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.     

Functional analysis of hybrid plasmids carrying genes for lambda site-specific recombination

A number of hybrid plasmids, carrying lambda genes involved in site-specific integrative recombination, have been constructed in vitro. Analysis of protein synthesis in Escherichia coli minicells has shown that Int protein is synthesized only when int gene is expressed constitutively. The plasmids RSF2124::lambda-CD, RSF2124::lambda-Cint-c57, and pInt lambda were able to integrate into the chromosome of E.coli at the attB. The integration of hybrid plasmids into the genome of bacteria has also been shown for polA1 strains restricting the autonomous replication of ColE1 type plasmids. Genetic markers of hybrid plasmids are maintained in polA1 bacteria for at least 50 generations under nonselective conditions. The Southern blotting experiments using [32P]pBR322 DNA and EcoRI fragments of E. coli polA1 chromosome carrying integrated plasmid pInt lambda demonstrated that in this strain hybrid plasmids can be observed only when integrated into the attB of the chromosome according to Campbell's model of integration. In the cells, where autonomous replication of plasmids is possible, they can be observed both in extrachromosomal and integrated states. The integration of the ColE1 replication origin into the chromosome of bacteria is not lethal for the cells. Only attP and the int gene of lambda are necessary for the integration of hybrid plasmids under conditions of effective int gene expression. If the level of Int protein synthesis is high enough, the prophage excision can be observed in the absence of Xis product. The six-fold decrease of Int protein concentration in the cell (in case of pInt lambda 2 as compared to pInt lambda 1) is critical both for integration and excision.     

Isolation of an autonomously replicating DNA fragment from the region of defective bacteriophage PBSX of Bacillus subtilis

We have isolated a 5.4-kilobase fragment of Bacillus subtilis DNA that confers the ability to replicate upon a nonreplicative plasmid. The B. subtilis 168 EcoRI fragment was ligated into the chimeric plasmid pCs540, which contains a chloramphenicol resistance determinant from the Staphylococcus aureus plasmid pC194 and an HpaII fragment from the Escherichia coli plasmid, pSC101. A recE B. subtilis derivative, strain BD224, is capable of maintaining this DNA as an autonomously replicating plasmid. In rec+ recipients, chloramphenicol-resistant transformants do not contain free plasmid. The plasmid is integrated as demonstrated by alterations in the pattern of chromosomal restriction enzyme fragments to which the plasmid hybridizes. The site of plasmid integration was mapped by PBS1-mediated transduction to the metC-PBSX region. A strain was a deletion in the region of defective bacteriophage PBSX differs in the hybridization profile obtained by probing EcoRI digests with this cloned fragment. This same deletion mutant, though proficient in normal recombinational pathways, permits autonomous replication of the plasmid apparently owing to the lack of an homologous chromosomal region with which to recombine. We believe that, like E. coli. B. subtilis contains at least one DNA fragment capable of autonomous replication when liberated from its normally integrated chromosomal site and that this cloned DNA fragment comes from the region of defective bacteriophage PBSX.     

Cloning chloroplast DNA in escherichia coli. I. Construction and selection of recombinant plasmids containing fragments of pea chloroplast DNA]

Fragments produced by digestion of Pisum sativum chloroplast DNA with EcoRI were examined by agarose gel electrophoresis. These EcoRI-fragments were joined in vitro to Apr-ColE1 RSF2124 plasmid and cloned in Escherichia coli. Methods of molecular cloning of plasmid chimeras by success gradient centrifugation and repeated transformation and selection of recombinant plasmids using mytomicin C were used for cloning hybrid plasmids with various EcoRI fragments of pea chloroplast DNA has been obtained.     

Amplification of the riboflavin operon genes of Bacillus subtilis in Escherichia coli cells]

Amplification of Bacillus subtilis DNA fragments was performed in Escherichia coli using plasmid RSF2124. The main principle of isolation and cloning hybrid plasmids was described using genes of riboflavin operon as a model. Bac. subtilis DNA was treated with restriction endonuclease EcoR; followed by the agarose gel electrophoretic separation of the resulting fragments. Gels were sliced, DNA was eluted from the corresponding slices and used to transform Bac. subtilis auxotrophs rib A72, rib S110 and rib D107. DNA fraction with the molecular weight 7 . 10(6) daltons restored prototrophy of these mutants. DNA of this fraction was ligated with EcoRI treated plasmid RSF2124 DNA and used for transformation of E. coli rk-mk+. Ampicillin resistant transformants which had lost the colicin production ability, were selected. The presence of riboflavin genes within the hybrid plasmids was detected by transformation of B. subtilis auxotrophs. Three hybrid plasmids (pPR1, pPR2 and pPR3), containing a fragment of Bac. subtilis DNA with the molecular weight 6.8 . 10(-6) daltons including riboflavin operon, were selected. The analysis of the transformation activity of Bac. subtilis DNA and plasmid pPR1 DNA revealed, that there was no restriction activity of Bac. subtilis cells against plasmid DNA amplified in E. coli. Heteroduplex analysis has shown that plasmids pPR1 and pPR2 differ in the orientation of Bac. subtilis DNA fragment. DNA of these plasmids restored prototrophy of the several studied E. coli riboflavin auxotrophs.     

TnA-directed deletion of the trp operon from RSF2124-trp in Escherichia coli

Plasmid pMT-trp was constructed by digestion of RSF2124-trp with restriction endonuclease PstI and ligation with T4 ligase. In pMT-trp about 78% of the DNA of transposon TnA from RSF2124-trp was deleted, and hence the gene for ampicillin resistance was lost. All Trp- segregants from pMT-trp carriers in Escherichia coli W3110 and its derivatives were found to have lost the entire plasmid. On the other hand, deletion plasmids which had lost the trp operon were found among Trp- segregants from RSF2124-trp carriers, particularly from the mutant strain trpAE1 trpR tnaA. The experimental fact that deletion occurred exclusively in RSF2124-trp suggests that the presence of TnA in the plasmid (RSF2124-trp) was responsible for the deletion.     

Characterization of a mini-F plasmid derived from an F mutant expressing incompatibility in the autonomous but not in the integrated state

Plasmid DNA from Escherichia coli F' ser/MA219 harboring an altered F' factor, which expressed incompatibility in the autonomous but not in the integrated state (DeVries and Maas, 1973, J. Bacteriol. 115, 213-220), was digested with the restriction endonuclease EcoRI and ligated to a nonreplicating trpED fragment. A miniplasmid was obtained containing a 5.7-kb EcoRI fragment capable of self-replication. This plasmid, designated pRE300, was incompatible with mini-F as well as with ColE1 derivatives. It represents a cointegrate formed in vivo between a 2.2-kb segment of the F replication region and a ColE1-type replicon of unknown derivation. The F-derived component of pRE300 corresponds to a minimalized F replicon (43.85-46.05 kb F) retaining oriII and the incB locus but missing the incC and incD functions. It is postulated that the Inc- mutation resulted from the insertion of a transposable DNA sequence into the incC locus of the parent F plasmid.     

Molecular cloning of an Escherichia coli plasmid determinant than encodes for the production of heat-stable enterotoxin.

A conjugative plasmid, ESF0041 was isolated from an enterotoxigenic strain of Escherichia coli from calves. ESF0041 was found to be 65 x 10(6) daltons in mass of a member of the F incompatibility complex. Acquisition of ESF0041 by E. coli K-12 was invariably associated with the capacity to produce heat-stable (ST) enterotoxin. ESF0041 and pSC101 deoxyribonucleic acids were cleaved with EcoRI, and the fragments were ligated with polynucleotide ligase. Transformation of E. coli K-12 with the ligation mixture led to the isolation of an ST+ clone. Further analysis of the plasmid deoxyribonucleic acid from this clone showed that a structural gene(s) associated with ST biosynthesis had been isolated as a 5.7 x 10(6)-dalton ESF0041 fragment in pSC101. In turn, 5.7 x 10(6)-dalton fragment was ligated to a multicopy COLE1 derivative, RSF2124, so that toxin synthesis was amplified about threefold.     

Restriction map of a capsule plasmid of Bacillus anthracis

The capsule plasmid pTE702 of Bacillus anthracis has been physically mapped with the restriction endonucleases HindIII, PstI, BamHI, SalI, and XhoI. A HindIII fragment map of pTE702 (96.5 kb) was obtained by analysis of the recombinant plasmids and cosmids containing overlapping fragments partially digested with HindIII. The physical map for PstI, BamHI, SalI, and XhoI was obtained by double digestion mapping of these sites in relation to the HindIII sites. The replication region of pTE702 was determined by in vitro genetic replicon labeling in B. subtilis.     

Recombination between compatible plasmids containing homologous segments requires the Bacillus subtilis recE gene product.

Plasmid pSL103 was previously constructed by cloning a Trp fragment (approximately 2.3 X 10(6) daltons) from restriction endonuclease EcoRI-digested chromosome DNA of Bacillus pumilus using the neomycin-resistance plasmid pUB110 (approximately 2.8 X 10(6) daltons) as vector and B. subtilis as transformation recipient. In the present study the EcoRI Trp fragment from pSL103 was transferred in vitro to EcoRI fragments of the Bacillus plasmid pPL576 to determine the ability of the plasmid fragments to replicate in B. subtilis. Endonuclease EcoRI digestion of pPL576 (approximately 28 X 10(6) daltons) generated three fragments having molecular weights of about 13 X 13(6) (the A fragment), 9.5 X 10(6) (B fragment, and 6.5 X 10(6) (C fragment). Trp derivatives of pPL576 fragments capable of autonomous replication in B. subtilis contained the B fragment (e.g., pSL107) or both the B and C fragments (e.g., pSL108). Accordingly, the B fragment of pPL576 contains information essential for autonomous replication. pSL107 and pSL108 are compatible with pUB110. Constructed derivatives of the compatible plasmids pPL576 and pUB110, harboring genetically distinguishable EcoRI-generated Trp fragments cloned from the DNA of a B. pumilus strain, exhibited relatively high frequency recombination for a trpC marker when the plasmid pairs were present in a recombination-proficient strain of B. subtilis. No recombination was detected when the host carried the chromosome mutation recE4. Therefore, the recE4 mutation suppresses recombination between compatible plasmids that contain homologous segments.     

Cloning the genetic determinant of alpha-hemolysin and obtaining a series of insertional mutations in this determinant by the transposon Tn1000

Functionally active genetic determinant of alpha-hemolysin was cloned. Hemolytic plasmid pHly195 was used as a donor of the determinant and pBR322 plasmid served as recipient. Cloning was done with a help of HindIII restriction endonuclease. The recombinant plasmid obtained represents pBR322 plasmid with the built-in fragment of 7.4 kb containing genes of functionally active determinant of alpha-hemolysin. Restriction map was constructed using HindIII, EcoRI, BamHI and SalI restriction endonucleases. Insertional mutagenesis was carried out with the help of the Tn1000 transposon. Plasmid DNAs were isolated from insertional mutants of Hly- phenotype and treated with EcoRI, SalI and BamHI. On the basis of the sizes of restriction fragments of the mutant plasmid DNAs localization and orientation of insertions of Tn1000 into the cloned determinant of alpha-hemolysin were determined.     

Hypersensitivity to Hg2+ and hyperbinding activity associated with cloned fragments of the mercurial resistance operon of plasmid NR1.

The region of plasmid NR1 concerned with resistance to Hg2+ and organomercurials consists of sequences found on restriction endonuclease fragments EcoRI-H and EcoRI-I. When both fragments were cloned together into a derivative of plasmid ColE1, the hybrid plasmid conferred properties indistinguishable from those of the parental plasmid, NR1: resistance to Hg2+ and to the organomercurials merbromin and fluoresceinmercuric acetate and the inducible synthesis of the enzyme mercuric reductase. When fragment EcoRI-I was cloned into plasmid ColE1, cells containing the plasmid was as sensitive to Hg2+ and organomercurials as plasmidless strains. When fragment EcoRI-H was cloned into ColE1, cells with the hybrid plasmid were hypersensitive to Hg2+ and organomercurials. This hypersensitivity was inducible by prior exposure to low, subtoxic Hg2+ or merbromin levels. It was associated with an inducible hyperbinding activity attributed to a gene governing Hg2+ uptake and found on fragment EcoRI-H (which contains the proximal portion of a mercuric resistance [mer] operon).     

Plasmid replication functions: two distinct segments of plasmid R1, RepA and RepD, express incompatibility and are capable of autonomous replication.

The genetic determinants for replication and incompatibility of plasmid R1 were investigated by gene cloning methods, and three types of R1 miniplasmid derivatives were generated. The first, exemplified by plasmid pKT300, consisted of a single BglII endonuclease-generated deoxyribonucleic acid fragment derived from the R1 region that is located between the determinants for conjugal transfer and antibiotic resistance. Two types of miniplasmids could be formed from PstI endonuclease-generated fragments of pKT300. One of these, which is equivalent to miniplasmids previously generated from plasmids R1-19 and R1-19B2, consisted of two adjacent PstI fragments that encode the RepA replication system of plasmid R1. The other type contained a segment of R1, designated the RepD replication region, that is adjacent to the RepA region and that has not been identified previously as having the capacity for autonomous replication. Plasmid R1, therefore, contained two distinct deoxyribonucleic acid segments capable of autonomous replication. The RepA-RepD miniplasmid pKT300 had a copy number about eightfold higher than that of R1 and hence lacked a determinant for the regulation of plasmid copy number. Like R1, it was maintained stably in dividing bacteria. RepA miniplasmids had copy numbers which were two- to fourfold higher than that of R1 (i.e., which were lower than that of pKT300) and were maintained slightly less stably than those of pKT300 and R1. The RepD miniplasmid was not maintained stably in dividing bacteria. Previous experiments have shown that incompatibility of IncFII group plasmids is specified by a plasmid copy control gene. Despite the fact that RepA miniplasmids of R1 were defective in copy control, they nevertheless expressed incompatibility. This suggests that two genes are responsible for plasmid copy control, one that specifies incompatibility and is located on RepA miniplasmids and another that is located outside of, but adjacent to, the RepA replication region. Hybrid plasmids composed of pBR322 and one PstI fragment from the RepA region, P-8, exhibited incompatibility towards R2 and RepA miniplasmids but not the RepD miniplasmid, whereas hybrids composed of pBR322 and the PstI fragment of the RepD region, P-3, exhibited incompatibility towards R1 and the RepD miniplasmid but not RepA miniplasmids. These results indicate that the two replication systems are functionally distinct and that, although the RepA system is the principal replication system of R1, the RepD system also plays a role in the maintenance of this plasmid.     

Restriction map of the 125-kilobase plasmid of Bacillus thuringiensis subsp. israelensis carrying the genes that encode delta-endotoxins active against mosquito larvae.

A large plasmid containing all delta-endotoxin genes was isolated from Bacillus thuringiensis subsp. israelensis; restricted by BamHI, EcoRI, HindIII, KpnI, PstI, SacI, and SalI; and cloned as appropriate libraries in Escherichia coli. The libraries were screened for inserts containing recognition sites for BamHI, SacI, and SalI. Each was labeled with 32P and hybridized to Southern blots of gels with fragments generated by cleaving the plasmid with several restriction endonucleases, to align at least two fragments of the relevant enzymes. All nine BamHI fragments and all eight SacI fragments were mapped in two overlapping linkage groups (with total sizes of about 76 and 56 kb, respectively). The homology observed between some fragments is apparently a consequence of the presence of transposons and repeated insertion sequences. Four delta-endotoxin genes (cryIVB-D and cytA) and two genes for regulatory polypeptides (of 19 and 20 kDa) were localized on a 21-kb stretch of the plasmid; without cytA, they are placed on a single BamHI fragment. This convergence enables subcloning of delta-endotoxin genes (excluding cryIVA, localized on the other linkage group) as an intact natural fragment.     

Determination of the functions of hemolytic plasmid pHly152 of Escherichia coli

The alpha-hemolytic Escherichia coli strain PM152 harbors three transmissible plasmids, which have molecular weights of 65 X 10(6) (pA152), 41 X 10(6) pHly152), and 32 X 10(6) (pC152). Plasmids pHly152 and pC152 belong to incompatibility groups J2 and N, respectively. By transforming E. coli K-12 with isolated plasmids, we showed that the genetic determinant required for hemolysis was located entirely on plasmid pHly152, and a physical map of this plasmid was constructed. By transposon mutagenesis, a deoxyribonucleic acid segment of about 3.5 X 10(6) daltons was identified as being essential for hemolysis. Most of the EcoRI and HindIII fragments of the hemolytic plasmid pHly152 were cloned by using pACYC184 and RSF2124 as vectors. Two classes of Tn3-induced hemolysis-negative mutants could be complemented by recombinant plasmids carrying fragments from the hemolysis region of pHly152, whereas a third class could be restored to hemolytic activity only by recombination between the mutant plasmids and a suitable recombinant deoxyribonucleic acid. These data suggest that there are at least three clustered cistrons which are required for hemolysis. Other EcoRI and HindIII fragments of pHly152 were identified as being essential for replication, incompatibility, transfer, and restriction.     

Cloning and endonuclease restriction analysis of argF and of the control region of the argECBH bipolar operon in Escherichia coli.

A 1.8 kb DNA fragment, liberated by endonuclease HindIII, contains the control region of the argECBH bipolar operon near one end and the weak secondary promoter of argH at the other extremity; it has been cloned in plasmid pBR322. The same plasmid vector has been used to clone the argF gene liberated from the chromosome by endonuclease BamHI. Restriction patterns for the two hybrid plasmids have been determined, using enzymes AluI, BglI, EcoRI, HaeIII, HincII, HindIII, HpaI and II, PstI and SalI. Two AluI sites situated on either side of and close to a HincII target delineate two short fragments covering the whole of the argECBH control region. The argF control elements are located in a region accessible to further dissection by BamHI, EcoRI, PstI and HindIII. Carriers of the argF plasmid produce extremely high amounts of ornithine carbamoyltransferase, a feature useful for purification of this enzyme.     

A specialized host-vector system for the in Vivo cloning of the trp operon of wild-type and mutant strains of Salmonella typhimurium by generalized transduction

Using in vitro methods, a 14.2-kb EcoRI fragment of the Salmonella typhimurium chromosome containing the trp operon plus associated flanking sequences from deletion mutant delta trpDCB763 was cloned into the EcoRI site of plasmid pBR322 in a S. typhimurium host. An in vivo cloning vector was constructed from the recombinant plasmid by the in vitro excision of a SalI fragment that contains the entire trp operon. The derived plasmid (pSTP21) carries a hybrid insert made up of the 5.4-kb EcoRI-SalI upstream flanking sequence and the 3.2-kb SalI-EcoRI downstream flanking sequence. Plasmid pSTP21 has been used as a receptor plasmid to clone a variety of mutant and wild-type trp operons by RecA-dependent in vivo recombination between the insert DNA of the plasmid and the homologous trp flanking sequences of transducing DNA fragments transferred into the cell by bacteriophage P22. The host-vector system developed for the in vivo cloning permits the differentiation of plasmid transductants from chromosomal transductants on the primary selective medium. Expression of the cloned trp operons is regulated normally by tryptophan. A substantial amplification of trp enzymes is attainable upon derepression. The recombinant plasmids are stably inherited in RecA+ and RecA- S. typhimurium hosts. However, conditions of high expression of the trp operon lead to a rapid loss of cellular viability and of plasmid stability.