Phage Trojan horses: a conditional expression system for lethal genes

The EcoRI restriction enzyme (ENase) cleaves DNA molecules within the sequence GAATTC. Cells expressing this lethal activity normally make a second enzyme, the M.EcoRI methyltransferase (MTase), which protects their chromosomal DNA by modifying the EcoRI recognition sites. To isolate mutants of the EcoRI ENase, its gene was cloned into a filamentous phage vector (M13mp18) under control of the lac promoter. Normally, filamentous phages (M13, f1 and their derivatives) form turbid plaques by impairing the growth of their host cell without killing it. In contrast, phages expressing the EcoRI ENase kill the host cell, but survive long enough to produce plaques which are very clear. Expression of the M.EcoRI MTase rescues the host and restores turbid plaque formation. EcoRI ENase mutants were isolated by screening for mutants that make turbid, instead of clear, plaques on an M- host. This conditional expression system may be useful for cloning and mutating genes for other toxic proteins.     

An exocytoplasmic endonuclease with restriction function in Streptomyces antibioticus.

Streptomyces antibioticus produces a strong endo-DNase which is located between the cytoplasmic membrane and the cell wall. All DNA substrates assayed, including the chromosomal DNA of this species and several bacteriophage DNAs, were completely degraded in vitro by the enzyme. The rate of synthesis of the nuclease depended on the growth medium. In NBG medium, in which the enzyme is not produced, the size of lytic plaques of several actinophages was larger than that in GYM or GAE medium, in which synthesis of the nuclease takes place late in growth. In addition, one of the phages assayed, phi A6, showed a diminution of its efficiency of plating in GYM medium with respect to that in NBG medium; another phage, phi A9, grew in NBG medium but not in the other two media. It is postulated that the presence of the host nuclease, together with the capability of the particular phage to absorb on S. antibioticus of different growth phases, determines the efficiency of growth and the plaque size of the phages on productive media. This hypothesis was confirmed when the growth of phi A6 and phi A9 in a mutant of S. antibioticus lacking the endonuclease activity was analyzed. It is concluded that the enzyme can assume, under some circumstances, a role in in vivo restriction.     

Lambdoid phages that simplify the recovery of in vitro recombinants

Summary Derivatives of phage λ are described for use as vectors for fragments of DNA generated with theHindIII andEcoRI restriction enzymes. With some vectors, hybrid molecules are recognised by a change from a turbid to a clear plaque morphology resulting from the insertion of a fragment of DNA into the λ gene coding for the phage regressor. Other vectors contain a central, replaceable fragment of DNA which imparts a readily recognisable phenotype. This central fragment may include either a gene for a mutant transfer RNA (suppressor) or a part of thelacZ gene ofE. coli able to complement alacZ host. The appropriatelacZ host and indicator plates permit the ready distinction between recombinant and vector phages by the colour of the plaques.     

Type III restriction is alleviated by bacteriophage (RecE) homologous recombination function but enhanced by bacterial (RecBCD) function

Previous works have demonstrated that DNA breaks generated by restriction enzymes stimulate, and are repaired by, homologous recombination with an intact, homologous DNA region through the function of lambdoid bacteriophages lambda and Rac. In the present work, we examined the effect of bacteriophage functions, expressed in bacterial cells, on restriction of an infecting tester phage in a simple plaque formation assay. The efficiency of plaque formation on an Escherichia coli host carrying EcoRI, a type II restriction system, is not increased by the presence of Rac prophage-presumably because, under the single-infection conditions of the plaque assay, a broken phage DNA cannot find a homologue with which to recombine. To our surprise, however, we found that the efficiency of plaque formation in the presence of a type III restriction system, EcoP1 or EcoP15, is increased by the bacteriophage-mediated homologous recombination functions recE and recT of Rac prophage. This type III restriction alleviation does not depend on lar on Rac, unlike type I restriction alleviation. On the other hand, bacterial RecBCD-homologous recombination function enhances type III restriction. These results led us to hypothesize that the action of type III restriction enzymes takes place on replicated or replicating DNA in vivo and leaves daughter DNAs with breaks at nonallelic sites, that bacteriophage-mediated homologous recombination reconstitutes an intact DNA from them, and that RecBCD exonuclease blocks this repair by degradation from the restriction breaks.     

Transduction of plasmid DNA in Streptomyces spp. and related genera by bacteriophage FP43.

A segment (hft) of bacteriophage FP43 DNA cloned into plasmid pIJ702 mediated high-frequency transduction of the resulting plasmid (pRHB101) by FP43 in Streptomyces griseofuscus. The transducing particles contained linear concatemers of plasmid DNA. Lysates of FP43 prepared on S. griseofuscus containing pRHB101 also transduced many other Streptomyces species, including several that restrict plaque formation by FP43 and at least two that produce restriction endonucleases that cut pRHB101 DNA. Transduction efficiencies in different species were influenced by the addition of anti-FP43 antiserum to the transduction plates, the temperature for cell growth before transduction, the multiplicity of infection, and the host on which the transducing lysate was prepared. FP43 lysates prepared on S. griseofuscus(pRHB101) also transduced species of Streptoverticillium, Chainia, and Saccharopolyspora.     

Development of a gene cloning system for Streptomyces hygroscopicus subsp. yingchengensis, a producer of three useful antifungal compounds, by elimination of three barriers to DNA transfer.

Streptomyces hygroscopicus 10-22 could not be transformed with any of the commonly used Streptomyces plasmid vectors and was resistant to plaque formation by the Streptomyces phages phi C31 and R4. Repeated selection resulted in the isolation of derivatives of S. hygroscopicus 10-22 that could be transformed with pIJ101- and pJV1-derived cloning vectors and of restriction-deficient derivatives that could accept DNA propagated in Streptomyces lividans 66. These new strains, which include three that still produce the original antibiotics, can be used as hosts for gene cloning. Insertion of nonreplicating vectors by homologous recombination and transposition of Tn4560 were demonstrated in S. hygroscopicus 10-22.     

Bacteriophages of Saccharopolyspora erythraea

Five bacteriophages infecting only Saccharopolyspora erythraea (formerly Streptomyces erythreus) among 43 Streptomyces spp. tested were classified into two groups by phage-host relationships, restriction enzyme mapping, cohesive-end determinations, and Southern hybridizations. phi SE6, the most frequently isolated phage, produced clear plaques on all hosts tested, while phi SE45, phi SE57, phi SE60, and phi SE69 produced turbid plaques. phi SE6 DNA was linear, had a molecular weight of (27.6 +/- 1) X 10(6) and, like the DNAs of phi SE45, phi SE57, and phi SE69, lacked cohesive ends. The characteristic patterns of of ClaI and HindIII restriction digests of phi SE6 DNA and the results of Southern hybridizations with three different ClaI fragments of phi SE6 DNA as probes indicated that phi SE6 DNA was partially circularly permuted and terminally redundant, suggesting that it was packaged by a headful packaging mechanism. Southern hybridization data also showed that phi SE45, phi SE57, and phi SE69 were closely related to phi SE6. phi SE60 DNA, in contrast, had cohesive ends, and restriction mapping plus Southern hybridization data showed that phi SE60 was unrelated to the other four phages.     

A site-specific endodeoxyribonuclease from Streptomyces albus CMI 52766 sharing site-specificity with Providencia stuartii endonuclease PstI.

A class II site-specific endodeoxyribonuclease (SalPI) was identified in cell-free extracts of Streptomyces albus CMI 52766 after high speed centrifugation and fractionation through Bio Gel AO.5M. SalPI cleaves lambda DNA into at least 18 fragments. Five cleavage sites were located in the linear lambda map by the use of double and triple restriction enzyme digests involving EcoRI, HindIII, SalGI and another new Streptomyces enzyme, SacI. The results were indistinguishable from those previously obtained for a Providencia stuartii enzyme, PstI, by Smith, Blattner & Davies (Nucleic Acids Res. 1976 3, 343). SalPI and PstI were shown by a double digest test to have the same site specificity. None of 34 phages tested was obviously restricted by S. albus CMI 52766, and correspondingly DNA from two of them was not cleaved in vitro by SalPI. DNA from Streptomyces phage that does not form plaques on S. albus CMI 52766, and plasmid SCP2 DNA from S. coelicolor A3 (2), were both cleaved.     

Characterization of bacteriophage phi C69 of Saccharopolyspora erythraea and demonstration of heterologous actinophage propagation by transfection of Streptomyces and Saccharopolyspora

A bacteriophage, designated phi C69, isolated from a culture of Saccharopolyspora erythraea was characterized. The phage propagates on Sac. erythraea NRRL 2338 but does not infect 10 Streptomyces or 3 Micromonospora species tested. It infects Sac. erythraea NRRL 2359 but does not produce infectious phage particles in this host. phi C69 is approximately 40 kb in length and contains cohesive ends. A cos fragment containing ligated phage DNA ends was cloned in Escherichia coli. Restriction maps of the phage DNA and the cos fragment for several enzymes are shown. Transfection of both Sac. erythraea and Streptomyces lividans with phi C69 resulted in approximately equal titres of infectious phage particles produced from approximately the same number of regenerating cells. Transfection of Sac. erythraea with DNA from Streptomyces phages SH10 and KC404 also resulted in the production of infectious phage particles. The basis for differences among hosts in susceptibility to infection by various actinophages is discussed.     

Characterization of temperate bacteriophages of Leuconostoc oenos and evidence for two prophage attachment sites in the genome of starter strain PSU-1

The close relatedness between 17 Leuconostoc oenos bacteriophages, induced with mitomycin C from strains isolated in different geographic regions, was inferred from their morphology, DNA homology and protein composition. The genome of all the phages had cohesive end termini and ranged in size from 36.4 to 40.9 kb. According to the restriction patterns obtained by digestion with five enzymes, the phages were divided in six groups. Lysogenization of a spontaneous phage-cured derivative of Leuc. oenos strain PSU-1 was achieved with 16 phages and the analysis of the lysogens showed that the phage DNA integrates in the host chromosome in one or two sites. The att B loci were located on the macrorestriction Asc I and Not I fragments of the recipient strain. A survey of Leuc. oenos strains with a phage DNA probe confirmed the lysogenic nature of several, but not all of the original phage hosts. These results are discussed in the light of evidence for the instability of some lysogenic PSU-1 derivatives.     

Isolation and characterization of two types of actinophages infecting Streptomyces scabies MR13

Two types of actinophages, phi S and phi L, were isolated from soil samples by using Streptomyces scabies MR13, a potato scab pathogen, as an indicator strain. The phages were partially characterized according to their physicochemical properties, plaques and particles morphology and their host-range. The host-range of these phages was narrow for phi S and wide for phi L. The adsorption rate constants of the phi S and phi L were 3.44 x 10(-9) and 3.18 x 10(-9) ml/min, and their burst sizes were 1.61 and 3.75 virions, respectively. One-step growth indicated that phi S and phi L have a latent period of 30 min followed by a rise period of 30 min. The temperate character of these phages was tested in other isolates of Streptomyces. Four of the phages (phi SS3, phi SS12, phi SS13 and phi SS17) were identified as temperate phages, since they were able to lysogenize SS3, SS12, SS13 and SS17. phi SS3, phi SS12 and phi SS13 were homoimmune, and they were heteroimmune with respect to phi SS17. The restriction barriers of lysogenic isolates (SS12, SS13 and SS17) interfered with the blockage of plaques formation by phages (phi SS12, phi SS13 or phi SS17) propagated on them, about 75% of lysogenic isolates had restriction systems. The exposure of the lysogenic isolates (SS12, SS13 and SS17) to UV-irradiation prevented the possible restriction barriers of these isolates, and these barriers could be overcome.     

Host Exopolysaccharide Quantity and Composition Impact Erwinia amylovora Bacteriophage Pathogenesis

Erwinia amylovora bacteriophages (phages) belonging to the Myoviridae and Podoviridae families demonstrated a preference for either high-exopolysaccharide-producing (HEP) or low-exopolysaccharide-producing (LEP) bacterial hosts when grown on artificial medium without or with sugar supplementation. Myoviridae phages produced clear plaques on LEP hosts and turbid plaques on HEP hosts. The reverse preference was demonstrated by most Podoviridae phages, where clear plaques were seen on HEP hosts. Efficiency of plating (EOP) was determined by comparing phage growth on the original isolation host to the that on the LEP or HEP host. Nine of 10 Myoviridae phages showed highest EOPs on LEP hosts, and 8 of 11 Podoviridae phages had highest EOPs on HEP hosts. Increasing the production of EPS on sugar-supplemented medium or decreasing production by knocking out the synthesis of amylovoran or levan, the two EPSs produced by E. amylovora, indicated that these components play crucial roles in phage infection. Amylovoran was virtually essential for proliferation of most Podoviridae phages when phage population growth was compared to the wild type. Decreased levan production resulted in a significant reduction of progeny from phages in the Myoviridae family. Thus, Podoviridae phages are adapted to hosts that produce high levels of exopolysaccharides and are dependent on host-produced amylovoran for pathogenesis. Myoviridae phages are adapted to hosts that produce lower levels of exopolysaccharides and host-produced levan.     

SAMase gene of bacteriophage T3 is responsible for overcoming host restriction.

Deletion and point mutants of T3 have been isolated and used to show that the early region of T3 DNA is organized in the same way as that of T7 DNA. Homologous early RNAs and proteins of the two phages have been identified by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Both phages have five early mRNA's, numbered 0.3, 0.7, 1,1.1 and 1.3 from left to right, although no T3 protein that corresponds to the 1.1 protein of T7 has yet been identified. In general, corresponding early RNAs and proteins of the two phages migrate differently on gels, indicating that they differ in molecular weight and/or conformation. In both T7 and T3, gene 0.3 is responsible for overcoming the DNA restriction system of the host, gene 0.7 specifies a protein kinase, gene 1 specifies a phage-specific RNA polymerase, and gene 1.3 specifies a polynucleotide ligase. The 0.3 protein of T3 is responsible for the S-adenosylmethionine cleaving activity (SAMase) induced after T3 (but not T7) infection. However, cleaving of S-adenosylmethionine does not appear to be the primary mechanism by which T3 overcomes host restriction, since at least one mutant of T3 has lost the SAMase activity without losing the ability to overcome host restriction.     

Parasites at the origin of life

This paper is concerned with parasitic virus-like particles and their hosts. It is proposed that parasitism must have occurred at an early stage of evolution, soon after the first self-reproducing systems had formed. When chemical building blocks for self-reproducing systems became scarce, current theories envision that some self-reproducing systems evolved the capability to synthesize materials for self-replication from chemical precursors in the environment. It is proposed that at about the same time parasitic systems (phages) arose that replicated at the expense of host systems by diverting host materials to the replication of their own genomes. With the aid of a mathematical model we demonstrate that host and phages can coexist in a stable equilibrium, depending upon the carrying capacity of the environment. If the latter falls below a threshold, then the parasites die out. A parasite that has the capability to integrate into the host genome is replicated along with it and thus escapes extinction during periods of population bottlenecks of the host population. The presence of phages creates evolutionary pressures favoring host defenses against them. Thus, modern bacteria are able to degrade most invading DNA (through restriction enzymes). Defense capabilities require a share of the genome, thus adding to the genetic complexity of organisms.     

Restriction/Modification systems and restriction endonucleases are more effective on lactococcal bacteriophages that have emerged recently in the dairy industry

Recently, eight lytic small isometric-headed bacteriophages were isolated from cheese-manufacturing plants throughout North America. The eight phages were different, but all propagated on one strain, Lactococcus lactis NCK203. On the basis of DNA homology, they were classified in the P335 species. Digestion of their genomes in vitro with restriction enzymes resulted in an unusually high number of type II endonuclease sites compared with the more common lytic phages of the 936 (small isometric-headed) and c2 (prolate-headed) species. In vivo, the P335 phages were more sensitive to four distinct lactococcal restriction and modification (R/M) systems than phages belonging to the 936 and c2 species. A significant correlation was found between the number of restriction sites for endonucleases (purified from other bacterial genera) and the relative susceptibility of phages to lactococcal R/M systems. Comparisons among these three phage species indicate that the P335 species may have emerged most recently in the dairy industry.     

The SalI (SalGI) restriction-modification system of Streptomyces albus G

The salIR and salM genes of Streptomyces albus G specify the SalGI (SalI) restriction enzyme and its cognate methyltransferase, respectively. These enzymes are responsible for restriction and modification of bacteriophages. Some phages carry genes that interfere with SalI-specific modification. The sal genes have been cloned in a Streptomyces host-vector system. Use of the cloned DNA as a hybridization probe reveals that sal mutants frequently arise from transposition of a DNA segment of approx. 1 kb into the sal genes. Some, but not all, other bacteria that produce SalGI isoschizomers contain nucleotide sequences that hybridize with sal DNA.     

Molecular evolution of bacteriophages: evidence of selection against the recognition sites of host restriction enzymes

Restriction enzymes produced by bacteria serve as a defense against invading bacteriophages, and so phages without other protection would be expected to undergo selection to eliminate recognition sites for these enzymes from their genomes. The observed frequencies of all restriction sites in the genomes of all completely sequenced DNA phages (T7, lambda, phi X174, G4, M13, f1, fd, and IKe) have been compared to expected frequencies derived from trinucleotide frequencies. Attention was focused on 6-base palindromes since they comprise the typical recognition sites for type II restriction enzymes. All of these coliphages, with the exception of lambda and G4, exhibit significant avoidance of the particular sequences that are enterobacterial restriction sites. As expected, the sequenced fraction of the genome of phi 29, a Bacillus subtilis phage, lacks Bacillus restriction sites. By contrast, the RNA phage MS2, several viruses that infect eukaryotes (EBV, adenovirus, papilloma, and SV40), and three mitochondrial genomes (human, mouse, and cow) were found not to lack restriction sites. Because the particular palindromes avoided correspond closely with the recognition sites for host enzymes and because other viruses and small genomes do not show this avoidance, it is concluded that the effect indeed results from natural selection.      

Partial characterization of coliphage WPK and a comparison with coliphage T3.

Coliphage WPK was originally isolated from sewage in Kiel, Germany, because its plaque diameter continued to expand for days. Electron microscopy revealed an isometric capsid with dimensions of 54 nm between opposite apices, and a short, noncontractile tail 16 nm long, placing phage WPK into morphogroup C1. The nucleic acid of phage WPK was linear double stranded DNA. The host ranges of phages WPK and T3 were identical. Of ten E. coli strains tested for host range, two were resistant and of eighteen other Enterobacteriaceae only four were susceptible. Seven gram-negative species which are not members of the Enterobacteriaceae were refractory. However, there were differences in plaque morphology and plaque expansion between the two phages. Phage T3 plaques expanded for at least seven days on E. coli B only, while phage WPK plaques expanded for at least seven days on four strains of E. coli. The buoyant density of WPK, determined by isopycnic density gradient centrifugation in CsCl, was 1,508 g/ml which was significantly different than that of T3 at 1.493 g/ml (P less than 0.05). Phage-encoded proteins were examined for each phage using [35S]methionine incorporation, SDS-PAGE, and autoradiography. Of thirty proteins identified in phage WPK and twenty-eight in phage T3, only fourteen were of the same size in both. We concluded that phage WPK was distinct, but related to T3.     

Properties of the streptomycete temperate bacteriophage FP43.

FP43 is a temperate bacteriophage for Streptomyces griseofuscus that forms plaques on many Streptomyces species. FP43 virions contain 56 kb of double-strand DNA that is circularly permuted and terminally redundant, and contains 65% G + C. A physical map of the FP43 genome was constructed, and the origin for headful packaging (pac) was localized to an 8.8-kb region of the genome (hft) that mediates high-frequency transduction by FP43 of plasmid pRHB101. The phage attachment site (attP), a replication origin (rep), a region that inhibits plaque formation (pin), and a 3-kb deletion (rpt) that caused a 100-fold reduction in plasmid transduction were mapped.     

Study of the diversity in a group of phages of Pseudomonas aeruginosa species PB1 (Myoviridae) and their behavior in adsorbtion-resistant bacterial mutants

A group of 12 Pseudomonas aeruginosa virulent bacteriophages of different origin scored with regard to the plaque phenotype are assigned to PB1-like species based on the similarity in respect to morphology of particles and high DNA homology. Phages differ in restriction profile and the set of capsid major proteins. For the purpose of studying adsorption properties of these phages, 20 random spontaneous mutants of P. aeruginosa PAO1 with the disturbed adsorption placed in two groups were isolated. Mutants of the first group completely lost the ability to adsorb all phages of this species. It is assumed that their adsorption receptors are functionally inactive or lost at all, because the attempt to isolate phage mutants or detect natural phages of PB1 species capable of overcoming resistance of these bacteria failed. The second group includes five bacterial mutants resistant to the majority of phages belonging to species PB1, These mutants maintain the vigorous growth of phage SN and poor growth of phage 9/3, which forms turbid plaques with low efficiency of plating. In the background of weak growth, phage 9/3 yields plaques that grew well. The examination of the progeny of phage 9/3, which can grow on these bacteria, showed that its DNA differed from DNA of the original phage 9/3 by restriction profile and is identical to DNA of phage PB1 with regard to this trait. Data supported a suggestion that this phage variant resulted from recombination of phage 9/3 DNA with the locus of P. aeruginosa PAO1 genome encoding the bacteriocinogenic factor R. However, this variant of phage 9/3 did not manifest the ability to grow on phage-resistant mutants of the first group. Possible reasons for the difference between phages 9/3 or SN and the remaining phages of PB1 species are discussed. A preliminary formal scheme of the modular structure for adsorption receptors on the surface of P. aeruginosa PAO1 bacteria was constructed based on the analysis of growth of some other phage species on adsorption mutants of the first type.