Transducing activity of the temperate SM bacteriophage of Pseudomonas aeruginosa

The temperate bacteriophage SM is not serologically related to the known transducing phages F116, G101, B3 of Pseudomonas aeruginosa. The strains with auxotrophic mutations within the wide ranges of the genetic map of P. aeruginosa strain PAO1 were used for studying the transducing activity of the SM phage. All of the 7 bacterial markers tested are transduced with SM phage grown on a prototrophic donor strain. The frequency of transduction of separate bacterial markers using the wild type SM phage is 2.3 to 4.6 X 10(-8). Linked ilv202+ - met28+ markers are cotransduced with SM phage at a frequency of about 1.5%.     

Isolation and propagation of phages naturally associated with the aizawai variety of Bacillus thuringiensis

This study describes the isolation of a phage, using mitomycin C and u.v. light, from each of four strains (HD67, HD130, HD228 and HD248) of Bacillus thuringiensis H-serotype 7 (var. aizawai). It also describes the isolation of two indicator strains (12.13 and HD 102) for these phages (φHD67, φHD130, φHD228 and φHD248) and the ideal conditions, using these indicator strains, for maximum phage production.     

Isolation and propagation of phages naturally associated with the aizawai variety of Bacillus thuringiensis

This study describes the isolation of a phage, using mitomycin C and u.v. light, from each of four strains (HD67, HD130, HD228 and HD248) of Bacillus thuringiensis H-serotype 7 (var. aizawai). It also describes the isolation of two indicator strains (12.13 and HD102) for these phages (phi HD67, phi HD130, phi HD228 and phi HD248) and the ideal conditions, using these indicator strains, for maximum phage production.     

短小芽孢杆菌的遗传转导

短小芽孢杆菌(Bacillus pumilus)噬菌体PP5在碱性蛋白酶生产菌珠B.pumilus 289中能进行普遍性转导。PP5对于B.pumilus 289的营养标记的转导频率为10~(-6)转导子/PFU。对于B.pumilus 1037和B.pumilus 289之间的链霉素抗性标记的转导频率为10~(-4)至10~(-7)转导子/PFU。从而建立了一个新的B.pumilus遗传转导系统。     

Two generalized transducing phages in Vibrio parahaemolyticus and Vibrio alginolyticus.

Two bacteriophages named phi VP253 and phi VP143 isolated after ultraviolet induction from lysogenic strains of Vibrio parahaemolyticus have been shown to be generalized transducing phages. So far, seven different auxotrophic markers of a V. parahaemolyticus strain could be transduced at the frequencies ranging from 2.2 x 10(-7) to 7.5 x 10(-5) per infected cell at the m.o.i. of approximately 1.0. The phage phi VP143, but not phi VP253, lysed 20 of the 28 strains of V. alginolyticus and the occurrence of generalized transduction by this phage in this Vibrio species has been confirmed. Molecular size of the genomes of both phages were estimated to be approximately 48 kb as judged from electrophoretic mobilities of the DNAs digested with HindIII endonuclease. The results and similarity of the two phages in morphology and other properties suggest very close relatedness of the phages.     

Phages naturally associated with the aizawai variety of insect pathogen Bacillus thuringiensis and their relevance to strain identification

Of 36 strains of the ' aizawai ' variety of Bacillus thuringiensis (H-serotype 7) 16 were naturally associated with bacteriophages, 11 of which were isolated at titres of 10⁶ plaque-forming units/ml and above. These 11 phages had varied host ranges among Bacillus cereus and five strains of the ' aizawai ' variety of B. thuringiensis. Host range, plaque morphology and differences in cold lability indicated dissimilarities between the phages, which could be used taxonomically to differentiate between strains of this bacillus.     

Transduction in Bacillus cereus by Each of Two Bacteriophages

The ability of phage CP-51 to mediate transduction both homologously and heterologously in some of its hosts was investigated. CP-51 was shown to transduce Bacillus cereus strains 6464, 9139, and T in addition to 569 which was reported earlier from this laboratory. Furthermore, CP-51 grown on B. thuringiensis was shown to transduce some mutants of B. cereus. During this investigation, a second transducing phage for B. cereus 569 was isolated from lysates of phage CP-51 grown on B. cereus 6464. This phage, designated CP-53, is carried by wild-type strain 6464 possibly as prophage. All auxotrophic mutants of B. cereus 569 tested, those requiring tryptophan, histidine, methionine, and leucine, were transduced to prototrophy by CP-53. Electron micrographs of the two phages revealed that CP-51 has a tail core surrounded by a contractile sheath and CP-53 has a long flexible tail without a contractile sheath. CP-53 is stable in the cold, whereas CP-51 is rapidly inactivated at 4 C.     

A generalised transducing bacteriophage for Rhodococcus erythropolis

Summary Q4, a bacteriophage isolated from soil, mediated the transduction of a number of unlinked markers in Rhodococcus erythropolis. Highest numbers of transductants were obtained at multiplicities of infection of over 100, transductants only being obtained because of the temperate nature of the phage. Under optimal conditions, transduction to prototrophy of auxotrophic markers was over 50 times the spontaneous reversion rate and transduction of some antibitic resistance markers was over 10 times the spontaneous mutation rate. Segregation of unselected, but linked, markers was observed and the phage was used to order loci in a three factor cross. The virus required magnesium ions. Highest phage titres and greatest transduction frequency were obtained with stationary phase cultures.     

Close association between shape alteration and loss of immunity to superinfection in a wild-type Klebsiella pneumoniae stable lysogen which can be both immune and nonimmune to superinfection.

Klebsiella pneumoniae MirM7 is a wild-type strain which grows as cocci at pH 7 and above and as rods at pH 6.5 and below. Cultures of this strain and an auxotrophic derivative, MirM7b, have been found to undergo spontaneous lysis after purification from possible contaminating viruses. Lysates always contained two phages, FR2 and AP3, most often at high titers. FR2 and AP3 plated with the same efficiency on both MirM7b and K59 (another K. pneumoniae strain sensitive to FR2 and AP3) and lysogenized 45 and 54% of the K59-infected cells, respectively. These findings raise the possibility that MirM7b is lysogenic for FR2 and AP3, although nonimmune to their superinfection. The fact that mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine can induce phages FR2 and AP3 from MirM7b confirmed this possibility. When MirM7b was infected with FR2 several strains immune to FR2 and AP3, which were all rod shaped, were obtained. Furthermore, 19 derivatives, rod shaped at all pH's have been isolated from MirM7b. They were all immune to both FR2 and AP3. From mating experiments between the MirM7b donor derivative, strain M720, and either K59 or MirCV5, a rod-shaped MirM7b derivative cured from the prophages, cysteine recombinants were obtained which were most often (80%) immune to FR2 and AP3. Nonimmune and still lysogenic recombinants were obtained by mating M720 with a rod-shaped immune MirM7b derivative; the majority of the non-immune strains maintained the rod shape. Five coccus-shaped recombinants were also isolated; they were nonimmune to superinfection. Several physiological properties of strain MirM7b and the other nonimmune coccal recombinants have been studied in comparison with those of the rod-shaped immune derivatives. All of the coccal strains have shown several alterations with respect to the rods. The role of possible derepressed prophage genes in the various physiological alterations of MirM7 is discussed, and the analogies between this system and those of vertebrate cells transformed by proviruses are stressed.     

Defective specialized SP beta transducing bacteriophages of Bacillus subtilis that carry the sup-3 or sup-44 gene

We isolated defective specialized transducing phages of SP beta that carry one of the extracistronic suppressors, sup-3 or sup-44. Lysates containing these phages can be used in a simple spot test to determine whether an auxotrophic mutation can be suppressed. The sup-3 and sup-44 mutations are distinct, in that their suppression patterns differ for the markers hisA1, metC3, and thr-5; and they are not alleles.     

Direct and general selection for lysogens of Escherichia coli by phage lambda recombinant clones.

We report a simple in vivo technique for introducing an antibiotic resistance marker into phage lambda. This technique could be used for direct selection of lysogens harboring recombinant phages from the Kohara lambda bank (a collection of ordered lambda clones carrying Escherichia coli DNA segments). The two-step method uses homologous recombination and lambda DNA packaging to replace the nonessential lambda DNA lying between the lysis genes and the right cohesive (cos) end with the neomycin phosphotransferase (npt) gene from Tn903. This occurs during lytic growth of the phage on a plasmid-containing host strain. Neomycin-resistant (npt+) recombinant phages are then selected from the lysates containing the progeny phage by transduction of a polA1 lambda lysogenic host strain to neomycin resistance. We have tested this method with two different Kohara lambda phage clones; in both cases, neomycin resistance cotransduced with the auxotrophic marker carried by the lambda clone, indicating complete genetic linkage. Linkage was verified by restriction mapping of purified DNA from a recombinant phage clone. We also demonstrate that insertion of the npt+ recombinant phages into the lambda prophage can be readily distinguished from insertion into bacterial chromosomal sequences.     

Genetic Analysis in Bacillus pumilus by PBS1-Mediated Transduction

Bacteriophage PBS1 mediates generalized transduction in Bacillus pumilus NRRL B-3275 (BpB1). Transduction frequencies for single auxotrophic markers are of the order of 10(-4) transductants per plaque-forming unit in crude phage lysates. The characteristics of PBS1 propagated on BpB1 and the properties of the system of transduction are similar to those reported for PBS1 propagated on Bacillus subtilis. By transduction, eight amino acid auxotrophic markers in BpB1 have been oriented into two linkage groups. One group contains the auxotrophic markers arginine A, leucine, and phenylalanine, and the other group contains the markers lysine, serine, tryptophan, isoleucine-valine, and isoleucine. The nature and relative order of the markers within each linkage group suggest that the arrangement of genes in these areas of the chromosome of BpB1 is similar to the arrangement of phenotypically comparable genes in two linkage groups (defined by PBS1 transduction) in B. subtilis. However, transduction of any of the above cited markers in BpB1 to prototrophy with PBS1 propagated on B. subtilis 168 could not be demonstrated. In addition to BpB1, seven other strains of B. pumilus can be infected with PBS1. Transduction has been demonstrated in three of these strains.     

Persistence of Infectious Shiga Toxin-Encoding Bacteriophages after Disinfection Treatments

In Shiga toxin-producing Escherichia coli (STEC), induction of Shiga toxin-encoding bacteriophages (Stx phages) causes the release of free phages that can later be found in the environment. The ability of Stx phages to survive different inactivation conditions determines their prevalence in the environment, the risk of stx transduction, and the generation of new STEC strains. We evaluated the infectivity and genomes of two Stx phages (Φ534 and Φ557) under different conditions. Infectious Stx phages were stable at 4, 22, and 37°C and at pH 7 and 9 after 1 month of storage but were completely inactivated at pH 3. Infective Stx phages decreased moderately when treated with UV (2.2-log₁₀ reduction for an estimated UV dose of 178.2 mJ/cm²) or after treatment at 60 and 68°C for 60 min (2.2- and 2.5-log₁₀ reductions, respectively) and were highly inactivated (3 log₁₀) by 10 ppm of chlorine in 1 min. Assays in a mesocosm showed lower inactivation of all microorganisms in winter than in summer. The number of Stx phage genomes did not decrease significantly in most cases, and STEC inactivation was higher than phage inactivation under all conditions. Moreover, Stx phages retained the ability to lysogenize E. coli after some of the treatments.     

Isolation and Characterization of φ80 Transducing Bacteriophage for a Ribonucleic Acid Polymerase Gene

A new method for isolating specialized transducing phages is described. It was used to isolate a group of phi80 transducing phages which carry various bacterial markers from the metB region of the Escherichia coli chromosome. Some of the phages selected for transduction of the supA36 marker were also shown to carry rif, a locus known to specify the beta subunit of ribonucleic acid polymerase. Expression of the prophage rif(r) gene in lysogens was demonstrated by its ability to confer rifampin resistance on part of the cellular ribonucleic acid polymerase pool.     

A novel transducing phage

Summary Two newly isolated generalized transducing phages, F126 and F130, are in many respects similar to the previously described phage F116 of P. aeruginosa strain PAO. They also share with F116 the property of not responding to host-specific restriction in strain PAO. However, while the transduction ability of phages F116 and F130 is also inert to PAO-specific restriction, transduction mediated by phage F126 is 8–120 fold reduced in restrictive conditions. In experiments designed to explore the conditions necessary to obtain restriction in F126 transduction it was found that it differed from those previously known to specify host-controlled restriction in P. aeruginosa PAO (Rolfe and Holloway, 1968). These observations suggest that more than one independent restriction system operates in strain PAO.     

Design and synthesis of indane-ureido-thioisobutyric acids: A novel class of PPARalpha agonists

A series of aminoindane derivatives were synthesized and shown to be potent PPARα agonists. The compounds were obtained as racemates in 12 steps, and tested for PPARα activation and PPARα mediated induction of the HD gene. SAR was developed by variation to the core structure as shown within. Oral bioavailability was demonstrated in a Sprague–Dawley rat, while efficacy to reduce plasma triglycerides and plasma glucose was demonstrated in db/db mice.     

Physical and genetical analysis of bacteriophage T4 generalized transduction

This report describes a comparison of the efficiency of transduction of genes in E. coli by the generalized transducing bacteriophages T4GT7 and P1CM. Both phages are capable of transducing many genetic markers in E. coli although the frequency of transduction for particular genes varies over a wide range. The frequency of transduction for most genes depends on which transducing phage is used as well as on the donor and recipient bacterial strains. Analysis of T4GT7 phage lysates by cesium chloride density gradient centrifugation shows that transducing phage particles contain primarily bacterial DNA and carry little, if any, phage DNA. In this regard transducing phages P1CM and T4GT7 are similar; both phages package either bacterial or phage DNA but not both DNAs into the same particle.     

Influencing the spontaneous and chemically induced mutability of E. coli through changing the genetic background

Summary The influence of a second auxotrophic marker to the spontaneous and chemical-induced mutability to prototrophy of a first auxotrophic marker in 7 monoauxotrophs and 31 biauxotrophs of E. coli K 12 was studied by growth layer technics. No case of influence of a second auxotrophy to the mutagen-induced mutability (7 mutagens tested) of the first auxotrophy among 172 possibilities was found. An influence to the spontaneous mutability seemed to be present in 4 cases out of 31. But 3 of them were shown to be imitated by influences of components of the medium to the growth of mutants or parent type. In one case a mutator mutation is responsible for the about 8 times higher rate of the mutation met 1+ in strain met 1/his 7 than in strain met 1. But backmutation and crossing experiments showed that the mutator (mum ⁺) was separate from the auxotrophic marker his 7 (recombination frequency 5/16). This mutator did not increase remarkably the spontaneous mutability of other markers tested (resistence to phages T1 or T4 or to Streptomycin 3, 5, 10, or 100 /ml, or to Chloramphenicol 2 /ml). It is assumed that the spontaneous mutations in the wild-type are at least partially different in their nature from the mutator promoted ones.     

Transduction of certain genes by an autonomously replicating Bacillus thuringiensis phage

A derivative of Bacillus thuringiensis subsp. kurstaki HD1 (HD1-9) released transducing phage (TP21) from late exponential cultures. Three of seven markers tested were transduced into Bacillus cereus, but only two of these (cysC and trpB/F) were transduced at a frequency of more than 100 times the reversion rates. A limited transduction capacity was given further support in that few chromosomal markers were carried in the HD1-9 lysate, as demonstrated by Southern hybridization. Restriction fragments from the phage DNA and from total B. thuringiensis DNA hybridized to an insertion sequence (IS231-like) probe, which may provide a region of homology for transduction. All of the B. cereus transductants contained the phage as a 44-kb plasmid, and each could transduce both the cys and trp genes to other B. cereus auxotrophs, albeit at lower frequencies than those for the B. thuringiensis transducing phage. In some cases, especially for cys, the transduced gene was integrated into the chromosome of the recipient, whereas the trp gene in many cases appeared to be lost with curing of the 44-kb plasmid. In addition, some B. cereus transductants lost prototrophy but retained a 44-kb plasmid, consistent with the presence of TP21 helper phage. These phage may mediate the subsequent transduction from B. cereus phototrophs. TP21 replicates as a plasmid and, at least under the conditions studied, selectively transfers markers to B. cereus.     

Transduction of certain genes by an autonomously replicating Bacillus thuringiensis phage.

A derivative of Bacillus thuringiensis subsp. kurstaki HD1 (HD1-9) released transducing phage (TP21) from late exponential cultures. Three of seven markers tested were transduced into Bacillus cereus, but only two of these (cysC and trpB/F) were transduced at a frequency of more than 100 times the reversion rates. A limited transduction capacity was given further support in that few chromosomal markers were carried in the HD1-9 lysate, as demonstrated by Southern hybridization. Restriction fragments from the phage DNA and from total B. thuringiensis DNA hybridized to an insertion sequence (IS231-like) probe, which may provide a region of homology for transduction. All of the B. cereus transductants contained the phage as a 44-kb plasmid, and each could transduce both the cys and trp genes to other B. cereus auxotrophs, albeit at lower frequencies than those for the B. thuringiensis transducing phage. In some cases, especially for cys, the transduced gene was integrated into the chromosome of the recipient, whereas the trp gene in many cases appeared to be lost with curing of the 44-kb plasmid. In addition, some B. cereus transductants lost prototrophy but retained a 44-kb plasmid, consistent with the presence of TP21 helper phage. These phage may mediate the subsequent transduction from B. cereus phototrophs. TP21 replicates as a plasmid and, at least under the conditions studied, selectively transfers markers to B. cereus.