Sensitivity of chromosomal and plasmid E. coli DNA to restriction endonuclease Eco RII]

It was shown that E. coli C, E. coli MRE 600 DNA, and also plasmid DNA of Col E1, RSF 2124 from E. coli K-12, and plasmid DNA from E. coli MRE 600 were completely resistant against restriction endonuclease R. Eco RII. Plasmid DNAs of Col E1, RSF 2124 amplificated for 4 hours in the presence of chloramphenicol are sensitive to R. Eco RII but after 16-hour amplification in the presence of chloramphenicol these DNAs acquire complete resistance against R. Eco RII. These data point to the slower rate of modification of DNA in vivo by DC-methylases of Eco RII type in comparison with DNA methylase Eco RII.     

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.     

Control of tryptophan synthetase amplified by varying the numbers of composite plasmids in Escherichia coli cells.

Using pSC101, RSF1010, RSF2124 and RP4 plasmids as vectors and bacteriophage lambdatrpD-A60-3 DNA as a source of the Escherichia coli whole tryptophan operon, composite plasmids of pSC101-trp, RSF1010-trp, RSF2124-trp and RP4-trp were constructed in vitro with EcoRI restriction endonuclease and DNA ligase. Each composite plasmid could be maintained stably in E. coli cells. The copy number of pSC101-trp, RSF1010-trp, RSF2124-trp and RP4-trp were 4.2, 11.2, 11.9 and 1.6 per chromosome respectively. The tryptophan synthetase activities in cells containing pSC101-trp, RSF1010-trp, RSF2124-trp aand RP4-trp plasmid were found to be 2.1, 6.0, 5.0 and 2.5 times compared with the level in chromosomal trp+ cells when they were grown in a minimal medium. By partial derepression with indolylacrylic acid, the enzyme levels were elevated to 10.1, 16.3, 15.3, 12.3 times, respectively, that of the control cells. The tryptophan synthetase activities did not increase in proportion to the copy number of the plasmids, but were strongly affected by the repression system of host cells.     

A multi-resistance plasmid isolated from commensal Neisseria species is closely related to the enterobacterial plasmid RSF1010

pFM739, an R plasmid from Neisseria sicca that encodes penicillin, streptomycin and sulphonamide resistance, and the enterobacterial IncQ(P-4) plasmid RSF1010, which encodes streptomycin and sulphonamide resistance, were incompatible, and were mobilized by the same conjugative plasmids. Restriction mapping confirmed a high degree of similarity between both R plasmids; pFM739 carried DNA fragments corresponding to the known replication and resistance regions of RSF1010. pFM739 also carried an extra segment with the same restriction map as that described for the beta-lactamase-coding region of transposon Tn3. It is suggested that the R plasmids isolated from commensal Neisseria sp. could have resulted from transposition of a Tn3-like genetic element to an RSF1010-like plasmid, and that they contain deletion derivatives of transposon Tn3.     

Characterisation of plasmids coding for the restriction endonuclease EcoRI

Summary The properties of two plasmids coding for the EcoRI restriction and modification enzymes are described. Both plasmids are non auto-transferring (NTP) but can be mobilised by transfer factors. Strains carrying NTP13 produce colicin E1 and the EcoRI enzymes. This plasmid has a molecular weight of 6x10⁶ daltons and is present as approximately 12 copies per chromosome. The second plasmid, NTP14, was detected after mobilisation of the EcoRI plasmid with the R factor R1–19. NTP14 codes for ampicillin resistance, synthesis of the EcoRI enzymes and colicin E1. The molecular weight of NTP14 is 10.7x10⁶ daltons and there are about 14 copies per chromosome. DNA-DNA reassociation experiments were performed to determine the interrelationships of NTP13, NTP14, ColE1 and the R factor R1–19. NTP13 and NTP14 continue to replicate when cellular protein synthesis is inhibited by the addition of chloramphenicol.     

SP02 particles mediating transduction of a plasmid containing SP02 cohesive ends

SP02 particles that mediate transduction of plasmid pPL1010, a 4.6-megadalton derivative of pUB110 containing an Eco RI endonuclease-generated fragment of SP02 deoxyribonucleic acid that spans the cohesive ends, exhibit three unusual features: the transducing particles have a lower buoyant density than infectious particles; the transduction of pPL1010 occurs at high efficiency; and the transducing activity of the particles is relatively resistant to ultraviolet irradiation when the recipient is recombination proficient. Evidence is presented which indicates that SP02(pPL1010) particles carry the plasmid predominantly as a linear multimer having a molecular mass comparable to that of infectious SP02 deoxyribonucleic acid (ca. 31 megadaltons). The plasmid monomers in the linear multimer appear oriented in the same polarity. The buoyant density difference between infectious and transducing particles appears to be due mainly to the buoyant density difference between pPL1010 (1.699 g/cm3) and SP02 deoxyribonucleic acid (1.702 gm/cm3).     

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.     

Conjugative R plasmids isolated from hospital strains of Pseudomonas aeruginosa]

It was shown that Pseudomonas aeruginosa hospital strains isolated from patients and environment in the Republican Centre of Burns in Tbilisi contained conjugative R plasmids. The plasmids were marked pM15 and pM19, respectively. The plasmid pM15 determined resistance to carbenicillin, kanamycin and tetracycline and plasmid pM19 determined resistance to carbenicillin, kanamycin, tetracycline, chloramphenicol, gentamicin and streptomycin. Plasmid pM15 had a molecular weight of 45.8 MD and seven sites for EcoRI, six sites for HindIII and five sites for Hpa-I-restrictase. This plasmid, as others, belongs to the Inc-P1 incompatibility group.     

Isolation and characteristics of a recombinant pOV13 plasmid as a vector for DNA cloning in a broad range of bacterial hosts

The hybrid plasmid pOV13 proposed as a potential vector for DNA cloning in a broad bacterial host range has been constructed on the basis of the broad host range plasmid RSF1010 and a shortened derivative of RP4, the plasmid pVZ115 serving a marker DNA fragment. The plasmid pOV13 contains the genes for streptomycin, kanamycin and tetracycline resistance and single cleavage sites for restriction endonucleases BamHI, BgIII, SalI, SmaI, PvuII, XhoI, as well as double cleavage sites for restriction endonucleases PstI and HindIII permitting one to clone DNA with insertional inactivation of genes. The physicogenetical map of the birepliconed plasmid pOV13 is presented.     

Construction of a hybrid bacteriophage-plasmid recombinant DNA vector.

A phage-plasmid hybrid was constructed for use as a recombinant DNA vector, allowing the propagation of cloned EcoRI restriction endonuclease fragments of about 2 X 10(6) to 11 X 10(6) daltons. The colicin E1 plasmid replicon was fused to the left arm of a lambdagt generalized transducing phage with a thermolabile repressor, yielding a genome which could be replicated either by phage lambda functions or via the colicin E1 plasmid replicon. At the nonpermissive temperature, phage functions were derepressed and phage growth occurred lytically. Alternatively, at the permissive temperature, lambda functions were repressed and the vector replicated as a covalently closed circular plasmid. The phage-plasmid hybrid vector could be maintained at a copy number determined by the colicin E1 plasmid replicon and was also sensitive to amplification after chloramphenicol treatment. An EcoRI fragment of Escherichia coli DNA encoding genes of the arabinose operon also was inserted into the central portion of the vector.     

Molecular cloning and mapping of a deletion derivative of the plasmid Rts 1

The plasmid pTW20 is a deletion derivative of the kanamycin resistance plasmid Rts1. By digesting pTW20 DNA with EcoRI endonuclease six fragments were generated, and each was cloned in the vector plasmid pACYC184. These cloned EcoRI fragments were further digested with various endonucleases, and the cleavage map of pTW20 was constructed. A SalI fragment (1.5 Md) in E1 (the largest EcoRI fragment; 11.5 Md) contained the genes kan (kanamycin resistance) and puv (uv sensitization of host). An electron microscopy study of a BamHI fragment containing kan revealed the presence of a transposon-like structure in the fragment. The smallest EcoRI fragment E6 (2.0 Md) was capable of autonomous replication in a polA host, indicating that E6 contained replication genes of pTW20. These genes were found to be located on a 1.1-Md HindIII fragment in E6. Two incompatibility genes were identified on the pTW20 genome, one located on each of the fragments E6 and E5 (3.5 Md), and expressed T incompatibility independently. The nature of the temperature sensitivity of pTW20 was discussed.     

Effect of temperature and 4,6'-diamidine-2-phenylindole on restriction of supercoiled Col E1 DNA by Eco RI endonuclease.

Supercoiled Col E1 DNA is split by Eco RI endonuclease at 37 degrees C without intermediate formation of open circular DNA. Accumulation of this restriction product is observed at low temperature. The fluorescent dye, 4,6'-diamidine-2-phenylindole (DAPI) inhibits restriction by Eco RI endonuclease. This effect is due to the DAPI:DNA rather than to the DAPI:Eco RI interactions.     

Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010

We present the complete nucleotide sequence of RSF1010, a naturally occurring broad-host-range plasmid belonging to the Escherichia coli incompatibility group Q and encoding resistance to streptomycin and sulfonamides. A molecule of RSF1010 DNA consists of 8684 bp and has a G + C content of 61%. Analysis of the distribution of translation start and stop codons in the sequence has revealed the existence of more than 40 open reading frames potentially capable of encoding polypeptides of 60 or more amino acids. To date, products of eleven such potential RSF1010 genes have been identified through the application of controlled expression vector systems, and for eight of them, the reading frame has been confirmed by N- and/or C-terminal amino acid sequence determinations on the purified proteins. The sequencing results are discussed in relation to the systems of replication, host range, conjugal mobilization and antibiotic resistance determinants associated with the RSF1010 plasmid.     

Transfer of plasmid RSF1010 by conjugation from Escherichia coli to Streptomyces lividans and Mycobacterium smegmatis.

The plasmid RSF1010 belongs to a class of plasmids (IncQ) that replicate in a range of bacterial hosts. Although non-self-transmissible, it can be mobilized at high frequency between different gram-negative bacterial species if transfer functions are supplied in trans. We report the transfer of RSF1010 by conjugation from Escherichia coli to the gram-positive actinomycetes Streptomyces lividans and Mycobacterium smegmatis. In its new hosts, the plasmid was stable with respect to structure and inheritance and conferred high-level resistance to streptomycin and sulfonamide. This is the first reported case of conjugative transfer of a naturally occurring plasmid between gram-negative and gram-positive bacteria.     

Ecological and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae.

Strains of Pseudomonas syringae pv. syringae resistant to copper, streptomycin, or both compounds were recovered from symptomless and diseased tissue of four woody hosts in three nurseries in Oklahoma. In strains resistant to copper and streptomycin (Cur Smr), resistance to both compounds was cotransferred with a single plasmid which was either 68, 190, or 220 kilobase pairs (kb). All Cus Smr strains contained a 68-kb conjugative plasmid. Cur Sms strains contained one plasmid which varied in size from 60 to 73 kb. All conjugative plasmids which transferred streptomycin resistance contained sequences homologous to the strA and strB Smr genes from the broad-host-range plasmid RSF1010. The Smr determinant was subsequently cloned from a 68-kb Cur Smr plasmid designated pPSR1. A restriction map detailing the organization of the homologous Smr genes from pPSR1 and RSF1010 and cloned Smr genes from P. syringae pv. papulans and Xanthomonas campestris pv. vesicatoria revealed the conservation of all sites studied. The Cur genes cloned from P. syringae pv. tomato PT23 and X. campestris pv. vesicatoria XV10 did not hybridize to the Cur plasmids identified in the present study, indicating that copper resistance in these P. syringae pv. syringae strains may be conferred by a distinct genetic determinant.     

High-frequency mobilization of broad-host-range plasmids into Neisseria gonorrhoeae requires methylation in the donor.

Antibiotic resistance in Neisseria gonorrhoeae has been associated with the acquisition of R plasmids from heterologous organisms. The broad-host-range plasmids of incompatibility groups P (IncP) and Q (IncQ) have played a role in this genetic exchange in nature. We have utilized derivatives of RSF1010 (IncQ) and RP1 (IncP) to demonstrate that the plethora of restriction barriers associated with the gonococci markedly reduces mobilization of plasmids from Escherichia coli into strains F62 and PGH 3-2. Partially purified restriction endonucleases from these gonococcal strains can digest RSF1010 in vitro. Protection of RSF1010-km from digestion by gonococcal enzymes purified from strain F62 is observed when the plasmid is isolated from E. coli containing a coresident plasmid, pCAL7. Plasmid pCAL7 produces a 5'-MECG-3' cytosine methylase (M.SssI). The M.SssI methylase only partially protects RSF1010-km from digestion by restriction enzymes from strain PGH 3-2. Total protection of RSF1010-km from PGH 3-2 restriction requires both pCAL7 and a second coresident plasmid, pFnuDI, which produces a 5'-GGMECC-3' cytosine methylase. When both F62 and PGH 3-2 are utilized as recipients in heterospecific matings with E. coli, mobilization of RSF1010 from strains containing the appropriate methylases into the gonococci occurs at frequencies 4 orders of magnitude higher than from strains without the methylases. Thus, protection of RSF1010 from gonococcal restriction enzymes in vitro correlates with an increase in the conjugal frequency. These data indicate that restriction is a major barrier against efficient conjugal transfer between N. gonorrhoeae and heterologous hosts.     

DnaG-dependent priming signals can substitute for the two essential DNA initiation signals in oriV of the broad host-range plasmid RSF1010.

Broad host-range plasmid RSF1010 contains in the oriV region two DNA initiation signals, ssiA(RSF1010) and ssiB(RSF1010), which are essential for plasmid replication. Each of ssiA and ssiB could be substituted functionally by either of the two G4-type (DnaG-dependent) priming signals, the oric of bacteriophage G4 and an ssi signal from plasmid pSY343 (an R1 plasmid derivative). Functions of the chimeric oriVs of RSF1010 thus constructed were dependent on the RSF1010-specific replication proteins, RepA, RepB' and RepC. When both of ssiA and ssiB were replaced by the G4-type ssi signals, functions of the chimeric oriVs were no longer dependent on RepB' (RSF1010-specific DNA primase). The replication activities of the chimeric oriVs of RSF1010 were not influenced markedly by the type of heterologous priming signals they contained. It is conceivable that DNA replication of RSF1010 does not need the priming mechanism for lagging strand synthesis and proceeds by the strand displacement mechanism.     

质粒RSF1010从大肠杆菌到稀有放线菌头状链轮丝菌和星状诺卡氏菌的？…

RSF1010 is a naturally occurring Escherichia coli broad host-range plasmid about 8.7 kb in size. It can be mobilized at high frequency between different gram-negative bacterial species when transfer functions are available in trans. Following the pioneering work of conjugational transfer of RSF1010 from E. coli to Streptomyces lividans and Mycobacterium smegmatis, the transfer of this plasmid by conjugation from E. coli S17.1 tp two gram-positive rare actinomycetes, Nocardia asteroides 3927 and Streptoverticillum caespitosus ATCC27422 was first time reported in this study. Southern blot analysis of the total DNA extracted from the actinomycetes' exconjugants proved that RSF1010 had been transferred from E. coli into the two new hosts and maintained staby in the exconjugants. Meanwhile, partial deletions of RSF1010 replicon loosing its antibiotics resistance makers were readily detected in E. coli. The implenmentation of this observation was discussed.