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.     

Defective Bacteriophage PBSH in Bacillus subtilis: I. Induction, Purification, and Physical Properties of the Bacteriophage and Its Deoxyribonucleic Acid

PBSH, a defective phage of Bacillus subtilis strain 168, is described. Conditions are given for optimal induction of the prophage with mitomycin C. After a latent period of 90 min, cells were lysed and phage-like particles were released with a burst size of approximately 100 to 400 phage per bacterium. Since no known host supports phage replication after infection, burst size was determined by electron microscope count. Purification procedures and criteria for purity are described. The molecular weight of deoxyribonucleic acid (DNA) extracted from PBSH was estimated by length measurement and sedimentation. No circular DNA molecules were found by either technique. PBSH DNA molecules are linear, double-stranded, and of homogeneous molecular weight, about 12 x 10(6) daltons. There is no evidence for single-strand breaks. The majority of PBSH DNA molecules show a sedimentation behavior dependent on ionic strength. It is inferred that most of the DNA molecules are less hydrodynamically rigid than native DNA having a similar average base composition and molecular weight. Possible reasons for the sedimentation behavior are discussed.     

Characterization of a Temperate Phage and Four Bacteriocins Produced by Nonpathogenic Clostridium Species

We previously reported the isolation of a temperate phage (named KT) and several bacteriocins (named clostocins) from strains of nonpathogenic Clostridium species. Later, the induction and some properties of the phage and four clostocins (A, B, C and D) were examined.

The phage was induced by UV light and mitomycin C. The phage had a polygonal head (about 85mμ in diameter) and a tail with contractile sheath (about 100mμ in length). Some other properties of the phage were also studied; plaque morphology, stability in salt solution, inactivation by UV light, pH stability, thermal inactivation, host-range and lysis of infected culture.

Clostocins A and D were partially induced by UV light and mitomycin C, whereas that of B and C were not. All clostocins failed to pass through a dialysis membrane, and were insensitive to UV light and to ribo- and deoxyribonuclease. They were destroyed by some proteolytic enzymes, but differences in degree of their susceptibility were observed among them. Clostocins A and D were very thermo-stable, whereas B and C were relatively thermo-labile. Clostocins A and D acted on some strains in the genus Clostridium, whereas B and C did on many strains in the family Bacillaceae.

There was no demonstrable serological relationship between phage KT and clostocin A, although they seemed to adsorb on the same bacterial receptor.     

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.     

Death of Bacillus subtilis Auxotrophs Due to Deprivation of Thymine, Tryptophan, or Uracil

Pritikin, William B. (University of California, Los Angeles), and W. R. Romig. Death of Bacillus subtilis auxotrophs due to deprivation of thymine, tryptophan, or uracil, J. Bacteriol. 92:291-296. 1966.-Auxotrophic mutants of Bacillus subtilis 168 that require either tryptophan, uracil, or thymine died rapidly when deprived of any of these compounds. Phage PBS1 was produced by infected B. subtilis 168 (thy try-2) deprived of thymine. Phage PBS1 was not produced by infected B. subtilis 168 (try-2) deprived of tryptophan or infected B. subtilis 168-15 (try-2 ura) deprived of uracil. B. subtilis 168 thy try-2 and 168-15 could be transduced by phage PBS1 after prolonged deprivation of tryptophan or uracil, respectively. When B. subtilis 168-15 was transduced to uracil independence by phage PBS1, the uracil-independent transductants became immune to uracil-less death within 10 min of exposure to phage, and began to multiply within 2 hr after exposure to phage at an incubation temperature of 46 C.     

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.     

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.     

Defective Bacteriophage PBSH in Bacillus subtilis: II. Intracellular Development of the Induced Prophage

Treatment of Bacillus subtilis strain 168 with mitomycin C caused induction of a defective prophage, PBSH. During induction, extensive deoxyribonucleic acid (DNA) synthesis took place. Concurrently, a change in marker frequency of the bacterial DNA was noticed. The frequency of only one marker, ade-16, the marker closest to the origin of the bacterial chromosome, was enhanced manyfold. DNA from whole phage particles transformed all bacterial markers at a frequency equal to that of DNA in the noninduced culture, except ade-16, the frequency of which was enhanced 30 to 100 times. Analysis of a double isotope experiment demonstrated that 14% of the phage DNA was derived from preinduction bacterial DNA. The other 86% of DNA in phage particles was DNA replicated after induction. Density label experiments with 5-bromodeoxyuridine showed that postinduction DNA synthesis took place preferentially at the origin region of the bacterial chromosome. Measurement of the molecular weight of DNA replicated after induction clearly showed that postinduction DNA replication is chromosomal. No evidence for prophage detachment and autonomous phage DNA replication was found. The data indicated that, after mitomycin C action, the bacterial chromosome under-went multiple reinitiation at the origin, while normal sequential DNA replication was stopped. The pool of replicated bacterial DNA was fragmented randomly. This DNA was packaged into PBSH particles which were released after cell lysis.     

A method for construction of specialized transducing phage rho 11 of Bacillus subtilis.

DNA from a temperate phage rho 11 and chromosomal DNA of Bacillus subtilis 168 were digested with endonuclease EcoRI and then ligated with T4 polynucleotide ligase. The ligated DNA fragments were used to transform a lysogenic strain, B. subtilis spoA12 lys21 hisA1 leuA8 p11, and Lys+, His+ or Leu+ transformants were selected. The cells of each type were then mixed, grown and treated with mitomycin C; the induced phages were tested for abilities abilities to form plaques and to tranduce the auxotrophic marker. Various types of plaque-forming or defective phages which transduce hisA or lys marker at considerably high frequencies were thus obtained.     

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.     

Restriction and modification in Bacillus subtilis: inducibility of a DNA methylating activity in nonmodifying cells.

The nonrestricting/nonmodifying strain Bacillus subtilis 222 (r-m-) can be induced to synthesize a DNA-modifying activity upon treatment with either mitomycin C (MC) or UV light. This is shown by the following facts. (i) Infection of MC-pretreated 222 cells with unmodified SPP1 phage yields about 3% modified phage that are resistant to restriction in B. subtilis R (r+m+). The induced modifying activity causes the production of a small fraction of fully modified phage in a minority class of MC-treated host cells. (ii) The MC-pretreated host cells contain a DNA cytosine methylating activity: both bacterial and phage DNAs have elevated levels of 5-methylcytosine. (iii) The MC-induced methylation of SPP1 DNA takes place at the recognition nucleotide sequences of restriction endonuclease R from B. subtilis R. (iv) Crude extracts of MC-pretreated 222 cells have enhanced DNA methyltransferase activities, with a substrate specificity similar to that found in modification enzymes present in (constitutively) modifying strains.     

Bacteriophages of Bacillus polymyxa

Virulent bacteriophages of colistin--producing Bacillus polymyxa strains were studied. The phages were found to differ in lytic spectrum and were active only against strains of B. polymyxa. They did not attack other strains of the genus Bacillus. The virulent bacteriophages belong to two morphological groups differing in size. The size of the DNA of the bacteriophages of both groups is similar and ranges from 74.9 X 10(6) to 87.8 X 10(6) daltons. The cells of different B. polymyxa strains were also found to carry various defective phages which could be shown after mitomycin C induction of cell cultures. The antibacterial activity of mitomycin C induced cell lysates was not detected. Strains of B. polymyxa most probably devoid of defective bacteriophages (delysogenized) were isolated.     

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.     

Clay minerals protect bacteriophage PBS1 of Bacillus subtilis against inactivation and loss of transducing ability by UV radiation

The effect of UV radiation on the survival of and transduction by phage PBS1 of Bacillus subtilis, free or adsorbed on the clay minerals montmorillonite (M) and kaolinite (K), was studied. After free or clay-associated phage (approximately 10(7) PFU.mL-1) was irradiated with UV light (254 nm) for 0, 1, 2, 5, 10, and 30 min and then allowed to infect B. subtilis FB300 (thiB4 metA29 argF4 Rfmr), the phage was titered, and Met+ transductants were enumerated on selective media. After 1 min of irradiation, the titer of free and clay-associated phage decreased significantly (approximately 1.6 times for free phage, and approximately 4.9 and 6.8 times for M and K, respectively), whereas the transduction frequency increased significantly (approximately 3 times for free phage and approximately 1.4 and 2.2 times for M and K, respectively). The titer and transduction frequency of clay-associated phage remain essentially constant between 1 and 10 min of irradiation, whereas the titer of free phage decreased by approximately 1 order of magnitude after 5 min of irradiation. When free phage was irradiated for 10 min, the titer and transduction frequency decreased by approximately 2 and 0.5 orders of magnitude, respectively, whereas 30 min of irradiation was necessary to obtain comparable decreases with clay-associated phage. These results indicated that phages are protected to some extent from UV radiation when adsorbed on clay minerals.     

Effects of Mitomycin C and Thymidine Deprivation on Lysogenic and Sensitive Strains of Bacillus megaterium

A triple auxotroph of Bacillus megaterium strain KM was lysogenized with a phage suspension from B. megaterium 899a. The lysogenic and phage-sensitive derivatives of KM were found to die at the same exponential rate during thymineless incubation, despite the fact that the lysogenic strain became induced. The lysogenic strain was also induced by mitomycin C, and died at an exponential rate which was approximately twice that of the sensitive strain. With both strains, the lethality of mitomycin C was the same in the presence and absence of thymidine; thymidine was required for maximal phage production. Mitomycin C preferentially inhibited deoxyribonucleic acid (DNA) synthesis of both strains for the first 60 min. The (DNA) synthetic ability of the lysogenic strain was subsequently restored, due to phage production. Since there was no evidence that sensitive strains of KM contained other inducible elements (prophage or probacteriocins), it is concluded that both thymineless death and mitomycin C death can occur via mechanisms not involving induction.     

Growth of Bacteriophage φ105 and Its Deoxyribonucleic Acid in Radiation-Sensitive Mutants of Bacillus subtilis

Growth of phage phi105 and its deoxyribonucleic acid (DNA) was studied in radiation-sensitive mutants of Bacillus subtilis. The recA gene is required for optimal prophage induction with mitomycin C and for infectivity of prophage DNA. rec B gene is required for marker rescue from mature DNA. The importance of bacterial genes for phage DNA activity seems to depend on phage DNA structure.     

Host-Bacteriophage Interaction in Agrobacterium tumefaciens III. Phage-Coded Endolysins

Endolysins were detected in a sensitive strain of Agrobacterium tumefaciens (B6) after infection with phage LV-1 and in the lysogen A. tumefaciens V-1 after induction with mitomycin C. A similar endolysin was found in mitomycin C-induced A. tumefaciens C-58, which apparently harbors a defective prophage.     

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.     

Study of lysogeny in Bacillus thuringiensis and B. cereus]

Forty-eight strains of Bacillus thuringiensis and 12 strains of B. cereus were treated with ultraviolet light and mitomycin C. The former agent was the more effective inducer. Bacillus thuringiensis produces at least seven different phage particles with long, non-contractile tails. The frequencies of lysogeny and polylysogeny are 83 and 25% respectively. Morphologically defective phages occur in 25% of strains, whereas five of them produce low molecular-weight bacteriocins. One strain of B. cereus harbors "killer-particles." There is no apparent correlation between the presence of phage-like particles, phage senstivity, and serotypes, biotypes, or the origin of B. thuringiensis strains.     

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.