Phage Synthesis Research Articles

Synthetic phage-based approach for sensitive and specific detection of Escherichia coli O157

Escherichia coli O157 can cause foodborne outbreaks, with infection leading to severe disease such as hemolytic-uremic syndrome. Although phage-based detection methods for E. coli O157 are being explored, research on their specificity with clinical isolates is lacking. Here, we describe an in vitro assembly-based synthesis of vB_Eco4M-7, an O157 antigen-specific phage with a 68-kb genome, and its use as a proof of concept for E. coli O157 detection. Linking the detection tag to the C-terminus of the tail fiber protein, gp27 produces the greatest detection sensitivity of the 20 insertions sites tested. The constructed phage detects all 53 diverse clinical isolates of E. coli O157, clearly distinguishing them from 35 clinical isolates of non-O157 Shiga toxin-producing E. coli. Our efficient phage synthesis methods can be applied to other pathogenic bacteria for a variety of applications, including phage-based detection and phage therapy.

PHEIGES: all-cell-free phage synthesis and selection from engineered genomes

Bacteriophages constitute an invaluable biological reservoir for biotechnology and medicine. The ability to exploit such vast resources is hampered by the lack of methods to rapidly engineer, assemble, package genomes, and select phages. Cell-free transcription-translation (TXTL) offers experimental settings to address such a limitation. Here, we describe PHage Engineering by In vitro Gene Expression and Selection (PHEIGES) using T7 phage genome and Escherichia coli TXTL. Phage genomes are assembled in vitro from PCR-amplified fragments and directly expressed in batch TXTL reactions to produce up to 1011 PFU/ml engineered phages within one day. We further demonstrate a significant genotype-phenotype linkage of phage assembly in bulk TXTL. This enables rapid selection of phages with altered rough lipopolysaccharides specificity from phage genomes incorporating tail fiber mutant libraries. We establish the scalability of PHEIGES by one pot assembly of such mutants with fluorescent gene integration and 10% length-reduced genome.

Inactivation of the Autolysis-Related Genes lrgB and yycI in Staphylococcus aureus Increases Cell Lysis-Dependent eDNA Release and Enhances Biofilm Development In Vitro and In Vivo.

Staphylococcus aureus ica-independent biofilms are multifactorial in nature, and various bacterial proteins have been associated with biofilm development, including fibronectin-binding proteins A and B, protein A, surface protein SasG, proteases, and some autolysins. The role of extracellular DNA (eDNA) has also been demonstrated in some S. aureus biofilms. Here, we constructed a Tn551 library, and the screening identified two genes that affected biofilm formation, lrgB and yycI. The repressive effect of both genes on the development of biofilm was also confirmed in knockout strains constructed by allelic recombination. In contrast, the superexpression of either lrgB or yycI by a cadmium-inducible promoter led to a decrease in biofilm accumulation. Indeed, a significant increase in the cell-lysis dependent eDNA release was detected when lrgB or yycI were inactivated, explaining the enhanced biofilm formed by these mutants. In fact, lrgB and yycI genes belong to distinct operons that repress bacterial autolysis through very different mechanisms. LrgB is associated with the synthesis of phage holin/anti-holin analogues, while YycI participates in the activation/repression of the two-component system YycGF (WalKR). Our in vivo data suggest that autolysins activation lead to increased bacterial virulence in the foreign body animal model since a higher number of attached cells was recovered from the implanted catheters inoculated with lrgB or yycI knockout mutants.

Phage Wrapping with Cationic Polymers Eliminates Nonspecific Binding between M13 Phage and High pI Target Proteins

M13 phage have provided scaffolds for nanostructure synthesis based upon self-assembled inorganic and hard materials interacting with phage-displayed peptides. Additionally, phage display has been used to identify binders to plastic, TiO(2), and other surfaces. However, synthesis of phage-based materials through the hybridization of soft materials with the phage surface remains unexplored. Here, we present an efficient "phage wrapping" strategy for the facile synthesis of phage coated with soluble, cationic polymers. Polymers bearing high positive charge densities demonstrated the most effective phage wrapping, as shown by assays for blocking nonspecific binding of the anionic phage coat to a high pI target protein. The results establish the functional group requirements for hybridizing phage with soft materials and solve a major problem in phage display-nonspecific binding by the phage to high pI target proteins.

Nitric oxide inhibits Shiga-toxin synthesis by enterohemorrhagic Escherichia coli.

Shiga-toxin (Stx) is the cardinal virulence factor of enterohemorrhagic Escherichia coli (EHEC). The genes encoding Stx are carried by a lambdoid phage integrated in the bacterial genome and are fully expressed after a bacterial SOS response induced by DNA-damaging agents. Because nitric oxide (NO) is an essential mediator of the innate immune response of infected colonic mucosa, we aimed to determine its role in Stx production by EHEC. Here we demonstrate that chemical or cellular sources of NO inhibit spontaneous and mitomycin C-induced stx mRNA expression and Stx synthesis, without altering EHEC viability. The synthesis of stx phage is also reduced by NO. This inhibitory effect apparently occurs through the NO-mediated sensitization of EHEC because mutation of the NO sensor nitrite-sensitive repressor results in loss of NO inhibiting activity on stx expression. Thus our findings identify NO as an inhibitor of stx expressing-phage propagation and Stx release and thus as a potential protective factor limiting the development of hemolytic syndromes.

Assembly of an RNA-Protein Complex: BINDING OF NusB AND NusE (S10) PROTEINS TO boxA RNA NUCLEATES THE FORMATION OF THE ANTITERMINATION COMPLEX INVOLVED IN CONTROLLING rRNA TRANSCRIPTION IN ESCHERICHIA COLI

Analytical ultracentrifugation and fluorescence anisotropy methods have been used to measure the equilibrium parameters that control the formation of the core subcomplex of NusB and NusE proteins and boxA RNA. This subcomplex, in turn, nucleates the assembly of the antitermination complex that is involved in controlling the synthesis of ribosomal RNA in Escherichia coli and that also participates in forming the N protein-dependent antitermination complex in lambdoid phage synthesis. In this study we determined the dissociation constants (K(d) values) for the individual binary interactions that participate in the assembly of the ternary NusB-NusE-boxA RNA subassembly, and we showed that multiple equilibria, involving both specific and nonspecific binding, are involved in the assembly pathway of this protein-RNA complex. The measured K(d) values were used to model the in vitro assembly reaction and combined with in vivo concentration data to simulate the overall control of the assembly of this complex in E. coli at two different cellular growth rates. The results showed that at both growth rates assembly proceeds via the initial formation of a weak but specific NusB-boxA complex, which is then stabilized by NusE binding. We showed that NusE also binds nonspecifically to available single-stranded RNA sequences and that such nonspecific protein binding to RNA can help to regulate crucial interactions in the assembly of the various macromolecular machines of gene expression.

Synthesis of bacteriophage ϕX174 in vitro: Mechanism of switch from DNA replication to DNA packaging

Replication of a replicative form DNA of bacteriophage ϕX174 initiates by rolling-circle synthesis of the viral DNA followed by discontinuous synthesis of the complementary DNA. Gene C protein of ϕX174, which is involved in DNA packaging, inhibits the rolling-circle DNA synthesis by binding to the initiation complex in vitro. The gene C protein-associated initiation complex can synthesize and package the viral DNA to produce infectious phage when supplemented with ϕX174 gene J protein and the prohead. Multiple rounds of phage synthesis occur without dissociation of the gene C protein from the complex. These results indicate that gene C protein is central in the switch from replication of a replicative form DNA to synthesis and concomitant packaging of viral DNA into phage capsid, which occurs in the late stage of infection.

Morphogenesis of phi X174: in vitro synthesis of infectious phage from purified viral components.

An in vitro system that synthesizes infectious phage phi X174 was developed. The synthesis depended on phi X174 supercoiled replicative form DNA, purified phi X174 gene A protein, gene C protein, gene J protein, prohead (phage head precursor composed of gene F, G, H, B, and D proteins), and uninfected host crude extract. The infectious phage synthesis was coupled with DNA synthesis. De novo initiation, elongation, and termination of phi X174 single-stranded DNA was observed. The phage synthesized in vitro cosedimented with in vivo phage in sucrose gradients and had the same buoyant density as in vivo phage in a CsCl gradient. Our results indicate that the in vitro system mimics the in vivo phi X174 assembly process.

Superinfection immunity and prophage repression in phage P1 and P7 III. Induction by virulent mutants

Two types of virulent mutants ( virB and virC) have been isolated for phage Pl; both induce a Pl prophage to replicate after superinfection of a lysogen. The genetic lesion of P1 virB (and P7 virB) is located in the immunity-specific region of the phage, and these mutants induce only a homoimmune prophage. The virB mutants appear to depend for prophage induction on titration of the phage-specific c4 immunity repressor in the lysogen, since DNA synthesis of the superinfecting phage is needed for this induction process. The P1 virC mutation, which is located at the left end of the phage map, induces both homoimmune and heteroimmune prophages. The virC mutant causes prophage induction in the absence of phage DNA replication and may induce because of constitutive synthesis of a vegetative repressor ( c1) antagonist.

Stability of the carrier state in bacteriophage M13-infected cells

Bacteriophage M13-infected carrier cells were shown to be unstable to prolonged growth under all conditions. Carrier Hfr cells were transferred in dilute culture (10(3) to 10(4)/ml), where reinfection was impossible and the physiology of the cell was minimally altered. After an initial period of about 10 generations, during which all cells in the culture remained infected, there was exponential decay in the proportion of infected cells in the culture. Uninfected cells that appeared were M13 sensitive. Hfr and F' males were also transferred serially at high cell densities (10(7) to 10(9)/ml), where high levels of phage should permit reinfection. The proportion of phage-producing cells in the cultures remained constant for 7 to 15 generations and then dropped exponentially on further growth. Non-phage-producing cells appearing in the culture were refractory to infection by M13; in some cases cells scored as non-phage producers for 20 generations were observed to produce phage on further growth in liquid culture. F'trp+ males infected with M13 lost trp+ function almost immediately; this was not regained in these experiments. Infected cells grown in dilute culture or on plates remained infected longer, produced more PFU per cell for a longer period, and retained trp+ function in F'trp+ males for over 90 generations. Non-phage-producing cells that appeared were sometimes phage resistant, sometimes phage sensitive. The existence of a phage-related material accumulating at high cell densities and affecting expression of free episomes, episomal expression in Hfr males, and phage synthesis itself is suggested.

Synthesis of phage- and host-specific RNA in Escherichia coli infected with a fragment of bacteriophage T5

Escherichia coli F bacteria were infected with an amber mutant of phage T5, which injects only 8% (the pre-early genes) of its genome into the host cell. This system allows study of the simultaneous transcription of host and phage DNA for an extended period of time. With increasing multiplicity of infection, the fraction of phage-specific RNA increases from 0.03 at m.o.i. = 1 to 1.0 m.o.i. →∞, while the fractional synthesis of bacterial RNA decreases correspondingly. This indicates that the pre-early T5 promoters compete with bacterial promoters for RNA polymerase. With increasing multiplicity, the synthesis rates of host mRNA and stable RNA (rRNA and tRNA) decrease in constant proportions. At low multiplicities, when the synthesis of phage RNA is proportional to the number of infecting phage, the initiation frequency at the average pre-early T5 promoter is 30 RNA chains/min; this promoter activity is comparable to that of bacterial rRNA promoters.

The role of Escherichia coli dna A gene and its integrative suppression in M13 coliphage DNA synthesis.

An F+ derivative of Escherichia coli E508 thermosensitive in dna A function (involved in DNA synthesis initiation), its revertant and an Hfr derivative of E508 (ts) in which the temperature-sensitive phenotype is suppressed by integrative suppression have been compared for their ability to support M13 phage DNA synthesis at the nonpermissive temperature. Upon infection at the nonpermissive temperature, both the revertant and the Hfr strain support normal phage replication while the temperature-sensitive mutant does not. However, when infection is carried out at a permissive temperature and the temperature is shifted up after infection, phage synthesis occurs in the temperature-sensitive mutant also, but in lesser quantity than in the revertant strain. Analysis of intracellular labeled phage DNA indicates: (a) parental replicative form DNA synthesis is not dependent on dna A function; (b) progeny replicative form DNA synthesis is strongly inhibited in the temperature-sensitive dna A mutant at the nonpermissive temperature; (c) progeny single-strand DNA synthesis does not absolutely require dna A function; (d) progeny single-strand DNA is present in the circular form. The implication of the host DNA replication in M13 DNA synthesis is discussed.

T4 DNA injection. I. Growth cycle of a gene 2 mutant.

Abstract When gene 2 amber mutants of T4 infect su − cells, whole particles are produced (2.Su − ) which appear normal in electron micrographs. 2.Su − particles plate less than 0.5% as efficiently as wild type phage on su + hosts. These particles adsorb, contract, and kill their host normally. Up to 40% of 2.Su − DNA is rapidly degraded to acid-soluble fragments after infection of Escherichia coli B, E. coli CR63 rgl − and E. coli B 40 SuI. 2.Su − particles provide gene products for in vivo complementation only 10–20% as efficiently as 2.Su + phage. 2.Su − DNA is glucosylated and unnicked inside the phage head. Morphogenesis of 2.Su − progeny is delayed and the burst of 2.Su − phage is decreased compared to wild-type infections. Thus, the absence of gene 2 product during phage synthesis affects both morphogenesis and particle infectivity. 2.Su − particles are defective at an early stage of infection, probably after DNA ejection and before gene expression.

A genetic analysis of bacteriophage λ head assembly

The sequence of steps involved in bacteriophage λ head assembly has been studied by characterizing second-site mutatons that can compensate for the reduced level of synthesis of a particular phage “head” protein. Such studies reveal these classes of protein interactions: (1) A reduced level of either pA synthesis or activity can compensate for the reduced amount of pD made when λD am mutants are grown in a supC host. This result implies that pA acts before pD during the packaging of phage DNA. (2) A reduced level of either pB synthesis or activity can compensate for the reduced amount of pE made when λE am mutants are grown in a supC host, and vice versa. This result suggests an interaction between these proteins that is stringently dependent on maintaining a proper ratio between them. An identical argument has been made to explain the properties of groE hosts (Sternberg, 1973). (3) A very specific class of mutations in phage gene E can compensate for the reduced amount of pC made when λC am mutants are grown in a supC host. This suggests that the products of these two genes directly interact. Using the techniques described in this report it is possible to easily isolate a variety of mutations ( am, oc, ts, and missense) in specific phage “head” genes (genes A, B and E).

Infection of Synechococcus cedrorum by the cyanophage AS-1M : I. Ultrastructure of infection and phage assembly

The replication of cyanophage ȦS-1M after infection of the blue-green alga Synechococcus cedrorum was monitored by electron microscopy. One-step growth and intracellular growth experiments were performed at high multiplicity of infection (m.o.i.) and the basic characteristics of phage growth determined. The eclipse period lasts until 3–4 hr after infection, the latent period continues until 8 hr, and complete lysis of the culture terminates about 12 hr after infection. The average burst size at high m.o.i. was about 40 plaque-forming units (PFU) per cell. Samples for electron microscopy were taken at 2, 4, 6, 8 and 10 hr after infection. The first noticeable evidence of phage infection was a degradation of the host nucleoplasm, which was manifest as a distinct lightening of the nucleoplasm and aggregation of ribosomes near the photosynthetic lamellae. All further phage synthesis takes place within the nucleoplasm. At 4 hr after infection, a large amount of fibrillar material, presumably the vegetative phage DNA, is evident. Beginning at this time, but particularly at 6 hr after infection, many phage precursors in the process of assembly can be seen; these include empty-head vesicles and “helical” structures which appear to be heads in the process of packaging the DNA. Based on the structures seen in the EM, we speculate that empty heads form first and are then filled with DNA. The completed capsid is then joined to the tail.

Bacteriophage SPO1 DNA- and RNA-directed protein synthesis in vitro: comparison with in vivo control.

A cell free protein synthesizing system, derived from E. coli, is shown to be a quantitiative assay system for messenger RNA extracted from B. subtilis infected with bacteriophage SPO1. DNA-directed protein synthesis in this system is shown to be limited mostly to those proteins whose messages are contained in early RNA. A phage induced enzyme, dCMP deaminase, is shown to be dependent on appearance of a class mRNA made in vivo in response to new initiations of transcription dependent on prior synthesis of phage induced protein. Control of the enzyme synthesized in the cell free system is contrasted with in vivo control, and an estimate of "read-through" by RNA polymerase in vitro is presented.

Cold-Sensitive Mutants of Bacteriophage φX174. II. Comparison of Two Cold-Sensitive Mutants

Cold-sensitive bacteriophage phiX174 mutants, another class of conditional lethals, were examined with regard to growth parameters, DNA synthesis, and particle properties. Two mutants, cs70 and cs82, were examined. Mutant cs70 was eclipse defective, showing altered eclipse kinetics at permissive temperature (40 C) and failing entirely to eclipse at restrictive temperature (25 C). Mutant cs70 replicated well at 25 C if allowed prior eclipse at 40 C. Mutant cs82 had wild-type eclipse at both temperatures but was defective in single-strand synthesis at 25 C, which led to delayed progeny phage appearance, decreased progeny phage synthesis rate, and greatly reduced burst size. The cs82 block could not be bypassed by temperature shift. Since complementation analysis of cs70 and cs82 was not feasible due to the unique properties of these mutants, those phiX174 properties affected by the virus coat were examined as an index of a mutation in a coat protein gene. Mutant cs70 had aberrant attachment kinetics at both 25 C and 40 C, evidence of a coat protein alteration. Mutant cs70 also exhibited significantly decreased thermal stability, further evidence of an altered virus structure. Mutant cs82 had increased thermal stability, but the difference was not sufficient to allow unequivocal assignment of this mutant to a coat protein gene. Both mutants had wild-type antiserum inactivation and host range, although cs70 was subject to less of (low-level) plating restriction by endogenous F(+) factors.

Transcriptional control of M13 phage DNA replication.

M13 belongs to the group of small filamentous coliphages containing single- stranded circular DNA of 2 × 106 molecular weight (1). The phage does not kill the host but leaks out of intact and multiplying cells (2,3). Thus it behaves like an autonomously multiplying lytic phage except for lysis of the host. Jaenisch et al. (4) showed that M13 messenger RNA has an unusually long half-life of at least 18 min. On the other hand, Roy (5) showed that rifampicin and actino-mycin D inhibit phage synthesis almost immediately after addition to the infected culture. It appeared reasonable to us to look into the effects of rifampicin on M13 DNA replication in wild-type and rifampicin-resistant strains of Escherichia coli.KeywordsReplicative FormLytic PhageAmino Acid IncorporationUridine IncorporationEquilibrium UltracentrifugationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Replication of Bacteriophage SH-133 in the Facultative Autotroph Hydrogenomonas facilis I. Bacteriophage Synthesis Under Heterotrophic, Autotrophic, and Mixotrophic Growth Conditions

The ability of bacteriophage SH-133 to replicate in heterotrophically (H-) and autotrophically (A-) grown Hydrogenomonas facilis was examined. Both the synthesis of infectious phage particles and the efficiency of plating (EOP) were reduced by 90% in A-grown cells. Adsorption of phage and lethal effects on H. facilis were identical in both systems. One-step growth experiments showed that cell lysis preceded the appearance of infectious particles in A-grown cells. Burst size studies with mixotrophically grown cells did not indicate the presence of an inhibitor of phage synthesis indigenous to autotrophic metabolism. DNA synthesis was identical in H- and A-grown infected cells; however, protein synthesis was significantly reduced in A-grown infected cells when compared with protein synthesis in H-grown infected cells. The data suggest that the reduction in EOP and phage synthesis in A-grown cells is caused by a defect in viral protein synthesis which results in the limited production of an essential viral protein at the time of cell lysis.

An extended phage growth cycle in T4 infected A. aerogenes

Spheroplasts of Aerobacter aerogenes infected with T4 produce about five times more phage than do spheroplasts of E. coli because they do not lyse at the usual time. This implies a role for the host in cessation of phage synthesis. In 1969 Wais and Goldberg reported that cultures of A. aerogenes spheroplasts infected with urea treated T4 phage particles produced more phage than did similarly infected cultures of E. coli spheroplasts. We thought it strange that phage T4 should grow better in spheroplasts of a resistant host than in its usual host. We now report that the A. aerogenes host supports an extended phage growth cycle resulting in the production of 1000 or more phage in singly infected, isolated spheroplasts. Augmented phage production appears to be due to failure of the phage lysis mechanism to lyse the membrane of the A. aerogenes spheroplast.