Lysogenic Strains Research Articles

Purification, Fine Structure and Characterization of Temperate Phages from Drug-resistant Staphylococcus aureus

Three kinds of temperate Staphylococcus aureus phages were differentiated by serological, sedimentation and electron microscopic studies. Phage S1 had a long hexagonal head and a long tail, a density in Cs2SO4 of 1.364 g/ml and belonged to serological group A; phage S2 had a short hexagonal head and a short tail, a density of 1.385 to 1.392 g/ml and belonged to serological group B; phage S3 had a shape similar to phage S2 except for some minor differences, but had a density of 1.416 g/ml and belonged to serological group F. S. aureus MS27 was found to be singly lysogenic for S2. However, S. aureus MS3878 was a doubly lysogenic strain carrying S1 and S2 and S. aureus E169 was a triply lysogenic strain carrying S1, S2 and S3. All these temperate phages showed similarity in shape to typing phages belonging to the same serological group. The temperate S. aureus phages revealed the presence of a hexagonal baseplate with spikes. The burst sizes of phages of S1, S2 and S3 were about 50, 110 and 120 respectively. The DNA from S1, S2 and S3 ranged from 29.4 to 30 megadaltons in size.

New waterborne bacteriophages active on Yersinia enterocolitica.

Seven bacteriophages active on Yersinia enterocolitica (YE) were isolated from surface water samples collected in Granada, Spain. A comparison of the respective host ranges of these new phages and of reference phages used for YE phage typing showed that YE strains belonging to various phage types, grown at either 37 or 25 degrees C, expressed susceptibility to reference sewage water phages whereas susceptibility to new waterborne phages, as well as to reference phages from lysogenic YE, was only demonstrated in YE strains grown at 25 degrees C. A YE strain isolated by stool culture from a pig was lysogenic for a bacteriophage which behaved like waterborne phages and reference phages from lysogenic YE strains. The possibility that the isolation of waterborne bacteriophages might, in certain circumstances, reflect the presence of lysogenic YE was raised.

Structure and restriction enzyme maps of the circularly permuted DNA of staphylococcal bacteriophage phi 11.

One restriction enzyme map of Staphylococcus aureus bacteriophage phi 11 DNA was established by reciprocal double digestions with the enzymes EcoRI, HaeII, and KpnI. The sequential order of the EcoRI fragments was thereafter established by a novel approach involving blotting of DNA partially cleaved with EcoRI and the probing the blots with nick-translated terminal fragments. A circular map of the phi 11 DNA was established, and the phage genome was circularly permuted based on the failure to end label mature viral DNA, restriction maps of replicating DNA, and finally, homoduplex analysis in the electron microscope. A restriction enzyme map of the prophage form of phi 11 DNA was obtained by analysis of chromosomal DNA from a lysogenic strain.

Resistance transfer in mixed cultures of Staphylococcus aureus

Transfer of resistance markers was regularly observed in mixed cultures of pairs of constructed strains of Staphylococcus aureus. This transfer was shown to be due to general transduction and required a lysogenic donor strain, carrying a transducing phage of serotype B and a transducible plasmid, and an acceptor strain without either strong restriction or an incompatible plasmid. Transfer was sometimes bidirectional when both strains carried a transducing phage. Unidirectional transfer occurred when the acceptor strain was immune. When the acceptor was sensitive to the donor phage, reversed transfer of a marker from the acceptor to the donor could take place. The kinetics of transfer are governed by the rate of phage production, the rate of phage adsorption and the frequency of transduction. A mathematical model has been developed to describe the interplay of these factors. The numbers of transductants observed in mixed cultures agreed with those expected from the model. Transfer was also observed in mixed cultures of constructed strains and wild-type strains. Transfer between wild-type strains is also possible, but was not observed in many combinations because of lack of transducing phage, strong restriction or other factors. Transfer was also observed in local lesions in mice infected with two strains and in mixtures inoculated on to human skin.

Curing of a Lysogenic Strain of Streptococcus cremoris and Characterization of the Temperate Bacteriophage

A derivative of Streptococcus cremoris MU0001 (R1) has been isolated which has been cured of an inducible prophage. This isolate, its parent strain and a relysogenized derivative of the cured strain have been compared with respect to their ultraviolet inducibility, production of plaque-forming units, bacteriophage immunity and milk coagulation properties. The bacteriophage (.MU1) harboured by strain MU0001 as a prophage has been characterized by isopycnic density gradient centrifugation, electron microscopy and sodium dodecyl sulphate gel electrophoresis. Antiserum raised against this bacteriophage was used to demonstrate true lysogeny in both the original strain and the relysogenized strain.

Induction of prophage and mutagenic effects by alkyl alkylaminosulfonates, ethyl aminosulfonate and alkyl methanesulfonates

Prophage induction and mutation by alkylaminosulfonates, ethyl aminosulfonate and alkyl methanesulfonates were examined comparatively. Prophage induction was carried out with a lysozyme lysis technique on the lysogenic strain Micrococcus lysodeikticus 53-40 (N5). The sulfonic ester derivatives show a slight lysogenic induction. At higher concentrations their toxicity seems to mask phage detection. Only methyl isopropylaminosulfonate and ethyl aminosulfonate exhibit no or negligible toxic effects, and with these compounds at higher concentrations a strong prophage induction is found. Alkyl sulfonate derivatives induce mutations in the tester strain of Salmonella typhimurium TA1535. Methyl methylaminosulfonate and ethyl N-methyl- N-2-chloroethyl aminosulfonate show a mutagenicity comparable to that of the well-known methyl methanesulfonate or ethyl methanesulfonate. With ethyl aminosulfonate, however, which does not show inactivation, no significant mutagenic effect was observed. DNA alterations were found in the polymerase-deficient strain E. coli P3478. The results of prophage induction and mutagenicity are compared and discussed.

Temperate coliphages: classification and correlation with habitats

Temperate coliphages were recovered from sewage, mammalian feces, and lysogenic strains of Escherichia coli. A total of 32 phages of independent origin were divided into six groups by applying the criteria of host range, antigenic homology, and the ultraviolet inducibility of the prophage. The demonstration of genetic interactions in some cases has confirmed the classification scheme. Nine phages were assigned to the P2 family and 19 to the lambda family. The remaining four isolates may represent some novel phylogenetic types. Phages recovered from the lysogenic strains of E. coli were all found to be P2 related, whereas a majority of the phages recovered as cell-free plaque-forming units were assignable to the lambda family. It is proposed that the biological attributes of the phages belonging to the two principal families are reflected in the distribution patterns observed. The virions of phage HK256 show multiple tail fibers and may thus represent a "new" virion form among the temperate coliphages.

Cloning of the immunity repressor determinant of bacteriophage P2 in the pBR322 plasmid.

Through in vitro recombination of DNA restriction fragments, we have constructed a plasmid, which expressed in vivo the immunity repressor gene (C) of bacteriophage P2. A bacterial strain carrying such a plasmid showed a high level of P2 specific immunity. It was lysogenized normally by an infecting P2, but the frequency of spontaneous phage production was reduced about 10(4) fold as compared to a normal P2 lysogen. Satellite phage P4, known to derepress P2 lysogens, was unable to derepress the plasmid-carrying lysogenic strain so to allow growth of coinfecting P2. Phage P4 multiplied on the plasmid-carrying, P2-lysogenic strain, but due to a prolonged latent period failed to form plaques on this strain.

Conditions for induction of bacteriophage from lysogenic Bacillus megaterium with aflatoxin B1.

The present study was conducted to determine whether or not aflatoxin B1 was an effective inducing agent for lysogenic bacteria and to characterize some of the parameters involved in induction. A lysogenic strain of Bacillus megaterium (NRRL-B-3695) and an indicator strain of this species (NRRL-B-3694) were used. Cultures of the lysogenic strain were incubated for various periods of time in the presence of aflatoxin B1. Plaque-forming units as well as colony-forming units were then determined. Results of the present study indicated that bacteriophage lysogenizing B. megaterium could be induced with aflatoxin B1. The optimum concentration for induction was 25 micrograms of toxin per ml of early-log-phase culture. Evidence suggested that: (i) higher concentrations of aflatoxin B1 formed hydrophobic complexes which would not efficiently induce B. megaterium; (ii) the toxic effect of aflatoxin B1 severely limited the number of cells which could be induced prior to killing action of the toxin; and (iii) concentrations less than 25 micrograms of aflatoxin B1 per ml were not efficient inducers of bacteriophage production nor did they demonstrate the toxic effect observed at higher concentrations.

A method for construction of specialized transducing phage ϱ11 of Bacillus subtilis

DNA from a temperate phage ϱ11 and chromosomal DNA of Bacillus subtilis 168 were digested with endonuclease EcoRI and then ligated with T4 polynucleotide ligase. The ligated DNA fragments were used to transform a lysogenic strain, B. subtilis spoA12 lys21 hisA1 leuA8 ϱ11, and Lys +, His + or Leu + transformants were selected. The cells of each type were then mixed, grown and treated with mitomycin C; the induced phages were tested for abilities abilities to form plaques and to transduce the auxotrophic marker. Various types of plaque-forming or defective phages which transduce hisA or lys marker at considerably high frequencies were thus obtained.

Selective expression of transduced or cloned DNA in minicells containing plasmid pKB280

DERIVATIVES of coliphage λ, carrying inserts of essentially any prokaryotic or eukaryotic DNA, can now be constructed using novel λ cloning vectors. Often, the reason for constructing these derivatives is to determine the polypeptides encoded by the inserted DNA. Phage expression can be analysed by infection of anucleate Escherichia coli minicells in which all host syntheses are absent1. However, analysis of the expression of the non-λ DNA insert in this system is complicated by the concurrent expression of λ DNA. Attempts to inhibit the expression of the λ genes by use of minicells from a λ lysogenic minicell-producing strain were unsuccessful, indicating that insufficient λ repressor was segregated into the minicells (Shaw, Epp, Pearson and J.N.R., in preparation). Recently a plasmid, pKB280, was constructed which carries the gene (cI) for the λ repressor protein and in which approximately 1% of the soluble protein of E. coli strains carrying this plasmid is λ repressor2. Minicells produced by a derivative of E. coli DS410 carrying plasmid pKB280 have been infected by transducing and cloning phages. We report here that only the non-λ DNA is expressed in these minicells.

Transfer of Erythromycin Resistance from Clinically Isolated Lysogenic Strains of Streptococcus pyogenes via Their Endogenous Phage

Endogenous bacteriophage from clinically isolated, erythromycin-resistant strains of Streptococcus pyogenes, upon induction by treatment with mitomycin C, were demonstrated to cause transduction of antibiotic resistance yielding transductants resistant to relatively high concentrations of erythromycin. Transduction of resistance to erythromycin via endogenous phage may play a significant role in the emergence of streptococci with multiple resistance in the clinical environment.

Bacteriophage P22-mediated specialized transduction in Salmonella typhimurium: high frequency of aberrant prophage excision

The temperate bacteriophage P22 mediates both generalized and specialized transduction in Salmonella typhimurium. Specialized transduction by phage P22 is different from, and less restricted than, the well characterized specialized transduction by phage lambda, due to differences in the phage DNA packaging mechanisms. Based on the properties of the DNA packaging mechanism of phage P22 a model for the generation of various types of specialized transducing particles is presented that suggests generation of substantial numbers of specialized transducing genomes which are heterogeneous but only some of which have terminally redundant ends. The primary attachment site, ataA, for phage P22 in S. typhimurium is located between the genes proA,B and supQ newD. (The newD gene is a substitute gene for the leuD gene, restoring leucine prototrophy of leuD mutant strains.) The proA,B and supQ newD genes are very closely linked and thus cotransducible by generalized transducing particles. Specialized transducing particles can carry either proA,B or supQ newD but not both simultaneously, and thus cannot give rise to cotransduction of the proA,B and supQ newD genes. This difference is used to calculate the frequency of generalized and specialized transducing particles from the observed cotransduction frequency in phage lysates. By this method, very high frequencies of supQ newD (10(-2)/PFU)- and proA,B (10(-3)/PFU)-specialized transducing particles were detected in lysates produced by induction of lysogenic strains. These transducing particles most of which would have been produced by independent aberrant excision events (which include in situ packaging), were of various types.

Bacteriophage P22-mediated specialized transduction in Salmonella typhimurium: identification of different types of specialized transducing particles

The temperate bacteriophage P22 mediates both generalized and specialized transduction in Salmonella typhimurium. Specialized transduction by phage P22 is different from, and less restricted than, the well characterized specialized transduction by phage lambda, due to differences in the phage DNA packaging mechanism. Phage lysates produced by induction of lysogenic strains contain very high frequencies of supQ newD- and proA,B-specialized transducing particles (10(-2)/PFU and 10(-3)/PFU, respectively), most of which are produced by independent aberrant excision events of various types. In a model, 12 different modes of transduction mechanisms were characterized by: (i) the structure of the specialized transducing genomes after injection into a new host cell, i.e., linear or circular, and (ii) the requirements for the transduction process, i.e., host recombination functions, phage integration functions, or presence of a prophage. By using different recipient strains and phage helper strains, it was possible to show that most specialized transducing particles (ca. 99%) contain linear genomes that cannot circularize upon injection into a new host cell and that require the presence of an integrated prophage as a site for a recombinational event to give rise to a transductant. Only 0.1% of all specialized transducing particles were shown to transduce by integration, suggesting that transducing genomes containing terminally redundant ends represent only a minor fraction of all transducing particles that are produced. However, it should be pointed out that the frequency (approximately 10(-5)/PFU) of these specialized transducing genomes that can circularize upon injection into a new host cell is as high as or even higher than the frequency of specialized transducing particles of phage lambda. The remaining approximately 1% of all specialized transducing particles can transduce by any one of the other mechanisms described.

Correlation between UV dose requirement for lambda bacteriophage induction and lambda repressor concentration

Escherichia coli K-12 wild type and a uvrA mutant derivative were used to construct isogenic strains bearing one, two, three, or more phage lambda cI genomes and containing increasing concentration of lambda repressor as measured by in vitro operator DNA-binding assays. The survival and phage induction in response to UV irradiation were determined. In both strains, dose-response relationships were obtained as a function of the cellular repressor concentration. The uvrA lysogens required one-tenth the UV fluence of the wild-type counterparts for induction. Lysogenic strains containing plasmids that overproduce the lambdaind+ repressor and the same lysogens with plasmids overproducing the lambdaind- repressor displayed the same survival curves as the nonlysogenic parental strain; however, only the former produced infectious centers (at a frequency of 2 x 10(-3) to 5 x 10(-4) in response to radiation.

Isolation of the L-phase variant from toxigenic Corynebacterium diphtheriae C7(beta).

The L-phase variant was isolated from lysogenic strain C7(beta) of Corynebacterium diphtheriae by the penicillin disk tecnnique on an agar medium containing 3.7% brain heart infusion, 3% NaCl, 1.25% Grycine, 1% Noble agar, and 10% horse serum. The L-phase formed a "fried egg" colony; it was resistant to antibiotics that inhibit biosynthesis of bacterial cell walls but was susceptible to antibiotics that act on the protein-synthesizing system. After several transfers of the L-phase initially isolated, the L-phase of strain C7(beta) became stable, forming a typical L-phase colony on the agar even without penicillin. The L-phase variant retained lysogenicity and toxigenicity. The toxin production by this L-phase variant was, like that by the bacterial phase, inhibited by excess iron.

Lysogeny Associated With Mucoid Variation in Mycobacterium Kansasii

Ten of 200 strains of Mycobacterium kansasii were found to produce very mucoid growth on Löwenstein-Jensen medium. By electronmicroscopy these 10 strains were found to be lysogenic, whereas no phage was observed in cultures of 30 non-mucoid strains. The cultural and biochemical properties of the lysogenic strains are compared with those of non-lysogenic strains, and the morphology of the phages is described.

Bacteriophage typing of canine staphylococci. II. Isolation of phage from lysogenic strains and establishment of a new phage set.

An attempt was made to isolate phages from staphylococci, so that phage typing of these organisms might be done. Of 150 coagulase-positive strains, 42 (28%) were proved to be lysogenic. All of them, exctpt seven, were found to carry phages of different host ranges. Of the phages detected, a few propagated to a titer of 100 RTD, and considerably many disappeared during passages for propagation. Multi-lysogenicity was demonstrated in four strains which produced two or three different Based on host ranges, 16 phages were selected for typing of staphylococci and called canine phages. They were divided into 7 groups according to their host ranges. Of them, five phages belonged to group I, six to group II, and one each to groups III to VII. Group II was further divided into sub-groups IIa and IIb, to which belonged three phages each.

Properties of lysogenic strains in Pseudomonas mori and their temperate phages

Properties of lysogenic strains in Pseudomonas mori and their temperate phages

Requirement of protein and RNA synthesis for λ repressor inactivation by tif-1: Effects of chloramphenicol, neomycin and rifampicin

The inactivation of lambda repressor was followed by the specific DNA binding assay during the course of lysogenic induction provoked by incubation at 42 degrees C of an E. coli tif-1 lysogenic strain. The presence of up to 400 microgram/ml chloramphenicol during the inducing treatment did not impair the loss of repressor binding activity, whilst concentrations of 200 microgram/ml neomycin and 100 microgram/ml rifampicin effectively inhibited the inactivation of lambda repressor. Residual protein synthesis in the presence of chloramphenicol, neomycin and rifampicin was 5%, 5% and 27% respectively of that observed in the drug-free control. This residual synthesis did not appear to involve amplification of the X-protein. These results suggest that tif-mediated inactivation of the lambda repressor requires the activation of some specific gene(s), the translation of which appears to be resistant to chloramphenicol.