Bacteriophage PBS2 Research Articles

New Deoxyribonucleic Acid Polymerase Induced by Bacillus subtilis Bacteriophage PBS2

The deoxyribonucleic acid (DNA) of Bacillus subtilis phage PBS2 has been confirmed to contain uracil instead of thymine. PBS2 phage infection of wild-type cells or DNA polymerase-deficient cells results in an increase in the specific activity of DNA polymerase. This induction of DNA polymerase activity is prevented by actinomycin D and chloramphenicol. In contrast to the major B. subtilis DNA polymerase, which prefers deoxythymidine triphosphate (dTTP) to deoxyuridine triphosphate (dUTP), the DNA polymerase in crude extracts of PBS2-infected cells is equally active whether dTTP or dUTP is employed. This phage-induced polymerase may be responsible for the synthesis of uracil-containing DNA during PBS2 phage infection.

Linkage groups in Bacillus pumilus determined by bacteriophage PBS1-mediated transduction.

Two linkage groups were established in Bacillus pumilus NRRL B-3275 by bacteriophage PBS1 transduction. Group L contains the auxotrophic markers gly, his, and met. Group M contains the markers argO, met, ura, and cys.

Biochemical and genetic characterization of a temperature-sensitive, tryptophanyl-transfer ribonucleic acid synthetase mutant of Bacillus subtilis.

A temperature-sensitive, 5-fluorotryptophan (5FT)-resistant mutant of Bacillus subtilis was isolated which forms an altered tryptophanyl transfer ribonucleic acid synthetase [l-tryptophan: sRNA ligase (AMP), EC 6.1.1.2]. The mutant grows well at 30 C but not at 42 C. At the latter temperature, protein and ribonucleic acid (RNA) synthesis are abolished while deoxyribonucleic acid (DNA) synthesis proceeds for a considerable time. Tryptophanyl-transfer RNA (tRNA) synthetase activity is not detectable in the extracts of the mutant grown at 30 C whether this activity is measured by the attachment of l-tryptophan to tRNA or the l-tryptophan-dependent exchange of (32)P-pyrophosphate with adenosine triphosphate. Mixing experiments with extracts from the wild type and the mutant have ruled out the presence of an inhibitor or the absence of an activator as possible causes. Attempts to retrieve enzyme activity in vitro by various means (different conditions for cell disruption, addition of l-tryptophan, and adenosine triphosphate to the extraction buffer containing glycerol) were unsuccessful. The mutation in the locus of the tryptophanyl tRNA synthetase (trpS) was mapped on the bacterial chromosome by transformation and transduction. It is located between argC and metA. All temperature-resistant transformants recover wild-type levels of tryptophanyl tRNA synthetase activity and sensitivity to 5FT. Spontaneous revertants to temperature resistance are 5FT sensitive, but their levels of tryptophanyl tRNA synthetase activity and the thermolability of this enzyme in cell-free extracts varies. These revertants do not support the growth of a presumed nonsense mutant of phase SPO-1. Transduction experiments with phage PBS-1 indicated that reversion must be the result of an event at the site of the original mutation or at a site extremely close to it.

Citric acid cycle: gene-enzyme relationships in Bacillus subtilis.

The genetic location of mutations affecting the citric acid cycle and the properties of mutants of Bacillus subtilis possessing these mutations have been examined. Genes coding for the component enzymes of the cycle were found to be unlinked to each other and thus do not form an operon. The mutational defect in a mutant lacking fumarase mapped between thr-5 and cysB3. Mutations causing inability to produce isocitrate dehydrogenase and succinate dehydrogenase were found to map between argA11 and leu-1. The alpha-ketoglutarate dehydrogenase mutations were mapped at the terminal end of the B. subtilis chromosome through a weak linkage in phage PBS-1 transduction of one class of these mutations of ilvA2 and metB4. A second class of alpha-ketoglutarate dehydrogenase mutations mapped closer to ilvA2 and metB4 but still terminal with respect to these markers. Aconitaseless mutants possessed mutations that could not be linked to any of the known transducing segments of the chromosome. An effect of mutation conferring loss of one enzyme of the cycle on the specific activity of the other enzymes in the cycle was observed.

Genetic analysis in Bacillus pumilus by PBSI-mediated transduction.

Bacteriophage PBS1 mediates generalized transduction in Bacillus pumilus NRRL B-3275 (BpB1). Transduction frequencies for single auxotrophic markers are of the order of 10(-4) transductants per plaque-forming unit in crude phage lysates. The characteristics of PBS1 propagated on BpB1 and the properties of the system of transduction are similar to those reported for PBS1 propagated on Bacillus subtilis. By transduction, eight amino acid auxotrophic markers in BpB1 have been oriented into two linkage groups. One group contains the auxotrophic markers arginine A, leucine, and phenylalanine, and the other group contains the markers lysine, serine, tryptophan, isoleucine-valine, and isoleucine. The nature and relative order of the markers within each linkage group suggest that the arrangement of genes in these areas of the chromosome of BpB1 is similar to the arrangement of phenotypically comparable genes in two linkage groups (defined by PBS1 transduction) in B. subtilis. However, transduction of any of the above cited markers in BpB1 to prototrophy with PBS1 propagated on B. subtilis 168 could not be demonstrated. In addition to BpB1, seven other strains of B. pumilus can be infected with PBS1. Transduction has been demonstrated in three of these strains.

Genetic mapping of antibiotic resistance in markers Bacillus subtilis.

Several antibiotic resistance markers in Bacillus subtilis have been mapped by three-factor transduction crosses using bacteriophage PBS1. These markers, which are thought to be located in genes coding for ribosomal proteins, are within a segment comprising about 5 per cent of the B. subtilis genetic map, close to the major group of rRNA cistrons. The order of genetic makers in this region was found to be cysA14 mic-1 dagger str-1 (ery-1, ole-2) spc-2 bry-2 lin-2. The positions of neo-2, nea-1, and kan-2 are uncertain, although they are located in this region to the right of cysA14.

Transformation and transduction in Bacillus subtilis: Evidence for separate modes of recombinant formation

In Bacillus subtilis strains 168 and W23 the specificity requirements of transduction with bacteriophage PBS1 are more stringent than those of transformation. Heterologous transduction between these strains occurs at a low frequency or not at all, whereas heterologous transformation occurs readily. The heterologous transduction which does occur in this system displays the loose genetic linkage characteristic of transformation and is sensitive to a rec † † Abbreviations used: rec, recombination deficient; ery, erythromycin; str, streptomycin; mic, micrococcin; leu, leucine; met, methionine; pur, adenine or guanine; arg, arginine; phe, phenylalanine; ura, uracil; trp, tryptophan; nia, niacin; ser, serine; aro, aromatic amino acids; his, histidine; tyr, tyrosine; thr, threonine; rib, riboflavin; ilv, isoleucine & valine; ile, isoleucine; amm, inability to assimilate NH 4 +; cys, cysteine; lys, lysine; thy, thymine. mutation which depresses transformation but hardly affects homologous transduction. It is suggested that two pathways for handling donor genetic material exist in B. subtilis. Pathway I predominates in transformation and is relatively permissive to foreign DNA, while pathway II predominates in transduction and is relatively non-permissive. The basis of the foreigness recognized in this system is most likely lack of DNA base sequence homology rather than DNA modification. Limited base-sequence inhomology is demonstrated between W23 and 168 DNA, but no evidence could be obtained for the existence of a restriction-modification system. A hypothesis is presented in which the difference in size between transforming and transducing DNA fragments is the most important factor determining the specificity difference between transformation and transduction in B. subtilis. This hypothesis is supported by heterologous transduction experiments using bacteriophage SP10, the DNA of which is smaller than that of PBS1. The level of heterologous transduction obtained with SP10 is intermediate between the level found with PBS1 and that obtained using heterologous transformation.

Chromosomal location of genes regulating resistance to bacteriophage in Bacillus subtilis.

Many of the viruses which infect Bacillus subtilis require glucosylated polyglycerol teichoic acid for adsorption. These mutants can be divided into three classes on the basis of enzymatic defects and growth on galactose-minimal medium. Transduction with phage PBS1 reveals that two of these, gtaA and gtaB, are linked to hisA1, whereas the gtaC locus is linked to argC. Analysis by deoxyribonucleic acid-mediated transformation indicates that these loci exist in a cluster between the hisA1 and argC4 loci. Anomalies in mapping in the group II region of the chromosome exist. The basis of these anomalies is discussed.

A novel enzyme, dCTP deaminase, found in Bacillus subtilis infected with phage PBS 1

A novel enzyme, dCTP deaminase is found in cells of Bacillus subtilis infected with phage PBS 1. Results indicate that the synthesis of this enzyme is induced specifically by phage infection. No other enzymes which deaminate deoxycytidylates are detectable. The dCTP deaminase acts only on dCTP and is inhibited noncompetitively by dTTP. In B. subtilis infected with PBS 1, it appears that this enzyme is primarily responsible for the formation of dUTP required in the synthesis of the phage DNA.

Transducing particles of PBS 1

A thymine-requiring strain of Bacillus subtilis was labeled with thymidine- 3H and infected with phage PBS 1 in the presence of 32P. As thymine is completely substituted by uracil in the phage DNA, transducing fragments (host DNA) are assumed to be labeled exclusively with 3H and the phage DNA with 32P. After centrifugation in a CsCl gradient, plaque-forming particles (PFP) and transducing particles (TP) banded at the peak positions of 32P and 3H, respectively. The density of TP was 0.010 g cm −3 lower than that of PFP. Plaque-forming and transducing activities were found only in these two bands. Electron microscopic examination revealed that bands other than those of TP and PFP contained phage heads or phage ghosts and debris. In CsCl density-gradient centrifugation, DNA- 3H extracted from TP banded at a position corresponding to the density of B. subtilis DNA. Zone sedimentation in neutral sucrose gradients of the DNA- 3H showed a broad distribution ranging from 4 × 10 6 to 1.3 × 10 8 in molecular weight, although the DNA was extracted by an extremely mild technique. Results indicate that TP contains only B. subtilis DNA and its total molecular weight per TP would be comparable with that of the PBS 1 genome.

A single amino acid substitution responsible for altered flagellar morphology

Mutants of Bacillus subtilis 168 lacking the long-period helix in their flagella have been isolated. Purified flagella from these cells have been studied by physical, immunological and chemical techniques. Straight flagella mutants are non-motile, yet their locomotor organelles appear to have the same fine structure and adsorption characteristics for phage PBS 1 as the wild type. Immunologically the two structures appear to be identical. A single peptide difference has been found in fingerprints of tryptic digests of flagellins from the mutants. The amino acid substitution responsible for this change was the exchange of alanine for valine in the altered dipeptide.

Primary adsorption site of phage PBS1: the flagellum of Bacillus subtilis.

The adsorption of Bacillus subtilis phage PBS1 was studied, and it was demonstrated that the primary adsorption site for this phage is the flagellum of B. subtilis. The capacity of flagella to function for motility may be lost without the loss of their capacity to adsorb the phage and permit infection. Deoxyribonucleic acid injection by the phage is inhibited by cyanide, suggesting the requirement for cellular energy in the infection process.

Incorporation of 5-fluorodeoxyuridine into the DNA of Bacillus subtilis phage PBS2 and its radiobiological consequences

Bacillus subtilis cells, infected with phage PBS2 and grown in synthetic medium supplemented with 50 µg [2-14C]5-fluorodeoxyuridine/m1., yield progeny phage in which their normal DNA constituent, deoxyuridine, has been replaced by 5-fluorodeoxyuridine as evidenced by (1) a buoyant density increase of the phage DNA in CsCl from 1·722 (normal DNA) to 1·737 to 1·745 g/cm3, and (2)14C incorporation from [2-14C]5-fluorodeoxyuridine specifically into this “heavy” DNA fraction with quantitative recovery of [2-14C]5-fluorouracil from perchloric acid digests of the DNA. [2-14C]5-Fluorodeoxyuridine incorporation data indicate that the replacement of 1 mole % of uracil by 5-fluorouracil is accompanied by an approximately 0·6 mg/cm3increase in the buoyant density of the DNA. Similar replacement of 1 mole % of uracil in PBS2 DNA by thymine (i.e., methylation of DNA) should result in a density decrease of 0·34 mg/cm3. Although its DNA contains no thymine, the PBS2 phage can undergo host-cell reactivation, since the ultraviolet sensitivity of PBS2 phage plated on non-host reactivating strains of Bacillus subtilis is greater than that for the same phage plated on wild-type strains. 5-Fluorodeoxyuridine incorporation into the PBS2 DNA enchances the ultraviolet sensitivity of the phage.

Genetic mapping in Bacillus subtilis

A map of the Bacillus subtilis genome has been constructed from the results of density transfer experiments carried out in strain W23, and from genetic linkage data obtained using DNA-mediated transformation and phage PBS1-mediated transduction in strain 168. Several new linkages have been detected and most of the known linkage groups have been located and oriented relative to the genome replication origin. All of the markers studied except one have been placed in four linkage groups, defined by cotransduction with phage PBS1. The size of the phage PBS1 transducing fragment has been estimated. Our map has been compared with two previously published maps, obtained using the marker-frequency method.

Transformation and transduction in recombination-defective mutants of Bacillus subtilis.

The effects on transformation and transduction of an ultraviolet sensitivity (uvr(-)) and two ultraviolet sensitivity-recombination deficiency (rec-1(-) and rec-2(-)) mutations in isogenic strains of Bacillus subtilis were investigated. Transformation frequency in the rec-1(-) and rec-2(-) strains was reduced to approximately 5 and 25%, respectively, of the parental strains. Normal kinetics of deoxyribonucleic acid dose response in transformation were found for the rec-1(+) and rec-2(-) strains. Biphasic curves were obtained with the rec-1(-) strains. Transduction frequency with bacteriophage SP-10 decreased parallel to transformation frequency in the rec-1(-) and rec-2(-) strains. This result suggests that transformation and SP-10 transduction share a common mechanism for genetic recombination. It also indicates that the reduction in transformation frequency of these strains was not due to altered competence. Transduction frequency with bacteriophage PBS-1 or 3NT, on the contrary, was not diminished in rec-1(-) strains. This frequency was reduced in rec-2(-) strains but not as severely as that of transformation or SP-10 transduction. Several hypotheses to interpret these differences are presented. Recombination frequency between linked markers was reduced more than 50% in transformation by the presence of the rec-1(-) mutation. Linkage was unaffected in the rec-2(-) strains. Neither the rec-1(-) nor the rec-2(-) mutation had an effect on linkage in PBS-1 or 3NT transduction. The uvr(-) strains were transformed at a frequency equal to or greater than that of the parental strains. These strains were transduced by all bacteriophage systems at frequencies about twofold higher than those of parental strains.

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.

Adsorption Specificity of Bacteriophage PBS1

Frankel, Ruth W. (University of Oregon Medical School, Portland), and Terence M. Joys. Adsorption specificity of bacteriophage PBS1. J. Bacteriol. 92:388-389. 1966.-By use of newly isolated nonflagellate mutants, the location of the receptor site for phage PBS1 is confirmed as being on the flagella of Bacillus subtilis. Tests with partially purified flagella isolated from a culture of susceptible organisms, and with a strain of B. subtilis possessing nonfunctional flagella, show that phage PBS1 has an adsorption specificity for active flagella.

Correlation between susceptibility to bacteriophage PBS1 and motility in Bacillus subtilis.

Joys, Terence M. (University of Minnesota Medical School, Minneapolis). Correlation between susceptibility to bacteriophage PBS1 and motility in Bacillus subtilis. J. Bacteriol. 90:1575-1577. 1965.-A correlation is shown to exist in Bacillus subtilis between susceptibility to phage PBS1 and motility, indicating that the receptor site for this phage is located on the flagellum.

LINKAGE RELATIONSHIPS OF GENES CONTROLLING ISOLEUCINE, VALINE, AND LEUCINE BIOSYNTHESIS IN BACILLUS SUBTILIS.

Barat, M. (Centre National de la Recherche Scientifique, Gif-sur-Yvette, Seine et Oise, France), C. Anagnostopoulos, and A.-M. Schneider. Linkage relationships of genes controlling isoleucine, valine, and leucine biosynthesis in Bacillus subtilis. J. Bacteriol.90:357-369. 1965.-In Bacillus subtilis, the genetic loci controlling isoleucine and valine biosynthesis are not all clustered. Some of them were located on two distinct transforming deoxyribonucleic acid "molecules." One of these molecules (the "ileilva(2-4)-met segment") carries the threonine deaminase and the dihydroxy acid dehydrase loci linked to methionine markers. The other (the "ilva(1-3)-leu segment") bears the reductoisomerase locus and one or more loci involved in leucine synthesis. A phenylalanine marker was also shown to be weakly linked to this latter group. In transduction mediated by phage PBS-1, these groups are transferred jointly with other gene clusters. The phage appears to convey chromosome fragments considerably longer than the transforming "molecules." The genetic maps of both the above segments were extended by transduction. Some groups previously studied by transformation can be placed in the following linear order: the ile-ilva(2-4)-met segment, the cluster of loci involved in aromatic amino acid synthesis (try segment), and a lysine locus. An arginine locus is cotransduced with the phe-ilva(1-2)-leu segment. Recombination frequencies between linked markers are much lower in transduction by this phage than in transformation.

BACILLUS SUBTILIS DEOXYRIBONUCLEIC ACID TRANSFER IN PBS2 TRANSDUCTION.

Mahler, I. (Brandeis University, Waltham, Mass.), M. Cahoon, and J. Marmur. Bacillus subtilis deoxyribonucleic acid transfer in PBS2 transduction. J. Bacteriol. 87:1423-1428. 1964.-Lysates of the general transducing bacteriophage PBS2 grown on Bacillus subtilis SB19 were fractionated by preparative CsCl density-gradient centrifugation. Five distinct and separate bands which varied in their ability to transduce three nutritional markers were obtained by this procedure. Deoxyribonucleic acid (DNA) samples prepared from unfractionated lysates and from each of the separate bands were examined by analytical CsCl density-gradient centrifugation. In addition to a major band (density, 1.723 g/cc) identified as PBS2 DNA, a satellite band of lighter density was detectable in the nucleic acids obtained from whole lysates and those bands that possessed transducing activity. The biological activity of the purified nucleic acids, determined by transformation experiments, was found to reside in the light satellite band (1.703 g/cc) characteristic of B. subtilis DNA. In view of the correlation between general transducing ability of phage particles and the presence of bacterial DNA which appears not to be physically associated with the phage DNA, it is suggested that transduction by bacteriophage PBS2 does not depend upon areas of homology between the phage genome and the host chromosome but resides in specific particles which have incorporated segments of bacterial DNA.