New replication mutant pnh602 and its relationship to plasmid pAS3, another deletion derivative of plasmid R6K

A stable deletion derivative pNH602 was obtained when the recently described higher-copy-number point mutant pNH601 of plasmid R6K was introduced to a minicells-producing strain of Escherichia coli. The size of plasmid pNH602 is 18.8 Mg/mol as determined by electron microscopy. The 7.2 Mg/mol fragment of R6K genome missing in pNH602 carries the Smr-determinant and the region finO and, according to the results of restriction analysis, it includes one EcoRI site. With its radioisotopically determined 33 copies of pNH602 per E. coli K-12 chromosome (npc), representing a 23% increase of the point mutant pNH601 and 150% enhancement of R6K npc, plasmid pNH602 differs from another closely related R6K deletion derivative pAS3 of the same size which exhibits only 20 npc. Both pNH602 and pAS3 plasmids are conjugative.     

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.     

Plasmids in Nitrobacter

A screening of nine Nitrobacter strains showed that Nitrobacter X14 and Nitrobacter Y possess plasmids designated as pNH1 and pNH2, respectively. The plasmids pNH1 and pNH2 had molecular weights of about 76 Mdal as estimated by agarose gel electrophoresis. They showed similar cleavage patterns when digested with EcoRI, HindIII or BamHI. Electron microscopic investigations exhibited that the plasmid pNH1 had a contour length of 36.9 m corresponding to a molecular weight of 76 Mdal.     

Cloned structural gene (ompA) for an integral outer membrane protein of Escherichia coli K-12

Summary pTU 100 is a hybrid plasmid constructed by cloning a 7.5 Kb EcoRI fragment (carrying the wildtype ompA gene) onto pSC 101 (Henning et al., 1979). This plasmid confers sensitivity to phages Tull^* and K3h1 when present in an ompA host strain, due to the expression of the phage receptor protein II^* from the plasmid ompA ⁺ gene. Plasmid mutants have been isolated that have become resistant to one or both of these phages. Restriction endonuclease analysis and DNA-sequencing studies in these plasmids demonstrate that a BamHI site and two PvuII sites are located within the ompA gene. BamHI cuts the gene at a site corresponding to residue 227 within a total of 325 amino acid residues.Neither the wildtype ompA gene nor the BamHI fragment encoding the NH₂-terminal part of the protein (residues 1–227) could be transferred to a high copy number plasmid, presumably due to lethal overproduction of the protein or its NH₂-terminal fragment. However, the NH₂-terminal fragment derived from one of the ompA mutants of pTU100 could be transferred to the high copy number plasmid pBR322, and was expressed in the presence of the amber suppressors supD or supF. Under these conditions two new envelope proteins with apparent molecular weights of 30,000 and 24,000 were synthesized, and the cells became sensitive to phage TuII^*, indicating the presence of phage receptor activity in the outer membrane. The major, 24,000 dalton protein has the molecular weight expected of a protein comprising residues 1–227 of protein II^*. DNA-sequencing studies demonstrated that no termination codons are present in the DNA region immediately downstream from the BamHI site at residue 227 in this hybrid plasmid, and it is therefore likely that the 24,000-dalton protein arises from the posttranslational proteolytic cleavage of a larger polypeptide. The 30,000-dalton protein is a likely candidate for such a larger polypeptide. These results also demonstrate that the 98 CO₂H-terminal residues of wildtype protein II^* (resisdues 228–325) are not required either for the activity of the protein as a phage receptor or for its incorporation into the outer membrane.     

Physical characterization of the plasmid pON5300

Summary The antibiotic resistance plasmid Rldrd19Km-which has spontaneously lost its kanamycin resistance marker, and its derivative pON5300, were analysed using the restriction endonucleases SalI, BamHI, HindIII and EcoRI. The fragment patterns were compared with that of the Rldrd19 and the fragments responsible for kanamycin resistance were found to be missing in Rldrd19Km ^– and pON5300. In these plasmids a 7 Mg/mol EcoRI fragment was observed instead of the D (6.3 Mg/mol) fragment of Rldrd19. Further a new 6 Mg/mol EcoRI restriction fragment was observed in pON5300. Using double digestions it was shown that the new fragment does not carry restriction sites for HindIII, BamHI and SalI endonucleases. The non-homology of the analysed plasmid was proved electron microscopically by heteroduplex techniques. The possibility of amplification in the regulatory region for the expression of R-determinants in pON5300 is discussed.     

Restriction endonuclease mapping and mutagenesis of the F sex factor replication region

Summary The plasmids pSC138 and pML31 each contain the EcoRI-generated f5 replicator fragment of the conjugative plasmid F in addition to an EcoRI fragment encoding antibiotic resistance: ampicillin resistance derived from Staphylococcus aureus in pSC138 and kanamycin resistance from Escherichia coli in pML31. We have mapped one HindIII and two BamHI restriction sites in the f5 region of these plasmids and one HindIII site in the antibiotic resistance region of each plasmid. The HindIII site in the Km region of pML31 occurs in the kan gene whereas the HindIII site in the Ap region of pSC138 appears to occur in an area important for the regulation of -lactamase production.By means of in vitro recombinant DNA manipulation of plasmids pML31 and pSC138, we have shown that 1.9x10⁶ daltons of the 6.0x10⁶ dalton f5 fragment can be deleted without disrupting plasmid stability. In addition, we have used these same techniques to isolate a novel F-controlled Ap plasmid cloning vehicle which contains a single restriction site for each of the enzymes EcoRI, HindIII, and BamHI. This cloning vehicle has been linked via either its EcoRI or HindIII site to a ColE1 plasmid replicon to yield stable recombinants.     

DNA sequence of a plasmid-encoded dihydrofolate reductase

Summary The sequence of the methotrexate-resistant dihydrofolate reductase (DHFR) gene borne by the plasmid R-388 was determined. The gene was subcloned and mapped by an in vitro mutagenesis method involving insertion of synthetic oligonucleotide decamers encoding the BamHI recognition site. Sites of insertion that destroyed the methotrexate resistance fell in two regions separated by 300 bp within a 1.2 kb fragment. One of these regions encodes a 78 amino acid polypeptide homologous to another drug-resistant DHFR. The second region essential for DHFR expression appears to be the promoter of the DHFR gene.     

Physical characterization of plasmids from Streptomyces kasugaensis MB273

Plasmid DNA of molecular weight 6.8 × 10⁶ was isolated from Streptomyces kasugaensis MB273. The plasmid DNA showed a single CsCl-ethidium bromide density gradient centrifugation, in neutral sucrose gradient centrifugation, and in agarose gel electrophoresis. When this DNA was digested with BamHI or SalI endonucleases, an unexpected number of fragments were found on agarose gel electrophoresis. Molecular weight summation of fragments obtained from double restriction enzyme digestions suggested that the plasmid DNA was a mixture of two different plasmids. This was confirmed by constructing recombinant plasmids between S. kasugaensis plasmid DNA and pBR322, and then by isolating two plasmids after SalI endonuclease treatment followed by sucrose gradient centrifugation. One of the plasmids (pSK1) had a single recognition site for BamHI, EcoRI, and SalI, and three sites for BglII. The other plasmid (pSK2) had a single recognition site for EcoRI and BglII, two recognition sites for BamHI, and no cleavage site for SalI. The cleavage maps of these plasmids were constructed using several restriction endonucleases.     

Cloning of a 1.1-kb Fragment Including srnB+ Gene in the F Plasmid and Isolation of an srnB Mutant

The srnB⁺ gene, promoting stable RNA degradation at 42 C in the presence of rifampin, was cloned by using pBR322 as a vector; it was located on a 1.1-kilobase (kb) EcoRI/BamHI fragment between 1.4 and 2.5 kb of the F plasmid. The region between 93.3 and 4.0 kb of the F plasmid was physically mapped by using restriction endonucleases EcoRI, HindIII, BamHI, PstI, and SmaI, with reference to a standard HindIII site in IS3. An srnB1 mutant was isolated from a chimeric plasmid, pOY54, after treatment of its DNA with hydroxylamine. The srnB1 allele on the F fragment of the mutant plasmid was recessive to the wild-type allele. Thermal elevation of cell cultures to 39 C was high enough to promote RNA degradation in strain YS12 carrying plasmid pOY54.     

Nucleotide sequence of BamHI family satellite DNA and its unit length polymorphism in bluegill sunfish Lepomis macrochirus

We have isolated and sequenced a member of tandem repetitive DNA containing BamHI site (BamHI family satellite DNA) from bluegill sunfish Lepomis macrochirus. PCR amplification with specific primers was performed to define the size of unit length repeat of the BamHI family satellite DNA, revealing that there were two distinct size of DNA fragments (0.9 kb and 1.3 kb) in the PCR products. The longer fragment (1.3 kb) consisted of internal sub-duplication of shorter fragment (0.9 kb). We have compared the size of PCR products among four fish populations, and found that both fragments co-existed in one population whereas the longer fragment was dominant in other three populations. The results may reflect ongoing homogenization of satellite DNA type over a short evolutionary time scale.     

The construction of a nopaline-inducible marker system for rapid detection of Agrobacterium strains

Summary An inducible marker system suitable for Agrobacterium tumefaciens was constructed to enable detection and enumeration of specific bacterial cells introduced into soil. A BamHI cassette carrying the catechol 2,3-dipxygenase (C230) gene, tdnC, fused to the nopaline-inducible Agrobacterium promoter Pi 2[noc] was constructed. This cassette was introduced into the broad host range vector pDSK5019 resulting in plasmid pTVNC2. Inducible C230 activity was observed in an A. tumefaciens strain carrying the plasmid pTVNC2 when nopaline was present. Colonies of bacteria tagged with the system could easily be identified by spraying agar plates containing nopaline with catechol, which is converted to the bright yellow compound 2-hydroxymuconic semialdehyde. The inducible tdnC cassette has also been introduced into the BamHI site of the transposon Tn5 carried by the pSUP1011 suicide vector which can be used as a delivery system for the stable introduction of the inducible marker into the chromosome of target cells.     

Cloning of mitochondrial DNA from the petite negative yeast Schizosaccharomyces pombe in the bacterial plasmid pBR322

Summary The entire mitochondrial (mt) genome of the yeast Schizosaccharomyces pombe (S. pombe) was cloned in the BamHI site of the Escherichia coli plasmid pBR322. Three lines of evidence demonstrate that the complete mtDNA molecule was amplified without rearrangement or partial loss. First, restriction of the hybrid plasmid with BamHI led to the recovery of two fragments corresponding to the linearized plasmid and the BamHI-cut mtDNA. Second, restriction of cloned and native mtDNA with HindIII revealed identical fragments. Third, mitochondrial ribosomal RNA hybridized to the same HindIII fragments from cloned mtDNA and from mtDNA isolated from mitochondria.     

Construction and analysis of plasmids containing the Escherichia coli serB gene

Summary Plasmid pserB59-1 carries the E. coli serB gene on a 5.2 kb BamHI fragment cloned into the BamHI site of plasmid pBR322. The results of genetic and biochemical experiments established that a functional serB gene is contained in the fragment. The location of the serB gene within the insert was determined by restriction endonuclease analysis of plasmids derived from pserB59-1 that carry the Tn5 element at sites that inactivate the serB gene, and by deletions of segments of the 5.2 kb insert that either inactivate or do not inactivate the serB gene. A 38,000 Mr serB ⁺ polypeptide was detected when plasmid pserB59-1 was used as template in a minicell system, but not when the serB gene was inactivated by insertion of a Tn5 element.     

Restriction analysis of lambda EMBL3 background recombinants: occurrence of lambda phages carrying a head to tail oriented left arm DNA sequence

Summary Eight representative recombinant background clones of λEMBL3 were analysed usingKpnI,BamHI,SalI,EcoRI andHindIII digestion. We found that λEMBL3 carries its own left arm in theBamHI cloning site. In this way, recombinant molecules were found to be generated which can grow onEscherichia coli strain NM539. In all cases analysed, the left arm DNA was inserted in a head to tail orientation. Seven clones carried a restoredBamHI site at thecos site-BamHI site connection. In the region where the inserted left arm and the right arm were ligated,BamHI cloning produces a large palindromic sequence consisting of two polylinkers. ThisBamHI site was incompletely cleaved in all cases analysed. We assume that a part of the λ DNA molecule in this region shows a cruciform structure prohibiting recognition or cleavage of this site by restriction endonucleaseBamHI.     

An integrative plasmid and multiple-sized plasmids of Pseudomonas syringae pv. phaseolicola have extensive homology

Summary Plasmids isolated from five strains of the bean pathogen Pseudomonas syringae pv. phaseolicola were characterized by restriction endonuclease and filter hybridization analyses. BamHI and EcoRI restriction patterns revealed that total plasmid DNA from each strain had a high level of sequence homology with pMC7105, a 148 kbp integrative plasmid found in a sixth strain. Only six BamHI fragments from the eight plasmids in these strains failed to hybridize with pMC7105 probe. Four of these fragments, three from pPP6520 and one from pPP6525 of strain PP652, hybridized strongly to plasmid DNA from a closely-related pathovar, P. syringae pv. glycinea. BamHI fragment 8, which is involved in the integration of pMC7105 into the host chromosome, contains a repeat sequence that was present on all the plasmids except pPP6120 (6.8 kbp), pPP6310 (40 kbp) and pPP6520 (45 kbp). Every plasmid but pPP6520 had fragments that showed weak hybridization to the small plasmid, pPP6120. This homology suggests that a second repetitive sequence is common to these plasmids. The large plasmids (148 to 151 kbp) were essentially identical to pMC7105. The intermediate plasmids (122 to 128 kbp) appeared to be derived mainly from pMC7105 or a related plasmid, whereas the smaller plasmids (6.8 to 45 kbp) appear to have been derived in part from sequences not present in pMC7105.     

Natural transformation of Pseudomonas stutzeri by plasmids that contain cloned fragments of chromosomal deoxyribonucleic acid

Natural transformation of plasmids by Pseudomonas stutzeri was found to depend on their bearing inserts of chromosomal DNA. A set of plasmids derived from the nonconjugative broad host range plasmid pSa151 was constructed by integrating various chromosomal DNA fragments into the single EcoR1 site of pSa151. Selection for the kanamycin resistance determined by pSa151 demonstrated that the derivative plasmids were taken into the cells by natural transformation and stably maintained; each could be reisolated unchanged. Thirty-two different derivative plasmids, 14 to 31 kbase pairs in size, all transformed. The frequency of transformation increased with the size of the chromosomal insert over a twenty fold range. These results suggest that the mechanism of transformation of plasmids by the Gram-negative P. stutzeri is similar to those proposed to operate in Gram-positive bacteria.Dedicated to Prof. Dr. H.-G. Schlegel on the occasion of his 60 th birthday     

Detailed restriction enzyme map of crown gall-suppressive IncW plasmid pSa, showing ends of deletion causing chloramphenicol sensitivity.

Detailed restriction enzyme maps of the antibiotic resistance plasmid pSa and of a chloramphenicol-sensitive spontaneous deletion mutant of pSa were constructed. The smaller plasmid contained one set of three restriction enzyme sites that appeared at both ends of the deleted region in the intact plasmid. The frequency of loss of chloramphenicol resistance was 1% after eight logs of growth and was dependent on the recA gene function of Escherichia coli. A previously published map lacks an 11-kilobase-pair SstII fragment present on this map.     

Tagging pathogenicity genes inUstilago maydis by restriction enzyme-mediated integration (REMI)

In the maize pathogenic fungusUstilago maydis integration of transforming DNA at homologous or heterologous sites is often accompanied by duplications of the DNA. We show that it is possible to generate single-copy integration events with high efficiency by restriction enzyme-mediated integration (REMI). In about 50% of cases, a plasmid that contains a singleBamHI site is integrated at chromosomalBamHI sites, ifBamHI is added to the transformation mixtures. In the other cases it appears that integration events have also occurred preferentially atBamHI sites, but without restoration of the recognition sites. Using REMI we have generated approximately 1000 insertion mutants. Pathogenicity tests demonstrated that about 1–2% of these mutants were unable to induce symptoms when testedin planta. For two of the mutants we have shown that the phenotype is linked to the insertion event.     

Isolation and characterization of a higher-copy-number mutant of plasmid R6K

A stable copy-number mutant (pNH601) of plasmid R6K was isolated by selection for increased resistance to ampicillin determined by this plasmid. The size of the mutant plasmid was found to be unchanged (26 Mg/mol) but it is present in 27 copies of pNH601 perE. coli K-12 chromosome which represents a two-fold increase of R6K copy number value. The following genetic properties of pNH601 are reported and compared with those of R6K: conjugative transfer, fertility inhibition of plasmids belonging to other incompatibility groups, incompatibility with plasmid R485 under both non-selective and selective conditions and the integrative suppression of thednaA ts mutation. The mutant plasmid pNH601 was found to be different from the original R6K in most of these properties.     

Anin vivo cointegrate of two plasmids from incompatibility group X

Formation of a recombinant plasmid designated pNH603 was observed when two plasmids from incompatibility group X, the multicopy plasmid pNH602 (a higher-copy-number deletion derivative of R6K) and the oligocopy plasmid R485, coexisted in a single Escherichia coli cell. According to its size and its restriction endonuclease cleavage pattern, plasmid pNH603 is a true cointegrate of pNH602 and R485. An insertion-sequence-like element coming from plasmid R485 is supposed to mediate the fusion of both replicons. The pNH603 copy number (1-2 per chromosome) indicates that the mechanism of replication of the low-copy-number plasmid is dominant in this cointegrate. No dissociation of pNH603 to parental plasmids was observed even in E. coli K-12 recA+ cells. On the other hand, deletion derivatives of four size classes originate from pNH603 in both recA+ and recA hosts. A miniplasmid designated pNH604, a representative of the most frequent 7 Mg/mol size class, was found, in a low number of copies per host chromosome.