Promoter-probe plasmid for Bacillus subtilis.

We have constructed a promoter-probe expression vector for Bacillus subtilis. This plasmid, pCED6, can be used to fuse various DNA sequences to the structural gene of Escherichia coli beta-galactosidase, permitting analysis of the promoter activity of such sequences. pCED6 replicates and confers drug resistances in both E. coli and B. subtilis.     

Analysis of plasmid deletional instability in Bacillus subtilis.

Using a model system, we have studied deletion formation in Bacillus subtilis. When the staphylococcal plasmids pSA2100 (7.1 kilobases) and pUB110 (4.5 kilobases) were ligated to one another at their unique XbaI sites and transformed into either rec+ or recE4 strains of B. subtilis, an intramolecular recombination event usually occurred. Two plasmids, one of 2.6 kilobases and the other of 9.0 kilobases, were consistently isolated and shown by restriction enzyme analysis to be derived by recombination occurring in the pSA2100-pUB110 cointegrate. Analysis of the sequence of the junctions of the recombinant plasmids and of the crossover regions of the parental plasmids suggested that a reciprocal, conservative, intramolecular recombination event had occurred between short 18-base-pair homologous sequences that were oriented as direct repeats and bounded by regions of dyad symmetry. Evidence is presented that the above illegitimate recombination event is biased to occur intramolecularly and that randomly chosen direct repeats of either 22 or 29 base pairs are not sufficient to support recombination. The recombination event occurs in recA1, recB2, recD3, recE5, recL16, recM13, polA59, polA13, uvr-22, uvr-13, and stb mutants of B. subtilis and does not require that the competent state be established.     

spoVG sequence of Bacillus megaterium and Bacillus subtilis.

We have sequenced the stage V sporulation specific gene spoVG in both Bacillus megaterium and Bacillus subtilis. The open reading frames encode polypeptides of 96 and 97 residues, respectively, and have an 88.6% amino acid identity. Both genes have putative rho-independent terminators. No significant amino acid or nucleotide homology of either gene was found when compared with sequences contained in either the Genbank or EMBL data bases.     

Generation of linear multigenome-length plasmid molecules in Bacillus subtilis.

Linear multigenome-length double and single stranded plasmid DNA was identified in a Bacillus subtilis ATP-dependent DNAase mutant strain (addA5) bearing plasmids pC194 or pBD95ts. Plasmid pBC30, a seg mutant of pC194, as well as some pUB110 derivatives with rearrangements external to the minimal replicon, produce high amounts of such a concatemeric DNA, even in Rec+ cells. The synthesis of this type of plasmid DNA occurs in the absence of an active plasmid-encoded Rep protein and is markedly affected in polA5 and recE4 genetic backgrounds. To account for these observations, we propose that the AddAB complex serves to prevent a sigma-type replication of plasmid DNA.     

Characterization of chimeric plasmid cloning vehicles in Bacillus subtilis.

Restriction endonuclease cleavage maps of seven chimeric plasmids that may be used for molecular cloning in Bacillus subtilis are presented. These plasmids all carry multiple antibiotic resistance markers and were constructed by in vitro molecular cloning techniques. Several of the antibiotic resistance markers were shown to undergo insertional inactivation at specific restriction endonuclease sites. Kanamycin inactivation occurred at the BglII site of pUB110 derivatives, erythromycin inactivation occurred at the HpaI and BclI sites of pE194 derivatives, and streptomycin inactivation occurred at the HindIII site of pSA0501 derivatives. A stable mini-derivative of pBD12 was isolated and characterized. By using these plasmids, we identified proteins involved in plasmid-coded kanamycin and erythromycin resistance. The properties and uses of these chimeric plasmids in the further development of recombinant deoxyribonucleic acid technology in B. subtilis are discussed.     

Introduction of a Streptococcus cremoris plasmid in Bacillus subtilis.

Streptococcus cremoris Wg2 plasmid pWV01 was introduced in Bacillus subtilis by protoplast transformation. The yield of pWV01 isolated from B. subtilis was low. pWV01 contains a unique site for the restriction endonuclease MboI.     

Complete nucleotide sequence of pTZ12, a chloramphenicol-resistance plasmid of Bacillus subtilis

The complete nucleotide sequence of pTZ12, a chloramphenicol-resistance (CmR) plasmid (2517 bp) derived from Corynebacterium xerosis plasmid pTZ10, has been determined after propagation in Bacillus subtilis. The nucleotide sequence of pTZ12 suggests that a recombination event may have occurred naturally within the open reading frames for the Rep protein of pT181 (or a pT181-like plasmid) and pC221 (or a pC221-like plasmid).     

Transformation of Bacillus subtilis by single-stranded plasmid DNA.

The single-stranded form of a pE194-based plasmid transformed Bacillus subtilis protoplasts at least as efficiently as did the double-stranded plasmid, but the single-stranded form did not detectably transform B. subtilis competent cells.     

Regulation of tetracycline accumulation in Bacillus subtilis bearing B. subtilis plasmid pNS1981

Abstract Accumulation of tetracycline (Tc) into Bacillus subtilis was studied by two methods, one involving a fluorescence assay and the other an absolute determination of accumulated drug. B. subtilis GSY908 harboring B. subtilis plasmid pNS1981 and the plasmidless host strain accumulated equivalent amounts of Tc. Prior exposure of the plasmid-harboring cells to subinhibitory concentration of Tc resulted in marked decrease in accumulation of the drug, indicating that pNS1981-determined Tc resistance is inducible. Of interest was the fact that the amount of Tc accumulated by the Tc-induced plasmid-harboring cells is increased somewhat by the addition of carboxylcyanide- m -chlorophenyl hydrazone (CCCP), an uncoupling reagent. This seems to show that energy-dependent accelerated Tc efflux is involved in decreased Tc accumulation.     

Bacillus subtilis (natto) plasmid pLS20 mediates interspecies plasmid transfer.

The 55-kilobase plasmid, pLS20, of Bacillus subtilis (natto) 3335 promotes transfer of the tetracycline resistance plasmid pBC16 from B. subtilis (natto) to the Bacillus species B. anthracis, B. cereus, B. licheniformis, B. megaterium, B. pumilus, B. subtilis, and B. thuringiensis. Frequency of pBC16 transfer ranged from 2.3 x 10(-6) to 2.8 x 10(-3). Evidence for a plasmid-encoded conjugationlike mechanism of genetic exchange includes (i) pLS20+ strains, but not pLS20- strains, functioned as donors of pBC16; (ii) plasmid transfer was insensitive to the presence of DNase; and (iii) cell-free filtrates of donor cultures did not convert recipient cells to Tcr. Cotransfer of pLS20 and pBC16 in intraspecies matings and in matings with a restriction-deficient B. subtilis strain indicated that pLS20 was self-transmissible. In addition to mobilizing pBC16, pLS20 mediated transfer of the B. subtilis (natto) plasmid pLS19 and the Staphylococcus aureus plasmid pUB110. The fertility plasmid did not carry a selectable marker. To facilitate direct selection for pLS20 transfer, plasmid derivatives which carried the erythromycin resistance transposon Tn917 were generated. Development of this method of genetic exchange will facilitate the introduction of plasmid DNA into nontransformable species by use of transformable fertile B. subtilis or B. subtilis (natto) strains as intermediates.     

Membrane association of a Staphylococcus aureus plasmid in Bacillus subtilis.

The Staphylococcus aureus plasmid pUB110 was found to be enriched in deoxyribonucleic acid-membrane complexes isolated from Bacillus subtilis containing pUB110.     

New plasmid expression vectors for Bacillus subtilis

The construction of new cloning vectors for Bacillus subtilis is described. They are derived from the in vitro joining of parts of pE194 and pUB110 DNAs. Their common feature is to present a cloning site immediately after the promoter and ribosome binding site of the erythromycin resistance gene, allowing the insertion and expression of either sticky or blunt ended DNA fragments coding for any heterologous gene. The cloning and expression of Escherichia coli beta-lactamase and EcoRI methylase are given as examples. The enzymes are efficiently synthesized by B. subtilis cells.     

Conjugal plasmid transfer in Bacillus subtilis in soil microcosms

Conjugal transfer of the small plasmid pUB110 between Bacillus subtilis strains was studied under conditions of microcosms with sterile and nonsterile soil. Plasmid transfer proved to be possible after soil inoculation with vegetative partner cells or with their spores. Plasmid transfer occurred at temperatures of 30 degrees C and 22-23 degrees C.     

Nucleotide sequence of threonine tRNA from Bacillus subtilis.

A threonine tRNA was purified from Bacillus subtilis W168 by a combined use of column chromatographic systems. The nucleotide sequence was determined to be pG-C-C-G-G-U-G-U-A-G-C-U-C-A-A-U-D-G-G-D(U)-A-G-A-G-C-A-A-C-U-G-A-C-U-mo5U-G-U-t6A-A-psi-C-A-G-U-A-G-m7G-U-U-G-G-G-G-G-T-psi-C-A-A-G-U-C-C-U-C-U-U-G-C-C-G-G-C-A-C-C-AOH, where about 40 % of D20 remained unmodified as U20. It consists of 76 nucleotides including a new minor nucleoside, 5-methoxyuridine (mo5U), which occupies the wobble position of anticodon.     

The Bacillus subtilis AddAB helicase/nuclease is regulated by its cognate Chi sequence in vitro

The AddAB enzyme is important to homologous DNA recombination in Bacillus subtilis, where it is thought to be the functional counterpart of the RecBCD enzyme of Escherichia coli. In vivo, AddAB responds to a specific five-nucleotide sequence (5'-AGCGG-3' or its complement) in a manner analogous to the response of the RecBCD enzyme to interaction with chi sequences. Here, we show that purified AddAB enzyme is able to load at a double-stranded DNA end and is both a DNA helicase and nuclease, whose combined action results in the degradation of both strands of the DNA duplex. During translocation, recognition of the properly oriented sequence 5'-AGCGG-3' causes attenuation of the AddAB enzyme nuclease activity that is responsible for degradation of the strand 3'-terminal at the entry site. Therefore, we conclude that 5'-AGCGG-3' is the B. subtilis Chi site and it is hereafter referred to as chi(Bs). After encountering chi(Bs), both the degradation of the 5'-terminal strand and the helicase activity persist. Thus, processing of a double-stranded DNA end by the AddAB enzyme produces a duplex DNA molecule with a protruding 3'-terminated single-stranded tail, a universal intermediate of the recombination process.     

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.     

Stability function in the Bacillus subtilis plasmid pTA 1060

Plasmid pBB2 (11.3 kb) was constructed by genetically labeling the cryptic Bacillus subtilis plasmid pTA 1060 with the pC194-derived CmR and the pUB110-derived KmR markers. In nonselective media pBB2 was segregationally almost completely stable (loss rates less than or equal to 0.02% per cell generation). In contrast, pBB3, obtained by deleting from pBB2 a region consisting of two ClaI fragments (1.45 and 0.20 kb, respectively), was unstable (loss rates greater than or equal to 0.5% per cell generation). This indicates that a genetic element required for stability is located on one or both of these fragments. In pBB3 cop, a mutant with a two- to threefold increased copy number, the rate of plasmid loss was reduced compared to that of pBB3. The insertion of a 4.2-kb Escherichia coli DNA fragment reduced the stability of pBB2 only slightly, suggesting that this vector may be useful for the cloning of relatively large fragments.