Transduction in Bacillus subtilis by Bacteriophage SPP1

Lysates of the virulent bacteriophage SPP1 were shown to be capable of mediating generalized transduction. Suppressible mutants of this bacteriophage (sus) were capable of transduction at a lower multiplicity of infection than virulent SPP1. Linkage analysis demonstrated that bacteriophage SPP1 transduced segments of the genome equal in size to that transferred by SP10. This bacteriophage should be useful in analyzing the regions of the genome where PBS1 appears to give anomalous results.     

Transcription After Bacteriophage SPP1 Infection in Bacillus subtilis

The role of the host polymerase in Bacillus subtilis infected with phage SPP1 was studied in vivo with regard to production of phage-specific and host-specific ribonucleic acid (RNA) and to phage yield. Evidence is presented that the subunit(s) of B. subtilis RNA polymerase which is sensitive to rifampin and streptolydigin is necessary at all times during infection for phage production. The synthesis of phage RNA and the phage yield in strains resistant to either antibiotic were unaffected by the drug. Host RNA synthesis continued throughout infection; phage-specific RNA never accounted for more than 20% of pulse-labeled RNA at any time during infection.     

Characterization of Temperate Bacillus Bacteriophage phi105

Temperate Bacillus phage 105 is serologically unrelated to previously described virulent Bacillus phages. Phage 105 is incapable of generalized transduction. Prophage 105 is inducible with mitomycin C. Phage 105 contains double-stranded deoxyribonucleic acid (DNA) with a molecular weight of about 25 × 10⁷ as determined by band sedimentation and electron microscopy. The per cent guanine plus cytosine of 105 DNA is 43.5 as determined by buoyant density in CsCl and by thermal denaturation. Phage 105 DNA may contain complementary single-stranded ends.     

Bacteriophage Interference in Bacillus subtilis 168

Strains of Bacillus subtilis lysogenic for temperate bacteriophage SPO2 inhibit the development of bacteriophage phi1. After infection by bacteriophage phi1, DNA and RNA synthesis in the lysogenic host terminates, culminating in cell death. Bacteriophage SPO2 also prevents the production of bacteriophage phi105. Mechanisms for these two types of bacteriophage interference are discussed.     

Initiation and Termination Mutants of Bacillus subtilis Bacteriophage SPO1

Mutants affected in cistrons 21 and 32 of bacteriophage SPO1 are defective specifically in the initiation of DNA replication. Mutations in cistron 32 also specifically affect the termination of replication.     

Bacteriophage SP50 as a marker for cell wall growth in Bacillus subtilis.

When grown under conditions of phosphate limitation, Bacillus subtilis W23 lacked wall teichoic acid and did not adsorb phage SP50. During transition from growth under conditions of phosphate limitation to those of potassium limitation, the bacteria developed an ability to adsorb phage which increased exponentially in relation to their content of wall teichoic acid. During transition in the reverse direction, the bacteria retained near-maximum phage-binding properties until their content of wall teichoic acid had fallen to a fairly low level. These observations suggest that newly incorporated wall material does not immediately appear at the cell surface in a structure to which phage can adsorb. Examination of the location of adsorbed phage particles showed that recently incorporated receptor material appeared at the cell surface first along the length of the cylindrical portion of the cell. The results are consistent with models of wall assembly in which newly synthesized wall material is intercalated at a large number of sites that are distributed along the length of the cell. This newly incorporated material may be located initially at a level underlying the surface of the cell and may become exposed at the surface only during subsequent growth. Incorporation of new material may also proceed rapidly into the developing septa, but new wall material is incorporated into existing polar caps more slowly, or perhaps not at all.     

Mapping of a Temperate Bacteriophage Active on Bacillus subtilis

Bacteriophage phi105 is a temperate bacteriophage for Bacillus subtilis 168. Temperature-sensitive and plaque mutants of phi105 were isolated. The results of two- and three-factor crosses with these mutants suggest the vegetative map of phi105 to be circular. The location of prophage phi105 between bacterial markers phe-1 and ilvA1 was shown by means of PBS1 transduction. Five markers in the prophage were linearly ordered with respect to the bacterial markers. Linkage between bacterial and prophage markers was demonstrated in transformation experiments with deoxyribonucleic acid extracted from lysogenic bacteria. The data demonstrate that prophage phi105 is linearly inserted into the bacterial chromosome.     

Viral mutation affecting bacteriophage phi 1 development in Bacillus subtilis 168.

Bacillus subtilis bacteriophage phi 1m, a host-range variant, was isolated after mutagenesis of virulent bacteriophage phi 1. Unlike its wild-type antecedent, phi 1m could not form plaques on lawns of B subtilis 168 at 37 C, although it adsorbed to, penetrated, and killed this bacterium. Experiments conducted in liquid medium at 37 C showed that B. subtilis 168 cells allowed reduced levels of phi 1m development at low multiplicities of infection, whereas high multiplicity infections of this strain by the phage were abortive. Certain mutants, derived originally from B. subtilis 168, were observed to be permissive for phi 1m at 37 C; moreover, their permissive phenotype could be duplicated by growing wild-type B. subtilis 168 cells at temperatures above 47 C. Studies on phi 1m and host nucleic acid synthesis under nonpermissive conditions demonstrated that transciption and DNA synthesis proceeded up to 20 min after infection, after which time there was a cessation of all nucleic acid production. These observations are discussed with respect to other abortive bacteriophage infections in B. subtilis.     

Temperature-Sensitive Induction of Bacteriophage in Bacillus subtilis 168

In a temperature-sensitive mutant of Bacillus subtilis 168, induction of the defective phage PBSX occurred at 48 C. Cell lysis began after 90 min of growth at 48 C, and cell viability began to decrease after 10 to 30 min. The loss in viability at the nonpermissive temperature was prevented by azide or cyanide. Deoxyribonucleic acid (DNA), ribonucleic acid, and protein synthesis were not inhibited at 48 C. Temperature induction of the temperate phage SPO2 also occurred in this mutant. The temperature-sensitive mutation, designated tsi-23, was linked by transduction to purB6 and pig, the order being purB6 pig tsi-23. Mutation tsi-23 was transformable to wild type by B. subtilis 168 DNA but not by DNA from the closely related strains W23 or S31. DNA from the latter two strains transformed auxotrophic markers of strain 168 at frequencies close to those found with 168 donor DNA. Upon temperature induction, cellular DNA was broken to a size of 22S, characteristic of DNA in PBSX particles. The DNA isolated from temperature-induced PBSX did not give an increased Ade(+)/Met(+) transformant ratio relative to cellular DNA nor contain preferential break points as determined by transformation of four closely linked markers.     

Nucleic Acid Synthesis in Bacteriophage SPO2c1-Infected Bacillus subtilis

The synthesis of host macromolecules was shut off very slowly and incompletely by bacteriophage SPO2c(1). No change in the rate of incorporation of radioactive precursors into protein and ribonucleic acid (RNA) could be detected after infection, and the rate of incorporation of thymidine was increased only slightly. The relative proportions of phage and host species of nucleic acids at various intervals in the latent period were determined by means of nucleic acid hybridization. Phage-specific RNA populations synthesized early were different from those synthesized late in the latent period. Host deoxyribonucleic acid (DNA) replication continued until 8 to 10 min after SPO2c(1) infection and then decreased markedly as phage-specific DNA synthesis was initiated. Host DNA was not degraded to trichloroacetic acid-soluble fragments, and its nucleotides were not found in either newly synthesized intracellular phage DNA or in progeny phage particles. The average burst size of SPO2c(1) was approximately 200 plaque-forming units per cell.     

Bacteriophage SPO1-induced macromolecular synthesis in minicells of Bacillus subtilis.

SPO1 bacteriophage injects its DNA into minicells produced by Bacillus subtilis CU403 divIVB1. The injected DNA is partially degraded to small trichloracetic acid-precipitable material and trichloroacetic acid-soluble material. The injected DNA is not replicated; however, it serves as a template for RNA and protein synthesis. The RNA produced specifically hybridizes to SPO1 DNA, and the amount of RNA hybridized can be reduced by competition with RNA isolated at all stages of the phage cycle from infected nucleate cells of the B. subtilis CU403 divIVB1. An unrelated phage, SPP1, also induces phage-specific RNA in infected minicells. Translation occurs in SPO1-infected minicells resulting in at least eight proteins which have been separated by gel electrophoresis, and two of these proteins have mobilities similar to proteins found only in infected B. subtilis CU403 divIVB1 nucleate cells. A large proportion of the polypeptide material synthesized in infected minicells is very small and heterogeneous in size.     

Bacteriophage lambda as a cloning vector

[This corrects the article on p. 582 in vol. 56.].     

Bacteriophage transformation of PBS2 in Bacillus subtilis.

Transformation of temperature-sensitive mutants of bacteriophage PBS2 for Bacillus subtilis was demonstrated. The number of transformants was linearly related to the concentration of DNA within a range of 0.01 to 1 mug/ml. No transformants were obtained when the DNA was pretreated with DNase. PBS2 DNA sheared to approximately 1% of the total chromosome length was centrifuged in Cs2SO4-Hg gradients to fractionate the DNA according to the base composition. Transformation experiments carried out with the fractionated DNA indicated the possibility of determining the base composition of different regions of the phage chromosome.     

Incorporation of Uridine into Bacillus subtilis and SPP1 Bacteriophage Deoxyribonucleic Acid

Tritiated uridine is incorporated into the deoxyribonucleic acid of Bacillus subtilis and bacteriophage SPP1; the tritium is recovered in the cytidine moiety of both deoxyribonucleic acids.     

Viral Protein Synthesis in Bacteriophage φ29-Infected Bacillus subtilis

Twenty-three (14)C-labeled phage phi29-specific proteins in lysates of UV-irradiated Bacillus subtilis have been resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and identified by autoradiography. Included in this group of proteins are the six major structural proteins of the virion. Analysis of the temporal sequence of viral protein synthesis indicates that three groups of proteins can be identified by time of appearance, beginning at 2 to 4, 4 to 6, or 8 to 10 min after infection, respectively. These proteins account for approximately 90% of the coding capacity of the phi29 genome.