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.     

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 PBSX-induced deletion mutants of Bacillus subtilis 168 constitutive for alkaline phosphatase

Mutants of Bacillus subtilis with deletions extending from the PBSX prophage, and in some cases removing pro(AB) and metC, have been found to be constitutive for vegetatively synthesized alkaline phosphatase. Such deletions were isolated by selecting for heat-resistant derivatives of a strain carrying a xhi-1479 mutation causing heat-inducibility of the defective phage PBSX. These deletions remove the phoS gene, a regulatory gene for alkaline phosphatase; it is concluded that the phoS gene product exerts negative control on alkaline phosphatase synthesis. Deletion mapping, combined with previously published linkage data, indicates a gene order of PBSX-phoS-pro(AB)-metC.     

Genetic Mapping of a Defective Bacteriophage on the Chromosome of Bacillus subtilis 168

A genetic marker responsible for the killing activity of PBSX, a defective phage carried by Bacillus subtilis 168, has been located on the bacterial chromosome. Two mutant strains of B. subtilis 168, which produced tailless phage particles upon mitomycin C induction, were shown to carry lesions, designated xtl-1 and xtl-2, which were linked by transformation and PBS1-mediated transduction to metC. The link-age relationship between xtl and adjacent auxotrophic markers was determined by three-factor PBS1 transduction, the suggested order of markers being argO 1 metA metC xtl.     

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.     

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.     

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.     

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.     

Construction and characterization of recombinant phage phi 105 d(Cmrmet) for cloning in Bacillus subtilis

A 1.6 kb fragment of DNA of plasmid pBD64, obtained after partial digestion with HpaII, carrying a chloramphenicol-resistance determinant and a single site for the enzyme Bg/II, was inserted into the genome of defective phage phi 105 d/ys. Two types of phage were subsequently isolated and both transduced cells of Bacillus subtilis to chloramphenicol resistance. One type contained 26 kb and the other 32 kb of DNA. Bacillus subtilis chromosomal DNA fragments generated by cleavage with Bg/II were ligated into the unique Bg/II site within the smaller phage genome. A specialized transducing phage was isolated which carried the metC gene on a 6 kb Bg/II fragment. This phage, denoted phi 105 d(Cmrmet), transduced B. subtilis strain MB79 pheA12 metC3 to Met+ and to chloramphenicol resistance, and the metC3 mutation was complemented in transductants.     

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.     

Genome instability of Pseudomonas aeruginosa phages of the EL species: examination of virulent mutants

The article continues a study of pseudolysogeny in Pseudominas aeruginosa infected with phiKZ-like phages of the EL species. Analysis was performed for several newly isolated virulent mutants of EL phages (EL and RU) that were virulent (capable of causing lysis of bacteria infected with the wild-type phage) and a lower extent of opalescence of negative colonies (NCs). Wile-type recombinants were detected in crosses of virulent mutants of phages EL and RU to confirm the polygenic control of virulence. Since a deletion mutation was found in one of the virulent EL mutants and high genetic instability was characteristic of another mutant, a mobile genetic element was assumed to play a role in mutagenesis. Pseudolysogeny of bacteria provides for horizontal gene transfer between different bacterial strains. Hence, sequencing of the phage genome and demonstration of the lack of toxic gene products are insufficient for the phage to be included into a therapeutic mixture. To use live phages, it is essential to study in detail the possible consequences of their interaction with host bacteria.     

Genome instability of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> phages of the EL species: Examination of virulent mutants

The article continues a study of pseudolysogeny in Pseudominas aeruginosa infected with phiKZ-like phages of the EL species. Analysis was performed for several newly isolated vir mutants of EL phages (EL and RU) that were virulent (capable of causing lysis of bacteria infected with the wild-type phage) and a lower extent of opalescence of negative colonies (NCs). Wile-type recombinants were detected in crosses of virulent mutants of phages EL and RU to confirm the polygenic control of virulence. Since a deletion mutation was found in one of the virulent EL mutants and high genetic instability was characteristic of another mutant, a mobile genetic element was assumed to play a role in mutagenesis. Pseudolysogeny of bacteria provides for horizontal gene transfer between different bacterial strains. Hence, sequencing of the phage genome and demonstration of the lack of toxic gene products are insufficient for the phage to be included into a therapeutic mixture. To use live phages, it is essential to study in detail the possible consequences of their interaction with host bacteria.     

Requirement for a functional host cell autolytic enzyme system for lysis of Escherichia coli by bacteriophage phi X174

Escherichia coli VC30 is a temperature-sensitive mutant which is defective in autolysis. Strain VC30 lyses at 30 degrees C when treated with beta-lactam antibiotics or D-cycloserine or when deprived of diaminiopimelic acid. The same treatments inhibit growth of the mutant at 42 degrees C but do not cause lysis. Strain VC30 was used here to investigate the mechanism of host cell lysis induced by bacteriophage phi X 174. Strain VC30 was transformed with plasmid pUH12, which carries the cloned lysis gene (gene E) of phage phi X174 under the control of the lac operator-promoter, and with plasmid pMC7, which encodes the lac repressor to keep the E gene silent. Infection of strain VC30(pUH12)(pMC7) with phage phi X174 culminated in lysis at 30 degrees C. At 42 degrees C, intracellular phage development was normal, but lysis did not occur unless a temperature downshift to 30 degrees C was imposed. Similarly, induction of the cloned phi X174 gene E with isopropyl-beta-D-thiogalactoside resulted in lysis at 30 degrees C but not at 42 degrees C. Temperature downshift of the induced culture to 30 degrees C resulted in lysis even in the presence of chloramphenicol. These results indicate that host cell lysis by phage phi X174 is dependent on a functional cellular autolytic enzyme system.     

SOS induction in a subpopulation of structural maintenance of chromosome (Smc) mutant cells in Bacillus subtilis

The structural maintenance of chromosome (Smc) protein is highly conserved and involved in chromosome compaction, cohesion, and other DNA-related processes. In Bacillus subtilis, smc null mutations cause defects in DNA supercoiling, chromosome compaction, and chromosome partitioning. We investigated the effects of smc mutations on global gene expression in B. subtilis using DNA microarrays. We found that an smc null mutation caused partial induction of the SOS response, including induction of the defective prophage PBSX. Analysis of SOS and phage gene expression in single cells indicated that approximately 1% of smc mutants have fully induced SOS and PBSX gene expression while the other 99% of cells appear to have little or no expression. We found that induction of PBSX was not responsible for the chromosome partitioning or compaction defects of smc mutants. Similar inductions of the SOS response and PBSX were observed in cells depleted of topoisomerase I, an enzyme that relaxes negatively supercoiled DNA.     

Loss of plasmids containing cloned inserts coding for novobiocin resistance or novobiocin sensitivity in Haemophilus influenzae.

Plasmids pNov1 and pNov1s , coding for resistance and sensitivity to novobiocin, respectively, were readily lost from wild-type Haemophilus influenzae but retained in a strain lacking an inducible defective prophage. The plasmid loss could be partly or wholly eliminated by a low-copy-number mutation in the plasmid or by the presence of certain antibiotic resistance markers in the host chromosome. Release of both phage HP1c1 , measured by plaque assay, and defective phage, measured by electron microscopy, was increased when the plasmids were present. The frequency of recombination between pNov1 and the chromosome, causing the plasmid to be converted to pNov1s , could under some circumstances be decreased from the normal 60 to 70% to below 10% by the presence of a kanamycin resistance marker in the chromosome. This suggested that a gene product coded for by the plasmid, the expression of which was affected by the kanamycin resistance marker, was responsible for the high recombination frequency. Evidence was obtained from in vitro experiments that the gene product was a gyrase.     

Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B.

A temperature-sensitive dnaK mutant (strain MT112) was isolated from Escherichia coli B strain H/r30RT by thymineless death selection at 43 degrees C. By genetic mapping, the mutation [dnaK7(Ts)] was located near the thr gene (approximately 0.2 min on the may). E. coli K-12 transductants of the mutation to temperature sensitivity were assayed for their susceptibility to transducing phage lambda carrying the dnaK and/or the dnaJ gene. All of the transductants were able to propagate phage lambda carrying the dnaK gene. When macromolecular synthesis of the mutant was assayed at 43 degrees C, it was observed that both deoxyribonucleic acid and ribonucleic acid syntheses were severely inhibited. Thus, it was suggested that the conditionally defective dnaK mutation affects both cellular deoxyribonucleic acid and ribonucleic acid syntheses at the nonpermissive temperature in addition to inability to propagate phage lambda at permissive temperature.     

Mechanism of phage-induced lysis in pneumococci

Earlier studies have suggested the possible role of host autolytic enzyme in the release of progeny phage from Dp-1 infected pneumococci. Several new experiments described here reinforce this notion. Specifically, the resistance of an autolysis-defective mutant to infection at low phage to cell ratios could be eliminated by prior 'coating' of the host bacteria with pneumococcal autolysin isolated from wild-type cells. Similar, productive infection was also possible by lowering the temperature of incubation to 30 degrees C, a condition that leads to a partial activation of the thermosensitive residual autolysin in the mutant cells. Other experiments, however, clearly indicate the role of the newly discovered phage-associated lysin (PAL), reported in the accompanying communication, in bacteriophage release and culture lysis; specifically, lysis was stimulated by reducing agents and inhibited by cardiolipin. It seems that both the host-related and the PAL activities are involved with Dp-1 induced lysis of pneumococci.     

Control of lysogeny and a genetic map of the temperate phage SM of Pseudomonas aeruginosa

76 mutants with impaired ability to lysogenize host cells were isolated in SM phage after mutagenesis using several chemical mutagens. By means of complementation test, these mutants were distributed into two groups, cI and cII. The mutants of the cI group were similar phenotypically to the cI mutants of phage lambda defective in synthesis of repressor. The mutants of the cII group establish and support the lysogenic state in infected cells with very low frequency. Temperature-sensitive mutants belonging to 13 complementation groups and nonlysogenizing mutants of the cI and cII groups were used in genetic mapping of SM phage. Mutual positions of markers and relative distances between them were determined by the method of two-factorial crosses. The greatest distance equal to 20 units of recombination was determined between ts 88 marker and one of early genes marked with ts 105 mutation. The genes cI and cII are closely linked to each other and also to ts 105 marker and are situated at one end of the genetic map.