Natural plasmid pSD89 (Cmr) from Salmonella derby K89, which increases the radiation tolerance of Escherichia coli strain K-12]

Several plasmids with molecular mass of 1.3-9 MDa were found in a clinical isolate of Salmonella derby K89 by electrophoresis in agarose gel. One of these plasmids, designated pSD89 (Cmr), was derived from the K89 strain via transformation of the plasmidless recipient S. derby K82 to chloramphenicol resistance. The plasmid-carrying strain K89 and the K82 strain completely cured of plasmids were equally sensitive to the lethal action of UV light, whereas the plasmid-carrying strain was even more sensitive to ionizing radiation than the plasmidless variant. Nevertheless, transformants carrying only plasmid pSD89 (Cmr) were found to be more resistant to gamma-rays and UV light than the recipient. By using an intermediate host Escherichia coli Z80 (r-m+), plasmid pSD89 (Cmr) was introduced into different E. coli K-12 strains: polA-, recA-, uvrA-, umuC-, and the wild-type strain. A slight increase in radioresistance of E. coli wild-type cells and a significant complementation of a repair defect in recA and polA mutants, but not in uvrA and umuC, were observed.     

R-factor from the natural strain of Salmonella derby carrying a DNA-polymerase gene

A R-factor which determines multiple stability to antibiotics (Cm, Pn, Sm) was found in a Salmonella derby strain isolated from the clinical material. The plasmid was eliminated by treatment with ethidium bromide; the DNA-polymerase activity in the antibiotic-sensitive derivatives measured under conditions optimal for DNA-polymerase I from E. coli was found to be decreased 10-50-fold. Plasmid DNA of S. derby K89 was fractionated by electrophoresis in agarose gel; individual zones I-IV were obtained, using a preparative technique. Upon transformation of S. derby K82 pol- cells, only plasmid DNA in zone II (designed as pSD Cm pol) gave Cm-resistant transformants, in which the DNA-polymerase activity decreased to the normal level. The experimental results pont to the binding of the DNA-polymerase gene to the S. derby plasmid.     

R plasmids with thermosensitive transferability in Salmonella strains isolated from humans.

Temperature dependence of transfer was examined with ten R plasmids originating from clinical isolates of Salmonella. Six of the plasmids were thermosensitive upon transfer, five of which were originally harbored in S. typhimurium and the remaining one in S. derby. One of these plasmids, pNR502, which conferred resistance to kanamycin, streptomycin (Sm) and tetracycline (Tc) on its host was stably maintained both in Salmonella and Escherichia coli at either 30, 37, or 43 C. Another plasmid, pNR516, which was resistant to chloramphenicol, sulfathiazole, Sm and Tc, was slightly unstable only at 43 C. The remaining four plasmids, pNR503, pNR510, pNR512 and pNR514, conferred resistance to Sm and Tc. Of these plasmids, the former two were stably maintained at both 30 and 37 C, but were unstable at 43 C. The latter two were slightly unstable at the lower temperatures and considerably unstable at 43 C. Kinetics of the transfer of the plasmid pNR503 revealed that the efficiency of transfer of the plasmid between E. coli strains was affected not only by the temperature of the conjugation but also by the preincubation temperature of the donor culture before the conjugation.     

Interrelation of the sensitivity of Salmonella derby cells to phage dp8 with the R plasmid of Salmonella derby

In the process of studying phage--Salmonella derby plasmid interaction effect, it has been found that the absence of the plasmid blocks the adsorption step and the step of phage penetration into the cell. However, the lytic functions and those responsible in maintenance of the lysogenic state are independent of plasmid. Probably, resistance of plasmidless cells of S. derby to extracellular dp8 phage is mediated by the absence of plasmid-borne genes in S. derby coding for membrane peptides which are indispensible for adsorption and DNA penetration into the cell.     

Molecular epidemiology of plasmid patterns in Shigella dysenteriae type I obtained from an outbreak in West Bengal (India)

Abstract Multiple antibiotic-resistant Shigella dysenteriae type 1 isolates from a recent epidemic in West Bengal (India) showed identical plasmid patterns. All isolates were resistant to ampicillin (Am), chloramphenicol (Cm), tetracycline (Tc), streptomycin (Sm) and trimethoprim (Tp) and contained 6 plasmids, ranging from 2.5–120 kb. The Am resistance determinant was located on the 120 kb plasmid. This plasmid was unstable when the S. dysenteriae strains were grown above 37°C. The Bangladesh strains of S. dysenteriae type 1 showed identical plasmid patterns, except that many isolates were Am-sensitive and lacked the 120 kb plasmid. In strains from both Bangladesh and West Bengal, predominantly group-B plasmids conferred resistance to Cm and Tc. Comparisons of Eco R1 fragments generated from the total plasmid DNA content of each strain support the view that the plasmids present in the S. dysenteriae type 1 strains isolated from all recent epidemics in India and Bangladesh were identical.     

Molecular-genetic organization of plasmid R89S of the incompatibility group Q

A new IncQ plasmid R89S has been analysed by molecular-genetic methods. A restriction map of this plasmid has been constructed and regions of homology with the plasmid RSF1010 have been identified. A genetic map of the plasmid R89S has been prepared based on the deletion and insertion plasmid derivatives. The phenotypic analysis of the derivatives has identified the location of genes coding for replication, incompatibility, mobilization for genetic transfer and resistance to streptomycin in the genome of R89S.     

Effect of R plasmid on cell membrane structure in Salmonella derby]

The structure of membranes of Salmonella derby cells both containing R-plasmid and free of plasmid was studied by small- and large-angle X-ray diffraction. Reflections with interplane distances of 8 and 11 A were detected, which are typical of plasmid-carrying S. derby cells. These reflections are assumed to be due to equidistant well ordered positions of the polar groups of phosphatidylcholine and phosphatidylethanolamine molecules on membrane surface. It is also suggested that the formation of these structures is determined by peculiar hydrophilic-hydrophobic interactions of the phospholipid in membranes.     

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.     

Pseudomonas aeruginosa plasmids that control streptomycin resistance]

Wide distribution of streptomycin resistance determinants (83 per cent) among the resistance plasmids of the clinical strains of Ps. aeruginosa isolated in several clinics of 2 towns was found. Nine plasmids determining resistance to this antibiotic, as well as some other antibiotics, sulfanilamides, metallic ions, hydroxyanions and UV radiation were studied. The frequency of the conjugation transfer in these plasmids was different, i.e. from 10(1) to 10(6). They belonged to the following incompatibility groups: P-1, P-2, P-5 and apparently P-3. Eight out of the 9 plasmids determined the synthesis of streptomycin phosphotransferase which was evident of wide distribution of the streptomycin inactivation mechanism by phosphorylation among the strains of Ps. aeruginosa. The strains carrying the plasmids significantly differed by the content of the enzyme. However, all the enzymes could inactivate only streptomycin and dihydrostreptomycin and had approximately the same molecular weight (about 20 000). The strain carrying plasmid pBSII had no enzyme inactivating streptomycin (by phosphorylation or adenylation). The antibiotic resistance determined by this plasmid must be connected with changes in permeability of the bacterial cell wall by streptomycin.     

Transposon Mutagenesis and Excision of R' Plasmids by Conjugative, Chimeric Plasmid pUW942 in Extra-Slow-Growing Rhizobium japonicum Strains

Transposons Tn501 (specifying mercury resistance) and Tn7 (specifying resistance to trimethoprim and streptomycin) were introduced into extra-slow-growing Rhizobium japonicum by conjugal transfer of the 82 kilobase chimeric plasmid pUW942. Mercury-resistant transconjugants were obtained at a frequency of 10 to 10. The transfer frequency of streptomycin resistance was lower than that of mercury resistance, and Tn7 was relatively unstable. pUW942 was not maintained as an autonomously replicating plasmid in R. japonicum strains. However, some of the Hg transconjugants from the RJ19FY, RJ17W, and RJ12S strains acquired antibiotic markers of the vector plasmid pUW942. Southern hybridization of plasmid and chromosomal DNA of R. japonicum strains with P-labeled pUW942 and pAS8Rep-1, the same plasmid as pUW942 except that it does not contain Tn501, revealed the formation of cointegrates between pUW942 and the chromosome of R. japonicum. More transconjugants with only Tn501 insertions in plasmids or the chromosome were obtained in crosses with strains RJ19FY and RJ17W than with RJ12S. These retained stable Hg both in plant nodules and under nonselective in vitro growth conditions. One of the RJ19FY and two of the RJ12S Hg transconjugants with vector plasmid-chromosome cointegrates conjugally transferred plasmids of 82, 84 or 86, and 90 kilobases, respectively, into plasmidless Escherichia coli C. These plasmids strongly hybridized to pUW942 and EcoRI digests of total DNA of each respective R. japonicum strain but not to indigenous plasmid DNA of the R. japonicum strains. These R' plasmids consisted of pUW942-specific EcoRI fragments and an additional one or two new fragments derived from the R. japonicum chromosome.     

Development of high-level streptomycin resistance affected by a plasmid in lactic streptococci

Some lactose-negative (Lac-) mutants of Streptococcus lactis C2 and ML3 exhibited development of very high level streptomycin resistance after incubation with subinhibitory concentrations of the drug for 18 to 22 h. These drug-resistant mutants showed no loss of resistance even after 6 months of subculturing in broth without any drug. The parental Lac+ strains did not show mutation to high-level streptomycin resistance. The Lac+ characteristic of the parental strain was conjugally transferred to Lac- derivatives of C2 and ML3, showing the ability to mutate to high-level resistance. When transconjugants were analyzed for this characteristic, they showed both mutable and nonmutable Lac+ types. The results suggested that genetic information for mutation to high-level streptomycin resistance in lactic streptococci resides on the chromosome, and its expression is affected by a plasmid. The plasmid profiles of strains C2, ML3, C2 Lac-, ML3 Lac-, and two kinds of transconjugants confirmed the presence of a plasmid of approximately 5.5 megadaltons in strains showing no mutation to high-level streptomycin resistance, while strains missing such a plasmid exhibited high-level streptomycin resistance after incubation with subinhibitory concentrations of the drug.     

Development of high-level streptomycin resistance affected by a plasmid in lactic streptococci.

Some lactose-negative (Lac-) mutants of Streptococcus lactis C2 and ML3 exhibited development of very high level streptomycin resistance after incubation with subinhibitory concentrations of the drug for 18 to 22 h. These drug-resistant mutants showed no loss of resistance even after 6 months of subculturing in broth without any drug. The parental Lac+ strains did not show mutation to high-level streptomycin resistance. The Lac+ characteristic of the parental strain was conjugally transferred to Lac- derivatives of C2 and ML3, showing the ability to mutate to high-level resistance. When transconjugants were analyzed for this characteristic, they showed both mutable and nonmutable Lac+ types. The results suggested that genetic information for mutation to high-level streptomycin resistance in lactic streptococci resides on the chromosome, and its expression is affected by a plasmid. The plasmid profiles of strains C2, ML3, C2 Lac-, ML3 Lac-, and two kinds of transconjugants confirmed the presence of a plasmid of approximately 5.5 megadaltons in strains showing no mutation to high-level streptomycin resistance, while strains missing such a plasmid exhibited high-level streptomycin resistance after incubation with subinhibitory concentrations of the drug.     

Spreading of streptomycin antibiotic resistance gene in Escherichia coli plasmids demonstrated by Southern blot analysis

Plasmids from E. coli strains of 38 donors were transconjugated to common recipient SY663 Escherichia coli K12. The restriction patterns of the isolated plasmids were highly heterogenous. However, the streptomycin (Sm) resistance genes of the plasmids were identical or closely homologous in 29 of the 33 plasmids conferring Sm resistance. These data were based on Southern blot analysis, using the Sm resistance gene (encoding aminoglycoside phosphoryl transferase) as probe cut out from pBP1 plasmid. Our data suggest an extensive spreading of streptomycin resistance gene of this type.     

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.     

Physicochemical properties of the pKMR plasmids controlling antibiotic resistance and the capacity to produce colonization antigen

KMR plasmids controlling antibiotic resistance and the capacity for production of the colonization antigen were identified in wild strains of E. coli (026, 0126, 0124) and S. sonnei isolated from patients with acute intestinal diseases. The strains of E. coli 026 and E. coli 0126 carried p KMR207-1 plasmid determining resistance to chloramphenicol and tetracycline and the adhesive properties. The molecular weight of the plasmid is 98 mD. The strain of S. sonnei carried p KMR 208-1 plasmid responsible for resistance to streptomycin, chloramphenicol and tetracycline and the adhesive properties. The molecular weight of this plasmid is 98 mD. The resistance to streptomycin and tetracycline and the capacity for the synthesis of the colonization antigen in E. coli 0214 was controlled by p KMR209 plasmid with the molecular weight of 2.66 mD. The restriction analysis suggests that p KMR207a-1 and p KMR 207b-1 plasmids detected in E. coli of different serotypes were identical, since they could be broken with BamH1 endonuclease into equal numbers of fragments similar by their molecular weights. p KMR207-1 and p KMR208-1 plasmids differed in their sensitivity to BamH-1 endonuclease. However, they were broken into 6 fragments similar by their molecular weights. p KMR207-1 and p KMR208-1 plasmids are probably closely related but not identical.     

Study of the transmissibility of multiple drug resistance and the capacity to ferment lactose in a clinical strain of Kl. pneumoniae]

The donor properties of K. pneumoniae PI 220 with multiple drug resistance were studied. It was shown that the above strain carried 2 plasmids, i.e. R-plasmid pPI 220 controling resistance to ampicillin, streptomycin, tetracycline, chloramphenicol, kanamycin and sulphanylamides and plasmid pPI 221 controlling lactose fermentation. Both plasmids can be transfered on conjugation to strain E. coli P678 at a temperture of 28 degrees C at a rate of 10(-5) for pPI 220 and 10(-4) for pPI 221. The drug resistance controlled by pPI 221 was transfered mainly in a "blocks" simultaneously to 6 drugs. Deletion of plasmid pPI 220 was observed rarely. The donor properties of the strain were defined by the conjugative plasmid pPI 220 controlling the self-transfer and mobilization of plasmid pPI 221 incapable of the self-transfer. E. coli P678 (pPI 220) (PPI 221) acquired the donor properties and transfered both plasmids to E. coli J62 on crossing simultaneously at a rate of 10(-2), as well as to S. typhimurium LT2 and P. rettgeri at a rate of 10(-5). In all the recipient strains studied the transfered plasmids were unstable and segregated also simultaneously at a rate being the highest for P. retgari PI 230. The clones with stable preservation of the plasmids could be obtained by selection.     

Antibiotic resistance study of clinical strains of Pseudomonas aeruginosa]

The study of 40 clinical strains of Ps. aeruginosa isolated from the wound surfaces of the patients showed that all the isolates were resistant to one or several antibiotics. The number of the strains resistant to 5, 4, 3, 2 or 1 drug was 5, 22.5, 25. 30 or 17.5 per cent respectively. Fifteen strains carried resistance plasmids capable of conjugative transfer. Eleven out of 21 plasmids controlled resistance to chloramphenicol, 7 plasmids controlled resistance to streptomycin and sulfanylamides, 1 plasmid controlled resistance to streptomycin and chloramphenicol. The presence of two types of the plasmids controlling resistance to chloramphenicol and streptomycin + sulfanylamides respectively was found. All the plasmids proved to be capable of conjugative transfer between the strains of Ps. aeruginosa ML (PAO). The frequency of the plasmid conjugative transfer in such crosses ranged from 10(-6) to 10(-3). Most of the plasmids belonged to the incompatibility groups P-2 and P-7. One plasmid belonged to the incompatibility group P-5. It should be noted that about a half of the plasmids (11 out of 21) belonged to the incompatibility group P-7 which up to the present time was conditional, since was represented by a single plasmid Rms 148.     

A new IncQ plasmid R89S: Properties and genetic organization

The new small (8.18 kb) streptomycin-resistant multicopy plasmid R89S of the Q group incompatibility is described. In contrast to other IncQ plasmids, replication of R89S is dependent on DNA polymerase 1 and proceeds in the absence of de novo protein synthesis. According to our data up to now, the host spectrum of the plasmid R89S is limited to Enterobacteriaceae. A genetic map of the plasmid R89S has been prepared through the construction of deletion and insertion derivatives. Phenotypic analysis of these derivatives has identified the location of genes encoding resistance to streptomycin, and the region essential for mobilization of R89S. The origin of vegetative replication has been located within a 0.7-kb fragment. Another region highly homologous to oriV of the plasmid RSF1010, but not functioning as an origin of replication, was localized. Two regions involved in the expression of incompatibility have also been identified. The data from the restriction analyses, DNA-DNA hybridization, and genetic experiments enable us to assume that the plasmid R89S is a naturally occurring recombinant between part of an IncQ plasmid and another narrow host range replicon of unknown incompatibility group.     

The ultrastructure of plasmid-containing and plasmid-free Salmonella derby cells

The comparative electron-microscopic study of S. derby plasmid-containing and plasmid-free cells has revealed certain differences in their structures: These structural differences are always accompanied by changes in the form and size of the cells, the form of the cell wall with the appearance of fimbria-like processes, depending on the presence or absence of S. derby R-plasmid in the cells. These differences in the morphology and ultrastructure of S. derby cells, associated with the R-plasmid, are of interest in the study of molecular mechanisms of plasmid action on the development of various forms of whole cells and individual bacterial structures, which play an important role in the cell function.