Restriction endonuclease mapping of bacteriophage phi105 and closely related temperate Bacillus subtilis bacteriophages rho10 and rho14.

Cleavage maps of the three similar Bacillus subtilis temperate bacteriophages, phi105, rho10, and rho14, were constructed by partial digestion analysis utilizing the restriction endonuclease EcoRI. Comparison of the topography of these maps indicates that all phage DNAs posses cohesive ends and a number of EcoRI restriction sites; the fragments are conserved, and the estimated base substitution/nucleotide divergence between these phages is 0.03 to 0.07 based on conserved fragments or between 0.03 and 0.11 based on conserved cleavage sites. These lines of evidence indicate that phi105, rho10, and rho14 are closely related. Double-enzyme digestion analysis reveals that rho14 DNA has unique SalGI and BglII restriction sites and phi105 DNA has a unique SalGI restriction site, making these phages possible cloning vectors for B. subtilis.     

枯草芽孢杆菌噬菌体载体的构建

以φ0105DI：It为原始株构建的重组噬菌体φ105S35和φ10 5S36具有自主侵染能力和溶源化特征。其基因组内插入的lkb片段上的cat,基因赋予二者所在宿主以氯霉素抗性,在两株噬菌体中插入位点相同,即原φ105DI ：It的smal酶切片段D、E之间,但插入片段在二者中的定向相反。与cat基因同时引入的单一BamHI和Xbal位点提供了外源DNA的插入位置。重组噬菌体DNA可高效转染枯草芽孢杆菌原生质体。因此φ105S35和币φ105S36可作为枯草芽孢杆随系统的载体而被利用。     

Cloning of the Bacillus subtilis recE+ gene and functional expression of recE+ in B. subtilis.

By use of the Bacillus subtilis bacteriophage cloning vehicle phi 105J23, B. subtilis chromosomal MboI fragments have been cloned that alleviate the pleiotropic effects of the recE4 mutation. The recombinant bacteriophages phi 105Rec phi 1 (3.85-kilobase insert) and phi 105Rec phi 4 (3.3-kilobase insert) both conferred on the recE4 strain YB1015 resistance to ethylmethane sulfonate, methylmethane sulfonate, mitomycin C, and UV irradiation comparable with the resistance observed in recE+ strains. While strain YB1015 (recE4) and its derivatives lysogenized with bacteriophage phi 105J23 were not transformed to prototrophy by B. subtilis chromosomal DNA, strain YB1015 lysogenized with either phi 105Rec phi 1 or phi 105Rec phi 4 was susceptible to transformation with homologous B. subtilis chromosomal DNA. The heteroimmune prophages phi 105 and SPO2 were essentially uninducible in strain YB1015. Significantly, both recombinant prophages phi 105Rec phi 1 and phi 105Rec phi 4 were fully inducible and allowed the spontaneous and mitomycin C-dependent induction of a coresident SPO2 prophage in a recE4 host. The presence of the recombinant prophages also restored the ability of din genes to be induced in strains carrying the recE4 mutation. Finally, both recombinant bacteriophages elaborated a mitomycin C-inducible, 45-kilodalton protein that was immunoreactive with Escherichia coli recA+ gene product antibodies. Collectively, these data demonstrate that the recE+ gene has been cloned and that this gene elaborates the 45-kilodalton protein that is involved in SOB induction and homologous recombination.     

Cloning of the Bacillus subtilis spoIIA and spoV A loci in phage phi 105DI:1t

A 6.95 kb HindIII-generated DNA fragment from Bacillus subtilis 168 was inserted into the DNA of phage phi 105DI:1t. The recombinant phage (phi 105DS1) contained DNA of 33.8 kb as compared with 35.2 kb for phi 105DI:1t and 39.2 kb for the wild-type phage. In the presence of helper phage, phi 105DS1 complemented both spoIIA and spoV A mutations in B. subtilis.     

Nucleotide sequence of the immunity region of Bacillus subtilis bacteriophage phi 105: identification of the repressor gene and its mRNA and protein products

A gene involved in the regulation of lysogeny in the temperate Bacillus subtilis phage phi 105 has been identified and isolated. A plasmid, pDC4, was constructed that contains a 740-bp HindIII-PvuII fragment that is derived from the phi 105 immunity region and is capable of rendering B. subtilis immune to infection by phi 105. Three different hybrid plasmids that contain the 740-bp fragment, pAG101 [Cully and Garro, J. Virol. 34 (1980) 789-791], pDC1 and pDC2, were found to synthesize a common 18-kDal polypeptide in B. subtilis minicells and Escherichia coli maxicells. The nucleotide (nt) sequence of this region revealed three open reading frames (ORFs) that predict proteins with Mrs of 16521, 7332, and 5516. In vivo synthesized phi 105 prophage RNA was mapped by primer extension and shown to be transcribed from the DNA strand coding for the Mr 16521 protein. The 5' end of the phi 105 lysogen RNA was mapped to a region that contains conserved sequences for RNA polymerase recognition.     

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.     

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.     

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.     

双启动子对重组溶源性枯草杆菌中外源蛋白表达的增强作用

探讨了双启动子对基于溶源性噬菌体构建的重组枯草杆菌中外源蛋白表达的影响。分别将不含或含有本身启动子的α-淀粉酶基因(来源于Bacillus amyloliquefaciens)和青霉素酰化酶基因(来源于Bacillus megaterium)克隆到溶源性枯草杆菌中,得到重组菌B.subtilisAMY1,B.subtilisAMY2,B.subtilisPA1以及B.subtilisPA2。由于同源重组,所克隆的片段整合到溶源性枯草杆菌中的噬菌体基因组上,并处于噬菌体强启动子的下游。在重组菌AMY1和PA1中,在热诱导的情况下外源基因的转录只受到噬菌体启动子的作用,而在重组菌AMY2和PA2中,在热诱导下外源基因的转录同时受到噬菌体启动子和基因本身所带启动子的作用。双启动子的应用使重组α-淀粉酶的表达量提高了133%,使重组青霉素酰化酶的表达量提高了113%。     

Growth of Bacteriophage φ105 and Its Deoxyribonucleic Acid in Radiation-Sensitive Mutants of Bacillus subtilis

Growth of phage phi105 and its deoxyribonucleic acid (DNA) was studied in radiation-sensitive mutants of Bacillus subtilis. The recA gene is required for optimal prophage induction with mitomycin C and for infectivity of prophage DNA. rec B gene is required for marker rescue from mature DNA. The importance of bacterial genes for phage DNA activity seems to depend on phage DNA structure.     

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.     

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.     

Genetic analysis of spo0A and spo0C mutants of Bacillus subtilis with a phi 105 prophage merodiploid system.

An 8.0-kilobase chromosomal fragment of Bacillus subtilis which contained an intact spo0A gene was recloned onto temperate phage phi 105 from the rho 11dspo0A+-1 transducing phage. A specialized transducing phage, phi 105-dspo0A+-1, was constructed and used to transduce the spo0A12 mutant strain 1S9. A Spo+ transductant which was a single lysogen of the phi 105dspo0A+-1 transducing phage was isolated. From competent cells of this Spo+ transductant was isolated a Spo- (Spo0A) strain which was immune to phi 105. It was used to prepare a lysate of the phi 105dspo0A12 phage. Transduction of the spo0C9V recE4 strain with the phi 105dspo0A12 and phi 105dspo0A+-1 phages was carried out. The phi 105dspo0A+-1 phage gave rise to a large number of heat-resistant cells, but the phi 105dspo0A12 phage formed no heat-resistant cells. These results indicate that the spo0A12 and spo0C9V mutant genes do not complement each other in the ability to sporulate and that the spo0C9V mutation is located within the spo0A gene. Although the spo0C9V strain was completely asporogenous, the spo0C9V/spo0C9V diploid strain produced heat-resistant cells at a frequency of ca. 10(-3) in the sporulation medium. This result indicates that two copies of the spo0C9V mutant gene partially restore the ability of these cells to sporulate.     

A conserved anti-repressor controls horizontal gene transfer by proteolysis

The mobile genetic element ICEBs1 is an integrative and conjugative element (a conjugative transposon) found in the Bacillus subtilis chromosome. The SOS response and the RapI-PhrI sensory system activate ICEBs1 gene expression, excision and transfer by inactivating the ICEBs1 repressor protein ImmR. Although ImmR is similar to many characterized phage repressors, we found that, unlike these repressors, inactivation of ImmR requires an ICEBs1-encoded anti-repressor ImmA (YdcM). ImmA was needed for the degradation of ImmR in B. subtilis. Coexpression of ImmA and ImmR in Escherichia coli or co-incubation of purified ImmA and ImmR resulted in site-specific cleavage of ImmR. Homologues of immR and immA are found in many mobile genetic elements. We found that the ImmA homologue encoded by B. subtilis phage phi105 is required for inactivation of the phi105 repressor (an ImmR homologue). ImmA-dependent proteolysis of ImmR repressors may be a conserved mechanism for regulating horizontal gene transfer.     

Heat Induction of Prophage φ 105 in Bacillus subtilis: Bacteriophage-Induced Bidirectional Replication of the Bacterial Chromosome

A mutant of Bacillus subtilis, dna-1, which cannot initiate new rounds of DNA replication (obtained from N. Sueoka) was lysogenized with wild-type phi 105 and with the heat-inducible mutant phi 105 cts23. Bacteria were incubated at the permissive temperature in the presence of chloramphenicol and then shifted to the nonpermissive temperature where induction of phi 105 cts23 occurs. DNA made after the shift was labeled with a density label, and the distribution of bacterial and phage markers in replicated and unreplicated DNA was determined. Similar experiments were performed with nonlysogenic dna-1 infected with phage phi 105 cts23 after the temperature shift. The results show that after induction of phi 105 cts23 prophage, bacterial markers on either side of the prophage replicate at an increased rate compared to more distant markers. No selective stimulation of bacterial DNA synthesis was observed on infection or after shifting bacteria lysogenic for noninducible phage to the higher temperature. Attempts to suppress the initiation mutation dna-1 by phage phi 105 were unsuccessful.     

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.