Transfer of plasmids RP4 and R68.45 and chromosomal mobilization in cowpea rhizobia

R-plasmids RP4 and its derivatives R68.45 were transferred from Escherichia coli to two cowpea rhizobia strains. The frequency of RP4 transfer in cowpea rhizobia strains JRC23-SM20 and IRC256-HA409 was 1,000-fold higher than transfer frequency of R68.45. The transconjugants were further used to transfer R-plasmids within (isogenic) and between (non-isogenic) cowpea rhizobia strains. The plasmid transfer frequency was higher in isogenic than non-isogenic strains. The ability of R-plasmids to mobilize chromosomal genes in cowpea rhizobia was also examined. R-plasmids mediated the chromosomal transfer; however, mobilization of chromosomal markers Sm^R and Met⁺ by RP4 in isogenic strains was more efficient than by R68.45. Chromosomal mobilization has not previously been reported in cowpea rhizobia.Abbreviations Ap ampicillin - Km kanamycin - Tc tetracycline - Rif rifampicin - TYS tryptone yeast-extract sodium chloride - YEMA yeast-extract mannitol agar - YEMB yeast-extract mannitol brothPart of the work was presented in 6th International Symposium on Nitrogen Fixation at Oregon State University, Corvallis, August 4–10, 1985     

Analysis of chromosomal integration and deletions of yeast plasmids.

Plasmid DNAs from six strains of Saccharomyces cerevisiae were compared. Three different plasmids were found, designated Scp 1, Scp 2 and Scp 3, with monomer lengths of 6.19, 6.06 and 5.97 kilobases as referenced to sequenced phiX174 DNA. DNA from each of the plasmids was inserted into a lambda vector DNA. Hybrid phage containing inserted DNA of the desired size were enriched by genetic selection and their DNAs analysed by rapid techniques. All three plasmids share the same organization, two unique sequences separated by two inverted repeats, and share basically the same DNA sequences. Scp 2 and Scp 3 differ from Scp 1 by missing a unique HpaI site and by having small overlapping deletions in the same region. The HpaI site in Scp 1 is, therefore, in a nonessential region and suitable for insertion of foreign DNA in the potential use of the yeast plasmid as a vector. Hybridization of labelled cloned plasmid DNA to restriction fragments of linear yeast DNA separated on agarose gels showed that the plasmid DNA was not stably integrated into the yeast chromosomal DNA.     

Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism.     

Replication of staphylococcal multiresistance plasmids

Based on structural and functional properties, three groups of large staphylococcal multiresistance plasmids have been recognized, viz., the pSK1 family, pSK41-like conjugative plasmids, and beta-lactamase-heavy-metal resistance plasmids. Here we describe an analysis of the replication functions of a representative of each of these plasmid groups. The replication initiation genes from the Staphylococcus aureus plasmids pSK1, pSK41, and pI9789::Tn552 were found to be related to each other and to the Staphylococcus xylosus plasmid pSX267 and are also related to rep genes of several plasmids from other gram-positive genera. Nucleotide sequence similarity between pSK1 and pI9789::Tn552 extended beyond their rep genes, encompassing upstream divergently transcribed genes, orf245 and orf256, respectively. Our analyses revealed that genes encoding proteins related to the deduced orf245 product are variously represented, in several types of organization, on plasmids possessing six seemingly evolutionarily distinct types of replication initiation genes and including both theta-mode and rolling-circle replicons. Construction of minireplicons and subsequent functional analysis demonstrated that orf245 is required for the segregational stability of the pSK1 replicon. In contrast, no gene equivalent to orf245 is evident on the conjugative plasmid pSK41, and a minireplicon encoding only the pSK41 rep gene was found to exhibit a segregational stability approaching that of the parent plasmid. Significantly, the results described establish that many of the large multiresistance plasmids that have been identified in clinical staphylococci, which were formerly presumed to be unrelated, actually utilize an evolutionarily related theta-mode replication system.     

Replication control of IncP plasmids

Clones of Escherichia coli with a chromosomally integrated RP4-prime plasmid were isolated and characterized. Chromosome transfer was increased about 50-fold and the Hfr still carried an autonomous plasmid indistinguishable from the original RP4-prime. This could be eliminated by pRP64 or R751, two distinguishably marked incompatible plasmids, giving rise to strains which stably retained the resistance patterns of both plasmids and which continued to transfer the chromosome at enhanced levels. In both cases, however, the copy number of the autonomous plasmid was reduced by the presence of a chromosomal RP4 such that the total number of P plasmid genomes (integrated and autonomous) remained constant. The results are consistent with the idea that copy number is controlled by diffusible inhibitors or initiators of replication.     

Replication regions of Sinorhizobium meliloti plasmids

The replication (rep) regions of small plasmids from three Sinorhizobium meliloti strains were cloned by marker rescue. Two unique replication regions were identified, one of which was common to two different strains. Plasmid pBB83 carried a 7.2 kbp rep region from a 42 kbp plasmid, and pBB84 carried a 4.5 kbp rep region from a 36 kbp plasmid. The cloned rep regions were of different compatibility types, and were capable of displacing their parent plasmids from S. meliloti. Neither could function in a PolA- strain of Escherichia coli. The cloned replication regions were less stable in S. meliloti than their parent plasmids. The rep genes for each plasmid were localized to less than 2.5 kbp segments. Sequencing data revealed that the pBB83 Rep protein is uncommon, with partial identity to a protein encoded by a plasmid from S. meliloti GR4 [Mercado-Blanco, J., Olivares, J., 1994. The large nonsymbiotic plasmid pRmeGR4a of Rhizobium meliloti GR4 encodes a protein involved in replication that has homology with the RepC protein of Agrobacterium plasmids. Plasmid 32, 75-79]. However, the cloned DNA fragment also contains a truncated segment of the common repABC genes, suggesting that the parent plasmid contained two sets of replication genes. Other genes and an IS-element within the insert are most closely related to sequences derived from the Rhizobiaceae family, suggesting that the plasmid has a limited host range. In contrast, the pBB84 rep region contained genes similar to those associated with several broad host-range plasmids, and its Rep protein is related to that of a Pseudomonas aeruginosa broad host-range plasmid, pVS1 [Heeb, S., Itoh, Y., Nishijyo, T., Schnider, U., Keel, C., Wade, J., Walsh, U., O'Gara, F., Haas, D., 2000. Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria. Mol. Plant-Microbe Interact. 13, 232-237]. The pBB84 rep region also includes a probable origin of replication, consisting of DNA boxes flanking a series of direct repeats and an AT-rich sequence.     

Occurrence of deletion plasmids at high rates after conjugative transfer of the plasmids RP4 and RK2 from Escherichia coli to Alcaligenes eutrophus H16

The broad host-range IncP-1 plasmids RP4 and RK2 were transferred by conjugation from Escherichia coli to Alcaligenes eutrophus H16. Among the transconjugants selected on media containing tetracycline, a considerable number did not express kanamycin resistance. By comparing restriction patterns of plasmids isolated from a large number of transconjugants a variety of different deletion derivatives were found. All of these possess more or less extended deletions always including parts of the tra 1-region. The plasmids RP4 and RK2, once established in A. eutrophus H16 showed a high stability and it can be concluded that deletion formation is connected with the conjugation process. Evidence is given that degradation of DNA entering an A. eutrophus recipient cell during the conjugative transfer process may be involved in deletion formation. Furthermore, the finding of a small deletion derivative of RP4 lacking the transacting replication function trfB and the entire kil-kor-system may allow the assumption that these gene functions are not essential for replication and maintenance of RP4 in A. eutrophus hosts.     

Construction of mobilizable vectors derived from plasmids RP4, pUC18 and pUC19

Mobilizable narrow-host-range plasmids were constructed from pUC18 and pUC19 by addition of a segment of pSUP2021 bearing the basis of mobilization (bom) site and origin of transfer (oriT) of RP4. One pair of expression vectors, pARO180 and pARO190, retains the beta-lactamase (bla) gene and twelve of the 13 restriction enzyme multiple cloning sites (MCS) of pUC18/19. Another pair was created by replacing the bla gene with the gene encoding kanamycin resistance (kan) from Tn5. The molecules replicate to high copy number in Escherichia coli and Enterobacter aerogenes. They can be transferred efficiently to other Gram- bacteria from the mobilizing strain, E. coli S17-1. In non-enteric strains, the new plasmids can be used as suicide vectors in site-specific insertional mutagenesis.     

Replication of plasmids in gram-negative bacteria.

Replication of plasmid deoxyribonucleic acid (DNA) is dependent on three stages: initiation, elongation, and termination. The first stage, initiation, depends on plasmid-encoded properties such as the replication origin and, in most cases, the replication initiation protein (Rep protein). In recent years the understanding of initiation and regulation of plasmid replication in Escherichia coli has increased considerably, but it is only for the ColE1-type plasmids that significant biochemical data about the initial priming reaction of DNA synthesis exist. Detailed models have been developed for the initiation and regulation of ColE1 replication. For other plasmids, such as pSC101, some hypotheses for priming mechanisms and replication initiation are presented. These hypotheses are based on experimental evidence and speculative comparisons with other systems, e.g., the chromosomal origin of E. coli. In most cases, knowledge concerning plasmid replication is limited to regulation mechanisms. These mechanisms coordinate plasmid replication to the host cell cycle, and they also seem to determine the host range of a plasmid. Most plasmids studied exhibit a narrow host range, limited to E. coli and related bacteria. In contrast, some others, such as the IncP plasmid RK2 and the IncQ plasmid RSF1010, are able to replicate in nearly all gram-negative bacteria. This broad host range may depend on the correct expression of the essential rep genes, which may be mediated by a complex regulatory mechanism (RK2) or by the use of different promoters (RSF1010). Alternatively or additionally, owing to the structure of their origin and/or to different forms of their replication initiation proteins, broad-host-range plasmids may adapt better to the host enzymes that participate in initiation. Furthermore, a broad host range can result when replication initiation is independent of host proteins, as is found in the priming reaction of RSF1010.     

Modified RP4 and Tn5-Mob derivatives for facilitated manipulation of large plasmids in Gram-negative bacteria

We have constructed a set of RP4 (NmS/TcS) and Tn5-Mob derivatives which have applications in experiments involving mobilization of replicons in many Gram-negative organisms. The different selection markers of the RP4 and Tn5-Mob derivatives include streptomycin, chloramphenicol, gentamicin, and spectinomycin resistance as well as mercury resistance, and a constitutively expressed lacZ gene. This choice of markers allows the use of these derivatives in bacteria which are naturally resistant to many antibiotics, and in strains which contain pre-existing resistance plasmids, transposons, or antibiotic cassette insertions. In addition, a RP4 derivative carrying the sacB gene of Bacillus subtilis was constructed. This allows the selection for the loss of RP4 after it has been used to mobilize other plasmids. A Tn5-Mob-sacB derivative with a new marker (Gm) was also developed, as were vectors which take advantage of the sacB gene to facilitate replacement of existing Tn5 inserts with other Tn5 derivatives. As an example of the use of these tools, three Rhizobium leguminosarum bv. viciae VF39 plasmids which have been shown to be involved in symbiosis were differentially tagged and mobilized (individually and in various combinations) to the plasmid-free Agrobacterium tumefaciens strain UBAPF2. None of the resultant Agrobacterium strains was able to fix nitrogen in symbiosis with peas.     

Transfer of Ti plasmids between Agrobacterium strains by mobilisation with the conjugative plasmid RP4

Summary The P type conjugative plasmid RP4 has been shown to be able to promote the transfer of the Agrobacterium Ti-plasmid. The results provide additional evidence that agrocin 84 sensitivity, exclusion of phage AP₁, ability to catabolize the guanidine derivatives octopine and nopaline and tumor inducing ability, are Ti-plasmid determined properties. Furthermore, the results strongly support the notion that at least part of the Ti-plasmid is transferred from the bacterium to the target plant cells, since it was demonstrated that Ti-plasmid linked genes specify the synthesis of octopine or nopaline by crown-gall tumor cells.     

Physical and functional mapping of two cointegrate plasmids derived from RP4 and TOL plasmid pDK1

Cointegrate plasmids were formed in vivo between the broad-host-range R-plasmid RP4 and two catabolic plasmids derived from Pseudomonas putida HS1. One of these was the wild-type plasmid pDK1 encoding the complete inducible toluene/xylene (TOL) catabolic pathway and one was pDKT1, a deletion derivative of pDK1 selected after growth of HS1 on benzoate and supporting growth on only toluene. The two plasmids formed, pDK2 and pDKT2 respectively, each consisted of a complete RP4 replicon in which was an insert of the parent plasmid DNA respectively 40 and 20 kbp in size. The detailed restriction maps of the two plasmids were determined and many of the catabolic genes were located by subcloning and enzyme assay of recombinant plasmids in Escherichia coli and Pseudomonas hosts. The insert in pDK2 contained both operons of the catabolic pathway, the 'upper pathway' operon (xylCAB) and the meta pathway operon (xylDLEGF(I,J,K)H), and a region identified as having the function of the regulator gene xylS. The insert in pDKT2 contained only the upper pathway operon and the regulatory region. Within each of the three coding regions there was great similarity with the same regions on TOL plasmids pWW0 and pWW53-4 apparent (a) by the same order of the genes, (b) by a similar pattern of restriction sites and (c) by hybridization studies. However, the order and orientations of the three coding regions differed from those previously described for both pWW0 and pWW53-4. The significance of these findings to the evolution of TOL plasmids is discussed.     

Restriction endonuclease analyses of the incompatibility group P-1 plasmids RK2, RP1, RP4, R68, and R68.45

The P-group plasmids RP1, RP4, RK2, R68 and R68.45 were analyzed by the following restriction endonucleases:BamHI,BglII,EcoRI,HindIII,PstI,PvuII,SalI, andSmaI. No differences between RP1, RP4, and RK2 were found, and the plasmid R68.45 was found to contain a direct duplication of an existing DNA sequence in R68. Our map of RK2 differs from the published map of RK2 in the corresponding region of the R68 map that is duplicated in R68.45.     

Relationship of group P1 plasmids revealed by heteroduplex experiments: RP1, RP4, R68 and RK2 are identical.

The molecular relationships of the IncP1 plasmids RP1, RP4, R68 and RK2 were tested by electron microscopic examination of heteroduplexes. In several hybridization experiments molecules were detected which had a 7.8% portion of incomplete reannealing. This 'heterologous region' could be explained by the typical renaturation behaviour of the transposon Tn1. The identity of the Tn1 transposon present in RP1 and RP4 was proved by heteroduplex experiments with lambda phage DNA containing this transposon. These results indicated that the plasmids RP1 and RP4 are identical. Additional heteroduplex experiments between plasmids R68.45 and RP8 and between R68.45 and RK2 were performed. R68.45, a derivative of R68, has a small DNA insertion and RP8 can be regarded as a large insertion mutant of RP4; both insertions were used as single-stranded hybridization markers. From the hybrid molecules formed, it was deduced that R68 and RK2 are identical with RP1 and RP4 as far as molecular structure is revealed by the technique used.     

Replication and conjugative mobilization of broad host-range IncQ plasmids

The IncQ plasmids have a broader host-range than any other known replicating element in bacteria. Studies on the replication and conjugative mobilization of these plasmids, which have mostly been focused on the nearly identical RSF1010 and R1162, are summarized with a view to understanding how this broad host-range is achieved. Several significant features of IncQ plasmids emerge from these studies: (1) initiation of replication, involving DnaA-independent activation of the origin and a dedicated primase, is strictly host-independent. (2) The plasmids can be conjugatively mobilized by a variety of different type IV transporters, including those engaged in the secretion of proteins involved in pathogenesis. (3) Stability is insured by a combination of high copy-number and modulated gene expression to reduce metabolic load.     

Mechanism of integration of the broad-host-range plasmid RP4 into the chromosome of Myxococcus xanthus

The site-specific recombination mechanism through which the plasmid RP4 has been previously shown to integrate into the chromosome of Myxococcus xanthus has been investigated further. Once integrated in one of the numerous chromosomal sites from two different strains, through a precise site on the plasmid, the latter can be excised either precisely or after a definite 14.5-kb deletion. In some cases, the integration is followed by different DNA rearrangements that yield a higher rate of excision and integration. A model for the site-specific integration and excision of the plasmid is proposed.     

Gene organization and nucleotide sequence of the primase region of IncP plasmids RP4 and R751.

The primase genes of RP4 are part of the primase operon located within the Tra1 region of this conjugative plasmid. The operon contains a total of seven transfer genes four of which (traA, B, C, D) are described here. Determination of the nucleotide sequence of the primase region confirmed the existence of an overlapping gene arrangement at the DNA primase locus (traC) with in-phase translational initiation signals. The traC gene encodes two acidic and hydrophilic polypeptide chains of 1061 (TraC1) and 746 (TraC2) amino acids corresponding to molecular masses of 116,721 and 81,647 Da. In contrast to RP4 the IncP beta plasmid R751 specifies four large primase gene products (192, 152, 135 and 83 kDa) crossreacting with anti-RP4 DNA primase serum. As shown by deletion analysis at least the 135 and 83 kDa polypeptides are two separate translational products that by analogy with the RP4 primases, arise from in-phase translational initiation sites. Even the smallest primase gene products TraC2 (RP4) and TraC4 (R751) exhibit primase activity. Nucleotide sequencing of the R751 primase region revealed the existence of three in-phase traC translational initiation signals leading to the expression of gene products with molecular masses of 158,950 Da, 134,476 Da, and 80,759 Da. The 192 kDa primase polypeptide is suggested to be a fusion protein resulting from an in frame translational readthrough of the traD UGA stopcodon. Distinct sequence similarities can be detected between the TraC proteins of RP4 and R751 gene products TraC3 and TraC4 and in addition between the TraD proteins of both plasmids. The R751 traC3 gene contains a stretch of 507 bp which is unrelated to RP4 traC or any other RP4 Tra1 gene.