Growth and Cultivation of the Unusual Generalized Transducing Bacillus Bacteriophage SP-15

Additional properties of SP-15, a generalized transducing bacteriophage notable for the ability to transfer an unusually large fragment of deoxyribonucleic acid (DNA) to Bacillus subtilis and B. licheniformis, are presented together with improved methods that enhance its utility. Simple means have been found to provide the rigid control over moisture that is necessary for the assay of plaque-forming units (PFU). Reproducible procedures for propagating transducing phage, which depend upon an appropriate mixing of PFU with uninfected bacteria, have replaced less reliable methods that utilized infected spores. Transduction of B. subtilis W-23 increased linearly when MgSO(4) in recipient cell-SP-15 mixtures was increased from 0.005 to 0.03 m. Methods have been developed that protect SP-15 from the damaging effects of CsCl and of osmotic shock subsequent to dilution. Evidence that the PFU and transducing particles of lysates decay at the same slow rate during extended storage suggests that the decay is a result of damage to protein rather than to DNA. One-step growth experiments, in which SP-15 was propagated on B. subtilis W-23-S(r)/1 mg, indicated a latent period of 100 min, a rise period of 60 min, and a burst size of 25 to 34 PFU per infected cell. These findings suggest explanations for some of the technical difficulties SP-15 has presented.     

Cotransduction and Cotransformation of Genetic Markers in Bacillus subtilis and Bacillus licheniformis

Bacteriophage SP-15, a large generalized transducing phage of Bacillus, was compared with phages PBS-1 and SP-10 for the ability to cotransduce pairs of genetic markers exhibiting different degrees of linkage. When auxotrophs of B. subtilis W-23 were used as recipients, SP-15 and PBS-1 effected a much higher frequency of cotransduction than did SP-10 with markers that were not closely linked. With more closely linked loci, the differences were not as great. SP-15 cotransduced linked markers at a higher mean frequency than PBS-1, suggesting that SP-15 is able to transfer a larger fragment of the Bacillus genome than any phage heretofore described. The frequency of the joint transfer of genetic markers in B. licheniformis was lower via transforming deoxyribonucleic acid than by transduction with phage SP-10. The availability of three procedures for genetic exchange-transduction by SP-15 and SP-10 as well as transformation-each of which reveals a different degree of linkage, makes B. licheniformis 9945A especially amenable to genetic analysis.     

Converting bacteriophage for sporulation and crystal formation in Bacillus thuringiensis.

Bacteriophage TP-13, a converting phage for sporulation and crystal formation in Bacillus thuringiensis, was isolated from soil. The phage converted anoligosporogenic (sporulation frequency, 10(-8), acrystalliferous mutant to spore positive, crystal positive at a high frequency. Each plaque formed by TP-13 in a lawn of sensitive cells contained spores and crystals. These spores were heat stable, and each one was capable of producing a plaque from which TP-13 could be reisolated. Conversion of cells to sporulation and crystal formation was independent of the ho-t used for TP-13 propagation. When converted cells were cured of TP-13, they lost the ability to produce spores and crystals. Incubation of TP-13 with antiserum prepared against purified phage particles prevented conversion. TP-13 has some characteristics similar to those of SP-15 and PBS-1, including large size, morphology, and adsorption specificity of motile cells. TP-13 mediated generalized transduction in several strains of B. thuringiensis at frequencies of 10(-6) to 10(-5). Comparison of cotransduction values indicated that TP-13 transduced considerably larger segments of deoxyribonucleic acid than CP-51 or TP-10, two other transducing phages for B. thuringiensis.     

Transductional selection of cloned bacteriophage phi 105 and SP02 deoxyribonucleic acids in Bacillus subtilis.

The Bacillus subtilis temperate bacteriophages phi 105 and SP02 are incapable of transduction of the small, multicopy drug resistance plasmids pUB110 and pCM194. Cloning endonuclease-generated fragments of phi 105 or SP02 DNA into each of the plasmids renders the chimeric derivatives susceptible to transduction specifically by the phage whose deoxyribonucleic acid is present in the chimera. The majority of phage deoxyribonucleic acid fragments identified that render plasmids transducible by phi 105 or SP02 appear to be internal fragments, not fragments containing the cohesive ends. However, the highest overall transduction frequency was observed in SP02-mediated transduction of a derivative of pUB110 containing a 1.6-megadalton EcoRI fragment that likely contains the SP02 cohesive ends (plasmid pPL1010). The transducing activity present in a phi 105 transducing lysate had a buoyant density slightly greater than infectious particles, whereas the majority of transducing particles in an SP02(pPL1010) transducing lysate had a buoyant density slightly less than infectious particles. Although no detectable change in plasmid structure resulted from transduction by phi 105 or SP02, deoxyribonucleic acid isolated from a purified SP02(pPL1010) transducing lysate contained no detectable monomeric pPL1010, but did contain a form of pPL1010 of higher molecular weight than the monomer.     

Organization of Gene Function in Bacillus subtilis Bacteriophage SP82G

A generalized assessment of the functions of 26 genes of SP82G, a bacteriophage of Bacillus subtilis, has been made. The production of phage-specific deoxyribonucleic acid (DNA), DNA-filled phage heads, completed phage particles, phage-specific antigen, and developmental aberrations has been examined in lysates of temperature-sensitive mutants grown under selective conditions. The genes show a tendency to occur on the genome in three groups of related function: genes involved with DNA synthesis, with tail synthesis, and with head synthesis.     

Isolation of Bacillus subtilis genes from a charon 4A library.

A library of Bacillus subtilis chromosomal deoxyribonucleic acid (DNA) was constructed, using lambda charon 4A as a cloning vector. Partially cleaved Bacillus subtilis DNA was prepared by partial methylation with EcoRI methylase, followed by complete EcoRI endonuclease digestion. More than 95% of the phage particles carried B. subtilis DNA inserts. When this library was screened for transforming activity, using competent cells, 70% of the genetic markers tested were found in a sample of 1,710 plaques. Cloned genetic loci were found to be about 100-fold more efficient in transforming activity than chromosomal DNA. Intact phage particles containing the pheA locus were found to be able to transform competent recipients with approximately the same efficiency as phage DNA. Transformation by intact particles was insensitive to deoxyribonuclease.     

Isolation and characterization of lambda transducing bacteriophages for the su1+ (supD minus) amber suppressor of Escherichia coli.

Specialized lambda transducing phages for the sul+ (supD-) amber suppressor in Escherichia coli K-12 have been isolated, using a secondary site lambda-cI857 lysogen in which we have shown the prophage to be closely linked to sul+.sul+ transducing particles were detected frequently, at 10-5 per plaque-forming unit, in lysates prepared from the secondary-site lysogens. High-frequency transducing lysates were obtained from several independently isolated sul+ transductants and were analyzed by CsCl equilibrium density gradient centrifugation. The transducing phages are defective; marker rescue analysis indicates that the lambda-N gene is not present. In lambda-cI857DELTANdSul+, a bio-type transducing phage, the genes specifying recombination and excision functions have been replaced by bacterial deoxyribonucleic acid.     

Mechanism of Transfection with Deoxyribonucleic Acid from the Temperate Bacillus Bacteriophage φ105

Bacteriophage phi105 is a temperate phage for the transformable Bacillus subtilis 168. The infectivity of deoxyribonucleic acid (DNA) extracted from mature phi105 phage particles, from bacteria lysogenic for phi105 (prophage DNA), and from induced lysogenic bacteria (vegetative DNA) was examined in the B. subtilis transformation system. About one infectious center was formed per 10(8) mature DNA molecules added to competent cells, but single markers could be rescued from mature DNA by a superinfecting phage at a 10(3)- to 10(4)-fold higher frequency. Single markers in mature DNA were inactivated at an exponential rate after uptake by a competent cell. Prophage and vegetative DNA gave about one infectious center per 10(3) molecules added to competent cells. Infectious prophage DNA entered competent cells as a single molecule; it gave a majority of lytic responses. Single markers in sheared prophage DNA were inactivated at the same rate as markers in mature DNA. Prophage DNA was dependent on the bacterial rec-1 function for its infectivity, whereas vegetative DNA was not. The mechanism of transfection of B. subtilis with viral DNA is discussed, and a model for transfection with phi105 DNA is proposed.     

A Bacteriophage of Bacillus subtilis Which Forms Plaques Only at Temperatures Above 50 C I. Physical and Chemical Characteristics of TSP-1

Bacteriophage TSP-1 was isolated from soil in a search for phage which would form plaques on Bacillus subtilis W168 at 53 C. It forms clear plaques only at temperatures from 50 to 55 C. Approximately 95% of the free phage adsorb after 2 min at 53 C. The lytic cycle is between 55 and 60 min long with a burst size of about 55 particles per infected bacterium. The phage was shown to contain double-stranded deoxyribonucleic acid with a base composition of 44.7% guanine plus cytosine. This deoxyribonucleic acid does not contain a base analogue for thymine and has a molecular weight estimated at 56 x 10(6) daltons.     

Unrelatedness of Bacillus amyloliquefaciens and Bacillus subtilis

Eight strains of highly amylolytic, sporeforming bacilli (hereafter referred to as Bacillus amyloliquefaciens) were compared with respect to their taxonomic relationship to B. subtilis. The physiological-biochemical properties of these two groups of organisms showed that B. amyloliquefaciens differed from B. subtilis by their ability to grow in 10% NaCl, characteristic growth on potato plugs, increased production of alpha-amylase, and their ability to ferment lactose with the production of acid. The base compositions of the deoxyribonucleic acid (DNA) of the B. subtilis strains consistently fell in the range of 41.5 to 43.5% guanine + cytosine (G + C), whereas that of the B. amyloliquefaciens strains was in the 43.5 to 44.9% G + C range. Hybrid formation between B. subtilis W23 and B. amyloliquefaciens F DNA revealed only a 14.7 to 15.4% DNA homology between the two species. Transducing phage, SP-10, was able to propagate on B. subtilis W23 and B. amyloliquefaciens N, and would transduce B. subtilis 168 (indole(-)) and B. amyloliquefaciens N-10 (arginine(-)) to prototrophy with a frequency of 3.9 x 10(-4) and 2.4 x 10(-5) transductants per plaque-forming unit, respectively. Attempts to transduce between the two species were unsuccessful. These data show that Bacillus amyloliquefaciens is a valid species and should not be classified as a strain or variety of B. subtilis.     

Cloning of sporulation gene spoIIG in Bacillus subtilis.

Two specialized transducing phages carrying a sporulation gene, spoIIG , of Bacillus subtilis were constructed from B. subtilis temperate phages p11 and phi 105 by the "prophage transformation" method. Restriction enzyme analysis and transformation experiments showed that the spoIIG gene was present on a 6.2 X 10(6)-dalton (6.2-Md) EcoRI fragment in both transducing phage genomes. Further analysis showed that spoIIG + transforming activity resides on a 2.25-Md EcoRI-BamHI fragment within the 6.2-Md EcoRI fragment. The 2.25-Md fragment was subcloned into the region between the EcoRI and BamHI sites of pUB110, and deletion plasmids lacking PstI or HindIII fragments within the 2.25-Md fragment were constructed. The recombinant plasmid carrying the intact spoIIG gene restored sporulation of strain HU1002 ( spoIIG41 recE4 ) to a frequency of 10(4) spores per ml and inhibited sporulation of strain 4309 ( spo + recE4 ) to a level of 10(3) spores per ml.     

Productive Infection of Bacillus subtilis 168, with Bacteriophage SP-10, Dependent upon Inducing Treatments

Strains of Bacillus that harbor defective phage PBSX were found to be insensitive to SP-10(C), although the phage adsorbed to these insensitive strains. Strains that did not carry the phage were sensitive to SP-10(C). B. subtilis 168 ind(-), which can be tranduced by SP-10(C) but is nonpermissive for the phage, was rendered phage-sensitive after treatment with ultraviolet (UV) light or mitomycin C. After induction with UV light, maximal sensitivity to SP-10(C) was obtained at a multiplicity of infection (MOI) of approximately 14; with mitomycin C induction, an MOI of approximately 1.0 was required. Phage maturation in sensitized cells was followed by plating infected streptomycin-sensitive cells in the presence of streptomycin at various stages during phase development. The latent period was estimated at 60 to 75 min. We suggest that the resistance of B. subtilis 168 to SP-10 is controlled, at least in part, by the presence of a defective prophage.     

Characterization of bacteriophage SPP1 transducing particles

Bacillus subtilis lysates produced by virulent bacteriophage SPP1 retained their transducing ability upon purification from contaminating PBSX particles. The buoyant density in CsC1 of the transducing activity was indistinguishable from that of the SPP1 plaque-forming units and the sedimentation behaviour in sucrose gradients of purified transducing particles was the same as that of SPP1 phage particles. Further, high concentrations of anti-SPP1 serum inactivated transducing particles and SPPl plaque-forming units at the same rate. The transduction process was resistant to DNAase treatment, but was enhanced by temperatures that did not allow transformation. It was concluded that particles of the size, shape, density and serum-sensitivity characteristic of SPP1, but carrying bacterial DNA, are vectors in a true transduction process. Cell survival upon SPP1 infection is discussed.     

Cloning of sporulation gene spoOB of Bacillus subtilis and its genetic and biochemical analysis.

A specialized transducing phage carrying a sporulation gene (spoOB) was constructed from Bacillus subtilis temperate phage rho 11 by in vitro and in vivo recombinations. Transformation experiments showed that the spoOB gene resides on a 1.4-megadalton fragment generated by EcoRI endonuclease treatment of the phage deoxyribonucleic acid (DNA). Mutants of this phage which lost transducing activity were isolated and used for genetic complementation tests and the analysis of protein(s) coded by the 1.4-megadalton fragment. The spoOB locus was shown to be composed of one cistron. Sodium dodecyl sulfate-polyacrylamide gel analysis of proteins synthesized in ultraviolet-irradiated cells infected with these phages showed that the 1.4-megadalton fragment codes at least one protein, of molecular weight 39,000, which is synthesized in both vegetative and sporulating cells. A cleavage map of the phage DNA was constructed by use of restriction endonucleases, EcoRI, BamHI, and SalI, and the site of integration of the 1.4-megadalton fragment was determined. Expression and function of the spoOB gene are discussed.     

Effect of Nalidixic Acid on Deoxyribonucleic Acid Synthesis in Bacteriophage SPO1-infected Bacillus subtilis

The effect of nalidixic acid on deoxyribonucleic acid (DNA) synthesis in Bacillus subtilis cells infected with bacteriophage SPO1 was studied. Nalidixic acid had little inhibitory effect on SPO1 DNA synthesis at concentrations that drastically inhibited B. subtilis DNA synthesis. Inhibition of DNA synthesis, appropriate to the concentration used, was imposed within 1 min after addition of nalidixic acid, suggesting that it acts directly on DNA synthesis in both infected and uninfected cells. The SPO1 DNA synthesized in the presence of high concentrations of nalidixic acid had a density characteristic of normal SPO1 DNA and was packaged into viable progeny phage particles, but its rate of synthesis was reduced and bacterial lysis was delayed.     

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.     

Genetic and physical studies of lambda transducing bacteriophage carrying the beta subunit gene of the Escherichia coli ribonucleic acid polymerase.

The prophage lambdac1857 was inserted into the bfe gene located near rif (the structural gene for the beta subunit of deoxyribonucleic acid [DNA]-dependent ribonucleic acid polymerase) on the Escherichia coli chromosome. Induced lysates (low-frequency transducing lysates) of such a lysogen contained defective lambda phage particles (lambdadrif+) that can specifically transduce the wild-type rif+ gene. Upon transduction into a recipient strain carrying recA, heterogenotes harboring both the wild-type and the mutant rif genes were isolated. Rec+ derivatives of these heterogenotes produce high-frequency transducing lysates that contain lambdadrif+ and normal active phages at a ratio of 1 to 2. The results of marker rescue experiments and of density determination with several transducing phages indicate that most of the late genes are deleted and replaced by a segment of the chromosomal DNA carrying the bfe-rif region. The length of the chromosomal segment seems to vary between approximately 0.5 and 0.6% of the total bacterial DNA among the three independently isolated lambdadrif+ phages. Electron microscopy of heteroduplex DNA consisting of one strand from lambdadrif+-6 and the other from lambdaimm-21 phages directly confirmed that most of the phage DNA of the "left arm" was replaced by the bacterial DNA. The heteroduplex study also demonstrated that the integration of prophage lambda into the bfe region occurred at the normal cross-over point within the phage attachment site.     

A Bacteriophage of Bacillus subtilis Which Forms Plaques Only at Temperatures Above 50 C III. Inhibition of TSP-1-Specific Deoxyribonucleic Acid Synthesis at 37 C and 45 C

The effect of temperature on phage-specific deoxyribonucleic acid (DNA) synthesis was studied in TSP-1-infected Bacillus subtilis. This was facilitated by selectively inhibiting host DNA synthesis with 6-(p-hydroxyphenylazo)-uracil. The results indicated that TSP-1 DNA synthesis did not continue at 37 C and was immediately shut down after transfer to this temperature. Incubation at 45 C greatly reduced TSP-1 DNA synthesis. Phage-specific DNA synthesis could resume at 53 C, however, when the infected culture was returned to 53 C after a 2-min incubation period at 37 C. The results suggest that the inhibition of phage DNA synthesis at 37 C is reversible. Since infected cultures returned to 53 C after 2 min at 37 C could not complete the replicative cycle, the irreversible inhibition of yet another intermediate step was suggested.     

Regulation of synthesis of alkaline phosphatase by deoxyribonucleic acid synthesis in a constitutive mutant of Bacillus subtilis

Hiraga, Sota (Osaka University, Osaka, Japan). Regulation of synthesis of alkaline phosphatase by deoxyribonucleic acid synthesis in a constitutive mutant of Bacillus subtilis. J. Bacteriol. 91:2192-2199. 1966.-It was found that synthesis of alkaline phosphatase (APase) correlated with deoxyribonucleic acid (DNA) synthesis in a partially constitutive mutant of Bacillus subtilis. When cultures of the mutant were made to undergo synchronous growth by germination of spores in an excess-phosphate medium, synthesis of APase was repressed at the beginning of DNA synthesis. If the initiation of DNA synthesis was inhibited by thymine starvation, the repression of APase was not observed. When DNA synthesis, previously initiated, was inhibited by thymine or uracil starvation, or by addition of mitomycin C, the repression was partially released at a later stage. In contrast, this correlation between repression and DNA synthesis was not observed in a repressible strain.     

Characterization of Inducible Bacteriophages in Bacillus licheniformis

Two morphologically distinct and physically separable defective phages have been found in Bacillus licheniformis NRS 243 after induction by mitomycin C. One of them (PBLB) is similar to the defective phage PBSX of B. subtilis, which has a density of 1.373 g/cm(3) in CsCl and a sedimentation coefficient of 160S. PBLB incorporates into its head mainly bacterial deoxyribonucleic acid (DNA) which has a sedimentation coefficient of 22S and a buoyant density in CsCl of 1.706 g/cm(3). The other phage (PBLA) has a morphology similar to the temperate phage phi105 of B. subtilis; the head diameter is about 66 nm, and it possesses a long and noncontractile tail. PBLA has a density of 1.484 g/cm(3) in CsCl and the phage-specific DNA, which is exclusively synthesized after induction by mitomycin C, has a density of 1.701 g/cm(3). PBLA DNA is double-stranded and has a sedimentation coefficient of 36S, corresponding to a molecular weight of 34 x 10(6) to 35 x 10(6) daltons. The phage DNA has one interruption per single strand, giving single-stranded segments with molecular weights of 13 x 10(6) and 4 x 10(6) daltons. Common sequences between the two phage DNA species and with their host DNA have been demonstrated by DNA-DNA hybridization studies. Both phage particles kill sensitive bacteria. However, all attempts thus far to find an indicator strain to support plaque formation have been unsuccessful.