R plasmid gene dosage effects in Escherichia coli K-12: copy mutants of the R plasmic R1drd-19.

Copy mutants of the R plasmid R1drd-19 were used to study gene dosage effects in Escherichia coli K-12. The specific activity of β-lactamase, chloramphenicol acetyltransferase, and streptomycin adenylylase, as well as ampicillin resistance, increased linearly with the gene dosage up to a level at least tenfold higher than that of the wild-type plasmid. This makes it possible to use ampicillin resistance to determine plasmid copy number and also to select for plasmid copy mutants with defined copy number. Chloramphenicol resistance, despite the increase in enzyme activity, reached a plateau level at a gene dosage less than twice that of the wild-type plasmid, presumably due to the high energy demand on the cells during inactivation of the antibiotic by acetylation with acetyl-coenzyme A. Similarly, resistance to streptomycin plateaued at a gene dosage about three times that of the wild-type plasmid, presumably because of a decreased efficiency of the cells' outer penetration barriers when carrying the R plasmid. The susceptibility of the cells to rifampicin was increased by the presence of plasmid copy mutants.     

Origin of Haemophilus influenzae R factors.

The Haemophilus influenzae R plasmids specifying resistance against one, two, or three antibiotics which have emerged in different parts of the world were shown to have closely related but not identical plasmid cores. The gene for ampicillin resistance in the H. influenzae plasmid pKRE5367 is part of a transposon similar to Tn3, which was transposed from pKRE5367 onto RSF1010 in Escherichia coli. An indigenous H. influenzae plasmid (pW266) was isolated. Its properties correspond to those of the H. influenzae R plasmids, except for the presence of a drug resistance transposon. The in vitro-generated H. influenzae R plasmids carrying an ampicillin resistance transposon, a tetracycline resistance transposon, and a transposon for combined tetracycline-chloramphenicol resistance resembled the natural isolates. The findings support the hypothesis that the R plasmids of H. influenzae are of multiclonal evolutionary origin.     

Mutations of temperature sensitivity in R plasmid pSC101.

Temperature-sensitive (Ts) mutant plasmids isolated from tetracycline resistance R plasmid pSC101 were investigated for their segregation kinetics and deoxyribonucleic acid (DNA) replication. The results fit well with the hypothesis that multiple copies of a plasmid are distributed to daughter cells in a random fashion and are thus diluted out when a new round of plasmid DNA replication is blocked. When cells harboring type I mutant plasmids were grown at 43 degrees C in the absence of tetracycline, antibiotic-sensitive cells were segregated after a certain lag time. This lag most likely corresponds to a dilution of plasmids existing prior to the temperature shift. The synthesis of plasmid DNA in cells harboring type I mutant plasmids was almost completely blocked at 43 degrees C. It seems that these plasmids have mutations in the gene(s) necessary for plasmid DNA replication. Cells haboring a type II mutant plasmid exhibited neither segregation due to antibiotic sensitivity nor inhibition of plasmid DNA replication throughout cultivation at high temperature. It is likely that the type II mutant plasmid has a temperature-sensitive mutation in the tetracycline resistance gene. Antibiotic-sensitive cells haboring type III mutant plasmids appeared at high frequency after a certain lag time, and the plasmid DNA synthesis was partially suppressed at the nonpermissive temperature. They exhibited also a pleiotrophic phenotype, such as an increase of drug resistance level at 30 degrees C and a decrease in the number of plasmid genomes in a cell.     

Relationships among some R plasmids found in Haemophilus influenzae.

Tetracycline resistance in a strain of Haemophilus influenzae isolated in the United Kingdom was found to be determined by an apparently non-selftransmissible plasmid of 31 X 10(6) daltons (31 MDal), designated pUB701. Deoxyribonucleic acid hybridization studies indicated that pUB701 shares about 70% base sequence homology with the 30-MDal ampicillin resistance R plasmid RSF007 isolated in the United States from H. influenzae, and 64% sequence homology with the 38-MDal tetracycline and chloramphenicol resistance R plasmid pRI234, isolated in the Netherlands. Heteroduplex studies between RSF007 and pUB701 confirmed the fact that these plasmids were largely homologous, except that pUB701 contained the tetracycline resistance transposon TnD, whereas RSF007 contained the ampicillin resistance transposon TnA. A strain of H. parainfluenzae resistant to both chloramphenicol and tetracycline carried two species of plasmid deoxyribonucleic acid of 2.7 and 0.75 MDal. We were unable to prove that either resistance was plasmid-borne in this strain. Hybridization studies with a [3H]thymine-labeled tetracycline resistance enteric plasmid suggested that the tetracycline transposon was integrated into the chromosome of H. parainfluenzae UB2832. We conclude either that the strains we studied received R factors of the same incompatibility group bearing different resistance genes, or that different resistance genes were translocated to a commom resident plasmid of H. influenzae.     

Optimal conditions for genetic transformation of the cyanobacterium Anacystis nidulans R2

Under optimal conditions, the cyanobacterium Anacystis nidulans R2 was transformed to ampicillin resistance at frequencies of greater than 10(7) transformants per microgram of plasmid (pCH1) donor DNA. No stringent period of competency was detected, and high frequencies of transformation were achieved with cultures at various growth stages. Transformation increased with time after addition of donor DNA up to 15 to 18 h. The peak of transformation efficiency (transformants/donor molecule) occurred at plasmid concentrations of 125 to 325 ng/ml with an ampicillin resistance donor plasmid (pCH1) and 300 to 625 ng/ml for chloramphenicol resistance conferred by plasmid pSG111. The efficiency of transformation was enhanced by excluding light during the incubation or by blocking photosynthesis with the electron transport inhibitor 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) or the uncoupler carbonyl cyanide-m-chlorophenyl hydrazone. Preincubation of cells in darkness for 15 to 18 h before addition of donor DNA significantly decreased transformation efficiency. Growth of cells in iron-deficient medium before transformation enhanced efficiency fourfold. These results were obtained with selection for ampicillin (pCH1 donor plasmid)- or chloramphenicol (pSG111 donor plasmid)-resistant transformants. Approximately 1,000 transformants per microgram were obtained when chromosomal DNA from an herbicide (DCMU)-resistant mutant was used as donor DNA. DCMU resistance was also transferred to recipient cells by using restriction fragments of chromosomal DNA from DCMU-resistant mutants. This procedure allowed size classes of fragments to be assayed for the presence of the DCMU resistance gene.     

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.     

Molecular and genetic studies of an R factor system consisting of independent transfer and drug resistance plasmids

Certain genetic, structural, and biochemical properties of a class 2 R-factor system consisting of the conjugally proficient transfer plasmid I and the naturally occurring non-conjugative tetracycline (Tc) resistance plasmid 219 are reported. I and 219 exist as separate plasmid deoxyribonucleic acid (DNA) species in both Escherichia coli and Salmonella panama, having molecular weights of 42 x 10(6) and 5.8 x 10(6), respectively. The buoyant densities of I and 219 are 1.702 and 1.710 g/cm(3), respectively, in neutral cesium chloride. Although the Tc resistance plasmid is not transmissible in a normal conjugal mating, it is mobilized in a three-component mating by plasmid I and by certain other conjugative plasmids of the fi(+) or fi(-) phenotype. Mobilization does not appear to involve intermolecular recombination between plasmids, and no covalent linkage of resistance markers and fertility functions is observed. Transformation of CaCl(2)-treated E. coli by plasmid DNA is shown to be a useful procedure for studying the biological properties of different plasmid molecular species that have been fractionated in vitro, and for selectively inserting non-self-transmissible plasmids into specific bacterial strains. The effects of tetracycline on the rate of protein synthesis carried out by plasmid 219 were studied by using isolated E. coli minicells into which this plasmid had segregated. Consistent with the results of earlier investigations showing the inducibility of plasmid-mediated Tc resistance in E. coli, the antibiotic was observed to stimulate protein synthesis in minicells carrying the plasmid 219 and totally inhibit (3)H-leucine incorporation by minicells lacking the Tc resistance marker. Five discrete polypeptide species were synthesized by minicells carrying plasmid 219; exposure of minicells or parent bacteria to Tc resulted in specific and reproducible changes in polypeptide synthesis patterns.     

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.     

Restriction endonuclease mapping of the HI2 incompatibility group plasmid R478.

A restriction map of the 272-kb IncHI2 plasmid R478 was constructed by using the enzymes ApaI, XbaI, SalI, and XhoI. The map was derived from cloned restriction fragments from R478 inserted into cosmid and plasmid vectors as well as from double-digestion analysis of R478 and R478 miniplasmids. All previously known resistance determinants were cloned from R478, and their positions were located on the restriction map. A region involved in incompatibility was cloned and mapped. The location of a previously unreported arsenite resistance gene was also determined. The genes encoding tellurite resistance, colicin B resistance, and phage inhibition were found to be associated with a 6.7-kb SalI fragment of R478.     

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.     

Genetic organization of the broad-host-range IncP-1 plasmid R751.

We have identified regions encoding conjugal transfer, plasmid maintenance, and trimethoprim resistance on the IncP-1 plasmid R751 by complementation tests with cloned deoxyribonucleic acid fragments and self-replicating derivatives constructed in vitro. The genes for replication and transfer show a scattered organization similar to that previously determined for RK2, another IncP-1 plasmid. Derivatives of RK2 are able to complement R751 derivatives defective in these functions. Restriction enzyme cleavage sites in R751 deoxyribonucleic acid are clustered in regions of the plasmid physical map. Neither region is required for plasmid maintenance or transfer, although one determines resistance to trimethoprim. A similar clustering of cleavage sites is seen with RK2, which nevertheless has a very different restriction map.     

Isolation and preliminary characterization of R6K plasmid deletion mutants.

Several deletion mutants of R6K have been isolated by mutagen treatment of bacterial host carrying wild type of the plasmid and search for clones that lost ampicillin or streptomycin resistance. The molecular weight of the mutants as estimated by agarose gel electrophoresis was 15 times 10(6) minus 23 times 10(6) compared to 26 times 10(6) for the parental plasmid. The mutants were characterized in respect of the level of resistance to ampicillin and frequency of conjugational transfer. Some of the mutants were found to differ in Eco RI digestion pattern from the wild type.     

Isolation of a nontransmissible antibiotic resistance plasmid by transductional shortening of R factor RP1.

A plasmid segregant carrying tetracycline and carbenicillin resistance markers has been isolated from R factor RP1 by transductional shortening with phage P22. The new plasmid RP1-S2, which has a molecular weight of 23 times 10-6, has lost the transfer, phage sensitivity, and neomycin resistance functions of RP1. It combines readily with a W group plasmid, R388, to form a transmissible carbenicillin and trimethoprim resistance plasmid, RWP1.     

Convenience of plasmid R6K higher-copy-number deletion derivative for cloning of larger DNA fragments

Vector properties of plasmid pNH602, a higher-copy-number deletion mutant of plasmid R6K, were tested by cloning the 6.5 Mg/molBam HI pSa fragment carrying determinants of resistance to four antibiotics in the uniqueBam HI site of pNH602. The resultingin vitro constructed recombinant plasmid pNH606 was found to be stable, conjugative, multicopy (20 copies of pNH606 perE. coli chromosome were estimated) and to ensure the increased expression of different genes responsible for the antibiotic resistance. The pSa fragment inserted in theBam HI site of plasmid pNH602 (located in Tn2660) was proved to be transposable to other replicons. Recombinant plasmid pNH606 was analyzed using restriction enzymesBam HI andEco RI and its physical and genetic map was constructed.     

Transduction of R plasmids by bacteriophages P1 and P22

Summary We demonstrated that bacteriophages P1 and P22 were able to form various types of hybrids with six out of seven different R plasmids tested. When the same R plasmid was used for isolation, P1-R hybrids usually carried more resistance markers than P22-R. Several genetical observations suggest that the hybrid prophages carried the resistance markers transposed to the phage genomes without loss of essential phage genes. Upon UV-irradiation the prophages produced phage lysates that transduced the relevant resistance markers at high frequencies by lysogenic conversion. The insertion of the resistance markers was even acquired by the P1 or P22 genomes during one-step growth in R⁺ cells. Some lytically prepared lysates grown on R⁺ cells contained the hybrids at a frequency of 10^-7 to 10^-6/plaqueforming unit. Analysis of P1-transductants for resistance markers of the R plasmids revealed in some cases more specialized transductants than generalized transductants. These results strongly indicate that a precise genetic map of an R plasmid can not be established only on the basis of co-transduction frequencies of the resistance markers of the R plasmid.     

Host Range and Properties of the Pseudomonas aeruginosa R Factor R1822

R1822, a plasmid specifying multiple drug resistances, has been transferred to a variety of species representative of related and unrelated genera. The host range of the plasmid includes Enterobacteriaceae, soil saprophytes, Neisseria perflava, and photosynthetic bacteria. With the acquisition of drug resistance(s), these strains became sensitive to a small, ribonuclease-sensitive bacteriophage, designated PRR1, isolated by enrichment from sewage.     

Plasmid R1 in Salmonella typhimurium: Molecular instability and gene dosage effects

Plasmid R1 drd-19 and two of its copy mutants (pKN102 and pKN103) were transferred from Escherichia coli to Salmonella typhimurium, where the expression of the copy mutations was studied further. The copy number (ratio of plasmid DNA to chromosomal DNA) was the same in S. typhimurium and in E. coli. The activities of the plasmid-coded antibiotic-metabolizing enzymes β-lactamase, chloramphenicol acetyltransferase, and streptomycin adenylyltransferase as well as the resistances to ampicillin and streptomycin were proportional to the gene dosage up to at least a threefold increase in the steady state plasmid copy number, whereas resistance to chloramphenicol showed no increase with increased number of plasmid copies per chromosome equivalent. Also the resistance to rifampicin was affected since S. typhimurium cells became more sensitive the higher the copy number of the resident plasmid. Furthermore, plasmid R1 showed molecular instability in S. typhimurium cells since there was a tendency to dissociate into resistance transfer factors and resistance determinants and also to form miniplasmids. This tendency to instability was more pronounced the higher the plasmid copy number.     

Expression of the gene for tetracycline resistance of plasmids R6 and RP4 in bacteria of the family Enterobacteriaceae]

It was found that manifestation of the tetracycline resistance gene depended on the type of the plasmid containing the gene. The tetracycline resistance gene was subject to less repression in plasmid R6 than in plasmid RP4. Sensitivity of the initial plasmid-free bacteria varied within lower dose ranges than that of the plasmid-carrying strains. Regulation of the tetracycline resistance gene manifestation in the given plasmid may change in different bacterial hosts, i.e. in different cytoplasmic environment at different gene background.     

Genetic properties of the Salmonella enteritidis R404 plasmid aggregate. II. Separation of plasmids by transformation.

Transformational separation of plasmids from R404 plasmid aggregate found in Salmonella enteritidis strain was performed. Three classes of transformants differing in their resistance patterns were isolated. Genetic properties of the transformants suggest that their resistance is determined by single plasmids. Plasmid pCK3 (Tra-ApCbCrSuSm) and pCK4 (Tra-ApCbCrCm) are nonconjugative while plasmid pCG1 (TraApCbCrSuSmTcKmNm) is conjugative. Separation of all plasmids of R404 plasmid aggregate allowed to determine their genetic properties and the manner of conjugational transfer of R404 plasmid aggregate R-determinants.     

Mobilization and location of the genetic determinant of chloramphenicol resistance from Lactobacillus plantarum caTC2R.

The mobilization of a nonconjugative plasmid (pCaT) that mediates chloramphenicol resistance in Lactobacillus plantarum caTC2R was achieved by comobilization with the conjugative plasmid pAM beta 1. The conjugation studies confirmed that the 8.5-kb pCaT in L. plantarum caTC2R contains the gene responsible for chloramphenicol resistance and that the plasmid has several unique restriction sites which make it useful for genetic studies in Carnobacterium spp. Cloning studies showed that the gene responsible for chloramphenicol resistance is located in the 2.6-kb EcoRV-SalI region of pCaT. This was confirmed by probing the 3.0-kb BglII fragment of pCaT with a biotin-labeled 1.6-kb BstEII-HpaII fragment from the streptococcal-derived plasmid pVA797(Cmr). Expression of chloramphenicol resistance in Carnobacterium as well as in other Lactobacillus species was achieved by electrotransformation using donor DNA from pCaT.