Restriction and modification of bacteriophage SP10 DNA by Bacillus subtilis Marburg 168: stabilization of SP10 DNA in restricting hosts preinfected with a heterologous phage, SP18.

SP10 phage cannot propagate in Bacillus subtilis Marburg 168 containing the wild-type allele of either gene nonA or gene nonB. The latter gene codes for the intrinsic cellular restriction activity. SP10 DNA was degraded in nonB+ derivatives of Marburg 168. The degree of degradation depended upon the previous host in which SP10 was propagated. In the case of SP10 grown in B. subtilis W23 (a nonrestricting, nonmodifying bacterium), 90% of the phage DNA was hydrolyzed to acid solubles, and the residual acid-precipitable material was recovered as 0.5- to 1-megadalton fragments. In contrast, if SP10 was propagated in B. subtilis PS9W7 (a nonA nonB derivative of Marburg 168 that retains modifying activity), 40 to 50% of the input DNA was degraded to acid solubles, and most of the remainder was recovered as 15- to 20-megadalton fragments. In nonA+ nonB cells, SP10 DNA was conserved as unit-length molecules (ca. 80 megadalton). Prior infection of nonB+ cells with SP18 protected superinfecting SP10 DNA, even when rifampin or chloramphenicol was added before the primary infection. The data are discussed in terms of the following conclusions. (i) The nonB gene product of B. subtilis Marburg 168 is required for restriction of SP10 DNA. (ii) Some sites on SP10 DNA are sensitive to both the restricting and modifying activities, whereas other sites are nonmodifiable even though they are sensitive to the restriction enzyme. (iii) In some manner, SP18 antagonizes the action of the nonB gene product.     

Restriction and modification in B. subtilis

Summary The content of 5-methylcytosine (5MC) and 6-methyladenine (6MA) in modified and nonmodified DNAs from B. subtilis and B. subtilis phage SPP1 were determined. Nonmodified SPP1 · O DNA contains about 15 5MC residues/molecule. Each modified SPP1 ·R DNA molecule carries 190 modification specific methyl groups. This number is sufficient to account for modification of the 80 restriction sites in SPP1 DNA (Bron and Murray, 1975) against endo R · Bsu R, assuming each modified site contains two 5MC residues. Resistance of SP01 DNA against endo R · Bsu R restriction both in vivo and in vitro is probably not due to methylation of endo R·Bsu R recognition sites.     

Bacteriophage infection in rod-shaped gram-positive bacteria: evidence for a preferential polar route for phage SPP1 entry in Bacillus subtilis

Entry into the host bacterial cell is one of the least understood steps in the life cycle of bacteriophages. The different envelopes of Gram-negative and Gram-positive bacteria, with a fluid outer membrane and exposing a thick peptidoglycan wall to the environment respectively, impose distinct challenges for bacteriophage binding and (re)distribution on the bacterial surface. Here, infection of the Gram-positive rod-shaped bacterium Bacillus subtilis by bacteriophage SPP1 was monitored in space and time. We found that SPP1 reversible adsorption occurs preferentially at the cell poles. This initial binding facilitates irreversible adsorption to the SPP1 phage receptor protein YueB, which is encoded by a putative type VII secretion system gene cluster. YueB was found to concentrate at the cell poles and to display a punctate peripheral distribution along the sidewalls of B. subtilis cells. The kinetics of SPP1 DNA entry and replication were visualized during infection. Most of the infecting phages DNA entered and initiated replication near the cell poles. Altogether, our results reveal that the preferentially polar topology of SPP1 receptors on the surface of the host cell determines the site of phage DNA entry and subsequent replication, which occurs in discrete foci.     

Effect of deoxyribonucleic acid replication inhibitors on bacterial recombination.

Two inhibitors of replicative deoxyribonucleic acid (DNA) synthesis, nalidixic acid (NAL) and 6-(p-hydroxyphenylazo)-uracil (HPUra), showed different effects on genetic recombination and DNA repair in Bacillus subtilis. Previous work (Pedrini et al., 1972) showed that NAL does not interfere with the transformation process of B. subtilis. The results reported in this work demonstrated that the drug was also without effect on the transfection by SPP1 or SPO-1 phage DNA (a process that requires a recombination event). The drug was also ineffective on the host cell reactivation of ultraviolet-irradiated SPP1 phage, as well as on transfection with ultraviolet-irradiated DNA of the same phage. HPUra instead markedly reduced the transformation process, as well as transfection, by SPO-1 DNA, but it did not affect the host cell reactivation of SPO-1 phage. In conclusion, whereas the NAL target seems to be specific for replicative DNA synthesis, the HPUra target (i.e., the DNA polymerase III of B. subtilis) seems to be involved also in recombination, but not in the excision repair process. The mutations conferring NAL and HPUra resistance used in this work were mapped by PBS-1 transduction.     

Functional analysis of the Bacillus subtilis bacteriophage SPP1 pac site.

Encapsidation of the DNA of the virulent Bacillus subtilis phage SPP1 follows a processive unidirectional headful-mechanism and initiates at a unique genomic location (pac). We have cloned a fragment of SPP1 DNA containing the pac site flanked by reporter genes into the chromosome of B. subtilis. Infection of such cells with SPP1 led to highly efficient packaging, initiated at the inserted pac site, of chromosomal DNA. The directionality in the packaging of this DNA was the same as observed with vegetative phage DNA. Mutagenizing the chromosomal pac insert defined an 83 base pair segment containing the pac cleavage site which is sufficient to direct phage specific DNA encapsidation. The packaging recognition signal as defined can also be utilized by the SPP1 related phages 41c, SF6 and rho 15.     

The genome of B. subtilis phage SSP1: the topology of DNA molecules.

DNA molecules of B. subtilis phage SPP1 exhibit terminal redundancy and are partially circularly permuted. This was established by the hybridization of selected EcoRI restriction fragments to single strands of SPP1 DNA and by an analysis of the distribution of denaturation loops in partially denatured SPP1 DNA molecules. Deletions in SSP1 DNA are not compensated by an increase in terminally repetitious DNA. This finding, which is unique to SPP1, is discussed in terms of a modification of the Streisinger/Botstein model of phage maturation.     

Restriction and modification in B. subtilis

Summary The effects of restriction in vivo by competent B. subtilis R cells and in vitro by purified endonuclease BsuR on transformation and transfection with native and denatured DNA were investigated.The results show that transformation by either native, or denatured DNA is not affected by restriction, whereas transfection both with native and denatured SPP1 DNA is severely restricted.In contrast to the results obtained in vivo, the biological activity of native and denatured transforming DNA is destroyed by BsuR in vitro, as is the transfecting activity of native and denatured SPP1 DNA. The sensitivity of denatured DNA, either with mixtures of the complementary strands or with separated single strands¹ alone, is significantly lower than that of native DNA.The results are discussed in the context of possible mechanisms underlying the different responses of transforming and transfecting DNA to in vivo restriction by B. subtilis R cells.Abbreviations EGTA ethyleneglycolbis-(aminoethylether)tetraacetic acid - m⁺ modified - m^- non-modified - moi multiplicity of infection - r⁺ m⁺ restricting and modifying - r^- m^- mon-restricting and non-modifying - SSC 0.15M NaCl+0.015 M trisodium citrate - SDS sodium dodecyl sulphate     

The minor capsid protein gp7 of bacteriophage SPP1 is required for efficient infection of Bacillus subtilis

Gp7 is a minor capsid protein of the Bacillus subtilis bacteriophage SPP1. Homologous proteins are found in numerous phages but their function remained unknown. Deletion of gene 7 from the SPP1 genome yielded a mutant phage (SPP1del7) with reduced burst-size. SPP1del7 infections led to normal assembly of virus particles whose morphology, DNA and protein composition was undistinguishable from wild-type virions. However, only approximately 25% of the viral particles that lack gp7 were infectious. SPP1del7 particles caused a reduced depolarization of the B. subtilis membrane in infection assays suggesting a defect in virus genome traffic to the host cell. A higher number of SPP1del7 DNA ejection events led to abortive release of DNA to the culture medium when compared with wild-type infections. DNA ejection in vitro showed that no detectable gp7 is co-ejected with the SPP1 genome and that its presence in the virion correlated with anchoring of released DNA to the phage particle. The release of DNA from wild-type phages was slower than that from SPP1del7 suggesting that gp7 controls DNA exit from the virion. This feature is proposed to play a central role in supporting correct routing of the phage genome from the virion to the cell cytoplasm.     

Restriction and Modification of Bacteriophage S2 in Haemophilus influenzae

The major conclusion from these studies is that variants of Haemophilus influenzae Rd which restrict and modify phage S2 are metastable and capable of giving rise to one another with high frequency. Nonrestrictive RdS cells segregate spontaneously to the restricting, modifying phenotype in about 5% of the progeny of a single clone. The restrictive cells derived from RdS revert to the nonrestrictive phenotype in 15 to 25% of the progeny of a single clone. These frequencies are not appreciably affected by treatment with acriflavine or ethidium bromide, compounds which affect plasmid stability, or by nitrosoguanidine, a powerful mutagen. The genetic locus for restriction and modification of bacteriophage S2 is found to have a chromosomal position between the biotin and proline loci. Restriction-modification of phage S2 has been shown to be a function of its deoxyribonucleic acid (DNA) in that transfection with S2 phage DNA or prophage DNA is subject to host restriction and modification. An enzyme preparation, which contains endodeoxyribonuclease but no appreciable exonuclease activity, from mutant H. influenzae com(-10) did not restrict phage S2.RdS DNA or prophage DNA transfecting activity, indicating that this endodeoxyribonuclease is not responsible for phage restriction. A new restriction enzyme isolated from H. influenzae Rd was found to be the major enzyme involved in the restriction of bacteriophage S2. The enzyme inactivated the transfecting activity of unmodified phage DNA but did not attack modified phage DNA. Unlike endodeoxyribonuclease R, this enzyme requires adenosine triphosphate and S-adenosylmethionine.     

Relationship Between Competence for Transfection and for Transformation

Deoxyribonucleic acid (DNA) extracted from phage SPP1 is highly infectious on Bacillus subtilis competent cells; the efficiency of infection is 5 x 10(3) to 6 x 10(3) phage equivalents per plaque-forming unit. This DNA was used to study the relationship between competence for transfection and for transformation. The experiments were concerned with the frequency of infection and transformation in mutants exhibiting different levels of competence, the effect of periodate on competence for infection and for transformation, the competition between phage and bacterial DNA, the transformation of cells preinfected with phage DNA, and the infection of cells pretreated with bacterial DNA. The data show that B. subtilis cells competent for transformation are also competent for transfection and vice versa; transfection with phage DNA represents, therefore, a simple way to measure the total number of competent cells in a culture. The fraction of competent cells, determined by SPP1 DNA infection, varied from 10(-2) to 7 x 10(-2).     

Restriction and modification in Bacillus subtilis: effects on transfection under marker rescue conditions

The role of homology between donor and recipient DNAs in the protection of transfecting DNA against restriction by competent Bacillus subtilis R cells was studied under marker rescue conditions with modified helper phage. By comparing restriction under conditions of preinfection marker rescue and superinfection marker rescue, the significance of DNA homology during the initial stages of DNA processing by competent cells could be studied. The results showed that both in preinfection and in superinfection, complete protection against restriction of transfectants produced via rescue by the modified homologous helper chromosome occurred. Even up to 90 min after entry, DNA entering the helper-mediated pathway of transfection was not affected by restriction. The significance of these findings is discussed in the general context of the role of DNA homology between donor and recipient on the fate of donor DNA in competent B. subtilis, in particular in relation to the effects on restriction.     

Bacteriophage SPO1-induced macromolecular synthesis in minicells of Bacillus subtilis.

SPO1 bacteriophage injects its DNA into minicells produced by Bacillus subtilis CU403 divIVB1. The injected DNA is partially degraded to small trichloracetic acid-precipitable material and trichloroacetic acid-soluble material. The injected DNA is not replicated; however, it serves as a template for RNA and protein synthesis. The RNA produced specifically hybridizes to SPO1 DNA, and the amount of RNA hybridized can be reduced by competition with RNA isolated at all stages of the phage cycle from infected nucleate cells of the B. subtilis CU403 divIVB1. An unrelated phage, SPP1, also induces phage-specific RNA in infected minicells. Translation occurs in SPO1-infected minicells resulting in at least eight proteins which have been separated by gel electrophoresis, and two of these proteins have mobilities similar to proteins found only in infected B. subtilis CU403 divIVB1 nucleate cells. A large proportion of the polypeptide material synthesized in infected minicells is very small and heterogeneous in size.     

DNA of Bacillus subtilis bacteriophage SPP1: physical mapping and localization of the origin of replication.

The genome of Bacillus subtilis bacteriophage SPP1, a linear, 28.5-megadalton DNA duplex, was mapped by analysis with the restriction endonucleases endo R.Sal I, Sma I, Xba I, Bgl I, Bgl II, and EcoRI. The SPP1 genome, like that of the Salmonella typhimurium phage, P22, was found to be a terminally repetitious, circularly permuted molecule. 6-(p-Hydroxyphenylazo)uracil, a selective, reversible inhibitor of SPP1 DNA synthesis, was exploited to synchronize the initiation of genome replication and to selectively label the site of its initiation with radioactive thymidine. Restriction endonuclease analysis of the distribution of the label located the origin of replicative synthesis at an area approximately 0.2 genome length from one molecular terminus.     

Effect of ultraviolet radiation on the Bacillus subtilis phages SPO2, SPP1 and phi 29 and their DNAs

A comparative study of the effects of ultraviolet radiation on three Bacillus subtilis phages is presented. Phages phi 29, SPP1 and SPO2c12 or their DNAs were irradiated by UVC (254 nm) and quantum yields for inactivation were calculated. For each phage, the purified DNA was found to be more sensitive than the intact virus when assayed in a uvr+ host. The data imply that this is because transfecting DNA is repaired less efficiently than DNA of the intact phage; rather than because of differences in sensitivity to lesion production. Even though phi 29 has the smallest target size of the three phages, phi 29 and its DNA are the most sensitive. Phages SPO2 and SPP1 code for gene products which complement the repair system of the host. The transfecting DNA of phage SPP1 is extremely sensitive to UV damage when assayed in a uvr-host. This is attributed to the fact that in transfection SPP1 DNA must undergo recombination for productive infection to occur. The recombination process strongly interferes with the repair of damaged DNA.     

The genome of Bacillus subtilis phage SPP1: structure of an early promoter

The strongest of five 'early' promoters of Bacillus subtilis phage SPP1 was localized in a DNA restriction fragment by analysis of RNA polymerase binding and R-loop formation. The nucleotide sequence of the promoter region was established. The signal structures identified were similar to those recognized by the sigma 55 RNA polymerase of B. subtilis. The promoter precedes an open reading frame with 51 codons. A protein with the Mr predicted from the nucleotide sequence was identified in minicells.     

Phage SPP1 reversible adsorption to Bacillus subtilis cell wall teichoic acids accelerates virus recognition of membrane receptor YueB

Bacteriophage SPP1 targets the host cell membrane protein YueB to irreversibly adsorb and infect Bacillus subtilis. Interestingly, SPP1 still binds to the surface of yueB mutants, although in a completely reversible way. We evaluated here the relevance of a reversible step in SPP1 adsorption and identified the receptor(s) involved. We show that reversible adsorption is impaired in B. subtilis mutants defective in the glucosylation pathway of teichoic acids or displaying a modified chemical composition of these polymers. The results indicate that glucosylated poly(glycerolphosphate) cell wall teichoic acid is the major target for SPP1 reversible binding. Interaction with this polymer is characterized by a fast adsorption rate showing low-temperature dependence, followed by a rapid establishment of an equilibrium state between adsorbed and free phages. This equilibrium is basically determined by the rate of phage dissociation, which exhibits a strong dependence on temperature compatible with an Arrhenius law. This allowed us to determine an activation energy of 22.6 kcal/mol for phage release. Finally, we show that SPP1 reversible interaction strongly accelerates irreversible binding to YueB. Our results support a model in which fast SPP1 adsorption to and desorption from teichoic acids allows SPP1 to scan the bacterial surface for rapid YueB recognition.     

Bacillus subtilis DNA polymerase III is required for the replication of DNA of bacteriophages SPP-1 and phi 105.

The replication of the Bacillus subtilis bacteriophages SPP-1 and phi 105 is sensitive to 6-(p-hydroxyphenylazo)-uracil (HPUra), a selective inhibitor of replicative DNA synthesis of B. subtilis which acts specifically at the levels of a replication-specific polymerase, DNA polymerase III (pol III). The origin of the HPUra-sensitive polymerase required for phage replication was examined by comparison of the drug sensitivity of phage development in a normosensitive host with that in a host carrying azp-12, a polC mutation that specifies production of an HPUra-resistant pol III. azp-12 specified HPUra-resistant phage host pol III. The host polIII requirement for SPP-1 replication also was confirmed by the demonstration that phage development was temperature sensitive in a host mutant carrying the polC mutation mut-1 (ts). Examination of the pol III activity of crude and purified cell-free preparations derived from phage-infected cells did not indicate any detectable changes in the specific activity, purification behavior, or drug sensitivity of the enzyme.