Excision process of integrated plasmid pBD12 from the Bacillus subtilis chromosome

We studied the behavior of pBD12 plasmid integrated into Bacillus subtilis chromosome via homologous recombination. One copy of the plasmid was integrated into the chromosome, it conferred resistance to low concentrations of antibiotics. Clones with enhanced resistance bearing autonomous plasmid DNAs appeared with a frequency 10(-6) in rec+ but not in recE strain with the integrated plasmid. By restriction and hybridization analysis of some excised plasmids, the sites of excision were determined, chromosomal location of pBD12 plasmid was found to be at the terminal fragment of prophage DNA, so that the att site of phi 105 phage is supposed to be situated on the EcoRI fragment of phage DNA.     

Integration of various plasmids into the Bacillus subtilis chromosome

Some features of integration of temperature-sensitive pE194, pGG10 and pGG20 plasmids into the Bacillus subtilis chromosome were studied. Several auxotrophic mutations were obtained using insertion of these plasmids into the chromosome. The sites of plasmids for illegitimate recombination were determined. It was shown that the integration into the Bac. subtilis chromosome is characteristic not only for the plasmid pE194 but is the property of Staphylococcus aureus plasmid pC194 and Escherichia coli pBR322 plasmid. The influence of different Bac. subtilis rec mutations on the frequency of integration was studied.     

Cloning of the purA16 locus in Rec+ cells of Bacillus subtilis

A portion of purA16 chromosomal locus of Bacillus subtilis was cloned into Rec+ cells of this microorganism with pBD12 plasmid (carrying chloramphenicol and kanamycin resistance determinants) serving as a vector. The hybrid plasmids were stably maintained in cells grown on media supplemented with antibiotics and were lost from cells in the absence of drugs. The cloned fragment could incorporate into the chromosome some with a frequency of 10(-2) per cell per generation. A clone carrying the hybrid plasmid inserted into the chromosome was detected.     

Construction of plasmids integrating into the Bacillus subtilis chromosome through homologous recombination and their use as integration vectors

We constructed a number of plasmids which integrate into the chromosome of Bacillus subtilis through homology recombination. Plasmids consist of pBR322 replicon, different fragments of Bac. subtilis chromosomal DNA, Cm resistance marker from pBD64 plasmid. Frequency of transformation was 10(-4) per bacterial cell. Foreign DNA (genes for tryptophan metabolism of Bac. mesentericus) was introduced into the chromosome of Bac. subtilis with the help of these plasmids.     

Interplasmid illegitimate recombination in Bacillus subtilis cells

The illegitimate recombination between S. aureus plasmids pE194 (or pGG20-the hybrid between pE194 and E. coli plasmid pBR322) and pBD17 (plasmid pUB110 without Hpa-II-C-fragment) in B. subtilis was studied. Plasmid cointegrates were generated with the frequency of 1-3.10(-8). Among the 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all the parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions has revealed that in 8 cases recombination occurred between short homologous regions (9-15 b.p.). One of the recombinants resulted from nonhomologous recombination. The similarity between nucleotide sequences of recombination sites of two types of contegrates and those used for pE194 integration into the B. subtilis chromosome (Bashkirov et al. 1987) was demonstrated. Possible mechanisms of illegitimate recombination are discussed.     

Direct selection of recombinant plasmids in Bacillus subtilis

A system is described which permits the direct, positive selection of recombinant plasmids in Bacillus subtilis. This system relies on the plasmid pBD214 which confers chloramphenicol (Cm) resistance and carries a thy gene, and on BD393, a highly competent B. subtilis thy A thy B host. Thy⁻ strains are resistant to trimethoprim (Tmp), and Thy⁺ strains are sensitive. Inactivation of the pBD214 thy determinant by insertion of a DNA fragment permits selection of Cm^r Tmp^r clones, all of which carry recombinant plasmids. This insertional inactivation can be accomplished using the unique EcoRl, Bell, Pvull, or EcoRV sites, all of which are located within the thy gene on pBD214. Some properties of this selective system are described, and its uses for molecular cloning are discussed     

Interplasmidic illegitimate recombination in Bacillus subtilis

Summary The illegitimate recombination between Staphylococcus aureus plasmids pE194 (or pGG20, the hybrid between pE194 and Escherichia coli plasmid pBR322) and pBD17 (plasmid pUB110 without HpaII C-fragment) was studied in Bacillus subtilis. Cointegrates were generated with the frequency of 1–3x10^-8. Among 22 hybrids analysed 9 types of recombinants were found. Nucleotide sequences of all three parental plasmids were involved in intermolecular recombination. Nucleotide sequencing of recombinant DNA junctions revealed that in 8 cases recombination occurred between short homologous regions (9–15 bp). One recombinant was formed using nonhomologous sites. The similarity was demonstrated between nucleotide sequences of the recombination sites of two types of cointegrates and those used for pE194 integration into the B. subtilis chromosome. Possible mechanisms of illegitimate recombination are discussed.     

Cointegrate formation between homologous plasmids in Escherichia coli.

Conjugation experiments were performed in which the donor was Escherichia coli K-12 strain KP245 containing either R plasmid NR1 plus an ampicillin-resistant derivative of ColE1 (*ColE1::Tn3, called RSF2124) or NR1 plus RSF2124 carrying a cloned EcoRI fragment of NR1. The recipient was the polA amber mutant JG112, in which RSF2124 cannot replicate. Ampicillin-resistant transconjugants can arise only when the genes for ampicillin resistance are linked to NR1 or are transposed to the host chromosome. When EcoRI fragment A of NR1 (20.5 kilobases) was cloned to RSF2124, the frequency of cotransfer of ampicillin resistance with tetracycline resistance was 25 to 60%. Plasmid DNA from these ampicillin-resistant transconjugant cells was analyzed by gel electrophoresis and was shown to be a cointegrate of NR1 and the RSF2124 derivative. Analysis of plasmid DNA isolated from donor cultures showed that the cointegrates were present before conjugation, which indicates that the mating does not stimulate cointegrate formation. When the cloned fragment was EcoRI fragment H of NR1 (4.8 kilobases), the frequency of cotransfer of ampicillin resistance with tetracycline resistance was about 4%, and the majority of the ampicillin-resistant transconjugants were found to contain cointegrate plasmids. When the donor contained NR1 and RSF2124, the frequency of cotransfer of ampicillin resistance was less than 0.1%, and analysis of plasmid DNA from the ampicillin-resistant transconjugants showed that Tn3 had been transposed onto NR1. These data suggest that plasmids which share homology may exist in cointegrate form to a high degree within a host cell.     

Potency factors in the delta-endotoxin of Bacillus thuringiensis var. aizawi and the significance of plasmids in their control

Mutants defective in delta-endotoxin crystal production from four closely related isolates of Bacillus thuringiensis var. aizawi with aizawi serotype crystals were as vigorous as the parents in terms of growth, extracellular protease production, sporulation and heat resistance of spores. Spores produced by mutants germinated faster than wild type spores possibly due to deficiency of protein, in the form of delta-endotoxin in the spore coat. Acrystalliferous (cry—) mutants were not active in Galleria mellonella or Pieris brassicae larvae. Mutants with small crystals (sm cry) lost activity or gained extra activity against either one or the other host, revealing the presence of different toxicity factors. Solubilized crystals of parent isolates were composed of two major polypeptides with Mr values of 130 000 and 138 000. Sm cry mutants lost either polypeptide irrespective of which insect potency had been lost. Some cry — and some sm cry mutants had the same plasmid pattern as the parent; others lost one plasmid sometimes gaining another of different size. No consistent correlation was found between plasmid loss in mutants and any loss or increase of potency indicated by bioassays. It is concluded that the delta-endotoxins of the isolates under investigation are composed of at least two toxins. The results suggest that genes coding for the production of toxic factors or for their expression may be carried on both the plasmids and the chromosome.     

Replication defective RP4 plasmids recovered after chromosomal integration

pHH6000 is a composite replicon made by the in vitro ligation of the IncP plasmid RP4 to a fragment of bacteriophage lambda capable of autonomous replication. Derivatives were selected in which it had integrated into the Escherichia coli chromosome by homologous recombination with the resident lambda prophage, and plasmids were subsequently regenerated from the integrated molecules. Although of the same molecular size as pHH6000, all had altered properties: those recovered from the chromosome of cells simultaneously carrying a distinguishable autonomous IncP plasmid showed a 100- to 1000-fold reduction in their ability to become established in a lambda lysogen; those regenerated from cells with no autonomous IncP plasmid were no longer RP4 replicons, now being dependent on replication functions encoded by the lambda DNA they carry and therefore unable to form a plasmid in a lambda lysogen. This second class of plasmids still exhibited normal RP4 incompatibility and stability even though neither property is encoded by the lambda replicator DNA. It was concluded that expression of RP4 incompatibility and partitioning control do not require an intact RP4 replicon. The data also suggest that the presence in the chromosome of a normal RP4 molecule may be deleterious to the host, although the manner in which the integrated molecules were obtained allows other explanations. The composite plasmids replicating from cloned lambda genes should be useful in analysis of the regulated distribution of RP4 molecules at cell division.     

Amplification of plasmid DNA inserted into the Bacillus subtilis chromosome

Amplification of plasmid pGG10 inserted into the Bacillus subtilis chromosome is described. The possibility of the 3.2 kb fragment of eucaryotic (wheat) DNA to be amplified within the bacterial genome is shown. The models explaining this phenomenon are discussed.     

Transfer of chromosomal genes and formation of R'-derivatives using PR4::D312cts15 plasmids in Pseudomonas aeruginosa cells

Several hybrid RP4 plasmids containing the genome of heat-inducible D3112cts15 phage integrated into 2 different sites of RP4 were selected. It was shown that the plasmids RP4::D3112cts15 mobilized the chromosome of Pseudomonas aeruginosa from many sites located in different chromosome regions. Chromosomal recombinants are, formed at frequencies of about 10(-4) per recipient cell. Analysis of coinheritance of unselected markers showed that the majority of recombinants inherited short donor chromosome fragments (about 5 min). R' plasmids can be easily selected by mating with a rec- recipient. For instance, the frequency of selection of R' plasmids containing argH+ locus was about 10(-5) per donor cell. Conjugative transfer of RP4::D3112cts15 into nonlysogenic strains PAO P. aeruginosa results in partial or complete loss of prophage from a hybrid plasmid. The RP4::D3112cts15 plasmids appear to have retained the broad host range of the original RP4 (they are maintained in P. putida and Escherichia coli).     

Cloning of sporulation gene spoIIG in Bacillus subtilis.

Two specialized transducing phages carrying a sporulation gene, spoIIG , of Bacillus subtilis were constructed from B. subtilis temperate phages p11 and phi 105 by the "prophage transformation" method. Restriction enzyme analysis and transformation experiments showed that the spoIIG gene was present on a 6.2 X 10(6)-dalton (6.2-Md) EcoRI fragment in both transducing phage genomes. Further analysis showed that spoIIG + transforming activity resides on a 2.25-Md EcoRI-BamHI fragment within the 6.2-Md EcoRI fragment. The 2.25-Md fragment was subcloned into the region between the EcoRI and BamHI sites of pUB110, and deletion plasmids lacking PstI or HindIII fragments within the 2.25-Md fragment were constructed. The recombinant plasmid carrying the intact spoIIG gene restored sporulation of strain HU1002 ( spoIIG41 recE4 ) to a frequency of 10(4) spores per ml and inhibited sporulation of strain 4309 ( spo + recE4 ) to a level of 10(3) spores per ml.     

Insertion of plasmids into the chromosome of Streptomyces griseofuscus

The results demonstrate a general method for randomly integrating plasmids into the genome by a single crossover between a cloned DNA fragment and homologous DNA in the chromosome. The integrated plasmid is flanked by directly repeated copies of the cloned homologous DNA sequence. Two protocols, "replica plating" and "liquid transfer," yielded strains with integrated plasmids.     

Properties ofthyP3-containing plasmids inBacillus subtilis

A series of hybrid plasmid molecules which contain both antibiotic resistance genes and the thyP3 gene of the Bacillus subtilis bacteriophage phi 3T have been constructed. Monomeric or restriction enzyme-cleaved plasmid DNA is capable of transforming competent cells to thymine prototrophy only. However, multimeric plasmid DNA can transform competent cells to both thymine prototrophy and antibiotic resistance. Cells which have been transformed to thymine prototrophy only do not contain extrachromosomal plasmid DNA but instead contain the thyP3 gene integrated into the host chromosome; the antibiotic resistance genes, however, do not become integrated into the chromosome. Although the thyP3-containing plasmids have extensive DNA sequence homology with the B. subtilis chromosome, they can be stably maintained, extrachromosomally, even in recE4+ hosts, in complex broth, and in the absence of antibiotics.     

Incorporation of the ampicillin resistance transposon into the Escherichia coli K-12 chromosome and into plasmids]

The mutant pEG1 of R-factor RP4 with temperature-sensitive defect in replication, carrying a transposable ampicillin resistance element Tn1 was used to define the frequency of insertion of this element into Escherichia coli K-12 chromosome and some other plasmids. Our results indicate that the frequency of colony forming by bacteria with pEG1-factor on ampicillin medium in non-permissive conditions corresponds to the frequency of Tn1 insertion into bacterial chromosome or some other plasmid (in case when the strains are carrying a second plasmid). The frequency of Tn1 insertion into the chromosome is about 4.10(-4). The defect in recA gene produce no serious change in the frequency of Tn1 insertion into the bacterial chromosome. The translocation of Tn1 element from pEG1-factor to R483, R6 and ColE1 plasmids occurs at 10 to 100-fold-higher frequency than from the plasmid to the chromosome. The insertion of Tn1 into the F'-factor KLF10 and R-factor R64-11 occurs at far lower frequency than that to plasmids R6, R483, or ColE1.     

Construction of recombinant plasmid carrying the lambda DNA fragment responsible for prophage integration.

The recombinant DNA molecules were constructed from plasmid RSF2124 and the EcoRI fragment of lambda DNA containing the genes responsible for prophage integration. The presence of these genes in recombinant plasmids was detected genetically. lambda int-gene was shown to be expressed in either orientation of insertion in the plasmid. We found that recombinant plasmid was able to integrate into chromosome of lambda lysogens. The integration of plasmid into host chromosome was demonstrated by contransduction of chromosome and plasmid markers using generalized transducer P1 and by specialized transduction with lambda phages.     

Effects of plasmids on chromosome metabolism in bacteria

In addition to many other functions of the cell, many bacterial plasmids are involved in repair, mutagenesis, replication, and recombination of the host chromosome. Numerous studies performed with wild-type strains and various mutants suggest that plasmids participate in these processes through three basic routes: (i) contribution to cell's regulatory systems; (ii) introduction of new pathways operating either independently of the existing ones or affecting the efficiency of the latter; these new pathways may or may not be subject to cellular regulation; (iii) replacement of defective proteins by functionally similar plasmid products or compensation for missing proteins by either activating existing pathways or introducing plasmid-born bypass pathways. The differences among individual plasmids in their effects on DNA metabolism are governed by intimate mechanisms of the metabolic process, the genetic background of the host, and the genetic constitution of the plasmid. The corresponding plasmid genetic determinants and the products thereof remain, for the most part, unidentified. However, the available evidence indicates that plasmids can confer on the cell additional resources which extend its DNA metabolism potential, thereby promoting evolutionary transformations.     

Characterization of eight excision plasmids of Pseudomonas syringae pv. phaseolicola

Summary Pseudomonas syringae pv. phaseolicola strain LR719 contains a 150 kilobase pair (kb) plasmid pMC7105, stably integrated into its chromosome. Occasionally, single colony isolates of this strain contain an excision plasmid. Eight unique excision plasmids were selected and characterized by BamHI restriction endonuclease and blot hybridization analyses. These plasmids ranged in size from 35 to 270 kb; the largest contained approximately 130 kb of chromosomal DNA sequences. Restriction maps of pMC7105 were developed to deduce the site of integration and to identify the fragments in which recombination occurred to produce each excision plasmid. The eight excision plasmids were arranged into five classes based on the sites where excision occurs. A 20 kb region of pMC7105, which includes BamHI fragment 9 and portions of adjacent fragments, is present in all excision plasmids and thought to contain the origin of replication. The site of integration on pMC7105 maps within BamHI fragment 8. This fragment shows homology with seven other BamHI fragments of pMC7105 and with five chromosomal fragments identified among the excision plasmids. The data strongly suggest that the integration of pMC7105 may have occurred at a repetitive sequence present on the chromosome and on the plasmid.     

Transformation of Bacillus licheniformis by plasmid DNA

A system for obtaining regenerating protoplasts of highly active Bacillus licheniformis 1001 strain was developed. Transformation of protoplasts by pUB110 and pminiKC plasmids (constructed from plasmids pUB110 and pC194) leading to the expression of kanamycine resistance, was demonstrated. It is supposed that in Bac licheniformis, the pminiKC plasmid is integrated into cellular chromosome, in contrast to pUB110 and parental Bac subtilis (pminiKC) strain. Still, the integrated plasmid seems to be not completely under control of the host chromosome. As a result of such integration, the plasmid conversion takes place, resulting in alteration of cytokinesis (filament formation) and sporulation, but not interfering with the ability to produce antibiotic bacitracin.