Concurrent changes in transducing efficiency and content of transforming deoxyribonucleic acid in Bacillus subtilis bacteriophage SP-10

Taylor, Martha J. (Fort Detrick, Frederick, Md.), and Curtis B. Thorne. Concurrent changes in transducing efficiency and content of transforming deoxyribonucleic acid in Bacillus subtilis bacteriophage SP-10. J. Bacteriol. 91:81-88. 1966.-Spores of Bacillus subtilis W-23-S(r) infected with transducing phage SP-10 served as convenient inocula for broth cultures from which transducing phage was harvested. Methods are described for producing highly infected spores. The inoculum level of infected spores in nutrient broth-yeast extract-glucose medium affected the transducing efficiency of SP-10 in lysates of these cultures. Phage in lysates of cultures inoculated with about 10(5) or fewer spores per milliliter transduced 20- to 350-fold more efficiently than did phage in lysates from cultures inoculated with 10(6) to 10(7) spores per milliliter. Transduction frequencies in the order of 10(-5) per plaque-forming unit were obtained routinely, and some infected-spore preparations yielded phage that gave frequencies as high as 10(-4). The combination of inoculum level and incubation time required to produce the best transducing phage had to be determined empirically for each batch of infected spores. Several possible explanations for the difference between lysates having high (HTE) and those having low (LTE) transducing efficiency were ruled out by special experiments. The hypothesis is presented that some cultural condition resulting from a relatively low inoculum of phage-infected spores favors the incorporation by phage particles of bacterial deoxyribonucleic acid (DNA) in the manner required for the production of transducing phage. Support for this hypothesis is a demonstration, through transformation experiments with DNA extracted from HTE and LTE phage particles, that populations of HTE phage particles yielded significantly more (7 to 27 times) transforming activity per microgram of DNA than did populations of LTE phage.     

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.     

Productive Infection of Bacillus subtilis 168, with Bacteriophage SP-10, Dependent upon Inducing Treatments

Strains of Bacillus that harbor defective phage PBSX were found to be insensitive to SP-10(C), although the phage adsorbed to these insensitive strains. Strains that did not carry the phage were sensitive to SP-10(C). B. subtilis 168 ind(-), which can be tranduced by SP-10(C) but is nonpermissive for the phage, was rendered phage-sensitive after treatment with ultraviolet (UV) light or mitomycin C. After induction with UV light, maximal sensitivity to SP-10(C) was obtained at a multiplicity of infection (MOI) of approximately 14; with mitomycin C induction, an MOI of approximately 1.0 was required. Phage maturation in sensitized cells was followed by plating infected streptomycin-sensitive cells in the presence of streptomycin at various stages during phase development. The latent period was estimated at 60 to 75 min. We suggest that the resistance of B. subtilis 168 to SP-10 is controlled, at least in part, by the presence of a defective prophage.     

SP-10 bacteriophage-specific nucleic acid and enzyme synthesis in Bacillus subtilis W23.

Bacillus subtilis W23 was infected with a clear-plaque variant of SP-10 phage, namely, SP-10c. Exogenous thymidine was not incorporated into phage DNA (even in the presence of deoxyadenosine), nor was there any transfer of thymidine nucleotides from bacterial to viral DNA. The lytic program was unaffected by concentrations of 5-fluorodeoxyuridine sufficient to reduce bacterial DNA synthesis by greater than 95%. Although these data are consistent with the interpretation that thymidine nucleotides are excluded from phage DNA, formic acid digests of SP-10c DNA contained what appeared to be the four conventional bases; however, adenine and thymine were not recovered in equimolar yields. DNA-RNA hybridization and hybridization competition experiments were done. Synthesis of host RNA started to wane moments postinfection and stopped completely by 36 min. SP-10c coded for discrete classes of early and late RNA. The possibility of discrete subclasses of early RNA exists. Replication of the bacterial genome appeared to terminate 12 min postinfection. Degradation of the host DNA to acid-soluble material started at 36 min and, by the end of the latent period, greater than 90% of the host chromosome was hydrolyzed. Four apparent phage-coded enzymes have been identified. A di- and triphosphatase degraded dUTP, dUDP, dTTP, and dTDP (and, to a lesser extent, dCDP and d CTP) to the corresponding monophosphates; the enzyme had no apparent activity on dATP and dGTP. SP10c also coded for a DNA-dependent DNA polymerase, lysozyme, and a nuclease that degrades native bacterial DNA. Judging from the dependence of enzyme synthesis on the time of addition of rifampin (an inhibitor of the initiation of RNA synthesis), messengers for the di- and triphosphatase, as well as the nuclease, are transcribed from promoters that start to function 6 min postinfection. Promoters for polymerase and lysozyme did not become functional until 8 and 16 min postinfection, respectively.     

Phi W-14 DNA inhibits transfection of Bacillus subtilis by SPP1 DNA.

The DNA of bacteriophage phi W-14 is unusual in that half of the thymine residues are replaced with the hypermodified pyrimidine alpha-putrescinylthymine (Kropinski et al., Biochemistry 12:151-157, 1973). Bacteriophage phi W-14 DNA and Bacillus subtilis DNA exhibited comparable competing abilities for the uptake of transfecting bacteriophage SPP1 DNA by competent cells of B. subtilis. B. subtilis DNA decreased transfection and uptake to the same extent, indicating that it merely competed with SPP1 DNA for uptake. Phi W-14 DNA, however, decreased transfection up to 30 times more effectively than it inhibited uptake. Phi W-14 DNA did not alter the kinetics of transfection. The degree of inhibition of transfection was dependent upon the time of addition of Phi W-14 DNA relative to the time of addition of SPP1 DNA. If failed to inhibit when added 30 min after SPP1 DNA. It had a fourfold-greater effect when added 10 min before, rather than simultaneously with, SPP1, but this enhancement was abolished by high concentrations of SPP1 DNA. The nature of the transfection process was not altered in those cells escaping inhibition by Phi W-14 DNA: two molecules of transfecting SPP1 DNA were required to form a transfectant with or without Phi W-14 DNA. Free putrescine did not affect transfection by SPP1 DNA. It was concluded that the putrescine groups covalently attached to phi W-14 DNA allowed this DNA to interfere with the transfection process at the intracellular level.     

Bacteriophage P22-mediated specialized transduction in Salmonella typhimurium: high frequency of aberrant prophage excision.

The temperate bacteriophage P22 mediates both generalized and specialized transduction in Salmonella typhimurium. Specialized transduction by phage P22 is different from, and less restricted than, the well characterized specialized transduction by phage lambda, due to differences in the phage DNA packaging mechanisms. Based on the properties of the DNA packaging mechanism of phage P22 a model for the generation of various types of specialized transducing particles is presented that suggests generation of substantial numbers of specialized transducing genomes which are heterogeneous but only some of which have terminally redundant ends. The primary attachment site, ataA, for phage P22 in S. typhimurium is located between the genes proA,B and supQ newD. (The newD gene is a substitute gene for the leuD gene, restoring leucine prototrophy of leuD mutant strains.) The proA,B and supQ newD genes are very closely linked and thus cotransducible by generalized transducing particles. Specialized transducing particles can carry either proA,B or supQ newD but not both simultaneously, and thus cannot give rise to cotransduction of the proA,B and supQ newD genes. This difference is used to calculate the frequency of generalized and specialized transducing particles from the observed cotransduction frequency in phage lysates. By this method, very high frequencies of supQ newD (10(-2)/PFU)- and proA,B (10(-3)/PFU)-specialized transducing particles were detected in lysates produced by induction of lysogenic strains. These transducing particles most of which would have been produced by independent aberrant excision events (which include in situ packaging), were of various types.     

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.     

Characterization of the N-acetylmuramic acid L-alanine amidase from Bacillus subtilis.

The N-acetylmuramic acid L-alanine amidase from Bacillus subtilis W-23 has been purified to apparent homogeneity. The enzyme is a monomer of molecular weight 51,000, which binds extremely tightly to homologous cell walls but not to heterologous cell walls, even of the closely related strain B. subtilis ATCC 6051. This difference in binding is only in part due to differences in teichoic acid between these two strains and to a large extent appears to represent differences in the arrangement of the peptidoglycan. A comparison of the amidase from B. subtilis W-23 and the enzyme previously purified from B. subtilis ATCC 6051 (Herbold and Glaser, 1975) shows that the two proteins, which cleave the same bond and are of the same size, do not cross-react immunologically and that the two enzymes are, therefore, not closely related in structure.     

Unique Properties of Nucleic Acid from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Phage SP-15

BACTERIOPHAGE SP-15 is a large, generalized transducing phage of Bacillus subtilis and B. licheniformis. The DNA extracted from the purified phage has unusual physical properties: its melting temperature (T_m) in 0.15 M NaCl, 0.015 M sodium citrate (SSC) is very low, 61.5° C and its buoyant density in neutral CsCl is very high, 1.761 g/ml.¹. We describe here additional unique features of SP-15 DNA: the presence of (1) a new modified pyrimidine which partially replaces the thymine; (2) a compound which reacts with orcinol as a pentose; (3) alkali-sensitive phosphodiester bonds; and (4) glucose.     

Revised restriction maps of Bacillus subtilis bacteriophage phi 105 DNA.

Physical mapping of Bacillus subtilis temperate phage phi 105 DNA was carried out by using restriction endonucleases EcoRI, SmaI, and KpnI, and a new revised EcoRI cleavage map is presented. In addition, the EcoRI cleavage maps of six specialized transducing phages carrying sporulation genes of B. subtilis were revised.     

Uptake and fate of bacteriophage phi W-14 DNA in competent Bacillus subtilis.

Phage phi W-14 DNA (in which one-half of the thymine residues are replaced by alpha-putrescinyl thymine) was taken up by competent Bacillus subtilis cells at a rate threefold higher than the rate of homologous DNA uptake. In contrast to other types of heterologous DNA, the amount of phi W-14 DNA taken up in 15 min exceeded the amount of homologous DNA taken up by a factor of two to three, as measured in terms of acid-precipitable material. The amount of phi W-14 DNA taken up was even greater than this analysis indicated if allowance was made for the fact that phi W-14 DNA was degraded more rapidly after uptake than homologous DNA. Competition experiments showed that the affinity of phi W-14 DNA for homologous DNA receptors was lower than the affinity of homologous DNA and was similar to the affinities of other types of heterologous DNA. The more rapid and more extensive uptake of phi W-14 DNA appeared to occur via receptors other than the receptors for homologous DNA, and these receptors (like those for homologous DNA) were an intrinsic property of competent cells. Uptake of phi W-14 DNA was affected by temperature, azide, EDTA, and chloramphenicol, as was uptake of homologous DNA. This was consistent with entry of both DNAs by means of active transport. After uptake, undegraded phi W-14 [3H]DNA was found in the cells in a single-stranded form, whereas a portion of the label was associated with recipient DNA, presumably as a result of incorporation of monomers resulting from degradation. Acetylation of the amino groups of the putrescine side chains in phi W-14 DNA decreased the affinity of this DNA for its receptors without affecting its ability to compete with homologous DNA.     

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.     

Purification of an SOS repressor from Bacillus subtilis.

We have identified in Bacillus subtilis a DNA-binding protein that is functionally analogous to the Escherichia coli LexA protein. We show that the 23-kDa B. subtilis protein binds specifically to the consensus sequence 5'-GAACN4GTTC-3' located within the putative promoter regions of four distinct B. subtilis DNA damage-inducible genes: dinA, dinB, dinC, and recA. In RecA+ strains, the protein's specific DNA binding activity was abolished following treatment with mitomycin C; the decrease in DNA binding activity after DNA damage had a half-life of about 5 min and was followed by an increase in SOS gene expression. There was no detectable decrease in DNA binding activity in B. subtilis strains deficient in RecA (recA1, recA4) or otherwise deficient in SOS induction (recM13) following mitomycin C treatment. The addition of purified B. subtilis RecA protein, activated by single-stranded DNA and dATP, abolished the specific DNA binding activity in crude extracts of RecA+ strains and strains deficient in SOS induction. We purified the B. subtilis DNA-binding protein more than 4,000-fold, using an affinity resin in which a 199-bp DNA fragment containing the dinC promoter region was coupled to cellulose. We show that B. subtilis RecA inactivates the DNA binding activity of the purified B. subtilis protein in a reaction that requires single-stranded DNA and nucleoside triphosphate. By analogy with E. coli, our results indicate that the DNA-binding protein is the repressor of the B. subtilis SOS DNA repair system.     

Synthesis of the Unusual DNA of Bacillus subtilis Bacteriophage SP-15

Cultures of Bacillus subtilis infected with phage SP-15 were examined to investigate the metabolic origin of two of the unique components of the phage DNA: the component responsible for the unusually high buoyant density in CsCl and the unusual pyrimidine, 5-(4', 5'-dihydroxypentyl) uracil (DHPU). Newly synthesized pulse-labeled DNA was light in buoyant density and shifted to the high density of mature phage DNA upon further incubation. Parental DNA was converted to a light-density intermediate form prior to replication. When labeled uracil, thymidine, or DHPU were added to infected cells, it was found that only uracil served as the precursor to DHPU and thymine in phage DNA. Analysis of the bases from hydrolyzed DNA of labeled phage or infected cells indicated that the uracil was incorporated into the DNA as such (presumably via deoxyuridine triphosphate) and later converted to DHPU and thymine at the macromolecular level. The sequence of events after phage infection appeared to be: (i) injection of parental DNA; (ii) conversion of parental DNA to a light form; (iii) DNA replication, yielding light DNA containing uracil; (iv) conversion of uracil to DHPU and thymine; and (v) addition of the heavy component.     

Increase in Lytic Activity in Competent Cells of Bacillus subtilis After Uptake of Deoxyribonucleic Acid

Extractable lytic activity in competent cells of Bacillus subtilis 168 was markedly increased after treatment with homologous or heterologous deoxyribonucleic acid (DNA). This increase was prevented by deoxyribonuclease, and did not occur with B. subtilis W23 or with noncompetent B. subtilis 168 cells, neither of which take up DNA. Although the deoxyribonuclease-sensitive step in DNA uptake was completed within 10 min, the increase in lytic activity did not begin until more than 30 min after the addition of DNA. The increase was prevented by any of several antibiotics. These results are discussed in relation to the mechanisms for the uptake of transforming DNA and the lysis of transfected cells.     

Synthesis of teichoic acids. VII. Synthesis of teichoic acids during spore germination

The synthesis of teichoic acids has been examined during germination in Bacillus licheniformis ATCC 9945 and in B. subtilis W-23. Teichoic acids are absent from the spores of both organisms. B. licheniformis spores lack the enzymes responsible for teichoic acid synthesis. The appearance of these enzymes during germination is correlated with the appearance of teichoic acids in the cell. The appearance of teichoic acid-synthesizing enzymes and of teichoic acids in the cell are inhibited by the addition of chloramphenicol to the germination medium. In B. subtilis W-23 the situation is similar for the synthesis of polyribitolphosphate. The synthesis of glucosyl polyribitolphosphate is only partially inhibited by chloramphenicol, puromycin, and penicillin, and uridine diphosphate-d-glucose polyribitol-phosphate glucosyl transferase can be demonstrated in spores. The possible implications of some of these observations are discussed.     

Characterization and genomic analysis of phage asccphi28, a phage of the family Podoviridae infecting Lactococcus lactis

Bacteriophage asccphi28 infects dairy fermentation strains of Lactococcus lactis. This report describes characterization of asccphi28 and its full genome sequence. Phage asccphi28 has a prolate head, whiskers, and a short tail (C2 morphotype). This morphology and DNA hybridization to L. lactis phage P369 DNA showed that asccphi28 belongs to the P034 phage species, a group rarely encountered in the dairy industry. The burst size of asccphi28 was found to be 121 +/- 18 PFU per infected bacterial cell after a latent period of 44 min. The linear genome (18,762 bp) contains 28 possible open reading frames (ORFs) comprising 90% of the total genome. The ORFs are arranged bidirectionally in recognizable functional modules. The genome contains 577 bp inverted terminal repeats (ITRs) and putatively eight promoters and four terminators. The presence of ITRs, a phage-encoded DNA polymerase, and a terminal protein that binds to the DNA, along with BLAST and morphology data, show that asccphi28 more closely resembles streptococcal phage Cp-1 and the phi29-like phages that infect Bacillus subtilis than it resembles common lactococcal phages. The sequence of this phage is the first published sequence of a P034 species phage genome.     

Effect of Nalidixic Acid on Deoxyribonucleic Acid Synthesis in Bacteriophage SPO1-infected Bacillus subtilis

The effect of nalidixic acid on deoxyribonucleic acid (DNA) synthesis in Bacillus subtilis cells infected with bacteriophage SPO1 was studied. Nalidixic acid had little inhibitory effect on SPO1 DNA synthesis at concentrations that drastically inhibited B. subtilis DNA synthesis. Inhibition of DNA synthesis, appropriate to the concentration used, was imposed within 1 min after addition of nalidixic acid, suggesting that it acts directly on DNA synthesis in both infected and uninfected cells. The SPO1 DNA synthesized in the presence of high concentrations of nalidixic acid had a density characteristic of normal SPO1 DNA and was packaged into viable progeny phage particles, but its rate of synthesis was reduced and bacterial lysis was delayed.