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.     

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.     

Revision of the linkage map of Bacillus subtilis 168: indications for circularity of the chromosome.

A revision of the linkage map of the Bacillus subtilis 168 chromosome has been undertaken with the use of the generalized transducing phage PBS1. The mapping of four new markers (narB1, mtlB1, aroI906, and tre-12) has allowed a determination of the relative orientation of the purB-dal segment and its linkage with the lin markers. The chromosomal segment comprised between the sacQ36 and gtaA12 markers has been linked with the narA1, ctrA1, and sacA321 markers. The recA1 marker has been mapped relative to the thyA and citB17 markers. Indications of linkage have been found between the tre-12 and catA markers and the aroG932 and sacQ36 markers. According to these results, a circular genetic map of the chromosome of B. subtilis 168 is presented. Taken together, the transduction data and the order of marker replication determined by Harford in the accompanying paper support strongly the hypothesis of a symmetrical and fully bidirectional mode of replication for the B. subtilis 168 chromosome.     

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.     

Genetic Analysis in Bacillus pumilus by PBS1-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.     

Bidirectional chromosome replication in Bacillus subtilis 168.

Density transfer analysis of deoxyribonucleic acid from Bacillus subtilis 168 thy spores germinating in 5-bromouracil medium shows the order of replication of genetic markers to be: purA16, cysA14, sacA, ctrA, (narB, arol), dal, (hisA1, purB6), (tre-12, thr-5), (argA, aroG, argC4), (metC, leu-8, pheA), (ura-1, aroD), lys-1, (trpC, metB, ilvA, citB, citK, gltA). The precise order of transfer of markers within parentheses could not be determined in these experiments. Taken together with new PBS1 transduction data presented here and in the accompanying paper of J. Lepesant-Kejzlarová, J.-A. Lepesant, J. Walle, A. Billaut, and R. Dedonder (1975), the results can be resolved in terms of a symmetric, fully bidirectional mode of chromosome replication with a replication origin close to the purA16 marker and a terminus in the region of the gltA, citK loci, diametrically opposed to the origin. A new genetic map of the B. subtilis 168 chromosome is presented.     

Bacteriophage PMB12 conversion of the sporulation defect in RNA polymerase mutants of Bacillus subtilis.

The pseudotemperate phage PMB12 was isolated from soil on the basis of its ability to enhance the rate of sporulation of Bacillus subtilis 168. PMB12 was subsequently shown to convert the sporulation defect in two genetically distinct classes of sporulation mutants. One class includes those rifampin-resistant mutants that are also spore-negative (mutated at the rif locus). The other class includes a strain carrying the sporulation mutation spoCM-1. The spoCM-1 mutation is linked to cysA15 by PBS1 transduction but is distinct from the rif locus. Several other sporulation mutants were not converted by PMB12. PMB12 is related to phage PBS1. However, PBS1 did not convert the above sporulation mutants. The replication of PBS2, a clear-plaquing derivative of PBS1, is rifampin insensitive, apparently due to a phage-induced rifampin-insensitive RNA polymerase. PMB12 replication is also rifampin insensitive.     

A new cloning system for Bacillus subtilis comprising elements of phage, plasmid and transposon vectors

A new cloning system for Bacillus subtilis was devised which makes use of a combination of Tn917-containing phage SP beta derivatives and Tn917-containing Escherichia coli-B. subtilis shuttle plasmids. This system allows the initial cloning of genes in single copy, via 'prophage transformation', with a selection for complementation of mutational defects in B. subtilis hosts and permits subsequent transfer of the cloned material by homologous recombination to low-copy and high-copy vectors that replicate in both B. subtilis and E. coli. Because cloned sequences are adjacent to pB322-derived DNA in the recombinant phages, inserts can also be 'rescued' directly from the phage DNA after digestion with appropriate restriction enzymes, circularization of the fragments by ligation and transformation of an E. coli recipient. Two genomic libraries of B. subtilis chromosomal Sau3A-generated partial-digest fragments in the size ranges of 5-8 kb and 8-10 kb were constructed and screened for the complementation of mutations aroI906, cysA14, dal-1, glyB133, metC3, purA16, purB33, thrA5, trpC2 and recE4. In all cases, specialized transducing phages carrying inserts that complemented the selected markers were recovered. Inserts complementing the dal-1 and trpC2 mutations could be transferred from recombinant phages to Tn917-containing plasmids by homologous recombination without in vitro subcloning. Another insert complementing the purB33 mutation was rescued directly into E. coli from a recombinant phage DNA.     

Effect of Nalidixic Acid on PBS2 Bacteriophage Infection of Bacillus subtilis

Infection of Bacillus subtilis by PBS2 phage, whose DNA contains uracil instead of thymine, is relatively unaffected by low concentrations of nalidixic acid which severely inhibit B. subtilis DNA synthesis. High concentrations of nalidixic acid do inhibit PBS2 DNA synthesis, but more severely reduce the burst size of PBS2 infections. Hydroxyurea blocks PBS2 DNA synthesis, preventing progeny phage production.     

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.     

Mechanism of Transfection with Deoxyribonucleic Acid from the Temperate Bacillus Bacteriophage φ105

Bacteriophage phi105 is a temperate phage for the transformable Bacillus subtilis 168. The infectivity of deoxyribonucleic acid (DNA) extracted from mature phi105 phage particles, from bacteria lysogenic for phi105 (prophage DNA), and from induced lysogenic bacteria (vegetative DNA) was examined in the B. subtilis transformation system. About one infectious center was formed per 10(8) mature DNA molecules added to competent cells, but single markers could be rescued from mature DNA by a superinfecting phage at a 10(3)- to 10(4)-fold higher frequency. Single markers in mature DNA were inactivated at an exponential rate after uptake by a competent cell. Prophage and vegetative DNA gave about one infectious center per 10(3) molecules added to competent cells. Infectious prophage DNA entered competent cells as a single molecule; it gave a majority of lytic responses. Single markers in sheared prophage DNA were inactivated at the same rate as markers in mature DNA. Prophage DNA was dependent on the bacterial rec-1 function for its infectivity, whereas vegetative DNA was not. The mechanism of transfection of B. subtilis with viral DNA is discussed, and a model for transfection with phi105 DNA is proposed.     

Bacillus subtilis 168 mutants resistant to arginine hydroxamate in the presence of ornithine or citrulline

Mutations in Bacillus subtilis 168 have been isolated that confer resistance to arginine hydroxamate in the presence, but not absence, of ornithine. Seven such Ahor mutants have been studied in detail. In common with certain classes of Ahr mutant (resistant to arginine hydroxamate in the absence of arginine precursors) described previously, these Ahor mutants showed little or no inducibility of enzymes of arginine catabolism. Mutants that showed no inducibility were unable to utilize arginine or ornithine as sole nitrogen source. The only biosynthetic enzyme to show any consistent differences in activity from the parent was ornithine carbamoyltransferase, whose level was slightly elevated in cells grown in the presence of ornithine or citrulline. PBS1 transduction crosses showed that two of the ahor mutations map at the ahrA locus, while a third (unique in its resistance to arginine hydroxamate in the presence of citrulline) mapped at a hitherto undescribed locus closely linked to metC, designated ahrD.     

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.     

Association of the replication terminus of the Bacillus subtilis chromosome to the cell membrane.

A chromosomal segment containing several genetic markers, from metB to thyA, near the replication terminus is associated with the membranous structure of Bacillus subtilis, but markers adjacent to this region, lys, ura, and metC, are not.     

Adsorption of Bacillus subtilis bacteriophage PBS 1.

Bacteriophage PBS 1 adsorbs initially on the flagella of its host, Bacillus subtilis (stage I). The phage can adsorb to both active and inactive flagella. Flagellar attachment is nonspecific as PBS 1 was shown to attach to the flagella of Bacillus species other than the normal host B. subtilis. The phage particle then quickly moves down the length of the flagellum to its base, the final adsorption site. Flagellar motion is required for flagellar base attachment (stage II). After proper attachment at the flagellar base, the phage tail sheath contracts sending the tail core through the final adsorption site (stage III). The phage DNA is then injected at this site (stage IV). Stage I adsorption does not cause loss of motility in PBS 1 -- resistant bacilli. The loss of motility observed upon infection of sensitive cells by PBS 1 may be associated with either stage II or stage III of adsorption.     

Interspecific transformation of Bacillus subtilis competent cells by chromosomal DNA in lysates of protoplasts of Bacillus amyloliquefaciens

Competent cells of Bacillus subtilis were transformed with chromosomal DNA in lysates of protoplasts of B. subtilis or B. amyloliquefaciens. The interspecific transformation frequency of B. subtilis by cysA in a conserved region was 3.1 x 10(4) transformants per microg DNA, 60 times higher than that for conventional transformation using purified DNA. Increased interspecific transformation frequencies of B. subtilis were also observed for arg-1, lys-1, leuB, aroG, thr-5, hisH, or metC markers outside the conserved region (3.1 x 10 approximately 5.2 x 10(2) transformants per microg DNA). An interspecific cotransformation ratio (33-50%) as high as an intraspecific one (46%) using purified DNA was also detected between cysA and rpsL markers, which are separated by 16 kb on the B. subtilis chromosome. Interspecific double transformation of the cysA-arg-1 or cysA-metC marker was observed, which have not been detected for conventional transformation. The involvement of mutS in the interspecific transformation was not significant.     

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.     

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.     

Joint transfer of genetic markers in Bacillus subtilis

Takahashi, I. (McMaster University, Hamilton, Ontario, Canada). Joint transfer of genetic markers in Bacillus subtilis. J. Bacteriol. 91:101-105. 1966.-To compare the processes of genetic incorporation in transduction and transformation in Bacillus subtilis, several groups of linked markers were selected and the degree of linkage was determined by the two means of genetic exchange. Bacteriophage PBS 1 was used in transduction experiments. In all cases, frequencies of joint transfer, as expressed by the cotransfer index or by percentage of joint transfer, were higher in transduction than in transformation. With a pair of closely linked markers, the frequency of joint transduction was only slightly higher than that of joint transformation. On the other hand, a considerably higher degree of linkage was obtained by transduction when loosely linked markers were examined. It appears that the size of donor chromosome transferred by transducing phage particles is much larger than that incorporated by recipient cells in transformation. It is suggested that transduction in B. subtilis may be a useful tool in extending further the linkage groups established by the transformation technique.     

Replacement of the Bacillus subtilis subtilisin structural gene with an In vitro-derived deletion mutation.

The entire subtilisin structural gene from Bacillus subtilis I168 has been cloned, and its nucleotide sequence has been determined. When expressed on a high-copy-number shuttle vector, a fivefold increase in serine protease activity was observed. The DNA sequence of the gene is 80% homologous to the Bacillus amyloliquefaciens subtilisin structural gene, and the translated mature coding sequence is 85% homologous to the published protein sequence of subtilisin BPN'. The chloramphenicol resistance determinant of a plasmid integrated at the subtilisin locus was mapped by PBS1 transduction and was found to be linked to glyB (83%) and argC (60%), but not with metC or purB . The chromosomal locus containing the wild-type subtilisin allele was replaced with an in vitro-derived allele of the gene (delta apr-684) that contained a 684-base-pair deletion. The technique used for introducing the deletion is a variation of the gene replacement methods used in Saccharomyces cerevisiae and Escherichia coli. When used in B. subtilis, deletion mutants could be directly screened among the transformants. Physiological characterization of the delta apr-684 mutation revealed no discernable effect on the formation of heat-resistant endospores, but strains carrying the mutation produced only 10% of wild-type serine protease activity. A model is presented that outlines the pathway for plasmid integration and deletion formation in B. subtilis.