Phage T4 Research Articles

Mechanisms of Very Long Abortive Transcript Release during Promoter Escape.

A phage T5 N25 promoter variant, DG203, undergoes the escape transition at the +16 to +19 positions after transcription initiation. By specifically examining the abortive activity of the initial transcribing complex at position +19 (ITC19), we observe the production of both GreB-sensitive and GreB-resistant VLAT19. This suggests that ITC19, which is perched on the brink of escape, is highly unstable and can achieve stabilization through either backtracking or forward translocation. Of the forward-tracked fraction, only a small percentage escapes normally (followed by stepwise elongation) to produce full-length RNA; the rest presumably hypertranslocates to release GreB-resistant VLATs. VLAT formation is dependent not only on consensus -35/-10 promoters with 17 bp spacing but also on sequence characteristics of the spacer DNA. Analysis of DG203 promoter variants containing different spacer sequences reveals that AT-rich spacers intrinsically elevate the level of VLAT formation. The AT-rich spacer of DG203 joined to the -10 box presents an UP element sequence capable of interacting with the polymerase α subunit C-terminal domain (αCTD) during the escape transition, which in turn enhances VLAT release. Utilization of the spacer/-10 region UP element by αCTD subunits requires a 10-15 bp hypertranslocation. We document the physical occurrence of hyper forward translocation using ExoIII footprinting analysis.

Characterization, Genome Sequence, and Analysis of Escherichia Phage CICC 80001, a Bacteriophage Infecting an Efficient L-Aspartic Acid Producing Escherichia coli.

Escherichia phage CICC 80001 was isolated from the bacteriophage contaminated medium of an Escherichia coli strain HY-05C (CICC 11022S) which could produce L-aspartic acid. The phage had a head diameter of 45-50 nm and a tail of about 10 nm. The one-step growth curve showed a latent period of 10 min and a rise period of about 20 min. The average burst size was about 198 phage particles per infected cell. Tests were conducted on the plaques, multiplicity of infection, and host range. The genome of CICC 80001 was sequenced with a length of 38,810 bp, and annotated. The key proteins leading to host-cell lysis were phylogenetically analyzed. One protein belonged to class II holin, and the other two belonged to the endopeptidase family and N-acetylmuramoyl-L-alanine amidase family, respectively. The genome showed the sequence identity of 82.7% with that of Enterobacteria phage T7, and carried ten unique open reading frames. The bacteriophage resistant E. coli strain designated CICC 11021S was breeding and its L-aspartase activity was 84.4% of that of CICC 11022S.

Subdiffusive motion of bacteriophage in mucosal surfaces increases the frequency of bacterial encounters

Bacteriophages (phages) defend mucosal surfaces against bacterial infections. However, their complex interactions with their bacterial hosts and with the mucus-covered epithelium remain mostly unexplored. Our previous work demonstrated that T4 phage with Hoc proteins exposed on their capsid adhered to mucin glycoproteins and protected mucus-producing tissue culture cells in vitro. On this basis, we proposed our bacteriophage adherence to mucus (BAM) model of immunity. Here, to test this model, we developed a microfluidic device (chip) that emulates a mucosal surface experiencing constant fluid flow and mucin secretion dynamics. Using mucus-producing human cells and Escherichia coli in the chip, we observed similar accumulation and persistence of mucus-adherent T4 phage and nonadherent T4∆hoc phage in the mucus. Nevertheless, T4 phage reduced bacterial colonization of the epithelium >4,000-fold compared with T4∆hoc phage. This suggests that phage adherence to mucus increases encounters with bacterial hosts by some other mechanism. Phages are traditionally thought to be completely dependent on normal diffusion, driven by random Brownian motion, for host contact. We demonstrated that T4 phage particles displayed subdiffusive motion in mucus, whereas T4∆hoc particles displayed normal diffusion. Experiments and modeling indicate that subdiffusive motion increases phage-host encounters when bacterial concentration is low. By concentrating phages in an optimal mucus zone, subdiffusion increases their host encounters and antimicrobial action. Our revised BAM model proposes that the fundamental mechanism of mucosal immunity is subdiffusion resulting from adherence to mucus. These findings suggest intriguing possibilities for engineering phages to manipulate and personalize the mucosal microbiome.

Single substitution in bacteriophage T4 RNase H alters the ratio between its exo- and endonuclease activities

The article describes substitutions in bacteriophage T4 RNase H which provide so called das-effect. Phage T4 DNA arrest suppression (das) mutations have been described to be capable of partially suppressing the phage DNA arrest phenotype caused by a dysfunction in genes 46 and/or 47 (also known as Mre11/Rad50 complex). Genetic mapping of das13 (one of the das mutations) has shown it to be in the region of the rnh gene encoding RNase H. Here we report that Das13 mutant of RNase H has substitutions of valine 43 and leucine 242 with isoleucines. To investigate the influence of these mutations on RNase H nuclease properties we have designed a novel in vitro assay that allows us to separate and quantify exo- or endonuclease activities of flap endonuclease. The nuclease assay in vitro showed that V43I substitution increased the ratio between exonuclease/endonuclease activities of RNase H whereas L242I substitution did not affect the nuclease activity of RNase H in vitro. However, both mutations were necessary for the full das effect in vivo. Molecular modelling of the nuclease structure suggests that V43I substitution may lead to disposition of H4 helix, responsible for the interaction with the first base pairs of 5′end of branched DNA. These structural changes may affect unwinding of the first base pairs of gapped or nicked DNA generating a short flap and therefore may stabilize the DNA-enzyme complex. L242I substitution did not affect the structure of RNase H and its role in providing das-effect remains unclear.

Structural remodeling of bacteriophage T4 and host membranes during infection initiation

The first stages of productive bacteriophage infections of bacterial host cells require efficient adsorption to the cell surface followed by ejection of phage DNA into the host cytoplasm. To achieve this goal, a phage virion must undergo significant structural remodeling. For phage T4, the most obvious change is the contraction of its tail. Here, we use skinny E. coli minicells as a host, along with cryo-electron tomography and mutant phage virions, to visualize key structural intermediates during initiation of T4 infection. We show for the first time that most long tail fibers are folded back against the tail sheath until irreversible adsorption, a feature compatible with the virion randomly walking across the cell surface to find an optimal site for infection. Our data confirm that tail contraction is triggered by structural changes in the baseplate, as intermediates were found with remodeled baseplates and extended tails. After contraction, the tail tube penetrates the host cell periplasm, pausing while it degrades the peptidoglycan layer. Penetration into the host cytoplasm is accompanied by a dramatic local outward curvature of the cytoplasmic membrane as it fuses with the phage tail tip. The baseplate hub protein gp27 and/or the ejected tape measure protein gp29 likely form the transmembrane channel for viral DNA passage into the cell cytoplasm. Building on the wealth of prior biochemical and structural information, this work provides new molecular insights into the mechanistic pathway of T4 phage infection.

Sequence-Dependent Promoter Escape Efficiency Is Strongly Influenced by Bias for the Pretranslocated State during Initial Transcription.

Abortive transcription initiation can be rate-limiting for promoter escape and therefore represents a barrier to productive gene expression. The mechanism for abortive initiation is unknown, but the amount of abortive transcript is known to vary with the composition of the initial transcribed sequence (ITS). Here, we used a thermodynamic model of translocation combined with experimental validation to investigate the relationship between ITS and promoter escape on a set of phage T5 N25 promoters. We found a strong, negative correlation between RNAP's propensity to occupy the pretranslocated state during initial transcription and the efficiency of promoter escape (r = -0.67; p < 10(-6)). This correlation was almost entirely caused by free energy changes due to variation in the RNA 3' dinucleotide sequence at each step, implying that this sequence element controls the disposition of initial transcribing complexes. We tested our model experimentally by constructing a set of novel N25-ITS promoter variants; quantitative transcription analysis again showed a strong correlation (r = -0.81; p < 10(-6)). Our results support a model in which sequence-directed bias for the pretranslocated state during scrunching results in increased backtracking, which limits the efficiency of promoter escape. This provides an answer to the long-standing issue of how sequence composition of the ITS affects promoter escape efficiency.

Phage T4‐induced DNA breaks activate a tRNA repair‐defying anticodon nuclease

The natural role of the conserved bacterial anticodon nuclease (ACNase) RloC is not known, but traits that set it apart from the homologous phage T4-excluding ACNase PrrC could provide relevant clues. PrrC is silenced by a genetically linked DNA restriction-modification (RM) protein and turned on by a phage-encoded DNA restriction inhibitor. In contrast, RloC is rarely linked to an RM protein, and its ACNase is regulated by an internal switch responsive to double-stranded DNA breaks. Moreover, PrrC nicks the tRNA substrate, whereas RloC excises the wobble nucleotide. These distinctions suggested that (i) T4 and related phage that degrade their host DNA will activate RloC and (ii) the tRNA species consequently disrupted will not be restored by phage tRNA repair enzymes that counteract PrrC. Consistent with these predictions we show that Acinetobacter baylyi RloC expressed in Escherichia coli is activated by wild-type phage T4 but not by a mutant impaired in host DNA degradation. Moreover, host and T4 tRNA species disrupted by the activated ACNase were not restored by T4's tRNA repair system. Nonetheless, T4's plating efficiency was inefficiently impaired by AbaRloC, presumably due to a decoy function of the phage encoded tRNA target, the absence of which exacerbated the restriction.

Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9.

ABSTRACTThe genomic DNAs of tailed bacteriophages are commonly modified by the attachment of chemical groups. Some forms of DNA modification are known to protect phage DNA from cleavage by restriction enzymes, but others are of unknown function. Recently, the CRISPR-Cas nuclease complexes were shown to mediate bacterial adaptive immunity by RNA-guided target recognition, raising the question of whether phage DNA modifications may also block attack by CRISPR-Cas9. We investigated phage T4 as a model system, where cytosine is replaced with glucosyl-hydroxymethylcytosine (glc-HMC). We first quantified the extent and distribution of covalent modifications in T4 DNA by single-molecule DNA sequencing and enzymatic probing. We then designed CRISPR spacer sequences targeting T4 and found that wild-type T4 containing glc-HMC was insensitive to attack by CRISPR-Cas9 but mutants with unmodified cytosine were sensitive. Phage with HMC showed only intermediate sensitivity. While this work was in progress, another group reported examples of heavily engineered CRISRP-Cas9 complexes that could, in fact, overcome the effects of T4 DNA modification, indicating that modifications can inhibit but do not always fully block attack.

Two mechanisms coordinate replication termination by the Escherichia coli Tus-Ter complex.

The Escherichia coli replication terminator protein (Tus) binds to Ter sequences to block replication forks approaching from one direction. Here, we used single molecule and transient state kinetics to study responses of the heterologous phage T7 replisome to the Tus–Ter complex. The T7 replisome was arrested at the non-permissive end of Tus–Ter in a manner that is explained by a composite mousetrap and dynamic clamp model. An unpaired C(6) that forms a lock by binding into the cytosine binding pocket of Tus was most effective in arresting the replisome and mutation of C(6) removed the barrier. Isolated helicase was also blocked at the non-permissive end, but unexpectedly the isolated polymerase was not, unless C(6) was unpaired. Instead, the polymerase was blocked at the permissive end. This indicates that the Tus–Ter mechanism is sensitive to the translocation polarity of the DNA motor. The polymerase tracking along the template strand traps the C(6) to prevent lock formation; the helicase tracking along the other strand traps the complementary G(6) to aid lock formation. Our results are consistent with the model where strand separation by the helicase unpairs the GC(6) base pair and triggers lock formation immediately before the polymerase can sequester the C(6) base.

30 Illuminating obscure states of the phage T3 DNA packaging motor

We use DNA packaging of the related phages, T3 and T7, as a model to develop analysis of biological structure-function relationships. A key objective is characterization of the DNA packaging motor ...

Phage Amplification and Immunomagnetic Separation Combined with Targeted Mass Spectrometry for Sensitive Detection of Viable Bacteria in Complex Food Matrices

We have developed and describe here for the first time a highly sensitive method for the fast and unambiguous detection of viable Escherichia coli in food matrices. The new approach is based on using label-free phages (T4), obligate parasites of bacteria, which are attractive for pathogen detection because of their inherent natural specificity and ease of use. A specific immunomagnetic separation was used to capture the progeny phages produced. Subsequently, T4 phage markers were detected by liquid chromatography coupled to targeted mass spectrometry. Combining the specificity of these three methodologies is of great interest in developing an alternative to conventional time-consuming culture-based technologies for the detection of viable bacteria for industrial applications. First, optimization experiments with phage T4 spiked in complex matrices (without a phage amplification event) were performed and demonstrated specific, sensitive, and reproducible phage capture and detection in complex matrices including Luria-Bertani broth, orange juice, and skimmed milk. The method developed was then applied to the detection of E. coli spiked in foodstuffs (with a phage amplification event). After having evaluated the impact of infection duration on assay sensitivity, we showed that our assay specifically detects viable E. coli in milk at an initial count of ≥1 colony-forming unit (cfu)/mL after an 8-h infection. This excellent detection limit makes our new approach an alternative to PCR-based assays for rapid bacterial detection.

Genome Sequence of Salmonella enterica Phage Det7

Det7 is a Myoviridae bacteriophage that gains entry into its Salmonella enterica serovar Typhimurium host cells by adsorbing to O-antigen polysaccharide. We report here the complete 157,498-bp sequence of its genome. Det7, together with its Vi01-like relatives, are distantly related to phage T4.

55.2, a phage T4 ORFan gene, encodes an inhibitor of Escherichia coli topoisomerase I and increases phage fitness.

Topoisomerases are enzymes that alter the topological properties of DNA. Phage T4 encodes its own topoisomerase but it can also utilize host-encoded topoisomerases. Here we characterized 55.2, a phage T4 predicted ORF of unknown function. High levels of expression of the cloned 55.2 gene are toxic in E. coli. This toxicity is suppressed either by increased topoisomerase I expression or by partial inactivation of the ATPase subunit of the DNA gyrase. Interestingly, very low-level expression of 55.2, which is non-lethal to wild type E. coli, prevents the growth of a deletion mutant of the topoisomerase I (topA) gene. In vitro, gp55.2 binds DNA and blocks specifically the relaxation of negatively supercoiled DNA by topoisomerase I. In vivo, expression of gp55.2 at low non-toxic levels alters the steady state DNA supercoiling of a reporter plasmid. Although 55.2 is not an essential gene, competition experiments indicate that it is required for optimal phage growth. We propose that the role of gp55.2 is to subtly modulate host topoisomerase I activity during infection to insure optimal T4 phage yield.

A decades-long journey with mobile introns.

A decades-long journey with mobile introns.

RNA dances to center stage.

RNA dances to center stage.

Low-temperature bacterial viruses VR - a small but diverse group of E. coli phages.

The complete genome sequences of four low-temperature Escherichia coli-specific tevenviruses, vb_EcoM-VR5, vb_EcoM-VR20, vb_EcoM-VR25 and vb_EcoM-VR26, were determined. Genomic comparisons including recently described genomes of vb_EcoM-VR7 and JS98 as well as phage T4 allowed the identification of two genetic groups that were consistent with defined host-range phenotypes. Group A included the broad-host-range phages vb_EcoM-VR5 and JS98, while group B included vb_EcoM-VR7, vb_EcoM-VR20, vb_EcoM-VR25 and vb_EcoM-VR26, which all had somewhat limited host ranges. All four sequenced phages had genomes that were similar in length (~170 kb) and GC content (~40 %), and, with the exception of vb_EcoM-VR5, at the nucleotide level, they were much more closely related to each other than either was to any other tevenvirus currently characterized. Nevertheless, the overall genome organization of vb_EcoM-VR5, vb_EcoM-VR20, vb_EcoM-VR25 and vb_EcoM-VR26 was comparable to that seen in tevenviruses.

Tail proteins of phage T5: Investigation of the effect of the His6-tag position, from expression to crystallisation

Upon binding to its bacterial host receptor, the tail tip of phage T5 perforates, by an unknown mechanism, the heavily armoured cell wall of the host. This allows the injection of phage DNA into the cytoplasm to hijack the cell machinery and enable the production of new virions. In the perspective of a structural study of the phage tail, we have systematically overproduced eight of the eleven T5 tail proteins, with or without a N- or a C-terminal His6-tag. The widely used Hi6-tag is very convenient to purify recombinant proteins using immobilised-metal affinity chromatography. The presence of a tag however is not always innocuous. We combined automated gene cloning and expression tests to rapidly identify the most promising constructs for proteins of phage T5 tail, and performed biochemical and biophysical characterisation and crystallisation screening on available proteins. Automated small-scale purification was adapted for two highly expressed proteins. We obtained structural information for three of the proteins. We showed that the presence of a His6-tag can have drastic effect on protein expression, solubility, oligomerisation propensity and crystal quality.

Evidence that the heterogeneity of a T4 population is the result of heritable traits.

Many bacteriophage populations display heterogeneity in their adsorption characteristics; a portion of the phage population remains free in solution throughout adsorption experiments (residual fraction). This residual fraction generally constitutes a minority of phages that exhibit significantly slower adsorption kinetics than the main phage stock (main fraction). While this phenomenon is likely the result of evolutionary driving forces, the present study demonstrates that the residual fraction is not always the result of phenotypic variations within a single genotype, as is generally thought. Experiments with phage T4 showed that two subgroups with distinct adsorption traits that were passed on to their progeny could be isolated from the original phage stock. Sequencing of genes involved in adsorption revealed two point mutations in gene 37 of residual fraction isolates, which resulted in modifications to the long tail-fiber, the organelle of attachment and host cell recognition. Adsorption studies consistently showed that T4 phage stocks amplified from residual fraction isolates had significantly lower adsorption efficiencies than those amplified from main fractions. The conducted experiments provide convincing evidence that the observed heterogeneity in T4 adsorption behavior is the result of conserved mutations to the phage genome and is not exclusively the result of phenotypic variations within the population. While it is believed high mutation rates exist to hasten phage adaptation, this study shows that this bet hedging strategy can also, in the short term, inadvertently handicap the phage's adsorption capabilities to a given host under normal infection conditions, resulting in the residual fraction observed in adsorption experiments.

Complete Genome Sequence of vB_EcoM_112, a T-Even-Type Bacteriophage Specific for Escherichiacoli O157:H7.

Bacteriophage vB_EcoM_112 (formerly e11/2) is an Escherichia coli phage with specificity for the O157:H7 serotype. The vB_EcoM_112 genome sequence shares high degrees of similarity with the phage T4 genome sequence.

An unconventional approach to impedance microbiology: detection of culture media conductivity variations due to bacteriophage generated lyses of host bacteria.

A novel and unconventional approach to impedance microbiology has been under investigation. In our approach, solution conductivity variations are generated from bacteriophage lyses of infected host cells and the consequent release of conductive endoplasmic material. To sensitively detect the lysis, low conductive growth media have been developed. A microchip has been fabricated to perform the analysis. The microchip is made of two bare gold electrodes and PDMS microchamber of 36 nL volume. Escherichia coli and selective phages T4 have been used as case study. Proof-of-principle experiments are here presented and discussed. The method was characterised in a wide range between 10(4) and 10(8) CFU/mL, where linear relation was found between conductivity variation and cell concentration in a log10 vs. log10 plot. The method is suited to integration with sample preparation based on phage-functionalised magnetic beads. It has a potential detection limit below 1 CFU/chamber and a total assay time of less than 1 h.