We first quantify a bias in the estimation of a critical parameter and consider whether that bias underlies the previously observed lack of concordance between theory and experiment. We then consider a seemingly ideal protocol that avoids this bias-mutagenesis of virions-but find that it is hampered by other problems. Finally, results that reveal difficulties in the mere interpretation of mutations assayed from double-strand genomes are derived. Our analyses expose unanticipated complexities in testing the theory. Nevertheless, the previous failure of the theory to predict experimental outcomes appears to reside in evolutionary mechanisms neglected by the theory (e.g., beneficial mutations) rather than from a mismatch between the empirical setup and model assumptions. This interpretation raises the specter that naive attempts at lethal mutagenesis may augment adaptation rather than retard it.

Overexpression of Escherichia coli udk mimics the absence of T7 Gp2 function and thereby abrogates successful infection by T7 phage.

Successful infection of Escherichia coli by bacteriophage T7 relies upon the transcription of the T7 genome by two different RNA polymerases (RNAps). The bacterial RNAp transcribes early T7 promoters, whereas middle and late T7 genes are transcribed by the T7 RNAp. Gp2, a T7-encoded transcription factor, is a 7 kDa product of an essential middle T7 gene 2, and is a potent inhibitor of the host RNAp. The essential biological role of Gp2 is to inhibit transcription of early T7 genes that fail to terminate efficiently in order to facilitate the coordinated usage of the T7 genome by both host and phage RNAps. Overexpression of the E. coli udk gene, which encodes a uridine/cytidine kinase, interferes with T7 infection. We demonstrate that overexpression of udk antagonizes Gp2 function in E. coli in the absence of T7 infection and thus independently of T7-encoded factors. It seems that overexpression of udk reduces Gp2 stability and functionality during T7 infection, which consequently results in inadequate inhibition of host RNAp and in the accumulation of early T7 transcripts. In other words, overexpression of udk mimics the absence of Gp2 during T7 infection. Our study suggests that the transcriptional regulation of the T7 genome is surprisingly complex and might potentially be affected at many levels by phage- and host-encoded factors.

Mapping of vascular ZIP codes by phage display.

Each organ and pathology has a unique vascular ZIP code that can be targeted with affinity ligands. In vivo peptide phage display can be used for unbiased mapping of the vascular diversity. Remarkably, some of the peptides identified by such screens not only bind to target vessels but also elicit biological responses. Recently identified tissue-penetrating CendR peptides trigger vascular exit and parenchymal spread of a wide range of conjugated and coadministered payloads. This review is designed to serve as a practical guide for researchers interested in setting up ex vivo and in vivo phage display technology. We focus on T7 coliphage platform that our lab prefers to use due to its versatility, physical resemblance of phage particles to clinical nanoparticles, and ease of manipulation.

The phenotype-fitness map in experimental evolution of phages.

Evolutionary biologists commonly interpret adaptations of organisms by reference to a phenotype-fitness map, a model of how different states of a phenotype affect fitness. Notwithstanding the popularity of this approach, it remains difficult to directly test these mappings, both because the map often describes only a small subset of phenotypes contributing to total fitness and because direct measures of fitness are difficult to obtain and compare to the map. Both limitations can be overcome for bacterial viruses (phages) grown in the experimental condition of unlimited hosts. A complete accounting of fitness requires 3 easily measured phenotypes, and total fitness is also directly measurable for arbitrary genotypes. Yet despite the presumed transparency of this system, directly estimated fitnesses often differ from fitnesses calculated from the phenotype-fitness map. This study attempts to resolve these discrepancies, both by developing a more exact analytical phenotype-fitness map and by exploring the empirical foundations of direct fitness estimates. We derive an equation (the phenotype-fitness map) for exponential phage growth that allows an arbitrary distribution of lysis times and burst sizes. We also show that direct estimates of fitness are, in many cases, plausibly in error because the population has not attained stable age distribution and thus violates the model underlying the phenotype-fitness map. In conjunction with data provided here, the new understanding appears to resolve a discrepancy between the reported fitness of phage T7 and the substantially lower value calculated from its phenotype-fitness map.

Microbial UV fluence-response assessment using a novel UV-LED collimated beam system.

A research study has been performed to determine the ultraviolet (UV) fluence-response of several target non-pathogenic microorganisms to UV light emitting diodes (UV-LEDs) by performing collimated beam tests. UV-LEDs do not contain toxic mercury, offer design flexibility due to their small size, and have a longer operational life than mercury lamps. Comsol Multiphysics was utilized to create an optimal UV-LED collimated beam design based on number and spacing of UV-LEDs and distance of the sample from the light source while minimizing the overall cost. The optimized UV-LED collimated beam apparatus and a low-pressure mercury lamp collimated beam apparatus were used to determine the UV fluence-response of three surrogate microorganisms (Escherichia coli, MS-2, T7) to 255 nm UV-LEDs, 275 nm UV-LEDs, and 254 nm low-pressure mercury lamps. Irradiation by low-pressure mercury lamps produced greater E. coli and MS-2 inactivation than 255 nm and 275 nm UV-LEDs and similar T7 inactivation to irradiation by 275 nm UV-LEDs. The 275 nm UV-LEDs produced more efficient T7 and E. coli inactivation than 255 nm UV-LEDs while both 255 nm and 275 nm UV-LEDs produced comparable microbial inactivation for MS-2. Differences may have been caused by a departure from the time-dose reciprocity law due to microbial repair mechanisms.

The role of the T7 Gp2 inhibitor of host RNA polymerase in phage development.

Bacteriophage T7 relies on its own RNA polymerase (RNAp) to transcribe its middle and late genes. Early genes, which include the viral RNAp gene, are transcribed by the host RNAp from three closely spaced strong promoters-A1, A2, and A3. One middle T7 gene product, gp2, is a strong inhibitor of the host RNAp. Gp2 is essential and is required late in infection, during phage DNA packaging. Here, we explore the role of gp2 in controlling host RNAp transcription during T7 infection. We demonstrate that in the absence of gp2, early viral transcripts continue to accumulate throughout the infection. Decreasing transcription from early promoter A3 is sufficient to make gp2 dispensable for phage infection. Gp2 also becomes dispensable when an antiterminating element boxA, located downstream of early promoters, is deleted. The results thus suggest that antiterminated transcription by host RNAp from the A3 promoter is interfering with phage development and that the only essential role for gp2 is to prevent this transcription.

Evolution at a high imposed mutation rate: adaptation obscures the load in phage T7.

Evolution at high mutation rates is expected to reduce population fitness deterministically by the accumulation of deleterious mutations. A high enough rate should even cause extinction (lethal mutagenesis), a principle motivating the clinical use of mutagenic drugs to treat viral infections. The impact of a high mutation rate on long-term viral fitness was tested here. A large population of the DNA bacteriophage T7 was grown with a mutagen, producing a genomic rate of 4 nonlethal mutations per generation, two to three orders of magnitude above the baseline rate. Fitness-viral growth rate in the mutagenic environment-was predicted to decline substantially; after 200 generations, fitness had increased, rejecting the model. A high mutation load was nonetheless evident from (i) many low- to moderate-frequency mutations in the population (averaging 245 per genome) and (ii) an 80% drop in average burst size. Twenty-eight mutations reached high frequency and were thus presumably adaptive, clustered mostly in DNA metabolism genes, chiefly DNA polymerase. Yet blocking DNA polymerase evolution failed to yield a fitness decrease after 100 generations. Although mutagenic drugs have caused viral extinction in vitro under some conditions, this study is the first to match theory and fitness evolution at a high mutation rate. Failure of the theory challenges the quantitative basis of lethal mutagenesis and highlights the potential for adaptive evolution at high mutation rates.

A novel convenient method for high bacteriophage titer assay.

The recent various applications of phages (bacteriophages) including phage therapy have brought about a revival of phage investigation. The phage titer assay is indispensable for phage experiments. However, the conventional standard method is a plaque counting method which requires a little skill with tedious repeating operation. Furthermore, it is not directly applicable to high phage titers. In this paper, we describe a novel convenient "cross streak and paper disk assay method" for high titer concentration without plaque counting.

Rescue of bacteriophage T7 DNA polymerase of low processivity by suppressor mutations affecting gene 3 endonuclease.

The DNA polymerase encoded by gene 5 (gp5) of bacteriophage T7 has low processivity, dissociating after the incorporation of a few nucleotides. Upon binding to its processivity factor, Escherichia coli thioredoxin (Trx), the processivity is increased to approximately 800 nucleotides per binding event. Several interactions between gp5/Trx and DNA are required for processive DNA synthesis. A basic region in T7 DNA polymerase (residues K587, K589, R590, and R591) is located in proximity to the 5' overhang of the template strand. Replacement of these residues with asparagines results in a threefold reduction of the polymerization activity on primed M13 single-stranded DNA. The altered gp5/Trx exhibits a 10-fold reduction in its ability to support growth of T7 phage lacking gene 5. However, T7 phages that grow at a similar rate provided with either wild-type or altered polymerase emerge. Most of the suppressor phages contain genetic changes in or around the coding region for gene 3, an endonuclease. Altered gene 3 proteins derived from suppressor strains show reduced catalytic activity and are inefficient in complementing growth of T7 phage lacking gene 3. Results from this study reveal that defects in processivity of DNA polymerase can be suppressed by reducing endonuclease activity.

Promiscuous usage of nucleotides by the DNA helicase of bacteriophage T7: determinants of nucleotide specificity.

The multifunctional protein encoded by gene 4 of bacteriophage T7 (gp4) provides both helicase and primase activity at the replication fork. T7 DNA helicase preferentially utilizes dTTP to unwind duplex DNA in vitro but also hydrolyzes other nucleotides, some of which do not support helicase activity. Very little is known regarding the architecture of the nucleotide binding site in determining nucleotide specificity. Crystal structures of the T7 helicase domain with bound dATP or dTTP identified Arg-363 and Arg-504 as potential determinants of the specificity for dATP and dTTP. Arg-363 is in close proximity to the sugar of the bound dATP, whereas Arg-504 makes a hydrogen bridge with the base of bound dTTP. T7 helicase has a serine at position 319, whereas bacterial helicases that use rATP have a threonine in the comparable position. Therefore, in the present study we have examined the role of these residues (Arg-363, Arg-504, and Ser-319) in determining nucleotide specificity. Our results show that Arg-363 is responsible for dATP, dCTP, and dGTP hydrolysis, whereas Arg-504 and Ser-319 confer dTTP specificity. Helicase-R504A hydrolyzes dCTP far better than wild-type helicase, and the hydrolysis of dCTP fuels unwinding of DNA. Substitution of threonine for serine 319 reduces the rate of hydrolysis of dTTP without affecting the rate of dATP hydrolysis. We propose that different nucleotides bind to the nucleotide binding site of T7 helicase by an induced fit mechanism. We also present evidence that T7 helicase uses the energy derived from the hydrolysis of dATP in addition to dTTP for mediating DNA unwinding.

Gene 1.7 of bacteriophage T7 confers sensitivity of phage growth to dideoxythymidine.

Bacteriophage T7 DNA polymerase efficiently incorporates dideoxynucleotides into DNA, resulting in chain termination. Dideoxythymidine (ddT) present in the medium at levels not toxic to Escherichia coli inhibits phage T7. We isolated 95 T7 phage mutants that were resistant to ddT. All contained a mutation in T7 gene 1.7, a nonessential gene of unknown function. When gene 1.7 was expressed from a plasmid, T7 phage resistant to ddT still arose; analysis of 36 of these mutants revealed that all had a single mutation in gene 5, which encodes T7 DNA polymerase. This mutation changes tyrosine-526 to phenylalanine, which is known to increase dramatically the ability of T7 DNA polymerase to discriminate against dideoxynucleotides. DNA synthesis in cells infected with wild-type T7 phage was inhibited by ddT, suggesting that it resulted in chain termination of DNA synthesis in the presence of gene 1.7 protein. Overexpression of gene 1.7 from a plasmid rendered E. coli cells sensitive to ddT, indicating that no other T7 proteins are required to confer sensitivity to ddT.

Effect of high hydrostatic pressure processing on freely suspended and bivalve-associated T7 bacteriophage.

The effectiveness of hydrostatic pressure processing (HPP) for inactivating viruses has been evaluated in only a limited number of studies, and most of the work has been performed with viruses freely suspended in distilled water. In this work, HPP inactivation of freely suspended and shellfish-associated bacteriophage T7 was studied. T7 was selected in hopes that it could serve as a model for animal virus behavior. Clams (Mercenaria mercenaria) and oysters (Crassostrea virginica) were homogeneously blended separately and inoculated with bacteriophage T7. The inoculated bivalve meat and the freely suspended virus samples were subjected to HPP under the following conditions: 2, 4, and 6 min at 241.3, 275.8, and 344.7 MPa pressure and temperatures of 29.4 to 35, 37.8 to 43.3, and 46.1 to 51.7 degrees C. Reductions of 7.8 log PFU (100% inactivation) were achieved for freely suspended T7 at 344.7 MPa for 2 min at 37.8 to 43.3 degrees C. At 46.1 to 51.7 degrees C, T7 associated with either clams or oysters was inactivated at nearly 100% (>4 log PFU) at all pressure levels and durations tested. These results indicate that T7 is readily inactivated by HPP under the proper conditions, may be made more susceptible to HPP by mixing with shellfish meat, and may serve as a viable model for the response of several animal viruses to HPP.

Inadequate inhibition of host RNA polymerase restricts T7 bacteriophage growth on hosts overexpressing udk.

Overexpression of udk, an Escherichia coli gene encoding a uridine/cytidine kinase, interferes with T7 bacteriophage growth. We show here that inhibition of T7 phage growth by udk overexpression can be overcome by inhibition of host RNA polymerase. Overexpression of gene 2, whose product inhibits host RNA polymerase, restores T7 phage growth on hosts overexpressing udk. In addition, rifampicin, an inhibitor of host RNA polymerase, restores the burst size of T7 phage on udk-overexpressing hosts to normal. In agreement with these findings, suppressor mutants that overcome the inhibition arising from udk overexpression gain the ability to grow on hosts that are resistant to inhibition of RNA polymerase by gene 2 protein, and suppressor mutants that overcome a lack of gene 2 protein gain the ability to grow on hosts that overexpress udk. Mutations that eliminate or weaken strong promoters for host RNA polymerase in T7 DNA, and mutations in T7 gene 3.5 that affect its interaction with T7 RNA polymerase, also reduce the interference with T7 growth by host RNA polymerase. We propose a general model for the requirement of host RNA polymerase inhibition.