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PubMed1990

Conservation of Salmonella typhimurium virulence plasmid maintenance regions among Salmonella serovars as a basis for plasmid curing.

The association of large plasmids with virulence in invasive Salmonella serovars has led to a number of studies designed to uncover the role of these plasmids in virulence. This study addresses two aspects of virulence-associated plasmids. The first is the distribution of the replication and maintenance regions among the plasmids of different Salmonella serovars, and the second is the use of the conserved virulence plasmid par region to provide a rapid method for eliminating the virulence plasmids specifically. Colony blots revealed that the par and repB regions of the S. typhimurium virulence plasmid hybridized with 80% of the isolates of S. choleraesuis, S. dublin, S. enteritidis, S. gallinarum, S. pullorum, and S. typhimurium, while the repC region was not detected in any of the isolates of S. dublin, S. gallinarum, or S. pullorum. None of these maintenance regions was found in any of the 30 additional serovars tested. The large plasmids of those serovars that hybridized with par were labeled with a Kmr insert within parA via P22HTint or P1L4 transduction, which destabilized the plasmids and allowed the rapid isolation of plasmid-free derivatives for all of the serovars, except for S. dublin, which exhibited weak homology with par.

Base Sequence↗

PubMed1977

Physical characterization of a plasmid cointegrate containing an F'his gnd element and the Salmonella typhimurium LT2 cryptic plasmid.

A recombinant plasmid (pAS19) isolated from a derivative of Salmonella typhimurium LT2, containing the strain LT2 cryptic plasmid and an F'his gnd element, has been physically characterized. The pAS19 plasmid contour length equals the sum of the contour lengths of the cryptic plasmid and F'his gnd element. Deoxyribonucleic acid (DNA)-DNA hybridization experiments demonstrated that whereas the pAS19 plasmid exhibits extensive DNA homology with both the cryptic plasmid and the F'his gnd element, there is little DNA homology between these latter two plasmids. The DNA fragmentation pattern of the pAS19 plasmid produced by the restriction endonuclease R-EcoRI is consistent with that expected for a composite plasmid cointegrate containing most, if not all, of the DNA sequences present in its two component plasmids.

Animals↗

PubMed1982

Marker rescue by a homologous recipient plasmid during transformation of gonococci by a hybrid Pcr plasmid.

A 42-kilobase hybrid Pcr plasmid (pFA14) was formed when the naturally occurring 7.2-kilobase Pcr plasmid pFA3 was introduced by transformation into a competent gonococcal recipient containing the 36-kilobase conjugative plasmid pFA2 (Sox et al., J. Bacteriol. 138:510-518). Analysis of the structure of pFA14 showed that it was a stable recombinant between pFA3 and pFA2. The transformation efficiency of pFA14 was increased 300- to 10,000-fold by the presence in isogenic recipients of the homologous plasmid pFA2. The presence of a homologous plasmid in the recipient also markedly increased the likelihood of recovery of intact donor-size Pcr plasmids in the transformants. The presence of pFA2 had no effect on the competence of piliated or nonpiliated gonococci for transformation by either linear chromosomal DNA or a nonhomologous Pcr plasmid. Increased transformation efficiency of the hybrid Pcr plasmid pFA14 may have been due to recombination between the nicked or linearized donor plasmid and the homologous recipient plasmid (marker rescue).

Chromosomes, Bacterial↗

PubMed2001

[Incompatibility among indigenous plasmids of Sinorhizobium fredii strains and its application for plasmid curing].

The indigenous plasmids of S. frediii HN01 and WWG18 were labeled with Tn5-MobsacB. Those plasmids were cured on TY medium containing 10% sucrose to identify its function. The pSymHN01b of HN01 was transferred into WWG18SR and C361SR. The results of plasmid detection, curing and pot plant experiment revealed that the incompatibility was existed between pSym-HN01b and symbiotic plasmid of WWG18SR or the third plasmid of C361SR. But it was compatible with the nonsymbiotic plasmid of WWG18SR and symbiotic plasmid of C361SR. Using plasmid incompatibility, The symbiotic plasmid of WWG18SR and one nonsymbiotic plasmid of C361SR were eliminated successfully.

Conjugation, Genetic↗

PubMed1989

Mobilization of R387 Inc K conjugative plasmid by the conjugative plasmid R64 Inc I.

Plasmid aggregate (R387, R64) was constructed in E. coli K12 strain. Plasmid R387 Inc K was stimulated to conjugational transfer by plasmid R64 Inc I. This stimulation was caused neither by recombination between both plasmids nor by trans-complementation of R387 conjugational systems by gene(s) product(s) of R64 plasmid. The observed phenomenon resembled rather mobilization of nonconjugative plasmids by conjugative ones. As in mobilization, the observed increase in R387 transfer frequency could take place only when both interacting plasmids were present in donor cells. Moreover, the entry exclusion system functioning in recipient cells, toward stimulating R64 plasmid affected strongly the conjugational transfer of stimulated R387 plasmid. Analogous phenomenon was observed during mobilization of nonconjugative plasmids by conjugative ones.

Citrobacter↗

PubMed1979

[Genetic control of the formation of plasmid F'. I. Effect of recA- and seg-2 mutations in an Hfr donor strain on the character of plasmid F' formation].

Assuming the similarity of the processes of illegitimate recombination, such as deletion formation, with the process of F' plasmid formation, we have undertaken the study of the influence of recA- and seg- alleles of Hfr donor on the F' plasmid formation. The data obtained demonstrate the strong influence of donor genotype on the frequency of F' plasmid formation and on the nature of F' plasmids formed, thus demonstrating that the most of F' plasmids have been formed via recombination in Hfr donor cells. The recA- mutation decreased the total yield of F' plasmids selected using both proximal and distal Hfr markers and affected drastically the distribution of the F' plasmids inheriting different proximal unselected markers. The existence of recA-dependent and recA-independent modes of F' plasmid formation was demonstrated. The Escherichia coli chromosome contains regions which involve preferentially in recA-dependent (between proA and gal, and clockwise from gal) or recA-independent (between leu and proA, and the region counterclockwise from argE) recombination. The seg-2 mutation causes only partial block of both recA-dependent and recA-independent recombination pathways, however it causes dramatic decrease of genetic exchanges leading to the formation of the type II F' plasmids. Both seg- and recA- mutations decrease the frequency of the formation of Tra+ F' transconjugants. The percent of Tra- transconjugants, which remain sensitive to MS2 and Q beta donor specific phages, also drops significantly under the influence of the recA- and seg- alleles. Thus, the recombination involving the F structure in wild type strains and seg- mutants occures preferentially in the points of F outside the regions essential for transfer and sensitivity to male specific phages, while in recA- and recA-ges- strains the points inside these regions (tra operon) frequently involved in F' plasmid looping out. There exist more strict correlation between the fertility and sensitivity to phage Q beta than to phage MS2.

Chromosome Deletion↗

PubMed1990

Characterization of plasmids and plasmid-borne macrolide resistance from Lactobacillus sp. strain 100-33.

Lactobacillus sp. strain 100-33 is resistant to macrolides, lincosamides, and streptogramin B-type antibiotics (MLSR) and appears to contain several major and minor plasmids. One of these plasmids, pLAR33, is approximately 18 kbp in size. When cells of strain 100-33 were protoplasted and regenerated, an MLSS isolate was derived. The derivative, designated strain ES1, contained a unique plasmid complement in which it had apparently lost the major plasmids of the parental strain, including pLAR33, and retained only a minor plasmid seen in low concentrations in strain 100-33. The MLSR determinant was cloned from plasmid DNA of strain 100-33 on a 3-kbp EcoRV fragment into pBR322 and localized to pLAR33. The determinant expressed macrolide and lincosamide resistance in Escherichia coli HB101, was localized to approximately 1 kbp on the cloned sequence, and is apparently under the control of its own promoter. MLSR electroporants were derived from strain ES1 electroporated with plasmid DNA from strain 100-33; these MLSR isolates had acquired a plasmid complement similar to that of strain 100-33, including pLAR33. Endonuclease digestion and Southern analysis of plasmid DNA from both strains indicated that the major plasmids are multimeric and deleted forms of one archetypal extrachromosomal element.

Anti-Bacterial Agents↗

PubMed1989

Distribution of plasmid maintenance regions among IncFIV plasmids.

The distribution of the IncFI basic replicons among IncFIV plasmids was assessed by DNA hybridization. In addition these and 20 other plasmids from 16 incompatibility groups were screened for the presence of IncIV, an incompatibility determinant recently found on the IncFIV plasmid R124. The IncIV determinant was found commonly but not universally among the IncFIV plasmids. It was also detected on the IncFI reference plasmid R386 and plasmids from IncB, IncI alpha and IncI gamma. The frequency and distribution of IncFI replicons among the IncFIV plasmids is similar to that observed in other F groups. The similarity of the IncFIV plasmids to plasmids of the other IncF groups and the failure to find replicons unique to IncFIV plasmids indicates that their division into a separate incompatibility group is not justified.

DNA Probes↗

PubMed1998

Salmonella virulence plasmid. Modular acquisition of the spv virulence region by an F-plasmid in Salmonella enterica subspecies I and insertion into the chromosome of subspecies II, IIIa, IV and VII isolates.

The spv operon is common to all Salmonella virulence plasmids. DNA hybridization analysis indicates that the spv region is limited in distribution to serovars of Salmonella enterica subspecies I, II, IIIa, IV, and VII and is absent from Salmonella bongori isolates. Among strains of subspecies II, IIIa, and VII, all isolates examined contained sequences that hybridized with the spv region. However, among isolates of subspecies I, DNA sequences capable of hybridizing with the spv region were found in some isolates of certain serovars. Furthermore, in isolates of subspecies I, the virulence plasmid was found in the same set of isolates as an F-related plasmid, as determined by the presence of the spv region of the virulence plasmid and the finO, traD, and repA sequences of the F-plasmid. The concordance of the virulence plasmid and all three F-plasmid sequences in subspecies I serovar Choleraesuis, Paratyphi, and Typhimurium is most easily explained if the spv region is carried in an F-related plasmid in these isolates. In contrast, among S. enterica subspecies II, IIIa, IV, and VII, the isolates that contain spv sequences did not hybridize with an F-related plasmid or any other identifiable plasmid. With the use of pulse-field gel electrophoresis, the spv region in subspecies II, IIIa, and VII was found to be encoded on the chromosome. Analysis of the phylogenetic distribution of spv among Salmonella isolates and comparative nucleotide sequence analysis of spvA and spvC suggests that the spv region was acquired very recently, after speciation of the salmonellae.

Base Sequence↗

PubMed2004

Antibiotic multiresistance plasmid pRSB101 isolated from a wastewater treatment plant is related to plasmids residing in phytopathogenic bacteria and carries eight different resistance determinants including a multidrug transport system.

Ten different antibiotic resistance plasmids conferring high-level erythromycin resistance were isolated from an activated sludge bacterial community of a wastewater treatment plant by applying a transformation-based approach. One of these plasmids, designated pRSB101, mediates resistance to tetracycline, erythromycin, roxythromycin, sulfonamides, cephalosporins, spectinomycin, streptomycin, trimethoprim, nalidixic acid and low concentrations of norfloxacin. Plasmid pRSB101 was completely sequenced and annotated. Its size is 47 829 bp. Conserved synteny exists between the pRSB101 replication/partition (rep/par) module and the pXAC33-replicon from the phytopathogen Xanthomonas axonopodis pv. citri. The second pRSB101 backbone module encodes a three-Mob-protein type mobilization (mob) system with homology to that of IncQ-like plasmids. Plasmid pRSB101 is mobilizable with the help of the IncP-1alpha plasmid RP4 providing transfer functions in trans. A 20 kb resistance region on pRSB101 is located within an integron-containing Tn402-like transposon. The variable region of the class 1 integron carries the genes dhfr1 for a dihydrofolate reductase, aadA2 for a spectinomycin/streptomycin adenylyltransferase and bla(TLA-2) for a so far unknown Ambler class A extended spectrum beta-lactamase. The integron-specific 3'-segment (qacEDelta1-sul1-orf5Delta) is connected to a macrolide resistance operon consisting of the genes mph(A) (macrolide 2'-phosphotransferase I), mrx (hydrophobic protein of unknown function) and mphR(A) (regulatory protein). Finally, a putative mobile element with the tetracycline resistance genes tetA (tetracycline efflux pump) and tetR was identified upstream of the Tn402-specific transposase gene tniA. The second 'genetic load' region on pRSB101 harbours four distinct mobile genetic elements, another integron belonging to a new class and footprints of two more transposable elements. A tripartite multidrug (MDR) transporter consisting of an ATP-binding-cassette (ABC)-type ATPase and permease, and an efflux membrane fusion protein (MFP) of the RND-family is encoded between the replication/partition and the mobilization module. Homologues of the macrolide resistance genes mph(A), mrx and mphR(A) were detected on eight other erythromycin resistance-plasmids isolated from activated sludge bacteria. Plasmid pRSB101-like repA amplicons were also obtained from plasmid-DNA preparations of the final effluents of the wastewater treatment plant indicating that pRSB101-like plasmids are released with the final effluents into the environment.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1979

Characterization of Plasmid Deoxyribonucleic Acid in Streptococcus lactis subsp. diacetylactis: Evidence for Plasmid-Linked Citrate Utilization.

The use of Streptococcus diacetylactis as a flavor producer in dairy fermentations is dependent upon its ability to produce diacetyl from citrate. Treatment of S. diacetylactis strains 18-16 and DRC1 with acridine orange resulted in the conversion of approximately 2% of the DRC1 population and 20% of the 18-16 population to citrate negative, which is indicative of the involvement of plasmid deoxyribonucleic acid (DNA). Growth in the presence of acridine orange also resulted in the appearance of 2% lactose-negative derivatives in S. diacetylactis 18-16 and 99% lactose-defective, proteinase-negative derivatives in S. diacetylactis DRC1. Cesium chloride-ethidium bromide equilibrium density gradients of cleared lysate material from each strain revealed the presence of covalently closed circular DNA. Samples of this covalently closed circular DNA were subjected to agarose gel electrophoresis to determine the plasmid composition of each strain. S. diacetylactis 18-16 was found to possess six plasmids, of approximately 41, 28, 6.4, 5.5, 3.4, and 3.0 megadaltons (Mdal). S. diacetylactis DRC1 contained six plasmids, of approximately 41, 31, 18, 5.5, 4.5, and 3.7 Mdal. Variants of S. diacetylactis 18-16 which failed to produce acetoin plus diacetyl from citrate (citrate negative) were missing a 5.5-Mdal plasmid. Lactose-negative mutants of the same strain were devoid of a 41-Mdal plasmid. Lactose-defective, proteinase-negative mutants of S. diacetylactis DRC1 were missing a 31-Mdal plasmid. The citrate-negative mutants of S. diacetylactis DRC1 isolated in this study did not possess a 5.5-Mdal plasmid. Thus, we have evidence that there is a correlation between the ability to utilize citrate and the presence of a 5.5-Mdal plasmid. A relationship was also noted between lactose fermentation and proteinase activity and plasmid DNA in S. diacetylactis.

Journal Article↗

PubMed1980

Consequences of interaction between F plasmid and a drug-resistance plasmid belonging to incompatibility group F1.

Two plasmids, pLK1 and pLK2, were derived from pIP218, an in vivo recombinant of plasmid F and the drug-resistance plasmid pIP176 (Cmr, Smr, Sur, Tcr). Of these two plasmids, pLK1 is 70 Mdaltons and carries the Tc-resistance determinant in a 7-Mdalton transposition element; pLK2 is 125 Mdaltons and carries Cm-, Sm- and Su-resistance determinants. The plasmid pLK1 resulted as a Tc-resistance segregaant of PIP218 during its coexistence with another plasmid, Co1E1-araC101, and pLK2 (125 Mdaltons) as a CmrSmrSur segregant during the conjugal transfer of pIP218. Both plasmids belong to the F1-incompatibility group, surface-exclude each other and Flac, and are derepressed for transfer. Incompatibility studies also indicated the preferential maintenance of pLK2 in hosts carrying either pLK2 and pLK1, or pLK2 and F'lac. An explanation of this phenomenon is provided. Furthermore, our data suggest the illegitimate recombination of the chromosomal lac genes with pLK1. In course of the incompatibility studies, the tet determinant was transposed from pLK1 into the chromosome, from the chromosome into the lac genes of an Flac plasmid, and from the Flac plasmid into another site on a second Flac plasmid.

Conjugation, Genetic↗

PubMed1985

Maintenance of multicopy plasmid Clo DF13 III. Role of plasmid size and copy number in partitioning.

To elucidate the mechanisms that operate in plasmid maintenance, we analysed the stability of different combinations of Clo DF13 derivatives present in the same bacterial cell. From the data described in this paper we conclude: (i) each Clo DF13 plasmid molecule has an equal chance of colonizing daughter cells upon cell division, (ii) the Clo DF13 minimal replicon harbours functions involved in plasmid segregation and incompatibility, (iii) in the case of cells harbouring plasmid replicons which differ in size, the smaller plasmid is gradually lost from the cell population, (iv) in the case of cells habouring plasmid replicons which differ in copy number, the lower copy number plasmid is always lost from the cell population. The effect of plasmid copy number is dominant over the effect of plasmid size.

Cell Division↗

PubMedDate not supplied

Characterization of cryptic plasmids from marine cyanobacteria and construction of a hybrid plasmid potentially capable of transformation of marine cyanobacterium, Synechococcus sp., and its transformation.

Among forty strains of marine cyanobacteria isolated in our laboratory, five strains had 1-3 different plasmids. The unicellular marine cyanobacterium, Synechococcus sp. NKBG 042902, contains at least three plasmids (pSY09, pSY10, and pSY11). However, these plasmids are cryptic. Therefore, a hybrid plasmid pUSY02 containing the 1.4-kb HindIII fragment of pSY11 and Escherichia coli plasmid pUC18 was constructed. The plasmid pUSY02 transformed both marine Synechococcus sp. NKBG042902-YG1116, which is a cured strain, and fresh water Anacystis nidulans R2 by dark incubation or Ca2+ treatment. However, the plasmid pSG111 constructed from the plasmid DNA of A. nidulans R2 failed to transform marine Synechococcus sp. Electroporation was also applicable to transformation of marine Synechococcus sp. and fresh water A. nidulans R2. The plasmid pUSY02 was rapidly introduced into marine Synechococcus sp.

Aspergillus nidulans↗

PubMed1985

Transformation of yeast with linearized plasmid DNA. Formation of inverted dimers and recombinant plasmid products.

The molecular products of DNA double strand break repair were investigated after transformation of yeast (Saccharomyces cerevisiae) with linearized plasmid DNA. DNA of an autonomous yeast plasmid cleaved to generate free ends lacking homology with the yeast genome, when used in transformation along with sonicated non-homologous carrier DNA, gave rise to transformants with high frequency. Most of these transformants were found to harbor a head-to-head (inverted) dimer of the linearized plasmid. This outcome of transformation contrasts with that observed when the carrier DNA is not present. Transformants occur at a much reduced frequency and harbor either the parent plasmid or a plasmid with deletion at the site of the cleavage. When the linearized plasmid is introduced along with sonicated carrier DNA and a homologous DNA restriction fragment that spans the site of plasmid cleavage, homologous recombination restores the plasmid to its original circular form. Inverted dimer plasmids are not detected. This relationship between homologous recombination and a novel DNA transaction that yields rearrangement could be important to the cell, as the latter could lead to a loss of gene function and lethality.

DNA Repair↗

PubMed1987

Regulation of mini-F plasmid DNA replication. A quantitative model for control of plasmid mini-F replication in the bacterial cell division cycle.

A quantitative model for the regulation of replication of plasmid mini-F in the Escherichia coli cell division cycle has been developed. The essential repE gene of mini-F encodes a polypeptide that serves both as a positive replication initiation protein and as a regulatory repressor protein. The mini-F regulatory processes include the interaction of repressor with an operator site in the autogenous control of transcription of the repE gene, and the binding of initiator to repeated DNA sequences located both downstream from the repE gene and at the replication origin. A statistical thermodynamic model was used to predict probable configurations of the regulatory processes in a single growing cell. These probabilities were coupled by a kinetic model to events of the cell cycle such as mRNA transcription and protein translation, and the initiation of plasmid DNA replication. Parameter values were chosen so that the simulated values for plasmid copy number and repressor and initiator protein concentrations of the model agreed with experimentally determined estimates for mini-F. Simulations of deviations from regular segregation of plasmid copies at cell division and of premature or delayed initiation of plasmid replication suggest that mini-F replication control responds rapidly and precisely to these perturbations. The simulations also accurately mimic the response of plasmid mini-F to various plasmid copy number mutations and to various mini-F regulatory elements provided in trans. These simulations predict a stable pattern of inheritance for plasmid mini-F despite its low plasmid copy number, in agreement with experimental observation.

Cell Division↗

PubMed1989

Plasmid-mediated lethality and plasmid multimer formation in an Escherichia coli recBC sbcBC mutant. Involvement of RecF recombination pathway genes.

Apparent plasmid instability, i.e. progressive plasmid loss in a bacterial culture growing in the absence of selection for the plasmid, in an Escherichia coli recBC sbcBC mutant was investigated with two different ColE1 derivatives (pMB9 and pBR322) and a mini-F plasmid. The instability was most striking for pMB9 and much less, but still significant, for pBR322 and the mini-F. It was also dependent upon a subset of the genes involved in the RecF recombination pathway: in addition to the previously reported recA, recF and recJ mutations, a recO and a recQ mutation showed a total and a partial suppression, respectively, of the instability. Other recF-family mutations, recN and ruv, were without such an effect. Population analyses of the recBC sbcBC strain carrying pMB9 or the mini-F, as carried out by plating and Coulter counting, revealed marked loss of viability in plasmid-carrying cells, strongly implicating plasmid-mediated cell death in the apparent defect in plasmid maintenance. Analysis of intracellular plasmid DNA by pulsed-field gel electrophoresis combined with the in-agarose cell lysis technique showed that the instability was associated with the formation of plasmid multimers, with a good correlation between the degree of the instability and the amount of the multimers. The multimer formation was also dependent on the same subset of the RecF pathway genes as in the instability phenomenon. These results strongly suggest that the lethality is somehow caused by the multimer formation. Various DNase treatments of cell lysates showed that such multimers of pMB9 DNA comprised molecules of exonuclease-sensitive and exonuclease-resistant types. It was inferred that the former class, which showed electrophoretic mobilities corresponding to plain linear duplexes of approximately 200 x 10(3) to 2200 x 10(3) base-pairs, represented linear multimers possibly carrying circular structures at one end. The latter class, which remained in the origin, was thought to consist of circular multimers and/or linear multimers protected by circular structures at both ends against exonucleolytic attack.

DNA Replication↗

PubMed1984

A mathematical model for lambda dv plasmid replication: analysis of wild-type plasmid.

A mathematical model for lambda dv plasmid replication in a growing single cell of Escherichia coli has been formulated and solved numerically. Quantitative description of the molecular control mechanism for initiation of lambda dv replication presumes regulatory functions of repressor and initiator proteins and transcriptional activation of the origin region. Random selection of a single plasmid for activation and replication is assumed, as is regular plasmid segregation to daughter cells. The model is capable of simulating the periodic changes in each regulatory element and the plasmid copy number during the cell cycle. The calculated average copy number, repressor concentration, and timing of plasmid replication agree well with experimental data. The simulated lambda dv plasmid replication rate is controlled primarily by transcription frequency. Initiation of plasmid replication is not related to variations in the levels of repressor or initiator proteins during the cell cycle. Simulation studies of perturbations in plasmid and repressor segregation indicate that replication regulation of the lambda dv plasmid compensates to readjust copy number to normal values in a few generations. Implications of these studies relative to the molecular mechanisms of replication control are discussed.

DNA Replication↗