Regions associated with the stable maintenance of plasmid pSC101 and its tetracycline resistance

Summary Two regions tentatively called unsA and unsR were identified on pSC101. One, unsA, corresponds to less than 650 bp of the N-terminal in the tetracycline resistance structural gene and seems to inhibit stable maintenance of pSC101. The other, unsR, is defined within the 1 kb XhoI-EcoRI region located upstream of the tetracycline resistance structural gene and is a regulatory gene clearly distinct from tetR (Unger et al. 1984); it serves as a suppressor of the unsA function.     

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.     

Construction and BamHL analysis of chimeric plasmids containing EcoRL DNA fragments of the F sex factor.

The construction of seven chimeric plasmids (pRS series) carrying EcoRI endonuclease-generated segments of the F sex factor cloned onto the vector pSC101 is described. BamHI endonuclease analysis of these seven plasmids, the six previously described pRS plasmids (Skurray, R. A., Nagaishi, H., and Clark, A. J. (1976) Proc. Nat. Acad. Sci. USA73, 64–68) and F plasmid DNA has enabled a partial BamHI map of F to be constructed; the orientation of insertion of F DNA segments into the pSC101 vector was also established for nine of the pRS plasmids. Results indicate that in the absence of their normal promoter, F cistrons cloned into the EcoRI site of pSC101 are expressed regardless of orientation of insertion although there is a preferred orientation for high levels of expression.     

Characterization of small plasmids from Staphylococcus aureus.

Small molecular weight plasmids from Staphylococcus aureus were characterized with respect to size, restriction enzyme cleavage pattern and transforming capacity. The plasmids pS194 and pC194 which encode streptomycin and chloramphenicol resistance respectively contained 3.0 and 2.0 megadaltons of DNA as determined by zonal rate centrifugation and electron-microscopy. Both plasmids transformed S. aureus with high efficiency. Plasmid pC194 contained only one cleavage site for endonuclease HindIII and pS194 contained single cleavage sites for HindIII and EcoRI. A natural recombinant between these two plasmids, pSC194, shared the high transforming capacity of the parental plasmids and contained one EcoRI site And two HindIII sites. pSC194 DNA also transformed B. subtilis with high efficiency. The recombinant plasmid pSC194 may be used as an EcoRI vector for construction and propagation of hybrid DNA in S. aureus as shown in the following paper (Löfdahl et al., 1978).     

Post-transformational rearrangement of an in vitro reconstructed group-A streptococcal erythromycin resistance plasmid

Cleavage of the group-A streptococcal macrolide, lincosamide, and streptogramin B (MLS) resistance plasmid pSM19035 yields 2 fragments [13 and 4 megadaltons (MD)] with EcoRI, and 15 fragments with HindIII, 12 of which are 6 pairs of identical fragments derived from the inverted repeats that comprise about 80% of the pSM19035 genome. The large EcoRI fragment was isolated, ligated, and used to transform the Challis strain of Streptococcus sanguis to erythromycin resistance. Plasmids (pDB101, pDB102, and pDB103) isolated from three different transformants had lower molecular masses than the original large EcoRI fragment. HindIII digestion of these molecules and subsequent analysis of fragment radioactivity distributions indicated the loss of plasmid segments of various sizes. The deletions, all of which occurred in the palindrome, did not affect the level and the inducible nature of pSM19035-determined antibiotic resistance. Only pDB101 retained the unique EcoRI cleavage site. The results of this analysis allowed the construction of an EcoRI and HindIII cleavage-site map of pSM19035 and promise to simplify future studies of genetic functions specified by streptococcal MLS resistance plasmids.     

Recombinant plasmids capable to replication in B. subtilis and E. coli.

Summary The plasmid pBC16 (4.25 kbases), originally isolated from Bacillus cereus, determines tetracycline resistance and can be transformed into competent cells of B. subtilis. A miniplasmid of pBC16 (pBC16-1), 2,7 kb) which has lost an EcoRI fragment of pBC16 retains the replication functions and the tetracycline resistance. This plasmid which carries only one EcoRI site has been joined in vitro to pBS1, a cryptic plasmid previously isolated from B. subtilis and shown to carry also a single EcoRI site (Bernhard et al., 1978). The recombinant plasmid is unstable and dissociates into the plasmid pBS161 (8.2 kb) and the smaller plasmid pBS162 (2.1 kb). Plasmid pBS161 retains the tetracycline resistance. It possesses a single EcoRI site and 6 HindIII sites. The largest HindIII fragment of pBS161 carries the tetracycline resistance gene and the replication function. After circularization in vitro of this fragment a new plasmid, pBS161-1 is generated, which can be used as a HindIII and EcoRI cloning vector in Bacillus subtilis.Hybrid plasmids consisting of the E. coli plasmids pBR322, pWL7 or pAC184 and different HindIII fragments of pBS161 were constructed in vitro. Hybrids containing together with the E. coli plasmid the largest HindIII fragment of pBS161 can replicate in E. coli and B. subtilis. In E. coli only the replicon of the E. coli plasmid part is functioning whereas in B. subtilis replication of the hybrid plasmid is under the control of the Bacillus replicon. The tetracycline resistance of the B. subtilis plasmid is expressed in E. coli, but several antibiotic resistances of the E. coli plasmids (ampicillin, kanamycin and chloramphenicol) are not expressed in B. subtilis. The hybrid plasmids seem to be more unstable in B. subtilis than in E. coli.     

Identification by deletion analysis of an inducible protein required for plasmid pSC101-mediated tetracycline resistance.

Plasmids containing small deletions within a tetracycline (Tc) resistance gene(s) of plasmid pHA121 were isolated. Plasmid pHA121 was formed by ligating the EcoRI-digested Tc resistance plasmid pSC101 and similarly digested mini-ColE1 plasmid pHA105. The DNA deletion plasmids were constructed by digesting plasmid pHA121 DNA with the restriction endonucleases BamH1 and Sal1 and, in addition, λ exonuclease. Two plasmids, designated pJT131 and pJT133, had small deletions of approximately 0.64 to 0.8 kb and a comparison of the radioactive polypeptides synthesized in plasmid-containing minicells revealed that a 34-kdal polypeptide was not specified by either pJT131 or pJT133. We conclude that the 34-kdal polypeptide is required for the expression of Tc resistance and that its structural gene probably maps in the deleted region of pSC101 DNA.     

The structures and fidelity of replication of mouse mitochondrial DNA-pSC101 EcoRI recombinant plasmids grown in E. coli K12

Recombinant DNAs containing the E. coli plasmid pSC101 and mouse cell (LA9) mitochondrial DNA (mtDNA) were formed in vitro via ligation of DNA fragments from limit EcoRI endonuclease digests and were used to transform E. coli K12. Four structurally different recombinant plasmid DNAs from transformed clones were characterized. Two of these were analyzed extensively and the mtDNA portions compared with mtDNA from LA9 cells. No differences were detected in the physical or chemical properties examined, except that the E. coli mtDNA lacked the alkali lability characteristic of animal mtDNAs.Heteroduplexes between the LA9 portions of the recombinant plasmids and LA9 mtDNA were analyzed by absorbance melting. The melting temperatures were indistinguishable from reannealed LA9 mtDNA homoduplexes, indicating that single-base replication errors occur at a frequency of fewer than 1 nucleotide in 300. Electron microscopic analyses of plasmid-LA9 mtDNA heteroduplexes and a comparison of agarose gel electrophoresis of restriction endonuclease fragments also indicated no differences. These results were independent of the order or the relative orientation of the pSC101 and mtDNA fragments.A third EcoRI fragment in LA9 mtDNA, not found in an earlier study (Brown and Vinograd, 1974), has been positioned in the LA9, EcoRI map. This fragment contains 165±10 nucleotide pairs.     

Cloning and analysis of pif, replication and leading regions of the F plasmid

Summary We describe the molecular cloning of BglII fragments of the hybrid plasmid pRS5 (pSC101 and EcoRI fragments of F; f7, f5, f3 and f6). The clones isolated were examined for the expression of F-specified replication, incompatibility, mobilization and inhibition of T7 bacteriophage multiplication. Proteins directed by the BglII clones were labelled in Escherichia coli K12 maxicells and analyzed by SDS-polyacrylamide gel electrophoresis. The sizes of previously reported proteins, encoded by the replication, incompatibility and leading regions encompassed by these plasmids have been confirmed in this study. In addition, the results demonstrate that a pif gene, which encodes an 80,000 dalton polypeptide essential for the inhibition T7 phage multiplication, is located on the BglII fragment that spans the junction of EcoRI fragments f7 and f5.     

Construction of a recombinant plasmid composed of B. subtilis leucine genes and a B. subtilis (natto) plasmid: its use as cloning vehicle in B. subtilis 168.

Summary Recombinant plasmids composed of Bacillus subtilis 168 leucine genes and a B. subtilis (natto) plasmid have been constructed in a recombination deficient (recE4) mutant of Bacillus subtilis 168. The process involved EcoRI fragmentation and ligation of a B. subtilis (natto) plasmid and a composite plasmid RSF2124-B · leu in which B. subtilis 168 leucine genes are linked to the R-factor RSF2124. A constructed plasmid (pLS102) was found to be composed of an EcoRI fragment derived from the vector plasmid and two tandemly repeated EcoRI fragments carrying the leucine genes. A derivative plasmid (pLS101 or pLS103) consisting of one molecule each of the EcoRI fragments was obtained by in vivo intramolecular recombination between the repeated leucine gene fragments in pLS102. pLS103 was cleaved once with BamNI, SmaI and HpaI. Insertion of foreign DNA (Escherichia coli plasmid pBR322) into the BamNI site inactivated leuA but not the leuC function which thus can serve as selective marker if the plasmid is used as vector in molecular cloning. The penicillin resistance carried in pBR322 was not functionally expressed in B. subtilis cells. By partial digestion of pLS103 with HindIII followed by ligation with T4-induced ligase, pLS107 was obtained which contained only one EcoRI site. However, insertion of exogenous DNA (pBR322) into this EcoRI site inactivated both leuA and leuC functions.     

Restriction endonuclease mapping of pSC101 and pMB9

Summary A restriction endonuclease analysis of the plasmids pSC101 and pMB9 has allowed a determination of the alterations that occurred in the tetracycline resistance locus during the construction of pMB9 from pSC101. The genes for four of the polypeptides involved in tetracycline resistance have been positioned on the restriction endonuclease map of pSC101.     

New cloning vectors using benomyl resistance as a dominant marker for selection inNeurospora crassa and in other filamentous fungi

We describe five new plasmid vectors derived from pBR322 that carry theNeurospora crassa β-tubulin gene conferring resistance to benomyl. The benomyl resistance gene has been modified to eliminate an internalEcoRI site to facilitate the cloning ofEcoRI restriction fragments. These plasmids allow rapid subcloning of fragments from one replicon to another without insert fragment purification due to the presence of different drug resistance markers (resistance to kanamycin, tetracycline, or chloramphenicol) carried on the plasmids. These vectors will allow rapid transformation ofN. crassa and other filamentous fungi to allow phenotypic characterization of subcloned fragments.     

On the nature of tetracycline resistance controlled by the plasmid pSC101.

In vitro enzymatic alteration of plasmid phenotype and in vitro construction of recombinant plasmids containing genetic information derived from the plasmid pSC101 have been used to investigate the mechanism of function of tetracycline resistance determined by the plasmid pSC101. The resistance has been shown to be inducible and involves the increased synthesis of membrane-associated polypeptides of 34,000, 26,000 and 14,000 daltons that are encoded for by the plasmid. The 34,000 dalton polypeptide along with another plasmid-encoded polypeptide of 18,000 daltons function in an ATP-independent manner to prevent the accumulation of tetracycline by the cell. These polypeptides are sufficient for resistance. A second component of plasmid-determined resistance involves the 14,000 dalton polypeptide and reduces the initial adsorption of tetracycline by sensitive cells, but is not alone sufficient for the generation of resistance. The role of the 26,000 dalton polypeptide in tetracycline resistance has not been identified.     

Construction of a bacterial artificial chromosome library containing large Eco RI and Hin dIII genomic fragments of lettuce

 Existing bacterial artificial chromosome (BAC) vectors were modified to have unique EcoRI cloning sites. This provided an additional site for generating representative libraries from genomic DNA digested with a variety of enzymes. A BAC library of lettuce was constructed following the partial digestion of genomic DNA with HindIII or EcoRI. Several experimental parameters were investigated and optimized. The BAC library of over 50,000 clones, representing one to two genome equivalents, was constructed from six ligations; average insert sizes for each ligation varied between 92.5 and 142 kb with a combined average insert size of 111 kb. The library was screened with markers linked to disease resistance genes; this identified 134 BAC clones from four regions containing resistance genes. Hybridization with low-copy genomic sequences linked to resistance genes detected fewer clones than expected from previous estimates of genome size. The lack of hybridization to chloroplast and mitochondrial sequences demonstrated that the library was predominantly composed of nuclear DNA. The unique EcoRI site in the BAC vector should allow the integration of BAC cloning with other technologies that utilize EcoRI digestion, such as AFLP^TM markers and RecA-assisted restriction endonuclease (RARE) cleavage, to clone specific large EcoRI fragments from genomic DNA. Received: 5 August 1996 / Accepted: 23 August 1996     

Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6

Summary DNA fragments generated by the EcoRI or HindIII endonucleases from the low copy number antibiotic resistance plasmids R6 and R6-5 were separately cloned using the high copy number ColEl or pML21 plasmid vectors and the insertional inactivation procedure. The hybrid plasmids that were obtained were used to determine the location of the EcoRI and HindIII cleavage sites on the parent plasmid genomes by means of electron microscope heteroduplex analysis and agarose gel electrophoresis. Ultracentrifugation of the cloned fragments in caesium chloride gradients localized the high buoyant density regions of R6-5 to fragments that carry the genes for resistance to streptomycin-spectinomycin, sulfonamide, and mercury and a low buoyant density region to fragments that carry the tetracycline resistance determinant. Functional analysis of hybrid plasmids localized a number of plasmid properties such as resistances to antibiotics and mercury and several replication functions to specific regions of the R6-5 genome. Precise localisation of the genes for resistance to chloramphenicol, kanamycin, fusidic acid and tetracycline was possible due to the presence of identified restriction endonuclease cleavage sites within these determinants.Only one region competent for autonomous replication was identified on the R6-5 plasmid genome and this was localized to EcoRI fragment 2 and HindIII fragment 1. However, two additional regions of replication activity designated RepB and RepC, themselves incapable of autonomous replication but capable of supporting replication of a linked ColE1 plasmid in polA^– bacteria, were also identified.     

Protein expression in E. coli minicells by recombinant plasmids.

The polypeptides synthesized in E. coli minicells from recombinant plasmids containing DNA fragments from cauliflower mosaic virus, Drosophila melanogaster, and mouse mitochondria were examined. Molecularly cloned fragments of cauliflower mosaic virus DNA directed the synthesis of high levels of three polypeptides, which were synthesized entirely from within the cloned virus DNA fragments independent of their insertion into the plasmid vehicles. Several fragments of D. melanogaster DNA were capable of initiating polypeptide synthesis; however, termination of these polypeptides was dependent upon the insertion into the plasmid vehicle. The majority of D. melanogaster DNA fragments examined did not direct the detectable synthesis of any polypeptides. Insertion of DNA into the Eco RI site of ColE1 and pSC101 plasmids resulted in the altered expression of plasmid-encoded polypeptides. In the case of ColE1, this site of insertion lies within the colicin E1 structural gene, and insertion of foreign DNA into the site results in the synthesis of an inactive truncated colicin E1 molecule. It is probable that the Eco RI site in pSC101 lies within the structural gene for a polypeptide involved in tetracycline resistance, and insertion of DNA into this site may also result in the synthesis of a truncated or elongated polypeptide.     

Physical mapping of plasmid pDB101: A potential vector plasmid for molecular cloning in streptococci

A physical map of the streptococcal macrolides, lincomycin, and streptogramin B (MLS) resistance plasmid pDB101 was constructed using six different restriction endonucleases. Ten recognition sites were found for HindIII, seven for HindII, eight for HaeII, and one each for EcoRI, HpaII, and KpnI. The localization of the restriction cleavage sites was determined by double and triple digestions of the plasmid DNA or sequential digestions of partial cleavage products and isolated restriction fragments, and all sites were aligned with a single EcoRI reference site. Plasmid pDB101 meets all requirements essential for a potential molecular cloning vehicle in streptococci; i.e., single restriction sites, a MLS selection marker, and a multiple plasmid copy number. The vector plasmid described here makes it possible to clone selectively any fragment of DNA cleaved with EcoRI, HpaII, or KpnI, or since the sites are close to each other in map position, any combination of two of these restriction enzymes.     

Mapping of the resistance genes of the R plasmid NR1.

Summary The drug resistance genes on the r-determinants component of the composite R plasmid NR1 were mapped on the EcoRI restriction endonuclease fragments of the R plasmid by cloning the fragments using the plasmid RSF2124 as a vector. The sulfonamide (Su) and streptomycin/spectinomycin (Sm/Sp) resistance genes are located on EcoRI fragment G of NR1. The expression of resistance to mercuric ions (Mer) requires both EcoRI fragment H and I of NR1. The expression of chloramphenicol (Cm) and fusidic acid (Fus) resistance requires EcoRI fragments A and J of NR1. The kan fragment of the related R plasmid R6-5 can substitute for EcoRI fragment J of NR1 in the expression of Cm and Fus resistance. The structural genes for Cm and Fus resistance appear to be a part of an operon whose expression is controlled by the same promoter.     

Replication of pSC101: effects of mutations in the E. coli DNA binding protein IHF

Summary We have shown that the plasmid pSC101 is unable to be maintained in strains of E. coli carrying deletions in the genes himA and hip which specify the pleitropic heterodimeric DNA binding protein, IHF. We show that this effect is not due to a modulation of the expression of the pSC101 RepA protein, required for replication of the plasmid. Inspection of the DNA sequence of the essential replication region of pSC101 reveals the presence of a site, located between the DnaA binding-site and that of RepA, which shows extensive homology with the consensus IHF binding site. The proximity of the sites suggests that these three proteins, IHF, DnaA, and RepA may interact in generating a specific DNA structure required for initiation of pSC101 replication.     

Mitochondrial DNA from Podospora anserina

Summary EcoRI fragments of the 94 kilobase mitochondrial DNA (mtDNA) from young, wild type Podospora anserina were cloned into the EcoRI site of the E. coli plasmid vector pBR325. A complete EcoRI clone bank was developed, containing all 16 of the EcoRI fragments from the native mtDNA. Restriction endonuclease maps for the enzymes SalI, XhoI, BamHI, EcoRI, BglII, and HaeIII were constructed from the analysis of single, double, and triple restriction digests of cloned and native mtDNA. In constructing the maps data were refined by extensive Southern analysis of the native genome hybridized to cloned DNA probes. Restriction maps were analyzed and permitted us to locate the origin of mtDNA derived from senescent cultures.Both the large and small rRNA genes were then localized on these restriction maps using Southern and Northern blot analysis. We have shown the large rRNA locus to lie within a 10.8 kb region of EcoRI fragments E5 and E7, and the small rRNA locus to lie on a 5 kb subfragment of EcoRI fragment E1. The limit of separation between these two loci was determined to be between 6 and 9 kb.Surprisingly, when electrophoresed in agarose-CH₃HgOH gels, the large rRNA was found to be 3.8 kb long, 500 bases longer than that from the very closely related Neurospora crassa, making it the largest rRNA yet described.