Production Of Progeny Phage Research Articles

From Host to Phage Metabolism: Hot Tales of Phage T4's Takeover of E. coli.

The mechanisms by which bacteriophage T4 converts the metabolism of its E. coli host to one dedicated to progeny phage production was the subject of decades of intense research in many labs from the 1950s through the 1980s. Presently, a wide range of phages are starting to be used therapeutically and in many other applications, and also the range of phage sequence data available is skyrocketing. It is thus important to re-explore the extensive available data about the intricacies of the T4 infection process as summarized here, expand it to looking much more broadly at other genera of phages, and explore phage infections using newly-available modern techniques and a range of appropriate environmental conditions.

A highly specific phage defense system is a conserved feature of the Vibrio cholerae mobilome.

Vibrio cholerae-specific bacteriophages are common features of the microbial community during cholera infection in humans. Phages impose strong selective pressure that favors the expansion of phage-resistant strains over their vulnerable counterparts. The mechanisms allowing virulent V. cholerae strains to defend against the ubiquitous threat of predatory phages have not been established. Here, we show that V. cholerae PLEs (phage-inducible chromosomal island-like elements) are widespread genomic islands dedicated to phage defense. Analysis of V. cholerae isolates spanning a 60-year collection period identified five unique PLEs. Remarkably, we found that all PLEs (regardless of geographic or temporal origin) respond to infection by a myovirus called ICP1, the most prominent V. cholerae phage found in cholera patient stool samples from Bangladesh. We found that PLE activity reduces phage genome replication and accelerates cell lysis following ICP1 infection, killing infected host cells and preventing the production of progeny phage. PLEs are mobilized by ICP1 infection and can spread to neighboring cells such that protection from phage predation can be horizontally acquired. Our results reveal that PLEs are a persistent feature of the V. cholerae mobilome that are adapted to providing protection from a single predatory phage and advance our understanding of how phages influence pathogen evolution.

A small, microRNA-size, ribonucleic acid regulating gene expression and development of Shiga toxin-converting bacteriophage Φ24Β.

A microRNA-size (20-nt long) molecule has been identified in Escherichia coli after induction of Shiga toxin-converting bacteriophage Φ24B. This small RNA, named 24B_1, is encoded in the lom-vb_24B_43 region of the phage genome, and apparently it is produced by cleavage of a larger transcript. A phage devoid of 24B_1 revealed decreased efficiency of lysogenization, quicker prophage induction after provoking the SOS response, higher efficiency of progeny phage production during the lytic cycle and less efficient adsorption on the host cells. Expression of most of phage genes was drastically increased after infection of E. coli by the Φ24BΔ24B_1 phage. Since 24B_1 may impair expression of the d_ant gene, coding for an anti-repressor, these results may explain the mechanism of regulations of the physiological processes by this small RNA due to impaired activity of the cI repressor and changed expression of vast majority of phage genes. To our knowledge, this is the first example of functional microRNA-size molecule in bacterial cells.

Differential efficiency of induction of various lambdoid prophages responsible for production of Shiga toxins in response to different induction agents

Shiga toxin-producing Escherichia coli (STEC) is a group of pathogenic strains responsible for bloody diarrhea and hemorrhagic colitis, with often severe complications. Shiga toxins are the main factors causing the phathogenicity of STEC. Production of these toxins depends on the presence of stx1 and stx2 genes, which are located on lambdoid prophages, and their expression is stimulated upon prophage induction. Therefore, a transition of the phage genome from the prophage state to an extrachromosomal genetic element, and its further propagation, is crucial for the pathogenic effects. However, our knowledge on specific conditions for induction of these prophages in bacteria occurring in human intestine is very limited. In this report we present results of our studies on five different phages, originally occurring in STEC strains, in comparison to bacteriophage lambda. We found that efficiencies of induction of prophages and their further development vary considerably in response to different induction agents. Moreover, efficiency of progeny phage production might be modulated by other factors, like temperature or bacterial growth rate. Therefore, it is likely that pathogenicity of different STEC strains may be significantly different under specific conditions in their natural habitats.

Evidence for the Intense Exchange of MazG in Marine Cyanophages by Horizontal Gene Transfer

BackgroundS-PM2 is a phage capable of infecting strains of unicellular cyanobacteria belonging to the genus Synechococcus. S-PM2, like other myoviruses infecting marine cyanobacteria, encodes a number of bacterial-like genes. Amongst these genes is one encoding a MazG homologue that is hypothesized to be involved in the adaption of the infected host for production of progeny phage.Methodology/Principal FindingsThis study focuses on establishing the occurrence of mazG homologues in other cyanophages isolated from different oceanic locations. Degenerate PCR primers were designed using the mazG gene of S-PM2. The mazG gene was found to be widely distributed and highly conserved among Synechococcus myoviruses and podoviruses from diverse oceanic provinces.Conclusions/SignificanceThis study provides evidence of a globally connected cyanophage gene pool, the cyanophage mazG gene having a small effective population size indicative of rapid lateral gene transfer despite being present in a substantial fraction of cyanophage. The Prochlorococcus and Synechococcus phage mazG genes do not cluster with the host mazG gene, suggesting that their primary hosts are not the source of the mazG gene.

Phage as a diagnostic – the use of phage in TB diagnosis

AbstractRapid TB diagnosis and drug susceptibility testing is limited by the slow growth of the causative organism, Mycobacterium tuberculosis (MTB). Effective and rapid methods for the detection and drug susceptibility testing of TB, that are suitable for implementation in low income countries, which bear the highest TB burden, are urgently needed for effective identification, treatment and control of the disease. Mycobacteriophages have the potential to become useful tools in the diagnosis of TB, as they are specific for mycobacteria and only replicate in, and hence detect, viable cells. Phage‐based techniques involve simple manual manipulations that typically require little capital expense and yield results rapidly. Two techniques have been developed recently which use mycobacteriophage for the antibiotic susceptibility testing and direct diagnosis of TB from sputum. Luciferase Reporter Phages (LRP) are recombinant phages that contain the gene for the fire‐fly luciferase enzyme. Upon infection of mycobacteria by these phages, this enzyme is produced within a cell. In the presence of this enzyme, light is produced by viable cells that also contain the substrates for the enzyme: ATP and luciferin (added exogenously). The ‘Bronx Box’ has been developed as a low cost means of detecting light signal from mycobacteria infected with LRP using photographic film. A MTB strain's susceptibility to an antibiotic can be assessed by comparing light production, as an indication of the presence of viable cells, in samples pre‐incubated in the presence and absence of the test antibiotic. Susceptibility testing of some of the front line drugs for TB chemotherapy has given excellent results (detection of all resistant isolates), using LRP and the Bronx Box. Phage amplification uses the production of progeny phage, instead of a reporter gene product, to reflect the presence of target bacteria. The technique has been successfully applied to drug susceptibility testing and for diagnosis to the TB from sputum specimens. Viable MTB cells within a specimen are uniquely infected by a bacteriophage reagent. A potent selective virucide destroys all the phage particles that remain outside the cells while remaining inactive against the MTB bacilli (and the phages that have infected them). After the virucide has been neutralised, and phages that are released from infected cells are indicative of TB cells being present in the original specimen. These phages can be easily detected by propagation on lawns of non‐pathogenic rapidly growing mycobacteria. A commercially available test, termed FASTPlaqueTB‐RIF, determines the rifampicin susceptibility of isolates by comparing the viability of cells pre‐treated with and without rifampicin. Results give excellent agreement (97–98% overall) with conventional ‘gold standard’ methods, but in 2 days rather than 3 weeks. Preliminary work has shown that it should be possible to reduce the assay time to 24 h. A variation to this method, FASTPlaqueTB, detects TB directly from sputum specimens and is capable of rapidly reporting the presence of viable MTB cells within 48 h. This technique has achieved excellent specificity (close to 100%) when evaluated in the clinical setting.© 2001 Society of Chemical Industry

Effect of Cadmium on the Infection of Lactobacillus lactis by Bacteriophage LL-H

The infectivity of Lactobacillus lactis bacteriophage LL-H was shown to be calcium-dependent. Of 10 different divalent cations screened, cadmium specifically decreased the infectivity of LL-H in the presence of calcium. At 1 to 2 mM, CdCl2 resulted in a decrease of the burst size of about 2.5- to 4-fold. Cd2+ was shown to reduce specifically the level of total phage DNA synthesis, resulting in a reduced progeny phage yield. Moreover, Cd2+ had the most profound irreversible effect on progeny phage production between 20 and 60 min after LL-H infection. This paralleled the beginning of phage DNA synthesis. Possible modes of action of Cd2+ on phage DNA replication are discussed.

Induction of prophage λ does not require full induction of RecA protein synthesis

In mitomycin C-treated lambda lysogens, even though the rate of synthesis of RecA protein was greatly reduced by a low concentration of rifampicin (4 microgram/ml), induction of prophage lambda occurred readily as assessed by (i) cell lysis of the lysogens, (ii) production of progeny phage, and (iii) extensive cleavage of lambda repressor. The extent and the rate of cleavage of lambda repressor were not significantly affected by the low rate of synthesis of RecA protein resulting from rifampicin action. However, the yield of phage progeny was reduced and lysis of the cells was slightly delayed. We conclude that in RecA+ bacteria, induction of prophage lambda does not require full induction of RecA protein synthesis.

The role of bacteriophage T7 gene 2 protein in DNA replication.

The in vivo function of the gene 2 protein of bacteriophage T7 has been examined. The gene 2 protein appears to modulate the activity of the gene 3 endonuclease in order to prevent the premature degradation of any newly-formed DNA concatemers. This modulation is not however a direct interacton between the two proteins. In single-burst experiments rifamycin can substitute for the gene 2 protein, allowing formation of fast-sedimenting replicative DNA intermediates and progeny phage production. This suggests that the sole function of the gene 2 protein is inhibition of the host RNA polymerase and that the latter enzyme directs or promotes the endonucleolytic action of the gene 3 protein.

Accumulation of bacteriophage T7 head-related particles in an Escherichia coli mutant

Upon infection with bacteriophage T7, a newly isolated mutant strain of Escherichia coli, Y49, produces T7-specific macromolecules including DNA almost normally. However, concatemeric T7 progeny DNA molecules, synthesized in Y49 cells, are later cleaved abnormally, resulting in an accumulation of DNA molecules shorter in size than the T7 genome and a poor production of progeny phage (Y. Yamada, J. Silnutzer, and D. Nakada, J. Mol. Biol. 121:95-111, 1978). The abnormal cutting of concatemeric T7 DNA in Y49 cells is accompanied by a simultaneous accumulation of large amounts of two types of phage head-related particles, proheads and newly found "X particles." Lysates from normal T7 infection of parental cells also contain X particles, although to a lesser amount. Electron microscopic examination of phage head-related particles (i.e., proheads, X particles, and empty heads), gel electrophoretic analysis of proteins in these particles, and kinetic studies on the appearance and fate of these particles suggest that X particles are likely to be intermediary structures between proheads and phage heads probably derived from proheads during the process of T7 DNA packaging. Our data also suggest that empty heads are not precursors to phage heads but are derived from proheads as by-products probably due to an abortive attempt to package T7 DNA. The host mutation in Y49 strain appears to block a step of T7 DNA processing and packaging pathway after generation of X particles from proheads.

Control of protein synthesis in Escherichia coli: control of bacteriophage Q beta coat protein synthesis after energy source shift-down

Escherichia coli Q13 was infected with bacteriophage Q beta and subjected to energy source shift-down (from glucose-minimal to succinate-minimal medium) 20 min after infection. Production of progeny phage was about fourfold slower in down-shifted cultures than in the cultures in glucose medium. Shift-down did not affect the rate of phage RNA replication, as measured by the rate of incorporation of [14C]uracil in the presence of rifampin, with appropriate correction for the reduced entry of exogenous uracil into the UTP pool. Phage coat protein synthesis was three- to sixfold slower in down-shifted cells than in exponentially growing cells, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The polypeptide chain propagation rate in infected cells was unaffected by the down-shift. Thus, the reduced production of progeny phage in down-shifted cells appears to result from control of phage protein synthesis at the level of initiation of translation. The reduction in the rate of Q beta coat protein synthesis is comparable to the previously described reduction in the rate of synthesis of total E. coli protein and of beta-galactosidase, implying that the mechanism which inhibits translation in down-shifted cells is neither messenger specific nor specific for 5' proximal cistrons. The intracellular ATP pool size was nearly constant after shift-down; general energy depletion is thus not a predominant factor. The GTP pool, by contrast, declined by about 40%. Also, ppGpp did not accumulate in down-shifted, infected cells in the presence of rifampin, indicating that ppGpp is not the primary effector of this translational inhibition.

Mutation in bacteriophage T3 affecting host cell lysis

Amber mutants of T3 phage defective in cell lysis were isolated. The mutational sites involve a new gene (called lys) between gene 17 and gene 19. Su − host cells infected with the mutant fail to lyse beyond the normal latent period though lysozyme is normally produced. Progeny phage production continues a little longer in mutant infected cells than in wild-type infected cells.

Heterologous transfection with bacteriophage ΦX174 DNA An improved system

A highly efficient and much more reproducible system for the heterologous transfection of several kinds of Gram-negative bacterial spheroplasts with bacteriophage ΦX174 DNA was established. By mild washing of the spheroplasts, the efficiency of transfection of all non-host heterologous bacterial species tested increased one or more orders of magnitude in producing the progeny phages and/or the infectious intermediates. Using the improved heterologous transfection systems, it has become clearer that a strong suppression system operates on the processes of ΦX174 progeny phage production and not on those of ΦX174 double-stranded replicative form DNA synthesis in the heterologous bacterial cells. Similar stimulatory effects of this washing procedure were observed in the homologous transfection. With this improved assay system, even less than 100 molecules of phage ΦX174 DNA can be detected and the number of molecules can be determined with accuracy.

Transfection of non-host bacterial spheroplasts with bacteriophage phi chi 174 DNA.

BACTERIOPHAGE ΦX174 containing circular single-stranded DNA (SS-DNA) can infect Escherichia coli C and Shigella1–4 Progeny phages are produced through synthesis of circular double-stranded DNA (replicative form, RF-DNA) as the intermediate5,6. ΦX174 cannot infect bacteria which have no specific receptors for phage adsorption on the cell wall. The host range in ΦX infection is thought to be determined primarily by success or failure of adsorption and if most of the cell wall is removed from the bacterium, the host range is extended and ΦX-DNA can infect E. coli strains other than the host, and produce progeny phages. But if the recipient bacterial spheroplasts are of species distantly related to the host, ΦX-DNA may not be able to penetrate into the spheroplasts and replicate and produce progeny phages as well as in the host spheroplast. It is known that ΦX phage replication in E. coli depends largely on host functions; for example, the ΦX-DNA replication is greatly affected by several genes responsible for host DNA synthesis7,8. On this basis, ΦX-DNA replication in bacteria distantly related to the natural host might on the one hand fail because of incompatibilities between ΦX-DNA and bacterial gene functions or on the other hand be successful because common gene functions are widespread throughout a wide range of bacterial species. We tried to transfect ΦX-DNA to various bacteria belonging to the Enterobacteriaceae, and Pseudomonas aeruginosa to examine the capability for phage production in bacteria other than the host, and found that ΦX-RF-DNA synthesis proceeded as efficiently as in the natural host in distantly related species of bacteria, but that progeny phage production was greatly reduced. Bacterial relationships determined on the basis of DNA homology.

Effect of nalidixic acid on PBS2 bacteriophage infection of Bacillus subtilis.

Infection of Bacillus subtilis by PBS2 phage, whose DNA contains uracil instead of thymine, is relatively unaffected by low concentrations of nalidixic acid which severely inhibit B. subtilis DNA synthesis. High concentrations of nalidixic acid do inhibit PBS2 DNA synthesis, but more severely reduce the burst size of PBS2 infections. Hydroxyurea blocks PBS2 DNA synthesis, preventing progeny phage production.

Resistance of bacteriophage PBS2 infection to rifampicin, an inhibitor of [formula omitted] RNA synthesis

The induction of viral enzymes, the synthesis of DNA, and the production of progeny phage during infection of Bacillus subtilis by bacteriophage PBS2 are all unaffected by the presence of rifampicin. Rifampicin, rifamycin SV, streptovaricin, and streptolydigin (all of which inhibit B . subtilis RNA synthesis by binding to DNA-dependent RNA polymerase) have no effect on PBS2 infection. However, actinomycin D and Lucanthone (which inhibit DNA-dependent RNA synthesis by binding to DNA) both prevent PBS2 replication. Therefore, PBS2 phage may utilize a rifampicin-resistant RNA polymerase to transcribe its uracil-containing DNA during phage infection.

Enlargement of Escherichia coli after bacteriophage infection. I. Description of the phenomenon.

Escherichia coli B/r and B(s-1) ceased division and increased in mean cell volume soon after infection with T-even phage. The effect was obtained with wild-type or rapid lysis mutants, as well as with ultraviolet light-killed phage and with bacteriophage ghosts which lack deoxyribonucleic acid. The cell response did not require the presence of phage genetic material or the production of progeny phage. A Poisson distribution of the fraction of adsorbed phage at different multiplicites of infection indicates that one phage per bacterium will produce maximum increase in cell volume. T-even phage-resistant E. coli mutants showed no enlargement response, and phage T1, T3, and T7 elicited neither abrupt termination of cell division nor host cell enlargement. Infection with baseplate-defective T4D 12(-) amber mutants, which bind reversibly to but do not penetrate the bacterium, also had no effect. In vitro restoration of normal baseplate function in these defective viruses allowed phage adsorption and penetration and caused host cell division arrest and enlargement. These findings indicate that arrest of division and increase in mean cell volume occur together when a sensitive strain of E. coli is infected with T-even phage that adsorb and penetrate normally.

Greater vulnerability of the infecting viral strand of replicative-form deoxyribonucleic acid of bacteriophage phi X174.

Four types of phiX-infected cells of Escherichia coli CR, a thymine-requiring strain of E. coli C, were prepared in which the parental replicative-form deoxyribonucleic acid (RF DNA) was labeled with same specific amounts of bromouracil in (i) both strands, (ii) only the infecting viral strand, (iii) only the complementary strand, and (iv) neither strand. The sensitivity of each type of infected cell toward irradiation by ultraviolet light, visible light, and X rays was measured. The results indicate that a certain amount of radiation damage in the infecting viral strand of the parental RF was more inhibitory to the production of progeny phage than when the damage was in the complementary strand. Similar conclusions were also drawn from "suicide" experiments of the phage-infected complexes containing (32)P of the same specific activity on either strand of the parental RF DNA. The results suggest that the beta decay occurring in the infecting viral strand was more effective in inactivating the plaque-forming ability of the complex.

Structural alteration of intracellular φX174 DNA following infection of host cells in which protein synthesis is inhibited

The structure of the intracellular form of φX174 DNA was studied in cells treated to inhibit protein synthesis before infection. Inhibition of protein synthesis was accomplished in Escherichia coli 15 THU by 90 minutes incubation in medium lacking histidine and uracil, and in E. coli THU or HF4704 by addition of 135 μg chloramphenicol/ml. five minutes before infection. φXDNA containing parental label was isolated from treated cells. Its sedimentation rate in a neutral high salt gradient was 5% faster than RFI † † Abbreviations used: RF, replicating form; BNC, benzoyl naphthoyl DEAE-cellulose. , and the buoyant density in neutral cesium chloride was more broadly distributed and somewhat denser than RFI. When the intracellular parental labeled DNA was fractionated by chromatography on benzoyl naphthoyl DEAE-cellulose 10 to 20% chromatographed as RFI or II, while 80 to 90% was tightly bound and could only be eluted by 1.0 m-NaCl plus 2% caffeine. The tightly bound DNA was infective in the spheroplast assay, but infectivity was lost after denaturation. In alkaline CsCl equilibrium gradients the parental label was found in the position of viral DNA but no infectivity was found in the region of viral or complementary strand. Hybrid ( 13C, 15N parental: 12C, 14N post-infection) DNA had a lower buoyant density than normal hybrid RF. Parental strands sedimented slower than viral DNA on alkaline CsCl gradients with a peak around ~ 8 s. Post-infection DNA had a heterogeneous sedimentation distribution in alkali, but a majority of the counts sedimented faster than viral DNA. Most of the parental label was membrane associated, and all infected cells were able to contribute to initial progeny phage production. The structural and biological significance of these findings is discussed.

Variations in genetic recombination due to amber mutations in T4D bacteriophage.

Recombination experiments were performed to assess the affect of amber mutations in 12 genes of T4D bacteriophage on genetic recombination. Crosses were performed in various suppressor-containing bacterial hosts to permit the production of progeny phage. Amber mutations in genes 32, 46, and 47 caused decreased recombination, amber mutations in genes 30, 41, 42, 43, 56, 61, and 62 caused increased recombination, whereas mutations in genes 63 and 37 showed no demonstrable effect on recombination.