RecE-dependent lysogenic induction in the absence of repressor in Bacillus subtilis non-complementing diploids

The RecE protein of Bacillus subtilis, known to be required for induction of the SOS response and of phi 105 prophage, was shown to be involved in mitomycin C induction of B. subtilis diploid lysogens carrying a silent phi 105 prophage in their unexpressed chromosome. These stable non-complementing diploid lysogens, formed by protoplast fusion and regeneration, did not synthesize repressor, so that the induction observed must have resulted from RecE-dependent activation of the prophage rather than from RecE-dependent inactivation of repressor. Mitomycin C treatment does not induce permanent expression of the silent chromosome, so the activation seems to be temporary, perhaps reflecting the action of an SOS function under RecE control.     

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.     

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.     

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.     

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.     

Use of the thermoinducible temperate phage Phi 105cts139 for cloning in Bacillus subtilis

The gene for alpha-amylase from Bacillus amyloliquefaciens having a foreign promoter providing gene expression in logarithmic growth phase and the cat gene of plasmid pC194 (AC fragment) were inserted into thermoinducible prophage phi 105 cts139. Possibility of amylolytic activity enhancement was studied after thermoinduction. When AC fragment and random PstI restricts of phage DNA were ligated and used to transform Bacillus subtilis 1A289 (phi 105 cts139) the Amy+ CmR transformants were obtained having the different levels of increased amylolytic activity (maximum--26 fold). Numerous phages without insert found in induced lysates suggest that insertions were unstable and (or) the clones were double lysogens for hybrid and original type phages. Stable insertion of AC fragment replacing the PstI-H-fragment of phage DNA revealed that all Amy+ CmR transformants were double lysogens. Inducibility depended on the insertion orientation.     

Construction of improved bacteriophage phi 105 vectors for cloning by transfection in Bacillus subtilis

A series of improved phage vectors have been constructed, based on Bacillus subtilis bacteriophage phi 105, which can be used to clone genes in B. subtilis by direct transfection of protoplasts. The new vectors, designated phi 105J23, phi 105J24, phi 105J27 and phi 105J28, show frequencies of plaque formation that are equal to those of wild-type phi 105. This represents at least a 10-fold improvement over phi 105J9, the vector used in previous cloning experiments. Two of the new vectors phi 105J27 and phi 105J28 incorporate a mutation, cts-52, that renders the prophage temperature inducible. This has made it possible to devise a rapid small-scale procedure for screening progeny phage for the presence of inserted DNA. The usefulness of the new vectors is illustrated in the accompanying paper by cloning more than 20 B. subtilis sporulation genes.     

Cloning and nucleotide sequence of phoP, the regulatory gene for alkaline phosphatase and phosphodiesterase in Bacillus subtilis.

Two DNA fragments which complement the alkaline phosphatase-negative mutation phoP of Bacillus subtilis were cloned from a B. subtilis chromosome with the prophage vector phi CM (a derivative of phi 105). One of the fragments contained the regulatory gene phoR in addition to phoP. Nucleotide sequence analysis of the phoP region revealed that the phoP gene product consists of 241-amino-acid residues and that the sequence of these amino acids is extensively homologous with the sequence of the phoB gene product. This protein is the positive regulator for the phosphate regulon in Escherichia coli. It therefore appears that phoP is a regulatory gene for alkaline phosphatase synthesis in B. subtilis.     

Interaction of the Bacillus subtilis phage phi 105 repressor DNA: a genetic analysis.

The Bacillus subtilis phage phi 105 repressor specifically recognizes a 14-bp operator sequence which does not exhibit 2-fold rotational symmetry. To facilitate a genetic analysis of this sequence-dependent DNA binding a B. subtilis strain was constructed in which mutations affecting the phi 105 repressor-operator interaction cause a selectable phenotype, chloramphenicol resistance. After in vivo mutagenesis, we isolated and mapped 22 different mutations in the repressor coding sequence, 15 of which are missense substitutions. These are exclusively located in the N-terminal part (positions 1-43) of the 144 residue long polypeptide. Two nonsense mutants, at positions 70 and 89, respectively, still show partial repressor activity. These data suggest that the phi 105 repressor consists of at least two independently folding structural domains, of which the N-terminal is involved in operator binding. Twelve missense mutations are clustered in a region extending from Gln-18 to Arg-37, which we propose to be the DNA-binding alpha-helix--beta-turn--alpha-helix motif, common to all lambda Cro-like repressors. The second ('recognition') helix shows significant homology with the corresponding sequence in Tn3 resolvase, and there is also a striking similarity between the phi 105 operator and the consensus sequence for a Tn3 res half-site. Based on these observations, and on the previously isolated phi 105 0c mutants, we tentatively assign some specific contacts between base pairs from the first half of a phi 105 operator site and amino acids from the repressor's 'recognition helix'.     

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%。     

Linked Transformation of Bacterial and Prophage Markers in Bacillus subtilis 168 Lysogenic for Bacteriophage φ105

Level of competence reached by Bacillus subtilis 168 lysogenic for temperate phage phi 105 was reduced compared to that reached by nonlysogenic cells. This effect was probably related to an alteration of the bacterial surface. Deoxyribonucleic acid extracted from phi 105 lysogenic bacteria was used to transform other lysogenic bacteria. About 25% linkage was found between the bacterial phe-1 marker and prophage marker ts N15. The order of a few prophage markers relative to phe-1 was established in three-factor crosses. The usefulness of this system for a study of the linkage between an integrated prophage genome and that of its host was discussed.     

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.