Isolation and characterization of prophage mutants of the defective Bacillus subtilis bacteriophage PBSX.

Bacillus subtilis mutants with lesions in PBSX prophage genes have been isolated. One of these appears to be a regulatory mutant and is defective for mitomycin C-induced derepression of PBSX; the others are defective for phage capsid formation. All of the PBSX structural proteins are synthesized during induction of the capsid defective mutants; however, several of these proteins exhibit abnormal serological reactivity with anti-PBSX antiserum. The two head proteins X4 and X7 are not immunoprecipitable in a mutant which fails to assemble phage head structures. In the tail mutant, proteins X5 and X6 are not immunoprecipitable, tails are not assembled, and a possible tail protein precursor remains uncleaved. The noninducible mutant does not synthesize any PBSX structural proteins after exposure to mitomycin C. The mutation is specific for PBSX since ø105 and SPO2 lysogens of the mutant are inducible. All of the known PBSX-specific mutations were shown to be clustered between argC and metC on the host chromosome. In addition, the metC marker was shown to be present in multiple copies in cells induced for PBSX replication. This suggests that the derepressed prophage replicates while still integrated and that replication extends into the adjacent regions of the host chromosome.     

Bacteriophage-specific protein synthesis during induction of the defective Bacillus subtilis bacteriophage PBSX.

Particles of PBSX, a defective, noninfectious phage which is inducible from strains of Bacillus subtilis 168, contain at least seven structural proteins resolvable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Five of these proteins are associated with the phage tail and two with the phage head. An eighth protein, which also may be coded for by the PBSX prophage, has been identified in cells derepressed for PBSX replication.     

A new defective phage containing a randomly selected 8 kilobase-pairs fragment of host chromosomal DNA inducible in a strain of Bacillus natto

A new defective phage, designated PBND8, was induced in Bacillus natto strain IAM1207 with bleomycin and mitomycin C. PBND8 particles contained a randomly selected 8 kilobase-pairs (kbp) fragment of the host chromosomal DNA. Electron microscopy showed that PBND8 has a small head with a complex tail structure like PBSX, a defective phage of Bacillus subtilis 168. The PBND8 head, however, is clearly smaller than that of PBSX which contains 13-kbp fragments of the host chromosomal DNA. SDS-polyacrylamide gel electrophoretic analysis revealed that the structural proteins of PBND8 are distinct from those of PBSX and PBSY (PBSZ) of B. subtilis W23. PBND8 exhibited a bacteriocin-like killing activity to the other Bacillus cells.     

Precursors of the T4 internal peptides.

The precursors of the two T4 internal peptides have been identified by in vitro cleavage of individual phage proteins eluted from sodium dodecyl sulfate-acrylamide gels. The precursor of internal peptide VII is p22, the product of T4 gene 22 and an essential component of the morphogenic core. The precursor of peptide II is a protein with a molecular weight of approximately 13,000, whose gene has yet to be defined by mutation. A newly detected protein of approximately 15,000 molecular weight is found to be cleaved and is, therefore, likely to be a component of precursor head structures.     

Internal proteins of bacteriophage T4D: Their characterization and relation to head structure and assembly

Three basic proteins of low molecular weight (about 8000, 10,000 and 18,000) were isolated from the T4D phage particle. Many molecules of each protein are located within the phage head, possibly in association with the DNA, and together with the proteins which form the head membrane comprise most of the head structural protein. The purified internal proteins were characterized by physicochemical and immunological techniques; a radio-immunoassay allowed measurement of their synthesis in phage infected bacteria. Each internal protein is synthesized at both early and late times after infection. Their structural genes are present in the phage genome, but do not appear to be among the known amber mutant-containing genes of T4D. No evidence was found to suggest that the internal proteins are formed from a common precursor molecule, nor are their origins related to those of the internal peptides; however, one of the internal proteins may be altered before its incorporation into the phage. Pulse-chase experiments with two of these proteins show that they are incorporated into certain defective T4D heads. Whether or not they are incorporated appears to depend on the degree of completion of these heads, perhaps with respect to DNA packaging.     

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.     

Purification and characterization of two phage PBSX-induced lytic enzymes of Bacillus subtilis 168: an N-acetylmuramoyl-L-alanine amidase and an N-acetylmuramidase

After heat-induction of the defective phage PBSX in a xhi-1479 mutant of Bacillus subtilis 168, the culture lysed rapidly even if the lyt-2 mutation was present (which greatly reduces the amount of the bacterial autolysins). Two lytic enzymes, an N-acetylmuramoyl-L-alanine amidase and an endo-N-acetylmuramidase, were purified from the culture supernatant. The amidase was readily distinguished from the bacterial amidase by its low molecular weight. In addition, it was not inhibited by antibody directed against the bacterial enzyme. These results indicate that PBSX does not rely on the bacterial autolysins to accomplish lysis.     

Prophage mutation causing heat inducibility of defective Bacillus subtilis bacteriophage PBSX.

A mutant of Bacillus subtilis 168 has been isolated in which the defective phage PBSX was heat inducible, whereas another phage, phi105, was not so induced. A culture of the mutant grown at 30 degrees C, when shifted to 45 degrees C, began to lyse after 45 min; cell viability began to decrease after 10 min. Heat-induced lysis of the mutant was prevented by chloramphenicol. DNA, RNA, protein, and peptidoglycan synthesis were normal at the nonpermissive temperature up to the time of lysis. The site of xhi-1479 mutation causing this phenotype was linked (50%) in phage PBS1-mediated transduction to the host marker metC and to another PBSX marker xtl and was thus thought to map within the PBSX prophage. The order of markers was argC-thiB-metA-xhi-metC. The xhi mutation was thus distinct from another mutation, tsi-23, causing a similar heat inducibility of PBSX (Siegel and Marmur, 1969), which was unlinked to the metC marker. tsi-23 is therefore thought to be a host mutation, and the available evidence for a scattered phage genome being the cause of the defective nature of PBSX is thus less tenable. It was shown that the mutant, besides carrying the xhi mutation, also carried another closely linked mutation, xki-1479, which caused the PBSX produced to have no killing activity on the sensitive strain W23. The xki mutation was separated from xhi by recombination.     

Genetic control of bacterial suicide: regulation of the induction of PBSX in Bacillus subtilis.

PBSX is a phage-like bacteriocin (phibacin) of Bacillus subtilis 168. Bacteria carrying the PBSX genome are induced by DNA-damaging agents to lyse and produce PBSX particles. The particles cannot propagate the PBSX genome. The particles produced by this suicidal response kill strains nonlysogenic for PBSX. A 5.2-kb region which controls the induction of PBSX has been sequenced. The genes identified include the previously identified repressor gene xre and a positive control factor gene, pcf. Pcf is similar to known sigma factors and acts at the late promoter PL, which has been located distal to pcf. The first two genes expressed from the late promoter show homology to genes encoding the subunits of phage terminases.     

Involvement of a Bacterial Factor in Morphogenesis of Bacteriophage Capsid

A new bacterial factor has been found necessary for the activity of T4 gene 31, the only catalytic factor in the early stage of phage head formation, to process the assembly of head precursor proteins. In a mutant missing this factor, the precursors of phage head aggregate on the bacterial membrane.     

Defective pages as an antagonistic factor in closely-related bacilli

The antagonistic effect produced by the detective phage PBSX during cocultivation of the mutant strain B. subtilis 168, in which this phage is heat-inducible, and strain B. subtilis NRS231, which also bears a defective phage, was investigated. As soon as in the first hours of cocultivation under conditions of PBSX induction, the number of viable cells of strain NRS231 decreased by two orders of magnitude. However, the effect was not observed if the temperature of cocultivation was noninducing. The results confirm the supposition that defective phages may play a role in the competition between closely related bacilli.     

Structural Aberrations in T-Even Bacteriophage V. Effects of Canavanine on the Maturation and Utilization of Specific Gene Products

Previous results have shown that when a T-even bacteriophage-infected cell was exposed to l-canavanine followed by an exposure to l-arginine, a monster phage particle, termed a lollipop, was formed. l-Canavanine was necessary for the induction event but l-arginine was required for the maturation of the particle. We now describe the effects of canavanine on the maturation of certain T4 proteins and their role in the induction of lollipops. The cleavage reactions of the head proteins P22, P23, P24, and IPIII are prevented by l-canavanine as shown by the accumulation of the precursor proteins and the failure of the cleaved products to appear. l-Canavanine also prevents the appearance of P12 (tailplate protein) and P20 (head protein) indicating that these proteins may undergo a proteolytic cleavage during normal assembly. The formation of P10 (tailplate protein) and P18 (tail sheath protein) is also affected by l-canavanine. The data suggest that P23 in conjunction with P20 plays a major role in the determination of the length of the phage head.     

Head length determination in bacteriophage T4: The role of the core protein P22

We have found that two different temperature-sensitive mutations in gene 22, tsA74 and ts22-2, produce high frequencies (up to 85%) of petite phage particles when grown at a permissive or intermediate temperature. Moreover, the ratio of petite to normal particles in a lysate depends upon the temperature at which the phage are grown. These petite phage particles appear to have approximately isometric heads when viewed in the electron microscope, and can be distinguished from normal particles by their sedimentation coefficient and by their buoyant density in CsCl. They are biologically active as detected by their ability to complement a co-infecting amber helper phage. Lysates of both mutants grown at a permissive temperature reveal not only a significant number of petite phage particles in the electron microscope, but also sizeable classes of wider-than-normal particles, particles having abnormally attached tails, and others having more than one tail.Striking protein differences exist between the purified phage particles of tsA74 or ts22-2 and wild-type T4. B1¹, a 61,000 molecular weight head protein, is completely absent from the phage particles of both mutants, and the internal protein IPIII¹ is present in reduced amounts as compared to wild type. The precursor to B1¹ is present in the lysates, but these mutations appear to prevent its incorporation into heads, so it does not become cleaved.The product of gene 22 (P22) is known to be the major protein of the morphogenetic core of the T4 head. Besides the mutations reported here, several mutations which affect head length have been found in gene 23, which codes for the major capsid protein (Doermann et al., 1973b). We suggest a model in which head length is determined by an interaction between the core (P22 and IPIII) and the outer shell (P23).     

DNA packaging by the Bacillus subtilis defective bacteriophage PBSX.

Defective bacteriophage PBSX, a resident of all Bacillus subtilis 168 chromosomes, packages fragments of DNA from all portions of the host chromosome when induced by mitomycin C. In this study, the physical process for DNA packaging of both chromosomal and plasmid DNAs was examined. Discrete 13-kilobase (kb) lengths of DNA were packaged by wild-type phage, and the process was DNase I resistant and probably occurred by a head-filling mechanism. Genetically engineered isogenic host strains having a chloramphenicol resistance determinant integrated as a genetic flag at two different regions of the chromosome were used to monitor the packaging of specific chromosomal regions. No dramatic selectivity for these regions could be documented. If the wild-type strain 168 contains autonomously replicating plasmids, especially pC194, the mitomycin C induces an increase in size of resident plasmid DNA, which is then packaged as 13-kb pieces into phage heads. In strain RB1144, which lacks substantial portions of the PBSX resident phage region, mitomycin C treatment did not affect the structure of resident plasmids. Induction of PBSX started rolling circle replication on plasmids, which then became packaged as 13-kb fragments. This alteration or cannibalization of plasmid replication resulting from mitomycin C treatment requires for its function some DNA within the prophage deletion of strain RB1144.     

Length of Deoxyribonucleic Acid of PBSX-like Particles of Bacillus subtilis Induced by 4-Nitroquinoline-1-Oxide

The formation of particles resembling PBSX phages was induced by 4-nitroquinoline-1-oxide in a Marburg strain of Bacillus subtilis. All particles were homogeneous in their morphology. Physical and biological analyses revealed that the deoxyribonucleic acid (DNA) carried by these particles are fragments of host-cell DNA. The contour length of the DNA is 4.25 mum, corresponding to a molecular weight of 8.1 x 10(6) daltons.     

Bacteriophage T4 tail assembly: structural proteins and their genetic identification

We have identified the structural proteins of phage T4 precursor tails. Complete tails, labeled with ¹⁴C-labeled amino acids, were isolated from cells infected with mutants blocked in head assembly. The proteins were characterized by sodium dodecyl sulfate-acrylamide gel electrophoresis and subsequent autoradiography. The complete tails are made up of at least fifteen different species of phage proteins.To identify the genes specifying these proteins we prepared ¹⁴C-labeled amino acid lysates made with amber mutants defective in each of the twenty-one genes involved in tail assembly. Comparison of the gel pattern of the amber mutant lysates with wild type lysates enabled us to identify the following gene products, with molecular weights in parentheses: P6 (85,000); P7 (140,000); P8 (46,000); P9 (34,000); P10 (88,000); P11 (26,000); P12 (55,000); P15 (35,000); P18 (80,000); P19 (21,000); P29 (77,000). These eleven species are all structural proteins of the tail. The genetically unidentified tail proteins have molecular weights of 42,000, 41,000, 40,000 and 35,000. They are likely to be the products of known phage genes which were not resolved in the crowded middle region of the whole lysate gel patterns. The major tail proteins are all synthesized during the late part of the phage growth cycle.The mobilities of the proteins derived from tails did not differ from the mobilities of the proteins when derived from the unassembled pools of subunits accumulating in mutant infected cells, or when derived from complete phage particles.The genes for at least seven of the structural proteins are contiguous on the genetic map. Genes for proteins needed in many copies seem to be clustered separ- ately from genes whose products are needed in only a few copies. Consideration of protein sizes and published mapping data on phage T4 also suggest that the phage structural proteins are, on the average, much larger than the non-structural proteins.The requirement that at least fifteen different species of proteins must come together in forming a phage tail emphasizes the complexity of this morphogenetic process.     

Characterisation of a repressor gene (xre) and a temperature-sensitive allele from the Bacillus subtilis prophage, PBSX

The defective prophage of Bacillus subtilis 168, PBSX, is a chromosomally based element which encodes a non-infectious phage-like particle with bactericidal activity. PBSX is induced by agents which elicit the SOS response. In a PBSX thermoinducible strain which carries the xhi1479 mutation, PBSX is induced by raising the growth temperature from 37 degrees C to 48 degrees C. A 1.2-kb fragment has been cloned which complements the xhi1479 mutation. The nucleotide sequence of this fragment contains an open reading frame (ORF) which encodes a protein of 113 amino acids (aa). This aa sequence resembles that of other bacteriophage repressors and suggests that the N-terminal region forms a helix-turn-helix motif, typical of the DNA-binding domain of many bacterial regulatory proteins. The ORF is preceded by four 15-bp direct repeats, each of which contains an internal palindromic sequence, and by sequences resembling a SigA-dependent promoter. The nt sequence of an equivalent fragment from the PBSX thermoinducible strain has also been determined. There are three aa differences within the ORF compared to the wild type, one of which lies within the helix-turn-helix segment. This ORF encodes a repressor protein of PBSX.     

Characterization of two Salmonella newport bacteriophages

Salmonella newport phages 16--19 and 7--11 have very long heads and are members of two rare and so far little-known phage groups. Both produce various morphological aberrations. Preparations of phage 7--11 contain numerous polyheads and about 0.4% short heads belonging to nine size classes. In addition, one giant phage particle was observed. The head of phage 7--11 seems to be an icosahedron which became elongated by adding successive rows of subunits. Phages 16--19 and 7--11 have buoyant densities in CsCl of 1.43 and 1.48 g/mL and particle weights of 103 and 204 x 10(6) respectively. Both viruses contain double-stranded DNA, internal proteins, and sugars. Phage 16--19 contains 46.5% DNA of 35 x 10(6) molecular weight, and glucose. Phage 7--11 contains 47.5% DNA of 108 x 10(6) molecular weight, and mannose. Base compositions of phage and S. newport DNAs were determined from buoyant densities, melting point, and acid hydrolysis. Phage 16--19 contains 5.4% 5-methylcytosine.     

Cleavage of head and tail proteins during bacteriophage T5 assembly: selective host involvement in the cleavage of a tail protein

Electrophoresis studies showed that at least three phage-specified proteins undergo proteolytic cleavage during the development of bacteriophage T5. One of these proteins has a molecular weight of about 135,000 and the product of this cleavage reaction is a minor component of the T5 tail, having a molecular weight of about 128,000. All of the tail-defective T5 mutants studied in this report failed to induce this cleavage reaction under restrictive conditions. This reaction also failed to occur in Escherichia coli groEA639 and groEA36 infected with wild type T5. Examination of lysates of infected groE cells in the electron microscope revealed the presence of filled and empty heads as well as tubular head structures, but no tails were detected. The filled heads were able to combine with separately prepared T5 tails in vitro to form infectious phage particles. Therefore, propagation of T5 in these groE mutants is prevented primarily by a specific block in tail assembly. A T5 mutant, T5?6, was isolated, which has the capacity to propagate in these groE hosts. The gene locus in T5?6 was mapped.The second T5 protein which is cleaved has a molecular weight of 50,000 and is related to head morphogenesis. Treatment of infected cells with l-canavanine (50 μg/ml) inhibited cleavage of this polypeptide. Only small quantities of the major head protein (32,000 mol. wt) were produced in these treated cells. Treatment with canavanine lead to production of tubular heads. The major protein component of partially purified tubular heads has a molecular weight of 50,000. Cells infected with T5 amber H30b, a mutant defective in head gene D20, does not produce the 50,000 and 32,000 molecular weight proteins. These findings suggest that the 50,000 molecular weight protein undergoes cleavage to form the major head polypeptide. A third T5 protein is cleaved to form a minor head component with a molecular weight of 43,000 and its cleavage is linked to that involving the major head protein.