Behavior of autonomous plasmids and plasmids integrated into the chromosome during the sporulation of Bacillus subtilis cells

The behaviour of plasmids in free and integrated states was studied upon sporulation of Bacillus subtilis cells. Autonomous plasmids pBD12 and pGG10 were shown to be either transmitted into spores in small copy numbers or completely eliminated from the sporulating cell. However, insertion of the autonomous plasmid into the host chromosome may occur with a certain degree of probability (about 10(-3)) during sporulation. When in the integrated state, pBD12 plasmid may either excise from the host chromosome or amplify within the genome with the probability 1.8-2.10(-3) in the course of sporulation. The pGG102 plasmid carrying the fragment of wheat DNA and integrated by this fragment into the chromosome was shown to enter spores without whichever intragenome rearrangements.     

Construction and characterization of recombinant phage phi 105 d(Cmrmet) for cloning in Bacillus subtilis

A 1.6 kb fragment of DNA of plasmid pBD64, obtained after partial digestion with HpaII, carrying a chloramphenicol-resistance determinant and a single site for the enzyme Bg/II, was inserted into the genome of defective phage phi 105 d/ys. Two types of phage were subsequently isolated and both transduced cells of Bacillus subtilis to chloramphenicol resistance. One type contained 26 kb and the other 32 kb of DNA. Bacillus subtilis chromosomal DNA fragments generated by cleavage with Bg/II were ligated into the unique Bg/II site within the smaller phage genome. A specialized transducing phage was isolated which carried the metC gene on a 6 kb Bg/II fragment. This phage, denoted phi 105 d(Cmrmet), transduced B. subtilis strain MB79 pheA12 metC3 to Met+ and to chloramphenicol resistance, and the metC3 mutation was complemented in transductants.     

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.     

Amplification of a major membrane-bound DNA sequence of Bacillus subtilis.

A membrane-bound DNA sequence from Bacillus subtilis was subcloned into a plasmid which can replicate in Escherichia coli but not in B. subtilis. This plasmid hybridized with an 11-kilobase HindIII fragment which is the major particle-bound fragment in lysates treated with HindIII. The plasmid integrated into the B. subtilis chromosome at the region of homology, conferring chloramphenicol resistance on the recipient. The inserted resistance was mapped close to purA by using the generalized transducing phage AR9. In one chloramphenicol-resistant strain, the pMS31 region was repeated at least 20 times. A large proportion of the copies of the cloned region were present in the particle fraction, indicating that the capacity to bind this region of the chromosome was substantially in excess of the normal dose of the region. The structure of the particle-bound region was sensitive to ionic detergents and high salt concentrations but was not greatly affected by RNase or ethidium bromide. The basis of a specific DNA-membrane interaction can now be studied by using the amplified region, without the complications of sequences required for autonomous plasmid replication.     

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.     

Recombination between bacteriophage T4 and plasmid pBR322 molecules containing cloned T4 DNA

Reciprocal recombination between T4 DNA cloned in plasmid pBR322 and homologous sequences in bacteriophage T4 genomes leads to integration of complete plasmid molecules into phage genomes. Indirect evidence of this integration comes from two kinds of experiments. Packaging of pBR322 DNA into mature phage particles can be detected by a DNA--DNA hybridization assay only when a T4 restriction fragment is cloned in the plasmid. The density of the pBR322 DNA synthesized after phage infection is also consistent with integration of plasmid vector DNA into vegetative phage genomes. Direct evidence of plasmid integration into phage genomes in the region of DNA homology comes from genetic and biochemical analysis of cytosine-containing DNA isolated from mature phage particles. Agarose gel electrophoresis of restriction endonuclease-digested DNA, followed by Southern blot analysis with nick-translated probes, shows that entire plasmid molecules become integrated into phage genomes in the region of T4 DNA homology. In addition, this analysis shows that genomes containing multiple copies of complete plasmid molecules are also formed. Among phage particles containing at least one integrated copy, the average number of integrated plasmid molecules is almost ten. A cloning experiment done with restricted DNA confirms these conclusions and illustrates a method for walking along the T4 genome.     

"Marker augmentation" phenomenon in the plasmid transformation and transduction of Bacillus subtilis

Transformation of Bacillus subtilis cells carrying pUB110 plasmid by DNA of homologous plasmid pBD12 results in the significant increase in the number of plasmid transformants. This phenomenon named "augmentation" was not observed when instead of intact cells, regenerating protoplasts were used, or if pBD12 DNA was introduced into the cells via transduction.     

Nature of the genetic control of antibiotic resistance in a Pseudomonas aeruginosa BS205 strain

The clinical strain BS205 of P. aeruginosa is characterized by a high level of resistance to streptomycin, kanamycin, chloramphenicol, sulfanilamide and mercuric chloride. These markers can be transferred to P. aeruginosa PAO by means of transduction with phage F116L or mobilization with plasmid RP4. In the same way as in the initial strain of P. aeruginosa BS205 no plasmid DNA is detected in transducers or transconjugants. After transference to the strains of the transducers or transconjugants containing markers Sm, Km, Cm, Su, and Hg. plasmid Rip 64 of the incompatibility group is eliminated from the cells of these strains when they are grown on the nonselective medium. The genes of resistance to streptomycin, kanamycin, chloramphenicol, sulfanilamide and mercury and the gene(s) of incompatibility specific of the plasmid of the incompatibility group P-3 are included in the DNA fragment of the size of about 24 megadalton. This fragment is probably a defective plasmid not capable of autonomic existence which is integrated into the bacterial chromosome of P. aeruginosa BS205.     

Transfer of chimeric plasmids among Salmonella typhimurium strains by P22 transduction

Salmonella typhimurium bacteriophage P22 transduced plasmids having P22 sequences inserted in the vector pBR322 with high frequency. Analysis of the structure of the transducing particle DNA and the transduced plasmids indicates that this plasmid transduction involves two homologous recombination events. In the donor cell, a single recombination between the phage and the homologous sequences on the plasmid inserted the plasmid into the phage chromosome, which was then packaged by headfuls into P22 particles. The transducing particle DNA contained duplications of the region of homology flanking the integrated plasmid vector sequences and lacked some phage genes. When these defective phage genomes containing the inserted plasmid infected a recipient cell, recombination between the duplicated regions regenerated the plasmid. A useful consequence of this sequence of events was that genetic markers in the region of homology were readily transferred from phage to plasmid. Plasmid transduction required homology between the phage and the plasmid, but did not depend on the presence of any specific P22 sequence in the plasmid. When the infecting P22 carried a DNA sequence homologous to the ampicillin resistance region of pBR322, the vector plasmid having no P22 insert could be transduced. P22-mediated transduction is a useful way to transfer chimeric plasmids, since most S. typhimurium strains are poorly transformed by plasmid DNA.     

Construction and characterization of plasmid vectors for cloning in the entomocidal organism Bacillus sphaericus 1593

A plasmid vector for cloning in Bacillus sphaericus 1593 was constructed in B. subtilis from two parent plasmids, pBC16 and pBD64. When characterized, the 3.9-MDa chimeric plasmid pNN101 was found to consist of the MspI fragment containing the chloramphenicol acetyltransferase (CAT) gene from pBD64 inserted into an MspI site in pBC16. pNN101 was shown to replicate, express, and be stably maintained in B. sphaericus 1593 without affecting the mosquito larvicidal activity of this organism. A derivative of this plasmid, pNN302, was constructed in which a unique HindIII site was introduced into the CAT gene without loss of chloramphenicol resistance.     

The metC gene in Escherichia coli K-12: Isolation and studies of relatedness in Enterobacteriaceae

Abstract A specialized transducing phage λ carrying the structural gene for β-cystathionase ( metC ) of Escherichia coli was isolated. The phage carries a 21-kb fragment of the E. coli K-12 chromosome, and its structure was analyzed using restriction enzymes. The metC gene was recloned into resistance plasmid pBR322, using Eco RI or Hin dIII. The information for the metC gene is contained in a 1.3-kb fragment, which shows a high degree of homology with representative strains of all tribes of Enterobacteriaceae.     

Identification of the sporulation gene spoOA product of Bacillus subtilis

A 2.4-kilobase fragment of the Bacillus subtilis chromosome containing the wild-type spoOA gene derived from the phi 105dspoOA+-Bc-1 transducing phage was cloned onto plasmid pBR322 in Escherichia coli. A recombinant plasmid harboring the mutant spoOA12 allele on the 2.4-kilobase insert was also constructed from the phi 105dspoOA12-1 phage DNA and pBR322. Protein products synthesized in response to plasmid DNA in a DNA-directed cell-free system derived from E. coli were analyzed by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. A protein of approximately 27,500 daltons synthesized with the recombinant plasmid DNA harboring the wild-type spoOA gene as template was not formed with the recombinant plasmid DNA harboring the spoOA12 allele. Since the spoOA12 mutation is a nonsense mutation, we conclude that the 27.5-kilodalton protein is the product of the spoOA gene.     

High-Frequency Plasmid Transduction by Lactobacillus gasseri Bacteriophage phiadh

The temperate bacteriophage phiadh mediates plasmid DNA transduction in Lactobacillus gasseri ADH at frequencies in the range of 10 to 10 transductants per PFU. BglII-generated DNA fragments from phage phiadh were cloned into the BclI site of the transducible plasmid vector pGK12 (4.4 kb). Phage phiadh lysates induced from Lactobacillus lysogens harboring pGK12 or the recombinant plasmids were used to transduce strain ADH to chloramphenicol resistance. The transduction frequencies of recombinant plasmids were 10- to 10-fold higher than that of native pGK12. The increase in frequency generally correlated with the extent of DNA-DNA homology between plasmid and phage DNAs. The highest transduction frequency was obtained with plasmid pTRK170 (6.6 kb), a pGK12 derivative containing the 1.4- and 0.8-kb BglII DNA fragments of phiadh. DNA hybridization analysis of pTRK170-transducing phage particles revealed that pTRK170 had integrated into the phiadh genome, suggesting that recombination between homologous sequences present in phage and plasmid DNAs was responsible for the formation of high-frequency transducing phage particles. Plasmid DNA analysis of 13 transductants containing pTRK170 showed that each had acquired intact plasmids, indicating that in the process of transduction a further recombination step was involved in the resolution of plasmid DNA monomers from the recombinant pTRK170::phiadh molecule. In addition to strain ADH, pTRK170 could be transduced via phiadh to eight different L. gasseri strains, including the neotype strain, F. Gasser 63 AM (ATCC 33323).     

Cloning of the gene of Saccharomyces cerevisiae yeasts that determines resistance to the toxic action of cadmium ions

A gene conferring resistance to cadmium in Saccharomyces cerevisiae was isolated from a yeast gene library created on the basis of the pL3 vector. The phenotype of resistance is only expressed in the yeast cells with cloned DNA inserted into a multicopy plasmid. Integration of the plasmid into chromosome or introduction of the centromeric region into the plasmid decreases the level of cadmium resistance. The cloned Sau3A I fragment of the yeast chromosome is 3.5 kbp in size. Restriction analysis and subcloning experiments showed the gene to be located within 1.6 kbp of the XhoI-Sau3A I fragment of DNA. Instability was observed in the vicinity of the XhoI-Sau3A I fragment of the yeast DNA in Escherichia coli.     

High copy number plasmid vectors for use in lactic streptococci

Abstract A 3.8 kb DNA fragment from plasmid pBD64 which encoded chloramphenicol and kanamycin resistance genes, but had no replication region, was used as a replicator probe to select for the replication region of the cryptic lactic streptococcal plasmid pSH71 using Bacillus subtilis as host. Three of the resultant recombinant plasmids, pCK1, pCK17 and pCK21 are described. They are vectors in Streptococcus lactis and can be used to clone Bgl II-compatible fragments into their kanamycin resistance gene. All the plasmids have single sites for restriction endonucleases Ava I, Bam HI, Eco RI, Pvu II and Xba I, while plasmids pCK17 and pCK21 have single sites for Cla I.     

Plasmid marker rescue transformation in Bacillus subtilis

We constructed an 18-megadalton plasmid (pBD221) carrying resistance determinants for kanamycin, chloramphenicol, and erythromycin, as well as the hisH determinant from the Bacillus licheniformis chromosome. This plasmid has a copy number of about one and can be stably maintained in Bacillus subtilis. Linear fragments of pBD221 DNA were used to transform competent cultures carrying mutant variants of the same plasmid. Rescue transformation did not proceed by recircularization and replication of the donor DNA. Rescue transformation exhibited first-order dependence on DNA concentration, and the concentration dependence curve was virtually identical to the curve obtained with chromosomal DNA. The donor DNA molecular weight dependence of plasmid marker rescue transformation obtained by using restriction fragments was not distinguishable from previously published data obtained by using fractionated sheared chromosomal DNA. Plasmid rescue transformation, like chromosomal transformation, was dependent on the recE, recA, recB, and recD gene products. Plasmid rescue transformation, like chromosomal transformation, proceeded with few exchanges. Linkage data obtained with the plasmid rescue system fit a quantitative model based on studies with chromosomal transformation. We conclude that plasmid marker rescue transformation probably proceeds by a mechanism similar to the mechanism used during the formation of chromosomal transformants and hence may be considered an appropriate general model for the study of transformational recombination.     

Cloning of Pf3, a filamentous bacteriophage of Pseudomonas aeruginosa, into the pBD214 vector of Bacillus subtilis.

The genome of Pf3, a filamentous single-stranded DNA bacteriophage of Pseudomonas aeruginosa (a gram-negative organism) was cloned into pBD214, a plasmid cloning vector of Bacillus subtilis (a gram-positive organism). Cloning in the gram-positive organism was done to avoid anticipated lethal effects. The entire Pf3 genome was inserted in each orientation at a unique Bc/I site within a thymidylate synthetase gene (from B. subtilis phage beta 22) on the plasmid. Additional clones were made by inserting EcoRI fragments of Pf3 DNA into a unique EcoRI site within this gene.     

The replication origin of Azotobacter vinelandii

The putative replication origin of Azotobacter vinelandii was cloned as an autonomously replicating fragment after ligation to an antibiotic resistance cartridge. The resulting plasmids could be isolated and labelled by Southern hybridisation with the antibiotic resistance cartridge as probe and also visualised by electron microscopy. These plasmids integrated into the chromosome after a few generations, even in the recA mutant of A. vinelandii. The integrated copy of the plasmid was re-isolated from the chromosome and the DNA and its subfragments were cloned in the plasmid vector pBR322. A 200-bp DNA fragment was sufficient to allow the replication of pBR322 in an Escherichia coli polA strain. Electron microscopic analysis of this plasmid showed that replication initiated mostly within the A. vinelandii DNA fragment. The nucleotide sequence of the putative replication origin and its flanking regions was determined. In the sequence of the 200-bp fragment many of the distinctive features found in other replication origins are lacking. A greater variation from the consensus DnaA binding sequence was observed in A. vinelandii. Direct sequencing of the relevant genomic fragment was also carried after amplifying it from A. vinelandii chromosomal DNA by PCR. This confirmed that no rearrangements had taken place while the cloned fragment was resident in E. coli. It was shown by hybridisation that the 200-bp chromosomal origin fragment of A. vinelandii was present in three other field strains of Azotobacter spp.     

cosmid克隆筛选的体内同源重组法——一个含有小鼠t复合体连锁DNA顺序的cosmid克隆的分离

一个从cosmid分子克隆库中筛选特别基因顺序的遗传学方法——体内同源重组(invlvo homologous recombination)法。即使探针DNA与分子克隆库中带有与探针同源顺序的克隆发生体内重组,然后以遗传学方法进行筛选。cosmid分子克隆库构建在rec宿主细胞内,经体内包装(in vivo Packaging)成λ噬菌体颗粒,把该噬菌体颗粒转入带有探针DNA的rec~+细胞内,探针是已被克隆在与cosmid载体没有同源顺序的质粒(如PUC8或PUC9)内的。经过一段时间(1—3小时),待重组发生后,把cosmid进行体内包装。此时探针DNA连同质粒已整合入cosmid基因组内,因此它带有原为两个载体所分别带有的双重抗性——Amp~r(氨苄青霉素,PUC8或PUC9)和Kan~r(卡那霉素,cosmid)。这种双重抗性菌落可在含有这2种抗菌素的培养平皿上选出,该重组cosmid借助于λ切除酶的作用将已被整合的探针质粒重新切除,再经体内包装后,该cosmid被还原并纯化,然后可用一含有Xgal的培皿识别和选出。本文用此法以有关DNA探针从cosmid分子克隆库中分离得到含有与小鼠t复合体连锁的基因组顺序的克隆,并对该克隆作了物理图谱分析。