Plasmid transformation in Bacillus subtilis: Fate of plasmid DNA

Summary Only multimeric, and not monomeric forms of B. subtilis plasmids can transform B. subtilis cells (Canosi et al. 1978). This finding prompted us to study the physico-chemical fate of plasmid DNA in transformation. Competent cells of B. subtilis were exposed to either unfractionated preparations or to preparations of multimeric plasmid DNA. Plasmid DNA was re-extracted from such cells and then analyzed by sedimentation and isopycnic centrifugation and also defined by its sensitivity to nuclease S1 degradation. No double-stranded plasmid DNA could be recovered from cells transformed with unfractionated plasmid preparations which contained predominantly monomeric covalently closed circular (CCC) DNA, Re-extracted plasmid DNA was single-stranded, had a molecular weight considerably smaller than monomer length DNA and had been subject to degradation to acid soluble products. However, when transformations were performed with multimeric DNA (constructed by in vitro ligation of linearized pC194 DNA), both double-stranded and partially double-stranded DNA could be recovered in addition to single-stranded DNA.We assume that plasmid DNA is converted to a single-stranded form in transformation, irrespective of its molecular structure. Double-stranded and partially double-stranded DNAs found in transformation with multimeric DNA would be the products of intramolecular annealing.Some of these results were presented at the 5th European Meeting on Bacterial Transformation and Transfection, September 1980, Florence     

Fate of plasmid DNA in transformation of Bacillus subtilis protoplasts

Summary Polyethylene glycol-treated protoplasts of B. subtilis can be transformed by plasmid DNA at very high frequencies (Chang and Cohen 1979). From analysis of plasmid mediated transformation of transformation-deficient mutants it appeared that mutants, reduced in the transformation by plasmid DNA in the competent state, were plasmid transformation-proficient when transformed as protoplasts. By means of CsCl-gradient centrifugation of re-extracted plasmid DNA it could be demonstrated that plasmid DNA enters the protoplasts in the double-stranded form. In addition, sucrose gradient centrifugation of the re-extracted plasmid DNA showed that the entered DNA is predominantly present as covalently closed circular DNA. The efficiency of plasmid transformation in protoplasts was found to be close to one (each plasmid molecule having entered into the protoplasts gives rise to a transformed cell). This is in good agreement with the observation that little, if any, damage is done to this DNA during or after entry into protoplasts.     

Polyethyleneglycol-induced transformation of Bacillus subtilis protoplasts by bacterial chromosomal DNA

Summary Bacillus subtilis protoplasts, which in the presence of polyethyleneglycol (PEG) are transformed by plasmid DNA (Chang and Cohen 1979) can also be transformed under these conditions by chromosomal DNA. Transformation in this case occurs at a much lower frequency, not fully accounted for by the heterogeneity of this DNA. Another unexpected feature of the transformation studied, which may explain why it previously went unnoticed, is that DNA concentrations higher than 1–2 g/ml decrease the yield of transformants, without showing signs of general toxicity.PEG-induced protoplasts (PIP) transformation for chromosomal markers operates normally with protoplasts prepared from a non-transformable bacterial mutant. The evidence indicates that both native linear and plasmid DNAs must somehow be forced into the cells as a result of PEG action. Denatured chromosomal DNA however is almost inactive in PIP transformation. No competition between chromosomal and plasmid DNAs could be detected, when the DNA tested as inhibitor was in tenfold excess.     

Novel plasmid marker rescue transformation system for molecular cloning in Bacillus subtilis enabling direct selection of recombinants

Summary A versatile plasmid marker rescue transformation system was developed for homology-facilitated cloning in Bacillus subtilis. It is based on the highly efficient host-vector system 6GM15-pHPS9, which allows the direct selection of recombinants by means of -galactosidase -complementation. The system offers several advantages over previously described cloning systems: (1) the convenient direct selection of recombinants; (2) the ability to effectively transform B. subtilis competent cells with plasmid monomers, which allows the forced cloning of DNA fragments with high efficiency; (3) the availability of 6 unique target sites, which can be used for direct clone selection, SphI, NdeI, NheI, BamHI, SmaI and EcoRI; and (4) the rapid segregational loss of the helper plasmid from the transformed cells.     

Stimulation of Bacillus subtilis transformation by spermidine

Summary Addition of spermidine in millimolar concentrations to Bacillus subtilis cells during competence development increases transformability. The spermidine must be added at least 30 min before DNA for maximum stimulation. An incubation period of about 30 minutes is also required for the maximum uptake of labeled spermidine. The amount of DNA initially attached and the rate of DNA uptake are increased to the same extent as transformation. The rate of protein synthesis is also equivalently increased. These observations are consistent with an increase in the number of competent cells in the cell population; this increase is mediated by a spermidine-stimulated protein synthesis.     

Plasmid transformation in Bacillus subtilis: Symmetry of gene conversion in transformation with a hybrid plasmid containing chromosomal DNA

Summary Allele transfer (conversion) was analyzed in transformations with a Cm^R determining hybrid plasmid, which contained a chromosomal gene controlling threonine prototrophy. In transformations, where a thr ⁺-cell was transformed with the thr ^- plasmid, the chromosomal allele was efficiently converted to the plasmid genotype. This process of gene conversion was rec dependent and greatly enhanced when monomeric rather than unfractionated plasmid DNA was used.     

Integration and excision of a plasmid in Bacillus subtilis

Summary We have studied the behaviour in Bacillus subtilis of a plasmid (pPV21) carrying the thymidylate synthetase gene of phage 3T (thyP3). The plasmid can transform efficiently the competent cells of all the strains tested. Polyethylene glycol (PEG)-mediated protoplast transformation is efficient only for recE, recD or recF mutants. When present in recombination proficient strains, the plasmid can be integrated into the chromosome, primarily at the thyA locus. This has been shown by genetic mapping and by blot-hybridization. A second less efficient site is at (or near to) the attachment site of phage 3T. Excision of the plasmid restores the EcoRI restriction pattern of the parental DNA, although with the loss of the defective thyA endogenotic allele and the retention of the thyP exogenotic gene.     

Molecular cloning with bifunctional plasmid vectors in Bacillus subtilis

Summary Cloning in Escherichia coli and Bacillus subtilis was carried out using the bifunctional plasmid pDH5060. B. subtilis chromosomal DNA and pDH5060 DNA were digested with either BamHI or SalI, then annealed, ligated, and transformed into E. coli SK2267. Transformants containing sequences ligated into the BamHI or SalI sites in the Tc^r gene of pDH5060 were selected directly using a modification of the fusaric acid technique. The BamHI and SalI clone banks contain about 250 and 140 B. subtilis fragments, respectively, with an average insert size of 8–9 Kbp in the BamHI and 4–5 Kbp in the SalI bank. The inserts ranged in size from 0.3 Kbp to greater than 20 Kbp. The vector used here therefore accepts inserts which are significantly larger than previously reported for other B. subtilis cloning systems. All individual cloned B. subtilis sequences examined were stably propagated in E. coli SK2267. Eight of eighteen B. subtilis auxotrophic markers tested (aroG, gltA, glyB, ilvA, metC, purA, pyrD, and thrA) were transformed to prototrophy with BamHI or SalI clone bank DNA. All or part of the hybrid plasmid DNA recombined at the sites of homology in the chromosome of these Rec⁺ recipients. Loss of sequences from hybrid plasmids was not prevented in a r ^- m ^- recE4 recipient strain of B. subtilis. Although the recE4 background prevented recombination between homologous chromosomal DNA, a variety of cloned fragments were shown to be unstable and undergo deletions of both insert and plasmid sequences. In addition, B. subtilis sequences propagated in E. coli transformed B. subtilis recE4 recipients with a 500-1,000-fold reduced efficiency.     

Isolation of a tetracycline-resistance plasmid excised from a chromosomal DNA sequence in Bacillus subtilis

When Bacillus subtilis GSY908 (recE4-) (H. C. Spatz and T. A. Trautner, 1971, Mol. Gen. Genet. 113, 174-190) protoplasts were infected with Staphylococcus aureus plasmid pNS1 specifying tetracycline resistance (Tcr) (N. Noguchi et al., 1983, Gene 21, 105-112), which was modified such that it either could not replicate or did not carry a functional Tcr gene, a plasmid with a molecular weight of 3.1 X 10(6) (4.9 kb) was generated in Tcr phenotypes. This plasmid, named Tcr pNS1981, exhibited completely different restriction endonuclease cleavage patterns to pNS1 and showed only negligible sequence homology in hybridization experiments. Southern hybridization experiments revealed that pNS1981 arises by excision of a B. subtilis chromosomal DNA sequence. No sequence corresponding to pNS1 was detectable on the chromosome of pNS1981-maintaining B. subtilis. The production of pNS1981 was also observed in B. subtilis RM125 (r-Mm-Mrec+) (T. Uozumi et al., 1977, Mol. Gen. Genet. 152, 65-69.) with almost the same frequency as B. subtilis GSY908. Since the recipient B. subtilis Marburg 168 derivatives stated above are sensitive to Tc, the results indicate that information essential for Tcr is under negative regulatory control in the integrated state on the chromosome. Restriction endonuclease analysis suggested that pNS1981 is essentially the same as pBC16, formerly found in B. cereus (K. Bernhard, H. Schrempf, and W. Goebel, 1978, J. Bacteriol. 133, 897-903).     

Fate of transforming DNA following uptake by competent Bacillus subtilis

Summary During transformation in Bacillus subtilis, donor and recipient DNA are initially associated by non-covalent bonds. The donor and recipient moieties later become covalently joined. The molecular weight of the donor component, when freed from the noncovalent complex by sucrose gradient sedimentation under alkaline conditions, ranges from 1 to 5×10⁶, with an average of about 2.5 to 3.0×10⁶. The latter values are in good agreement with previous measurements of the size of the integrated donor fragment.     

Study of the phenomenon of marker rescue in plasmid transformation and transduction of intact cells, protoplasts and different rec mutants of Bacillus subtilis

Summary Marker rescue in plasmid transformation of competent cells of different rec mutants of B. subtilis was studied. In most cases the value of marker rescue decreased proportionally to reduction of plasmid transformation efficiency (although there were certain exceptions). Marker rescue was not observed either in plasmid transformation of protoplasts or in plasmid transduction of intact cells.Abbreviations Km ^R Kanamycin-resistant - Cm ^R Chloramphenicol-resistant     

Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology

Summary To determine the minimal DNA sequence homology required for recombination in Bacillus subtilis, we developed a system capable of distinguishing between homologous and illegitimate recombination events during plasmid integration into the chromosome. In this system the recombination frequencies were measured between is pE194 derivatives carrying segments of the chromosomal -gluconase gene (bglS) of various lengths and the bacterial chromosome, using selection for erythromycin resistance at the non-permissive temperature. Homologous recombination events, resulting in disruption of the bglS gene, were easily detected by a colorimetric assay for -gluconase activity. A linear dependence of recombination frequency on homology length was observed over an interval of 77 bp. It was found that approximately 70 bp of homology is required for detectable homologous recombination. Homologous recombination was not detected when only 25 by of homology between plasmid and chromosome were provided. The data indicate that homology requirements for recombination in B. subtilis differ from those in Escherichia coli.     

Peptide syntheses mediated by Bacillus subtilis protease

Bacillus subtilis protease (Amano protease N) was examined as a catalyst for peptide bond formation via both the kinetically and thermodynamically controlled approaches. In general, the latter approach proved to be superior to the former, and a series of dipeptide syntheses and several segment condensations were achieved in good to high yields using the immobilized enzyme on Celite in acetonitrile with low water content.     

Structural plasmid instability in Bacillus subtilis: Effect of direct and inverted repeats

Summary Using precise excision as a model system, we have quantified the effect of direct repeats, inverted repeats and the size of the spacer between the repeats in the process of deletion formation in Bacillus subtilis. Both in the presence and absence of inverted repeats, the frequency of precise excision was strongly dependent on the direct repeat length. By increasing the direct repeat length from 9 bp to 18 and 27 bp, the precise excision frequency was raised by 3 and 4 orders of magnitude, respectively. In addition, irrespective of the direct repeat length, the presence of flanking inverted repeats enhanced the excision frequency by 3 orders of magnitude. Varying the inverted repeat length and the spacer size over a wide range did not significantly affect the excision frequencies. These results fit well into a model for deletion formation by slipped mispairing during replication of single-stranded plasmid DNA.     

Analysis of structural and biological parameters affecting plasmid deletion formation in Bacillus subtilis

Summary Deletions generated following stimulation by the deletion hot spot of plasmid pHV15-1 were studied in Bacillus subtilis. Nucleotide sequencing showed that deletions extend between short direct repeat sequences. Such direct repeat sequences may have homology to the sequence of the hot spot. Deletion formation is recE-independent, but requires an active exonuclease V (AddAB) enzyme. Other structural parameters like plasmid size and structure influence deletion formation.     

In vitro degradation of zearalenone by Bacillus subtilis

Zearalenone (ZEN) is a non-steroidal estrogen produced by many Fusarium species in cereals and other plants, and is frequently implicated in safety of foods and feeds. A ZEN-degrading microorganism has been isolated and identified as a Bacillus subtilis subspecies. It degraded 99% ZEN (1 mg kg⁻¹) in liquid medium after 24 h and more than 95% of ZEN (0.25 mg kg⁻¹) could be degraded after 48 h in a solid-state fermentation. This isolate can thus be used to decontaminate raw materials, like grains, to reduce the mycotoxin concentration.     

Sequence homologies of glucose-dehydrogenases of Bacillus megaterium and Bacillus subtilis

The sequence homologies of the glucose dehydrogenase subunits of B. megaterium and B. subtilis are compared. From the known B. megaterium aminoacid sequence and the base sequence of the cloned B. subtilis structural gene we predict the B. megaterium structural glucose dehydrogenase gene. Assuming the minimal mutational changes to convert one gene into the other 23 transitions, 30 transversions, 1 inversion, 3 insertion-deletions, but no frameshifts are postulated necessary to interconvert the structural genes. The homology of both enzyme subunits of 85% reflects the close evolutionary distance between B. subtilis and B. megaterium.     

DNA fixation and development of transformability and transfectability in Bacillus subtilis

Summary After pre-competent cells of Bacillus subtilis are placed in the medium in which competence develops, peak competence for transformation is found to occur earlier than competence for transfection by DNA from phages whose DNA is dissimilar to that of the cells. There is a nonlinear dependence of transfection on the concentration of DNA from phage SP 82 despite a linear dependence of DNA fixation on DNA concentration. Both results support the idea that fixation of DNA is a poor indicator of the actual competence of the cells for both transformation and transfection.