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At least 19 recordsLinked to original sources
PubMed1984

A comparison of virulence determinants in an octopine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants.

Transposon-insertion mutants with vir- Ti plasmids were characterized and then used in complementation experiments. One of the mutants (LBA 1517) had a mutation in a newly discovered vir locus called virF. The virF mutation led to a strongly diminished virulence on tomato and tobacco, but not on certain other plant species. Also a mutant (LBA 1505) was isolated with a mutation somewhere in the bacterial genome but outside the octopine Ti plasmid that caused a restriction in host range for tumor induction. Introduction of a nopaline Ti plasmid or an Ri plasmid into LBA 1505 did not restore normal virulence, showing that the vir gene affected in LBA 1505 determines a factor which is essential for normal tumor induction both by different types of Ti plasmids and by the Ri plasmid. The introduction of R primes containing part or all of the octopine Ti plasmid virulence region led to a restoration of virulence in strains with a vir- nopaline Ti plasmid. Also the transfer of an Ri plasmid to a large number of different vir- octopine or nopaline Ti plasmid mutants rendered these strains virulent. These results indicate that the octopine Ti plasmid, the nopaline Ti plasmid, and the Ri plasmid each have a similar virulence system which can mediate the transfer of T-DNA to plant cells from different types of Ti or Ri plasmids. In complementation experiments between vir- octopine Ti plasmid mutations and vir- nopaline Ti plasmid mutations it was found that equivalent functions are determined by the areas of DNA homology in the virulence regions of these two types of Ti plasmids. The previously defined octopine Ti plasmid virC locus appeared to consist of two different loci. One of these loci was found to be in a region of the octopine Ti plasmid which does not share DNA homology with the nopaline Ti plasmid, and was therefore called virO (octopine Ti plasmid specific). For the other locus the name virC was retained. Whereas mutations in the virC locus were avirulent on all plant species tested, mutations in virO were avirulent on tomato and pea, but virulent on sunflower and Nicotiana rustica. VirO- mutants produced rooty tumors on Kalanchoë tubiflora.

Arginine↗
PubMed2004

Plasmid evolution and interaction between the plasmid addiction stability systems of two related broad-host-range IncQ-like plasmids.

Plasmid pTC-F14 contains a plasmid stability system called pas (plasmid addiction system), which consists of two proteins, a PasA antitoxin and a PasB toxin. This system is closely related to the pas of plasmid pTF-FC2 (81 and 72% amino acid identity for PasA and PasB, respectively) except that the pas of pTF-FC2 contains a third protein, PasC. As both pTC-F14 and pTF-FC2 are highly promiscuous broad-host-range plasmids isolated from bacteria that share a similar ecological niche, the plasmids are likely to encounter each other. We investigated the relative efficiencies of the two stability systems and whether they had evolved apart sufficiently for each pas to stabilize a plasmid in the presence of the other. The three-component pTF-FC2 pas was more efficient at stabilization of a heterologous tester plasmid than the two component pas of pTC-F14 in Escherichia coli host cells (+/- 92% and +/- 60% after 100 generations, respectively). The PasA antidote of each pas was unable to neutralize the PasB toxin of the other plasmid. The pas proteins of each plasmid autoregulated their own expression as well as that of the pas of the other plasmid. The pas of pTF-FC2 was more effective at repressing the pas operon of pTC-F14 than the pas of pTC-F14 was able to repress itself or the pas of pTF-FC2. This increased efficiency was not due to the PasC of pTF-FC2. The effect of this stronger repression was that pTF-FC2 displaced pTC-F14 when the two plasmids were coresident in the same E. coli host cell. Plasmid curing resulted in the arrest of cell growth but did not cause cell death, and plasmid stability was not influenced by the E. coli mazEF genes.

Antidotes↗
PubMed1996

The conjugal transfer system of Agrobacterium tumefaciens octopine-type Ti plasmids is closely related to the transfer system of an IncP plasmid and distantly related to Ti plasmid vir genes.

We have determined the DNA sequences of two unlinked regions of octopine-type Ti plasmids that contain genes required for conjugal transfer. Both regions previously were shown to contain sequences that hybridize with tra genes of the nopaline-type Ti plasmid pTiC58. One gene cluster (designated tra) contains a functional oriT site and is probably required for conjugal DNA processing, while the other gene cluster (designated trb) probably directs the synthesis of a conjugal pilus and mating pore. Most predicted Tra and Trb proteins show relatively strong sequence similarity (30 to 50% identity) to the Tra and Trb proteins of the broad-host-range IncP plasmid RP4 and show significantly weaker sequence similarity to Vir proteins found elsewhere on the Ti plasmid. An exception is found in the Ti plasmid TraA protein, which is predicted to be a bifunctional nickase-helicase that has no counterpart in IncP plasmids or among Vir proteins but has homologs in at least six other self-transmissible and mobilizable plasmids. We conclude that this Ti plasmid tra system evolved by acquiring genes from two or three different sources. A similar analysis of the Ti plasmid vir region indicates that it also evolved by appropriating genes from at least two conjugal transfer systems. The widely studied plasmid pTiA6NC previously was found to be nonconjugal and to have a 12.65-kb deletion of DNA relative to other octopine-type Ti plasmids. We show that this deletion removes the promoter-distal gene of the trb region and probably accounts for the inability of this plasmid to conjugate.

Agrobacterium tumefaciens↗
PubMed1985

[Role of the conjugative transfer of R-plasmids in competitive interactions between plasmid-containing and plasmid-free strains of Escherichia coli in continuous culture].

Competition of the plasmid-containing strain C600 (RP4) (pBS94) of E. coli and the plasmid-free strain C600 rifr of E. coli in chemostatic and pH-static continuous cultures resulted in displacement of the plasmid-free strain in spite of its higher specific growth rate. Conjugative transfer of RP4 and pBS94 plasmids into the cells of the plasmid-free strain was observed in the experiments. Competition of strains C600 (pBS94) (RP4 Tra-) and C600 rifr in the chemostatic culture in the absence of the plasmid conjugative transfer resulted in displacement of the plasmid-containing strain from the population, while in the pH-static culture, the initial ratio of the plasmid-containing and plasmid-free bacteria was preserved. The conjugative transfer of the plasmids lowered the number of the plasmid-free cells in the population and might be considered as a factor stabilizing the plasmids in the bacterial population of the continuous cultures.

Conjugation, Genetic↗
PubMed1995

Salmonella typhimurium DT 193: differentiation of an epidemic phage type by antibiogram, plasmid profile, plasmid fingerprint and salmonella plasmid virulence (spv) gene probe.

Of over 2000 isolates of Salmonella typhimurium DT 193 from humans examined in the 2 year period 1991-92, 93% were antibiotic-resistant with the most common R-types being ASSuT (38%) and T (29%). Fourteen plasmid profiles were identified in DT 193 R-type ASSuT with the majority of isolates being characterized by a single plasmid of 80 MDa (pDEP 34) which in addition to coding for ASSuT, also hybridized with a spv gene probe prepared from the 50 MDa Salm. dublin serovar-specific plasmid. On the basis of restriction fragment length polymorphisms, two variant lines of pDEP 34-like plasmids were identified and a third line which had lost the genes coding for resistance to ampicillin, streptomycin and sulphonamides, was recognized. Although 18 plasmid profile types were identified in DT 193 R-type T, all isolates carried a high mol. wt plasmid which coded for tetracycline resistance only. Further discrimination was achieved on the basis of hybridization of tetracycline resistance plasmids with the spv gene probe and restriction enzyme fingerprinting. These results demonstrate that Salm. typhimurium DT 193 can be rapidly subdivided by antibiogram and that further subdivision can be achieved on the basis of plasmid profile, plasmid fingerprint and hybridization with a spv gene probe.

Animals↗
PubMed1991

Thermosensitive plasmid replication, temperature-sensitive host growth, and chromosomal plasmid integration conferred by Lactococcus lactis subsp. cremoris lactose plasmids in Lactococcus lactis subsp. lactis.

Evidence is presented that lactose-fermenting ability (Lac+) in Lactococcus lactis subsp. cremoris AM1, SK11, and ML1 is associated with plasmid DNA, even though these strains are difficult to cure of Lac plasmids. When the Lac plasmids from these strains were introduced into L. lactis subsp. lactis LM0230, they appeared to replicate in a thermosensitive manner; inheritance of the plasmid was less efficient at 32 to 40 degrees C than at 22 degrees C. The stability of the L. lactis subsp. cremoris Lac plasmids in lactococci appeared to be a combination of both host and plasmid functions. Stabilized variants were isolated by growing the cultures at 32 to 40 degrees C; these variants contained the Lac plasmids integrated into the L. lactis subsp. lactis LM0230 chromosome. In addition, the presence of the L. lactis subsp. cremoris Lac plasmids in L. lactis subsp. lactis resulted in a temperature-sensitive growth response; growth of L. lactis subsp. lactis transformants was significantly inhibited at 38 to 40 degrees C, thereby resembling some L. lactis subsp. cremoris strains with respect to temperature sensitivity of growth.

DNA Replication↗
PubMed1994

Mathematics of microbial plasmid instability and subsequent differential growth of plasmid-free and plasmid-containing cells, relevant to the analysis of experimental colony number data.

Differential growth of plasmid-containing and plasmid-free microbial cells occurs in many and probably most plasmid systems. Misinterpretation of differential growth as replicational, recombinational, or segregational stability or instability can unfortunately result in grossly erroneous conclusions about replication, recombination, or segregation in plasmid model systems for studies of such phenomena. The differential growth rate should ideally be measured every time that the rate of loss due to instability per se is measured. Unfortunately, the possibility of differential growth has been ignored in most plasmid model systems, since the mathematics of instability and differential growth has not usually been dealt with in ways that are intuitively understandable to experimental microbiologists. Nevertheless, rapid diagnosis of differential growth, and accurate estimation of differential growth rate and rate of loss due to instability per se, can be done by analysis of colony number data using only the relatively simple mathematics described in this review. This review is intended for experimental microbiologists rather than for theoretical population geneticists or for pure mathematicians. However, the same mathematics described in this review is also applicable to certain simple model systems for plasmid ecology or evolution in natural or clinical environments.

Bacteria↗
PubMed1991

Antiparallel plasmid-plasmid pairing may control P1 plasmid replication.

The copy number of the P1 plasmid replicon is stringently controlled, giving only one or two copies per newborn cell. Control is achieved by the action of the copy-control locus incA, which contains nine repeats of the 19-basepair binding site for the plasmid-encoded initiator protein RepA. A set of five similar repeats are present in the replication origin where RepA acts to trigger initiation. Using an in vitro replication system consisting of an Escherichia coli extract, the P1 origin as a template, and purified RepA protein, we show that supercoiled DNA circles containing the incA locus block origin function in trans. Shutdown becomes complete at a 1:1 ratio of origin to incA sequences. This is not due to titration of the RepA protein, as an excess of RepA can be added without restoring activity. Rather, the incA sequences appear to block the origin by direct contact in a plasmid-plasmid pairing event. When both the origin and the incA locus are present on one plasmid, trans contacts with daughter molecules appear to predominate over cis looping. The results are consistent with a model for replication control where daughter plasmids block their own replication by a pairing in which each origin is in contact with the incA locus of its partner.

Bacterial Proteins↗
PubMed2005

Conjugative plasmid pAW63 brings new insights into the genesis of the Bacillus anthracis virulence plasmid pXO2 and of the Bacillus thuringiensis plasmid pBT9727.

BACKGROUND: Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis belong to the genetically close-knit Bacillus cereus sensu lato group, a family of rod-shaped Gram-positive bacteria. pAW63 is the first conjugative plasmid from the B. cereus group to be completely sequenced. RESULTS: The 71,777 bp nucleotide sequence of pAW63 reveals a modular structure, including a 42 kb tra region encoding homologs of the Type IV secretion systems components VirB11, VirB4 and VirD4, as well as homologs of Gram-positive conjugation genes from Enterococcus, Lactococcus, Listeria, Streptococcus and Staphylococcus species. It also firmly establishes the existence of a common backbone between pAW63, pXO2 from Bacillus anthracis and pBT9727 from the pathogenic Bacillus thuringiensis serovar konkukian strain 97-27. The alignment of these three plasmids highlights the presence of well conserved segments, in contrast to distinct regions of high sequence plasticity. The study of their specific differences has provided a three-point reference framework that can be exploited to formulate solid hypotheses concerning the functionalities and the molecular evolution of these three closely related plasmids. This has provided insight into the chronology of their divergence, and led to the discovery of two Type II introns on pAW63, matching copies of the mobile element IS231L in different loci of pXO2 and pBT9727, and the identification on pXO2 of a 37 kb pathogenicity island (PAI) containing the anthrax capsule genes. CONCLUSION: The complete sequence determination of pAW63 has led to a functional map of the plasmid yielding insights into its conjugative apparatus, which includes T4SS-like components, as well as its resemblance to other large plasmids of Gram-positive bacteria. Of particular interest is the extensive homology shared between pAW63 and pXO2, the second virulence plasmid of B. anthracis, as well as pBT9727 from the pathogenic strain B. thuringiensis serovar konkukian strain 97-27.

Bacillus anthracis↗
PubMed1985

Genetic Rearrangement of Plasmids: In Vivo Recombination between a Dehalogenation Plasmid and Multiple-Resistance Plasmid RP4 in Pseudomonas sp.

When Moraxella plasmid pUO1 encoding haloacetate dehalogenase and mercury resistance coexisted with IncP-1 plasmid RP4 in Pseudomonas sp., genetic exchange between the plasmids often occurred, probably by site-specific recombination. The recombinant plasmids obtained were classified into four groups on the basis of phenotype. Representative plasmids for each group were analyzed for DNA composition and function, and the mechanism for the formation of these plasmids was sought. They were inherited stably in Escherichia coli and a Pseudomonas sp.

Journal Article↗
PubMed1994

Construction of plasmids, estimation of plasmid stability, and use of stable plasmids for the production of poly(3-hydroxybutyric acid) by recombinant Escherichia coli.

Plasmids containing the Alcaligenes eutrophus poly(3-hydroxybutyric acid) (PHB) biosynthetic genes were constructed for the production of PHB in Escherichia coli and plasmid stability was investigated by repeated subculturing without antibiotic pressure. Both pSYL101 (high copy) and pSYL102 (medium copy) were unstable during the subcultures. Higher instability was observed when cells were accumulating PHB. Segregational instability was aggravated by the faster growth of plasmid-free cells and by appearance of non-dividing cells harboring large amount of PHB during the fed-batch culture. Two derivatives, pSYL103 and pSYL104, were then developed by cloning the parB locus of plasmid R1 into pSYL102 and pSYL101, respectively. They showed 100% stability even during PHB synthesis and accumulation over 110 generations. All four plasmids were structurally stable. The final cell mass, PHB concentration, and PHB per dry cell weight (P/X, w/w, %) of 101.4 g l-1, 81.2 g l-1, and 80.1%, respectively, were obtained in 39 h by high cell density culture of XL1-Blue (pSYL104). The final PHB concentration was lower using XL1-Blue (pSYL103), which suggested that high gene dosage was required for the synthesis and accumulation of PHB to a high concentration in E. coli.

Escherichia coli↗
PubMed2005

A new family of mobilizable suicide plasmids based on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPalpha) conjugative machineries and their cognate Escherichia coli host strains.

We describe the construction of the pSW family of conditionally replicating plasmids which are based on the IncX oriV origin (oriV(R6Kgamma)) of replication that is dependent on the pir-encoded protein. We constructed several Escherichia coli derivatives expressing pir from different chromosomal loci, and the pir gene could be transduced by phage P1 to any E. coli strain. These chromosomal constructions generate dapA and thyA knockouts, which lead to diaminopimelate or thymidine auxotrophies, respectively, and they serve to provide absolute counterselection even in rich media. These strains can be easily counterselected if used in plasmid transfer experiments into markerless recipients, and they have been demonstrated to work efficiently in E. coli xVibrio or E. coli xBartonella matings. We constructed different pSW plasmids carrying either the oriT(RP4) or the oriT(R388), and we demonstrated that these derivatives can be efficiently transferred using RP4 and R388 conjugation machineries, respectively. We also constructed two plasmids expressing the R388 conjugation machinery, but lacking the oriT(R388). We demonstrated that these plasmids enabled efficient and exclusive transfer of a pSW-oriT(R388) derivative from E. coli to V. cholerae, and we offer an alternative to the popular RP4-based delivery system.

Conjugation, Genetic↗
PubMed1994

Transposon-mediated mobilization of chromosomally located catabolic operons of the CAM plasmid by TOL plasmid transposon Tn4652 and CAM plasmid transposon Tn3614.

The CAM (camphor degradation) plasmid is integrated into the chromosome of Pseudomonas putida PaW-line strains and is not self-transferable as a plasmid via conjugation. Our results show that the mobilization of chromosomally located CAM and the integration of cam-operons into the chromosome of the new Cam+ transconjugants is a recA-independent process mediated by transposons Tn4652 (17 kbp) and Tn3614 (7.2 kbp). Transposon Tn3614 is apparently identical to the left-hand and the right-hand sequences of the TOL plasmid pWW0 transposon Tn4654. The insertion of Tn401 inside the left-hand terminal IR of Tn4652 completely inhibited the mobilization of CAM. According to our data transposons Tn4652 and Tn3614 together with CAM plasmid catabolic operons are integrated into the chromosome. We propose that in pseudomonads the transposons Tn4652 and Tn3614 play a key role in the evolution and spread of new catabolic plasmids in nature.

Chromosomes, Bacterial↗
PubMed1987

[Design of hybrid plasmids based on the actinomycete plasmid pSB24.1 and plasmid prBR325 and a study of their stability in the Streptomyces-E. coli system].

Actinomycete plasmid pSB24.1 was cloned on vector of the E. coli pBR325 system. The following bireplicon plasmids were obtained: pSU501 and pSU502 (by XhoI site of pSB24.1 and SalGI site of pBR325), pSU503 (by Bg1II (c) site of pSB24.1 and BamHI site of pBR325) and pSU504 (by Bg1II(b) site of pSB24.1 and BamHI site of pBR325). In the cells of E. coli C600 plasmids pSU501-504 determined phenotype AprCmrTcs and were stable. In the cells of Str. lividans the initial structure pSU501 selected by Ltz+ phenotype was maintained at a frequency of 12.5 per cent. Analysis of the deletion variants of pSU501 isolated from Str. lividans showed that the deletions were induced by both the pBR325 region and the pSB24.1 DNA fragment near XhoI site. The region from SacII(a) site to Bg1II(b) site clockwise in the map of plasmid pSB24.1 was not significant for its replication and maintenance in Str. lividans. There were detected unique sites of pSB24.1 and its derivatives useful for cloning. Possible shortening of plasmid pSB24.1 by 567 kb (the length between the terminator of the open frame reading translation and XhoI site) was revealed.

Chromosome Deletion↗
PubMed1987

A new vector derived from Agrobacterium rhizogenes plasmids: a micro-Ri plasmid and its use to construct a mini-Ri plasmid.

A new binary vector system has been constructed, based on agropine-type root-inducing plasmid (pRi) left transferred-region border sequences cloned in a plasmid containing the replication origin of another A. rhizogenes plasmid (pArA4a). This micro-pRi has been used to introduce a chimeric kanamycin resistance gene into tobacco plants, vir functions being provided by either octopine or nopaline tumor-inducing plasmids deleted of their own transferred regions. In addition, we show that cloning of pRi EcoRI fragment 15, which contains three open reading frames (which may correspond to loci rolA, B and C), in the micro-Ri vector generates a mini-pRi capable of inducing the proliferation of transformed roots.

Cloning, Molecular↗
PubMed1999

A comparison of the kinetics of plasmid transfer in the conjugation systems encoded by the F plasmid from Escherichia coli and plasmid pCF10 from Enterococcus faecalis.

Quantitative measurements of horizontal DNA transfer are critical if one wishes to address questions relating to ecology, evolution and the safe use of recombinant bacteria. Traditionally, the efficiency of a conjugation system has been described by its transfer frequency. However, transfer frequencies can be determined in many ways and may be sensitive to physical, chemical and biological conditions. In this study the authors have used the mechanistic similarity between bacterial conjugation and simple enzyme catalysis in order to calculate the maximal conjugation rate (Vmax) and the recipient concentration (K(m)) at which the conjugation rate is half its maximal value, for two different conjugation systems: the F plasmid from Escherichia coli and plasmid pCF10 from Enterococcus faecalis. The results are compared with the data obtained from the aggregation-mediated conjugation system encoded on pXO16 from Bacillus thuringiensis. The conjugation systems analysed are fundamentally different; however, they have some characteristics in common: they are able to sustain conjugative transfer in liquid medium and the transfer efficiencies are very high. Conjugation encoded by the F plasmid in E. coli involves the formation of small aggregates (2-20 cells), established by sex pili, and the plasmid's maximal conjugation rate was estimated to be approximately 0.15 transconjugants per donor per minute. Pheromone-induced conjugation in Ent. faecalis, which involves the formation of large aggregates, was found to proceed at a maximal conjugation rate of 0.29 transconjugants per donor per minute. Also, the K(m) value differed significantly between these conjugation systems; this may reflect the inherent differences in mating pair formation and transfer mechanisms. In these conjugation systems, the donors underwent a 'recovery period' between rounds of conjugative transfer and newly formed transconjugants required a period of about 40-80 min to mature into proficient donors.

Conjugation, Genetic↗
PubMed1994

High-frequency homologous plasmid-plasmid recombination coupled with conjugation of plasmid SCP2* in Streptomyces.

Non-transmissible derivatives of the Streptomyces multi-copy plasmid plJ101 were mobilized, by cointegrate formation, at frequencies approaching 100% (measured per recipient) by derivatives of the conjugative, low-copy-number Streptomyces coelicolor A3(2) plasmid SCP2*. Efficient co-integrate formation required that the plasmids shared at least 112 bp sequence identity, and it occurred only during conjugation. An SCP2* plasmid gene is involved in the process. Co-integrates were presumably formed in the donor cells and transported to the recipient cells. This is a new phenomenon, not known in other bacteria.

Conjugation, Genetic↗
PubMed1995

Replication control of plasmid pLS1: efficient regulation of plasmid copy number is exerted by the combined action of two plasmid components, CopG and RNA II.

Two elements, the products of genes copG and rnaII, are involved in the copy-number control of plasmid pLS1. RNA II is synthesized in a dosage-dependent manner. Mutations in both components have been characterized. To determine the regulatory role of the two genes, we have cloned copG, rnaII or both elements at various gene dosages into pLS1-compatible plasmids. Assays of incompatibility towards wild-type or mutant pLS1 plasmids showed that: (i) the rnaII gene product, rather than the DNA sequence encoding it, is responsible for the incompatibility, and (ii) CopG and RNA II act in trans and are able to correct up fluctuations in pLS1 copy number. A correlation between the gene dosage at which the regulatory elements were supplied and the incompatibility effect on the resident plasmid was observed. The entire copG-rnaII circuit has a synergistic effect when compared with any of its components in the correction of pLS1 copy-number fluctuations, indicating that, in the homoplasmid steady-state situation, the control of pLS1 replication is exerted by the co-ordinate action of CopG and RNA II.

Base Sequence↗