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.     

Plasmids with temperature-dependent copy number for amplification of cloned genes and their products

Miniplasmids (pKN402 and pKN410) were isolated from runaway-replication mutants of plasmid R1. At 30°C these miniplasmids are present in 20–50 copies per cell of Escherichia coli, whereas at temperatures above 35°C the plasmids replicate without copy number control during 2–3 h. At the end of this period plasmid DNA amounts to about 75% of the total DNA. During the gene amplification, growth and protein synthesis continue at normal rate leading to a drastic amplification of plasmid gene products. Plasmids pKN402 (4.6 Md) and pKN410 (10 Md) have single restriction sites for restriction endonucleases EcoRI and HindIII; in addition plasmid pKN410 has a single BamHI site and carries ampicillin resistance. The plasmids can therefore be used as cloning vectors. Several genes were cloned into these vectors using the EcoRI sites; chromosomal as well as plasmid-coded β-lactamase was found to be amplified up to 400-fold after thermal induction of the runaway replication. Vectors of this temperature-dependent class will be useful in the production of large quantities of genes and gene products. These plasmids have lost their mobilization capacity. Runaway replication is lethal to the host bacteria in rich media. These two properties contribute to the safe use of the plasmids as cloning vehicles.     

Temperature-dependent and amber copy mutants of plasmid R1drd-19 in Escherichia coli.

The isolation of conditional mutants with an altered copy number of the R plasmid R1drd-19 is described. Temperature-dependent as well as amber-suppressible mutants were found. These mutant plasmids have been named pKN301 and pKN303, respectively. Both types of mutations reside on the R plasmid. No difference in molecular weight could be detected by neutral sucrose gradient centrifugation for any of the mutant plasmids when compared with the wild-type plasmid. The number of copies of the plasmids was determined by measurement of the specific activity of the R plasmid-mediated β-lactamase and by measurement of covalently closed circular (CCC) DNA in alkaline sucrose gradients and dye-CsCl density gradients. Below 34 °C the temperature-dependent mutant, pKN301, had the same copy number as the wild type, while this was four times that of the wild type above 37 °C. The amber mutant pKN303 had a copy number indistinguishable from that of the wild-type plasmid in a strain containing a strong amber suppressor and a copy number about five times that of the wild-type plasmid in a strain lacking an amber suppressor. In a strain containing a temperature-sensitive amber suppressor, the amber mutant's copy number increased with the decrease in amber suppressor activity. Thus, the existence of the temperature-dependent and the amber-suppressible R-plasmid copy mutants indicates that the system that controls the replication of plasmid R1drd-19 contains an element with a negative function and that this element is a protein.     

Plasmid pGB301, a new multiple resistance streptococcal cloning vehicle and its use in cloning of a gentamicin/kanamycin resistance determinant

Summary Streptococcal plasmid pGB301 is an in vivo rearranged plasmid with interesting properties and potential for the molecular cloning of genes in streptococci. Transformation of S. sanguis (Challis) with the group B streptococcal plasmid pIP501 (29.7 kb) gave rise to the deletion derivative pGB301 (9.8 kb, copy number 10) which retained the multiple resistance phenotype of its ancestor (inducible MLS-resistance, chloramphenicol resistance). Among the eight restriction endonucleases used to physically map pGB301 were four that cleaved the plasmid at single sites yielding either sticky (HpaII, KpnI) or bluntends (HpaI, HaeIII/BspRI). Passenger DNA derived from larger streptococcal plasmids (pSF351C61, 69.5 kb; pIP800, 71 kb) was successfully inserted into the HpaII site and, by blunt-end cloning, into the HaeIII/BspRI site. The gentamicin/kanamycin resistance gene of pIP800 was expressed by recombinant plasmids carrying the insert in either orientation. Insertion of passenger DNA into the HaeIII/BspRI site (but not the HpaII site) caused instability of adjacent pGB301 sequences which were frequently deleted, thereby removing the chloramphenicol resistance phenotype. The vector pGB301 has a remarkable capacity for passenger DNA (inserts up to 7 kb) and the property of instability and loss of a resistance phenotype following insertion of passenger DNA into the HaeIII/BspRI site should facilitate the identification of cloned segments of DNA when using this plasmid in molecular cloning experiments.     

Klebsiella aerogenes Strain Carrying Drug-Resistance Determinants and a lac Plasmid

In addition to carrying determinants conferring resistance to at least two antibiotics, chloramphenicol and streptomycin, a Klebsiella aerogenes strain contains a plasmid responsible for increased β-galactosidase activity. The plasmid can be transferred to Escherichia coli and Salmonella typhimurium strains. K. aerogenes segregants without the plasmid grow on lactose one-half as fast as the parent strain and contain only one-tenth to one-fifth as much β-galactosidase.     

Incidence of resistance to ampicillin,chloramphenicol, kanamycin,tetracycline and trimethoprim of Salmonella strains isolated in The Netherlands during 1975–1980

From 1975–1980, about 130 000 Salmonella strains isolated from various sources were tested for resistance to ampicillin, chloramphenicol, kanamycin, tetracycline and trimethoprim. Following the ban on incorporation of tetracycline in animal feeds for nutritive purposes, tetracycline resistance in S. typhimurium and S. panama strains of porcine origin dropped from about 90% in 1974 for both species, to about 34% and 1%, respectively, in 1980. The incidence of resistance in human strains concurrently decreased from about 80% in 1974 to 25% and 1%, respectively, in 1980.The build-up of multiple resistance in bovine S. dublin and S. typhimurium strains, already started in 1973–1974, has continued. Recently, phage type 193 S. typhimurium strains have become predominant and they are invariably resistant to ampicillin, chloramphenicol, tetracycline, kanamycin, neomycin, streptomycin, sulphonamide and trimethoprim. Up to now, type 193 strains were hardly encountered in human patients, but the number of human isolates is slowly increasing.A fairly large number of multiply resistant strains belonging to S. oranienburg, S. schwarzengrund, S. typhimurium and, recently, S. krefeld have been isolated from adoptive children from the Far East.     

Mapping andfi character determination of R factors ofEnterobacter cloacae DF13

In conjugation experiments betweenEnterobacter cloacae DF13 andEscherichia coli K12, resistances against tetracycline, sulfanilamide, streptomycin, and chloramphenicol were nearly always transferred simultaneously. These properties could be transferred fromE. coli exconjugants by transduction to a drug-sensitiveE. coli K12 strain with bacteriophage P1kc. It may be inferred thatEnt. cloacae DF 13 harbours a multiple R factor, which promotes its own transfer. This R factor was found to be of thefi ⁺ type. The molecular nature of this R factor was studied by labelling the DNA of an exconjugant with³H-thymidine, careful lysis, sedimentation of the chromosomal DNA, and characterization of the circular DNA by sucrose-gradient centrifugation, equilibriumdensity centrifugation in CsCl containing ethidium bromide and by electron microscopy. By these methods the multiple R factor was identified as a circular DNA molecule with a contour length of 22.6 Μm, corresponding to a molecular weight of 45 × 10⁶ daltons. A segregant R factor harbouring resistance against tetracycline only, was found to have a contour length of 16.0 Μm and a sedimentation constant of 58 S. In addition to the multiple R factor, the wild-type strain harboured a plasmid with a sedimentation constant of 38 S, corresponding to a molecular weight of 16 × 10⁶ daltons. The function of this plasmid is unknown. After many transfers on agar slants spontaneous segregation of the R factor was observed and several types of segregants were obtained. In most segregants, resistance against streptomycin could not be transferred by conjugation and could not be mobilized by other sex factors. Some of these segregants had acquired a requirement for methionine; in these, the streptomycin-resistance determinant may be integrated into the chromosome. The resistance pattern of the various types of segregants and exconjugants allowed to draw a circular map of the R factor. The order of markers is ---tet---rtf---sul---str---cml-. After short-term conjugation experiments most exconjugants were found to have received resistance against sulfanilamides only. This resistance determinant does not promote its own transfer by conjugation but could be mobilized by other sex factors. An exconjugant become resistant against tetracycline and sulfanilamide, was found to harbour two independent plasmids of which only that carrying resistance against tetracycline promoted its own transfer. Consequently a second R factor, determining resistance against sulfanilamide alone must be present inEnt. cloacae DF13. This R factor was identified as a circular DNA molecule with a sedimentation constant of 26 S, a contour length of 2.6 Μm and a buoyant density of 1.709. From a strain harbouring the independent R(SA) plasmid and an R(TC) fragment of the multiple R factor, transductants resistant against sulfanilamide were obtained. These were found to harbour an R(SA) plasmid with properties of a defective Rfi ⁺ transfer factor. Most probably these plasmids resulted from recombination between the R(SA) plasmid and the Rtf region of the R(TC) fragment. The author published previously under the name of “G. A. Tieze”. The technical assistance of Miss J.T.M.P.A. Havermans, Mrs. A. Mak-Zuidervaart, and Mr. M. V. M. Lafleur is gratefully acknowledged. The authors thank Dr. E. F. J. van Bruggen and Dr. D. Ellens for the electronmicroscopical measurements.     

Cloning of rpsO, the gene for ribosomal protein S15 of Escherichia coli

Summary The gene for Escherichia coli ribosomal protein S15 (rpsO) was cloned on the vector pBR322 from F-prime JCH55 DNA. The recombinant plasmid was transformed to Serratia marcescens cells and it was proved that E. coli S15 was synthesized and incorporated into ribosome particles in S. marcescens cells. A DNA fragment containing rpsO was also inserted into the vector pRF3, which changes its copy number depending on the growth temperature in a temperature-sensitive polA host. By use of this recombinant plasmid it was shown that the relative synthesis rate of S15 increased about twice even when the copy number of the plasmid increased more than twenty-fold.     

Translocation of sequences encoding antibiotic resistance from the chromosome to a receptor plasmid in Salmonella ordonez

Summary Salmonella ordonez strain BM2000 carries kanamycin (Km), ampicillin (Ap), spectinomycin (Sp), chloramphenicol (Cm), tetracyline (Tc), and sulfonamide (Su) resistance and production of colicin Ib (Cib). The Km and Cib characters were carried by a 97kb IncI1 plasmid (pIP565). In addition to the Km and Cib traits, all or part of the other antibiotic resistance (R) determinants could be transferred by conjugation from S. ordonez to Escherichia coli where all the acquired characters are borne by an IncI1 plasmid, designated complete or partial composite plasmid respectively. DNA from pIP565 and composite plasmids and total DNA from strain BM2000 were studied by agarose and polyacrylamide gel electrophoresis following digestion with restriction endonucleases, and by Southern hybridization. These comparative analyses enabled us a) to show that acquisition by pIP565 of resistance to all or some of the antibiotics was due to the insertion of a single DNA fragment into the receptor plasmid; b) to detect two types of composite plasmids with regard to the specificity of insertion into pIP565 and the mapping of the inserts; c) to demonstrate that the ApCmSpSuTc resistance determinants were integrated into S. ordonez BM2000 chromosomal DNA; d) to map the restriction fragments of the translocatable sequence integrated into strain BM2000 chromosome or into pIP565.The results obtained suggest that two distinct mechanisms for the translocation of the R determinants coexist in S. ordonez BM2000. Recombination between two of the four directly repeated copies of the IS-like sequence (IS1522) present in S. ordonez chromosome leads to the circularisation of all or part of the AmCmSpSuTc R determinants and is followed by either 1) a second recombination with the copy of IS1522 in pIP565 (Type I composite plasmids), or 2) transposition of precise groups of characters in various sites of pIP565 (Type II composite plasmids).     

Discontinuity of homology of Escherichia coli and Salmonella typhimurium DNA in the lac region

Summary Partial homology of Salmonella typhimurium DNA to Escherichia coli DNA was demonstrated by Southern hybridization blots to exist on either side of the lac operon of E. coli but no homology was detected between S. typhimurium DNA and about 12 kb of E. coli DNA including the lac genes as well as about 5 kb of E. coli DNA between lac and proC. Thus portions of DNA seem to have been either added to the E. coli genome or deleted from the S. typhimurium genome since their divergence from a common ancestor. Although an IS1 element was located near the lac operon of E. coli, the insertional element was shown not to be near any of the junctures of discontinuity of E. coli - S. typhimurium homology near lac.     

Partitioning of plasmid R1 in Escherichia coli,: I. Kinetics of loss of plasmid derivatives deleted of the par region

The stability of inheritance of plasmid R1drd-19 was tested. The copy number of the plasmid was determined in two different ways: As the ratio between covalently closed circular DNA and chromosomal DNA, and by quantitative determination of single-cell resistance to ampicillin. In the latter case, strains carrying the R1 ampicillin transposon Tn3 on prophage λ was used as standard. The values were transformed to copy number per cell by using the Cooper-Helmstetter model for chromosome replication as well as by determination of chromosomal DNA per cell by the diphenylamine method. The copy number was found to be five to six per cell (or about four per newborn cell). Nevertheless, plasmid R1drd-19 was found to be completely stably inherited. This stability was shown not to be due to retransfer of the plasmid by the R1 conjugation system, since transfer-negative derivatives of the plasmid were also completely stably inherited. Smaller derivatives of plasmid R1drd-19 were found to be lost at a frequency of about 1.5% per cell generation. The copy-number control was not affected in these miniplasmids, since their copy numbers were the same as that of the full size plasmid. Quantitatively, the instability of the miniplasmids was in accord with random partitioning. It is, therefore, suggested that the plasmid R1drd-19 carries genetic information for partitioning (par) of plasmid copies at cell division, and that the par mechanism is distinct from the copy number control (cop) system. Finally, the par gene maps on the resistance transfer part of the plasmid, but far away from the origin of replication and the so-called basic replicon; this is in accord with the approximate location of the repB gene (Yoshikawa, 1974, J. Bacteriol.,, 118, 1123–1131).     

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.     

Cloning of the regulatory locus ompB of Salmonella typhimurium LT-2

Summary We have cloned the complete functional ompB locus of Salmonella typhimurium LT-2 into Escherichia coli K-12 using a cosmid vector and in vitro packaging into . The ompB locus of Salmonella was found to complement both envZ and ompR mutations in E. coli as well as an ompR mutation of Salmonella. The ompR part of the ompB locus was further subcloned into the multicopy plasmid pKN410 as a 1.3 kb fragment. This fragment coded for a single 28.5 kd protein corresponding to about 820 bp in length. Furthermore, the OmpR proteins of S. typhimurium and E. coli were shown to be structurally and functionally highly similar.Abbreviations SDS sedium dodecyl sulfate - kb kilobase pairs - bp base pairs - kd kilodaltons     

The 20 bp, directly repeated DNA sequence of broad host range plasmid R1162 exerts incompatibility in vivo and inhibits R1162 DNA replication in vitro

Summary The broad host range plasmid R1162 contains a directly repeated, 20 bp DNA sequence in the region of the plasmid required in cis for replication and maintenance. This sequence has been chemically synthesized and cloned, and shown to be sufficient for expression of plasmid incompatibility. The sequence also inhibits replication of R1162 DNA in a cell-free system. The strengths of both these effects are determined by the number of direct repeats (DRs) present, and are also affected to similar degrees by different mutations within the repeated sequence. Several of the mutations were tested for their effect in cis on plasmid maintenance in the cell, and one was found to cause an increase in plasmid copy number. The results suggest that the direct repeats exert incompatibility by inhibiting DNA replication, presumably because they are the binding sites for a limiting essential protein.Abbreviations bp base pairs - Cb^r, Km^r, Sm^r resistance to carbenicillin, kanamycin, streptomycin, respectively - DR direct repeat     

Functional complementation between chromosomal and plasmid mutagenic DNA repair genes in bacteria

Summary The umuDC operons of Escherichia coli and Salmonella typhimurium and the analogous plasmid operons mucAB and impCAB have been previously characterized in terms of their roles in DNA repair and induced mutagenesis by radiation and many chemicals. The interrelationships of these mutagenic DNA repair operons were examined in vivo in functional tests of interchangeability of operon subunits in conferring UV resistance and UV mutability phenotypes to wild-type S. typhimurium and umu mutants of E. coli. This approach was combined with DNA and protein sequence comparisons between the four operons and a fifth operon, samAB, from the S. typhimurium LT2 cryptic plasmid. Components of the E. coli and S. typhimurium umu operons were reciprocally interchangeable whereas impCA and mucA could not function with umuC in either of these species. mucA and impB could also combine to give a mutagenic response to UV. These active combinations were associated with higher degrees of conservation of protein sequence than in other heterologous gene combinations and related to specific regions of sequence that may specify subunit interactions. The dominance of the E. coli umuD44 mutation over umuD was revealed in both wild-type E. coli and S. typhimurium and also demonstrated against impCAB. Finally interspecies transfer showed that the apparently poor activity of the S. typhimurium umuD gene in situ is not the result of an inherent defect in umuD but is due to the simultaneous presence of the S. typhimurium umuC sequence. It is suggested that the limitation of umuD activity by umuC in S. typhimurium is the basis of the poor induced mutability of this organism.     

Identification of a globally distributed clinical streptomycin-resistance plasmid and other resistance determinants in a coastal bay of China

Aims: To study streptomycin‐resistant bacteria isolated from Jiaozhou Bay and their molecular determinants of resistance. Methods and Results: Twenty‐seven tetracycline‐resistant and 49 chloramphenicol‐resistant bacterial isolates from surface seawater of Jiaozhou Bay were selected for investigation. More than 88% of these isolates were resistant to streptomycin. Half of the streptomycin‐resistant bacteria harboured the strA–strB gene pair, and six isolates carried Tn5393‐like transposons by PCR detection. The p9123‐related plasmids containing the sul2–strA–strB gene cluster were characterized in two environmental Escherichia coli isolates. Transposon Tn5393 was first identified on a Klebsiella pneumoniae plasmid, which also carried Tn1721, estP and umu genes responsible for antimicrobial and insecticide resistance. Conclusions: Coresistance to streptomycin and tetracycline or chloramphenicol was found with high frequency. p9123‐related plasmid and Tn5393 transposon may contribute to the wide distribution and spread of the strA–strB gene pair in Jiaozhou Bay. The detection of streptomycin‐resistance plasmid pQ1‐1 from Jiaozhou Bay seawater bacteria and human bacterial pathogens from USA indicates its global dissemination and transmission, across different components of the microbiota on earth. Significance and Impact of the Study: Streptomycin resistance can be recognized as an important bioindicator of environmental quality, owing to its association with anthropogenic pollution and the multidrug‐resistant microbiota.     

Molecular studies of an fi+ plasmid from strains of Salmonella typhimurium

Summary Plasmid DNA has been isolated from five fi ⁺ strains of Salmonella typhimurium of independent origin, including type 36 and LT2. The mean contour length of the plasmids was between 27.3 and 29.3 m. A variant line of S. typhimurium type 36 which was fi ^- yeilded no plasmid DNA. These results support the hypothesis that the fi ⁺ property of S. typhimurium is coded by a plasmid. In S. typhimurium 36 this plasmid, designated MP10₃₆, also appears to code for restriction of non-donor-specific phages. Molecular studies indicate that superinfection of S. typhimurium 36 with the kanamycin resistance determinant K, which results in loss of the fi ⁺ property, is correlated with loss of MP10₃₆. Reassociation experiments demonstrate a high degree of homology between the DNA of all five S. typhimurium plasmids, and between MP10₃₆ and K. MP10₃₆ has some homology with F and F-like R factors, but not with plasmids of other compatibility groups. A recombinant between an ampicillin resistance determinant and MP10₃₆ is autotransferable at low frequency. The significance of these findings is discussed.     

Visualization of pathogenicity regions in bacteria

We show here how pathogenicity islands can be analysed using GenomeAtlases, which is a method for visualising repeats, DNA structural characteristics, and base composition of chromosomes and plasmids. We have applied this method to the E. coliplasmid pO157, and the Y. pestisplasmid pPCP1. In both cases pathogenic genes were shown to differ in A+Tcontent and structural properties. Furthermore, examination of an antibiotic resistance gene cluster from S. typhimuriumshowed that the same was true for genes encoding antibiotic resistance.     

recB recJ mutants ofSalmonella typhimurium are deficient in transductional recombination, DNA repair and plasmid maintenance

recB recJ mutants ofSalmonella typhimurium are deficient in transduction of chromosomal markers and ColE1-derived plasmids, and also in the maintenance of ColE1 and F plasmids. Plasmid instability is less severe inrecD recJ strains; ColE1 plasmid DNA preparations from these strains show an increased yield of high molecular weight (HMW) linear multimers and a concomitant reduction in plasmid monomers compared to the wild type. Plasmids remain unstable inrecA recD recJ mutants; since these do not produce HMW linear concatemers, we propose that a decrease in monomer production leads to plasmid instability.recB recJ strains also display decreased viability, a component of which may be related to their deficiency in DNA repair. In contrast to their severe defects in recombination, DNA repair and plasmid maintenance,recB recJ mutants ofS. typhimurium behave similarly to the wild type in the segregation of chromosome duplications. The latter observation suggests that neither RecBCD nor RecJ functions are required for chromosomal recombination events that do not involve the use of free ends as recombination substrates.     

A Useful Cloning Vector for Bacillus subtilis

We have constructed plasmid pDN1050 a new small cloning vector for Bacillus subtilis . pDN1050 harbors the origin of replication of Staphylococcus aureus plasmid pUB110 and the chloramphenicol resistance gene of S. aureus plasmid pC194. The plasmid is segregationally and structurally stable. Plasmid pDN1370, a low copy number mutant of pDN1050 was isolated and shown to harbor a mutation in the repA gene of the replication protein.