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'     

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'.     

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.     

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.     

Nucleotide sequence of the thymidylate synthetase gene (thyP3) from the Bacillus subtilis phage phi 3T

The thyP3 gene, encoding thymidylate synthetase, from the Bacillus subtilis phage phi 3T has been cloned and the nucleotide sequence determined. The derived amino acid sequence indicates a subunit Mr of 32 748. The primary amino acid sequence is compared with the sequences of the analogous proteins specified by Escherichia coli (thyA), Lactobacillus casei, (thyA) and phage T4 (td). Extensive conservation exists in all four sequences implying a shared tertiary structure.     

The temperate B. subtilis phage phi 105 genome contains at least two distinct regions encoding superinfection immunity

Summary Two different PstI fragments of temperate phage 105 DNA are shown to confer superinfection immunity upon Bacillus subtilis when inserted into the multicopy cloning vector pE194 cop-6. The 2.3 kb PstI fragment I is located almost entirely within EcoRI fragment F and encompasses a region previously known to encode a repressor. The other fragment, PstI-E (4.3 kb) maps inside the EcoRI-B fragment, and allows an explanation of the clear-plaque phenotype of the deletion mutant 105DII:6c. The two regions can be distinguished functionally, since only the PstI fragment I product interacts with a specific 105 promoter-operator site.     

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.     

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.     

Nucleotide sequence of the Bacillus subtilis phoR gene.

The nucleotide sequence of phoR, the positive and negative regulatory gene for alkaline phosphatase and phosphodiesterase formation in Bacillus subtilis, was determined. The sequence data predicted an open reading frame of 1,740 base pairs (579 amino acids) which overlaps the 5 base pairs of the preceding phoP coding sequence. The deduced amino acid sequence was significantly homologous with that of the Escherichia coli phoR gene product, which is the sensory element for the pho regulon.     

Nucleotide sequence of Bacillus phage phi 29 genes 14 and 15: homology of gene 15 with other phage lysozymes.

The nucleotide sequence of Bacillus phage phi 29 genes 14 (g14) and 15 (g15) have been determined and shown to encode proteins with molecular weights of 15,014 and 28,022, respectively. The g14 open reading frame (ORF) was confirmed by sequencing a sus14(1241) mutant. Gene product 15 (gp15) has considerable homology with Salmonella phage P22 lysozyme and lesser homology with Escherichia coli phage T4 lysozyme. Putative translation signals are identified. In addition, the role of a previously described promoter, B2, is discussed.