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.     

Correlation between lethality and DNA single-strand breaks in Bacillus subtilis cells treated with N-methyl-N'-nitro-N-nitrosoguanidine

The effects of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment two Bacillus subtilis strains which exhibit different UV sensitivities were monitored at both the cellular (survival) and subcellular (DNA strand break) levels. The MNNG-induced single strand DNA breaks (SSB) in either strain, as measured by alkaline sucrose gradient centrifugation, were shown to be well correlated with lethality. These DNA lesions were shown by a computer simulation to be randomly induced. Cell survival after MNNG treatment is inversely related to the number of replication forks per cell which in turn depends upon the doubling time of the culture and the growth phase. The production of single-strand breaks and cell killing are proportional to the log of the initial MNNG concentration and may imply that decomposition of the mutagen is (pseudo) second order.     

Direct selection of complementing diploids from PEG-induced fusion of Bacillus subtilis protoplasts

Summary A minimal medium containing horse serum is described on which Bacillus subtilis protoplasts revert to bacillary forms at high frequency (ca. 30%). Used as a plating medium for a mixture of polyethyleneglycol-treated protoplasts from two complementary polyauxotrophic parental strains, it selects the prototrophic fusion products efficently, and also allows isolation of various auxotrophic recombinants. These prototrophs and recombinants amount respectively to 1% and 10% of the regenerated bacteria.We confirm that two types of prototrophs can be isolated after fusion: stable recombinants and complementing diploids, the latter segregating into various types of recombinants. Based on easily recognized colonial aspects, an approximate estimation of the proportion of the two types becomes possible when a spoOA mutation has been introduced in one of the parents. At least 50% of the prototrophic fusion products are complementing diploids. Incidently, the data also settle a controversy by showing the dominance of spoOA mutations in heterozygotic bacteria.This work was supported by the Centre National de la Recherche Scientifique (Contrat L.A. 136).     

Direct fractionation of genes by preparative electrophoresis of Bacillus subtilis DNA

Discontinuous electrophoresis through agarose has been shown to be a satisfactory method for preparation of biologically active restriction fragments from milligram quantities of DNA. The DNA is obtained in sufficient quantity for: (1) direct use in genetic transformation, (2) the production of multiple-dimensional restriction analyses, or (3) use as a high-resolution hybridization probe.     

Identification of a sporulation locus in cloned Bacillus subtilis deoxyribonucleic acid.

A cloned deoxyribonucleic acid from the purA-cysA region of the Bacillus subtilis chromosome was shown to contain the spoVC locus, a gene whose product is required for sporulation. This is the first demonstration of a spo locus in cloned B. subtilis deoxyribonucleic acid.     

Essential structure in the cloned transforming DNA that induces gene amplification of the Bacillus subtilis amyE-tmrB region.

Bacillus subtilis B7, a mutant which acquired gene amplification of the amyE-tmrB region, showed, as a result, hyperproductivity (about a 5- to 10-fold increase) of alpha-amylase and tunicamycin resistance. The mutational character was transferred to recipient cells by competence transformation. A 14-kilobase (kb) EcoRI chromosomal DNA fragment of strain B7 was found to have the transforming activity. We cloned a 6.4-kb EcoRI fragment on a phage vector lambda Charon 4A through a spontaneous deletion of 7.6 kb from the 14-kb fragment and subcloned a 1.6-kb HindIII fragment on pGR71. The cloned 6.4-kb EcoRI and 1.6-kb HindIII fragments retained the transforming activity of inducing gene amplification of the amyE-tmrB region. At the junction point (J) of the repeating units (16 kb), the tmrB gene was linked to a DNA region (M) located 4 kb upstream of amyE. The essential structure of the cloned, transforming (gene amplification-inducing) DNA was deduced to be that around J. The subcloned 1.6-kb HindIII fragment that retained the transforming activity was shown to be almost solely composed of the tmrB-J-M region. In addition, the DNA sequence around J was determined.     

DNA synthesis in competent Bacillus subtilis cells.

Competent cells of Bacillus subtilis incorporate degradation products from transfecting DNA into their chromosomal DNA. The sensitivity of this incorporation to inhibitors of bacterial DNA synthesis [phage infection or 6-(p-hydroxyphenylazo)-uracil] suggests that semiconservative DNA synthesis can occur in competent cells.     

Intrastrand self-complementary sequences in Bacillus subtilis DNA.

Intrastrand self-complementary sequences have been isolated from the DNA of Bacillus subtilis by hydroxyapatite (HA) chromatography following thermal renaturation of strands separated by chromatography on methylated albumin kieselguhr (MAK). The instrastrand structures derived from the MAK H strand (HA HII) were biologically active showing transforming activity for a wide variety of markers, as well as hybridization to both pulse-labelled and ribosomal RNA. Removal of regions of single-strand DNA with S1 nuclease did not significantly alter the biological activity of the self-annealed molecules. The overall efficiency of transformation and hybridization of the intrastrand self-annealing DNA was low suggesting that many sequences in the population are neither active in transformation to prototrophy nor transcribed into RNA.     

Activity of two strong promoters cloned into Bacillus subtilis

Two DNA fragments, one encoding the Escherichia coli trc promoter and the other encoding a sequence from the early region of Bacillus subtilis phage SPO1, were cloned into the B. subtilis promoter-probe vector pPL603. Both fragments effected strong in vivo promoter activity in vegetative B. subtilis cells.     

DNA repair in competent cells of Bacillus subtilis.

A series of isogenic transformable strains of Bacillus subtilis carrying the uvr-19 or rec-43 mutation or both were constructed. Both mutations made competent cells defective in repairing UV-irradiated cellular or transforming DNA, and their effects were additive in a doubly deficient strain, suggesting that two repair processes, requiring uvr-19+ and rec-43+ gene products, are independently functional in competent cells of B. subtilis.     

Mutation and killing of Escherichia coli expressing a cloned Bacillus subtilis gene whose product alters DNA conformation.

Expression of the Bacillus subtilis gene coding for SspC, a small, acid-soluble protein, caused both killing and mutation in a number of Escherichia coli B and K-12 strains. SspC was previously shown to bind E. coli DNA in vivo, and in vitro this protein binds DNA and converts it into an A-like conformation. Analysis of revertants of nonsense mutations showed that SspC caused single-base changes, and a greater proportion of these were at A-T base pairs. Mutation in the recA gene abolished the induction of mutations upon synthesis of SspC, but the killing was only slightly greater than in RecA+ cells. Mutations in the umuC and umuD genes eliminated most of the mutagenic effect of SspC but not the killing, while the lexA mutation increased mutagenesis but did not appreciably affect the killing. Since there was neither killing nor mutation of E. coli after synthesis of a mutant SspC which does not bind DNA, it appears likely that the binding of wild-type SspC to DNA, with the attendant conformational change, was responsible for the killing and mutation. A strain containing the B. subtilis gene that is constitutive for the RecA protein at 42 degrees C showed a lower frequency of mutation when that temperature was used to induce the RecA protein than when the temperature was 30 degrees C, where the RecA level is low, suggesting that at the elevated temperature the high RecA level could be inhibiting binding of the B. subtilis protein to DNA.     

Plasmid DNA transduction in Bacillus subtilis

Transduction of Bacillus subtilis pUB110 plasmid by AR9 phage is described. Some aspects of this process are studied. Plasmid transduction depended on multiplicity of infection similar to cases of chromosomal markers transduction, though optimal multiplicity of infection was achieved using low number of phage particles. No cotransduction of plasmid and chromosomal markers was demonstrated. The transduction frequencies of plasmid and chromosomal markers increased after UV irradiation of phage suspensions within the range of definite doses.     

Characterization of a cloned Bacillus subtilis gene that inhibits sporulation in multiple copies.

We have isolated a 1.0-kilobase fragment of the Bacillus subtilis chromosome which, when present in high-copy-number plasmids, caused a sporulation-proficient strain to become phenotypically sporulation deficient. This is referred to as the sporulation inhibition (Sin) phenotype. This DNA fragment, in multicopy, also inhibited the production of extracellular protease activity, which normally appears at the beginning of stationary growth. The origin of the fragment was mapped between the dnaE and spo0A genes on the B. subtilis chromosome, and its complete DNA sequence has been determined. By analysis of various deletions and a spontaneous mutant the Sin function was localized to an open reading frame (ORF) predicted from the DNA sequence. Inactivation of this ORF in the chromosome did not affect the ability of cells to sporulate. However, the late-growth-associated production of proteases and alpha-amylase was elevated in these cells. The predicted amino acid sequence of the protein encoded by this ORF had a DNA-binding domain, typically present in several regulatory proteins. We propose that the sin ORF encodes a regulatory protein that is involved in the transition from vegetative growth to sporulation.