Restriction enzyme analysis of Bacillus subtilis bacteriophage phi 105 DNA

The recognition sites on phi 105 DNA for the restriction endonucleases EcoRI, Bg/II, SmaI, KpnI, SstI, SalI, XhoI, NcoI, PstI, HindIII, ClaI, EcoRV and MluI have been mapped. The sites for EcoRI are shown to be different from those published earlier. The DNA from phi 105 contains no recognition sites for the endonucleases BamHI and XbaI.     

Deletion mutants of temperate Bacillus subtilis bacteriophage phi105.

Summary Six deletion mutants of temperate Bacillus subtilis phage 105 have been isolated on the basis of their increased resistance to chelating agents. The size and position of the deletions was determined by electronmicroscopy of DNA heteroduplexes. All deletions are located in a region about 55–70% from one end of the DNA molecule, in the right half of the known genetic map of the phage. The segment 55–65% does not contain any genes essential for lytic growth or lysogenization. A gene(s) for immunity is located in a segment 65–70% from the left end.By electronmicroscopy of partially denatured 105 DNA two A-T rich regions have been localized in the right half of the molecule. One of these regions falls within the non-essential 55–65% DNA segment.     

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.     

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.     

Location of the Bacillus subtilis temperate bacteriophage phi 105 attP attachment site.

Chromosomal DNAs of lysogens of phi 105 and phi 105 DI:1t were digested with restriction enzymes EcoRI and HpaI and were probed with nick-translated mature phi 105 DNA. Altered bacteriophage-specific bands in the lysogens were detected, indicating that the phage integrates into the host chromosome at a single site, probably via a Campbell-type circular intermediate. The phage attachment site is centrally located in the phage genome and lies between the phage immunity region and the nonessential deletable region of phi 105.     

Evidence for circular permutation of the prophage genome of Bacillus subtilis bacteriophage phi 105.

Analysis of DNA extracted from Bacillus subtilis lysogenic for bacteriophage phi 105 was performed by restriction endonuclease digestion and Southern hybridization using mature phi 105 DNA as a probe. The data revealed that the phi 105 prophage is circularly permuted. Digests using the enzymes EcoRI, SmaI, PstI, and HindIII localized the bacteriophage attachment site (att) to a region 63.4 to 65.7% from the left end of the mature bacteriophage genome. The phi 105 att site-containing SmaI C, PstI J, and HindIII L fragments were not present in digests of phi 105 prophage DNA. phi 105-homologous "junction" fragments were visualized by probing digests of prophage DNA with the purified PstI J fragment isolated from the mature bacteriophage genome. The excision of the phi 105 prophage was detected by observing the appearance of the mature PstI J fragment and the concomitant disappearance of a junction fragment during the course of prophage induction.     

Correlated genetic and EcoRI cleavage map of Bacillus subtilis bacteriophage phi105 DNA.

The seven previously identified EcoRI cleavage fragments of phi 105 DNA were ordered with respect to their sites of origin on the phage genome by marker rescue. One fragment, H, did not carry any determinants essential for replication. This fragment was totally missing in a deletion mutant which exhibited a lysogenization-defective phenotype. There is a nonessential region on the phi 105 genome which begins in fragment B, spans fragment H, and ends in fragment F. The size of the nonessential region, as estimated by alterations observed in the fragmentation patterns of deletion mutant DNAs, is approximately 2.7 X 10(6) daltons. Two new EcoRI cleavage fragments with molecular weights of approximately 0.2 X 10(6) were detected by autoradiography of 32P-labeled DNA. These small fragments were not located on the cleavage map.     

Reorienting and expanding the physical map of temperate Bacillus subtilis bacteriophage phi 105.

During the course of extending the physical map of Bacillus subtilis temperate bacteriophage phi 105 to include SstI, XhoI, and HpaI, we recognized that the previous physical map for EcoRI was incorrect. The new enzyme maps were determined by single, double, and partial enzyme digestions, redigestion of purified phage fragments, end joint analysis, and DNA-DNA hybridization. The EcoRI physical map was corrected by double digestion of isolated fragments, DNA hybridization, and physical mapping by partial digestion of end-labeled fragments. EcoRI fragment G was repositioned to give the order D-G-I-E-B-H-F-C.     

Cloning in Bacillus subtilis by transfection with bacteriophage vector phi 105J27: isolation and preliminary characterization of transducing phages for 23 sporulation loci

Bacteriophage cloning vector phi 105J27, the construction of which is described in an accompanying paper, has been used for shotgun cloning of sporulation genes in Bacillus subtilis. Various genomic libraries have been constructed and screened for the presence of recombinant phages capable of transducing strains containing sporulation (spo) mutations to Spo+. Of a total of 30 spo loci tested, transducing phages have been isolated for 23, more than half of the known spo loci. Included are nine loci (spo0D, spo0J, spoIIIA, spoIIIE, spoIIIF, spoIVF, spoVB, spoVH and spoVJ) that do not appear to have been cloned previously. Preliminary genetic characterization of some of the new clones by a rapid screening procedure has enabled the status of various sporulation loci to be clarified.     

Nucleotide sequence of the cohesive single-stranded ends of Bacillus subtilis temperate bacteriophage phi 105.

The cohesive single-stranded ends of temperate Bacillus subtilis phage phi 105 were analyzed with the exonuclease activities of the Klenow fragment of DNA polymerase I and with exonuclease III and were found to be 3' extensions. Chemical sequencing of 3'-end-labeled fragments showed that the ends are 7-base extended 3' single strands and have the sequence: 5'-GCGCTCC-3'. 3'-CGCGAGG-5'     

Transductional selection of cloned bacteriophage phi 105 and SP02 deoxyribonucleic acids in Bacillus subtilis.

The Bacillus subtilis temperate bacteriophages phi 105 and SP02 are incapable of transduction of the small, multicopy drug resistance plasmids pUB110 and pCM194. Cloning endonuclease-generated fragments of phi 105 or SP02 DNA into each of the plasmids renders the chimeric derivatives susceptible to transduction specifically by the phage whose deoxyribonucleic acid is present in the chimera. The majority of phage deoxyribonucleic acid fragments identified that render plasmids transducible by phi 105 or SP02 appear to be internal fragments, not fragments containing the cohesive ends. However, the highest overall transduction frequency was observed in SP02-mediated transduction of a derivative of pUB110 containing a 1.6-megadalton EcoRI fragment that likely contains the SP02 cohesive ends (plasmid pPL1010). The transducing activity present in a phi 105 transducing lysate had a buoyant density slightly greater than infectious particles, whereas the majority of transducing particles in an SP02(pPL1010) transducing lysate had a buoyant density slightly less than infectious particles. Although no detectable change in plasmid structure resulted from transduction by phi 105 or SP02, deoxyribonucleic acid isolated from a purified SP02(pPL1010) transducing lysate contained no detectable monomeric pPL1010, but did contain a form of pPL1010 of higher molecular weight than the monomer.     

Structurally stable Bacillus subtilis cloning vectors

Cloning of long DNA segments (greater than 5 kb) in Bacillus subtilis is often unsuccessful when naturally occurring small (less than 10 kb) plasmids are used as vectors. In this work we show that vectors derived from the large (26.5 kb) plasmids pAM beta 1 and pTB19 allow efficient cloning and stable maintenance of long DNA segments (up to 33 kb). The two large plasmids differ from the small ones in several ways. First, replication of the large plasmids does not lead to accumulation of detectable amounts of ss DNA, whereas the rolling-circle replication typical for small plasmids does. In addition, the replication regions of the two large plasmids share no sequence homology with the corresponding regions of the known small plasmids, which are highly conserved. Taken together, these observations suggest that the mode of replication of the large plasmids is different from that of small plasmids. Second, short repeated sequences recombine much less frequently when carried on large than on small plasmids. This indicates that large plasmids are structurally much more stable than small ones. We suggest that the high structural stability of large plasmids is a consequence of their mode of replication and that plasmids which do not replicate as rolling circles should be used whenever it is necessary to clone and maintain long DNA segments in any organism.     

Construction of plasmid vectors for gene cloning in Escherichia coli and Bacillus subtilis

The construction and some properties of new hybrid plasmids which are able to replicate in both Escherichia coli and Bacillus subtilis are presented. A 5.5 Md hybrid plasmid pJP9 was constructed from pBR322 (Tc, Ap) and pUB110 (Nm) plasmids. pIM1 (7.0 Md) and pIM3 (7.7 Md) plasmids are its different erythromycin resistant derivatives. Tetracycline, ampicillin, neomycin and possibly erythromycin resistance genes are expressed in E. coli while neomycin and erythromycin resistance genes are expressed in B. subtilis. Insertional inactivation of only one gene is possible using the pJP9 plasmid as a vector in B. subtilis. However, insertional inactivation of at least two different genes can be achieved and monitored in E. coli and B. subtilis transformants in cloning experiments with PIM1 and pIM3 plasmids. Insertional inactivation of antibiotic resistance genes present in pJP9 plasmid was achieved by cloning of Streptococcus sanguis DNA fragments generated by appropriate restriction endonucleases. The pJP9 plasmid and its derivatives were found to be stable in both hosts cells.     

Upper limit for DNA packaging by Bacillus subtilis bacteriophage phi 105: isolation of phage deletion mutants by induction of oversized prophages

Summary We have determined the upper size limit for DNA packaging in Bacillus subtilis bacteriophage 105 by examining the plaque-forming and transducing capabilities of lysates made from strains containing prophages of various sizes. The upper size limit for efficient packaging of the phage genome appears to be about 40.2 kb, which is about 1 kb larger than the wild-type genome. This places an upper limit of about 5 kb on the size of insertions that can be accommodated in 105 transfection cloning vectors, such as 105J27. Induction of prophages that exceed the upper limit, followed by selection for plaque formation or transduction, provides a powerful means of isolating phage deletion mutants. A comparison of the location of each deletion with the resultant phenotype has enabled us to identify non-essential regions of the phage genome, and regions that are required for tail biosynthesis and for host cell lysis.     

Expression of superinfection immunity to bacteriophage phi 105 by Bacillus subtilis cells carrying a plasmic chimera of pUB110 and EcoRI fragment F of phi 105 DNA.

A 2.1-megadalton, EcoRI-generated fragment of Bacillus subtilis phage phi 105 DNA was cloned into plasmid pUB110. The hybrid plasmid produces a biologically active product which renders B. subtilis immune to infection by phi 105.     

Transfection of Bacillus subtilis protoplasts by bacteriophage phi do7 DNA.

DNA from the Bacillus subtilis temperate bacteriophage phi do7 was found to efficiently transfect B. subtilis protoplasts; protoplast transfection was more efficient than competent cell transfection by a magnitude of 10(3). Unlike competent cell transfection, protoplast transfection did not require primary recombination, suggesting that phi do7 DNA enters the protoplast as double-stranded molecules.