Replication of the broad-host-range plasmid RK2: isolation and characterization of a spontaneous deletion mutant that can replicate in Agrobacterium tumefaciens but not in Escherichia coli

Two spontaneous deletions of a derivative of the broad-host-range plasmid RK2 were isolated from Agrobacterium tumefaciens. The two deletions have lost 56 and 505 bp, respectively, near the origin of replication (oriV). Of the eight 17-bp repeats present in the RK2 oriV, the smaller deletion has lost the first two while the larger one has lost the first three. The deletions led to a significant increase (3- to 7-fold) in plasmid copy number in A. tumefaciens, indicating their importance in copy number control. While the smaller deletion could replicate in Escherichia coli, the larger one could not. The role of the oriV sequences in the replication of pRK2 in A. tumefaciens and in E. coli is discussed.     

OriVRK2 replicon function in the absence oftrfA inAzotobacter vinelandii

The oriV_RK2 does not need the function of either trfA⁺ ortrfA Operon for replication and maintenance of an oriV_RK2-containing plasmid inAzotobacter vinelandii.     

A Potential New Gene (tccA) on IncP Plasmid RK2 and Transposon Tn1721:Relationship of Its Product to the TrwC Relaxase/Helicase of IncW Plasmid R388

Computational analysis of the fully sequenced 60-kb genome of broad-host-range IncPα plasmid RK2 revealed a previously unreported potential protein-coding sequence, an 80-codon open reading frame (tccA), located in the region between the vegetative origin of replication (oriV) and thetetRgene of the tetracycline resistance determinant. The coding region is also present in the transposon Tn1721 tetregion, which is nearly identical to thetetregion of RK2. Remarkably, the predicted polypeptide product of the coding region displays 56% identity and 72% similarity with the C-terminal domain of the TrwC relaxase/helicase protein of IncW plasmid R388.     

Replication and incompatibility properties of segments of the origin region of replication of the broad host range plasmid RK2

Summary Replication and incompatibility properties in Escherichia coli of DNA segments from the replication origin region of plasmid RK2 have been investigated. A 393 bp HpaII fragment, derived from the region of the RK2 origin of replication, carries an active origin when essential RK2 encoded functions are provided in trans and will form a mini RK2 replicon when linked to a non-replicating selective fragment. This small ori _RK2 plasmid cannot stably coexist with other functional RK2 replicons and is thus incompatible with RK2 replicons. However, the 393 bp ori _RK2 segment when cloned into a high copy number plasmid, where plasmid maintenance is no longer dependent on ori _RK2, does not interfere with maintenance of a resident RK2 replicon. This is in contrast to larger segments from the origin region that, when cloned at high copy number, cause the loss of a resident RK2 replicon. The apparent ability of the small HpaII ori_RK2 plasmid to displace resident RK2 replicons may indicate the turning on of one incompatibility mechanism only when replication from ori _RK2is required or may simply reflect the strong selective pressure for establishment of the incoming ori _RK2 plasmid and poor ability of the HpaII ori _RK2 plasmid to replicate in the presence of another RK2 replicon. The incompatibility expressed by the functional HpaII ori _RK2 may be designated inc ₁. The activity of a segment of RK2, cloned at high copy number, to eliminate a resident RK2 plasmid has been localized to a region of RK2 DNA, designated the inc ₂ region, to distinguish it from inc ₁, above, that overlaps but does not coincide with the 393 bp HpaII ori _RK2. This inc ₂ region also appears to be involved in segregation of RK2 derivatives since removal of a portion of this region results in both higher copy number and increased instability of the RK2 derivative. In addition to defining the region of the RK2 origin of replication, these results indicate that the ability to eliminate a resident RK2 replicon can be expressed by fragments, cloned at high copy number, that do not contain the complete ori _RK2. Also, only part of the inc ₂ region that appears to be responsible for efficient elimination of RK2 replicons by fragments cloned at high copy number is required for ori _RK2.     

Roles of Escherichia coli heat shock proteins DnaK, DnaJ and GrpE in mini-F plasmid replication

Summary A subset of Escherichia coli heat shock proteins, DnaK, DnaJ and GrpE were shown to be required for replication of mini-F plasmid. Strains of E. coli K12 carrying a missense mutation or deletion in the dnaK, dnaJ, or grpE gene were virtually unable to be transformed by mini-F DNA at the temperature (30° C) that permits cell growth. When excess amounts of the replication initiator protein (repE gene product) of mini-F were provided by means of a multicopy plasmid carrying repE, these mutant bacteria became capable of supporting mini-F replication under the same conditions. However, the copy number of a high copy number mini-F plasmid was reduced in these mutant bacteria as compared with the wild type in the presence of excess RepE protein. Furthermore, mini-F plasmid mutants that produce altered initiator protein and exhibit a very high copy number were able to replicate in strains deficient in any of the above heat shock proteins. These results indicate that the subset of heat shock proteins (DnaK, DnaJ and GrpE) play essential roles that help the functioning of the RepE initiator protein in mini-F DNA replication.     

Properties of plasmids from Methylomonas clara

Three independently isolated clones of the obligate methylotrophic bacterium Methylomonas clara (ATCC 31226) were tested for their plasmid content. Strains B45-BE-2 and B45-BE-3 were shown to harbor one class of plasmid molecules each, with molecular sizes of 28 MDa (46 kb) and 10 MDa (16 kb), respectively. Strain B45-BE-1 does not contain extrachromosomal DNA. Restriction maps of plasmid pBE-2 from strain B45-BE-2 and of plasmid pBE-3 from strain B45-BE-3 were established and the smaller plasmid pBE-3 shown to be a linear deletion derivative of plasmid pBE-2. Two other deletions were characterized. When subcloned into the Escherichia coli vector pBR322, plasmid pBE-3 was unable to complement for the polymerase A dependence of the pBR322 replicon. Additional experiments indicate a general failure of the M. clara specific replicon to function in E. coli.     

Cloning of heterologous genes specifying detrimental proteins on pUC-derived plasmids inEscherichia coli

A system is described that enables the cloning of genes specifying detrimental proteins inEscherichia coli. The system is based on pUC plasmids and was developed for the expression of theBacillus subtilis csaA gene, which is lethal when expressed at high levels. Suppressor strains that tolerate the presence of plasmids for high-level expression ofcsaA were isolated, which contained small cryptic deletion variants of the parental plasmid in high copy numbers. The cryptic plasmids consisted mainly of the pUC replication functions and lacked thecsaA region and selectable markers. The co-resident, incompatible, cryptic plasmids enabled the maintenance of thecsaA plasmids by reducing their copy number 20-fold, which resulted in a concomitant 3- to 7-fold reduction in the expression of plasmid-encoded genes. Strains carrying these cryptic endogenous plasmids proved to be useful for the construction of pUC-based recombinant plasmids carrying other genes, such as theskc gene ofStreptococcus equisimilis, which cannot be cloned in high copy numbers inE. coli. Several strategies to reduce production levels of heterologous proteins specified by plasmids are compared.     

Development of a Native Plasmid as a Cloning Vector in Clavibacter xyli subsp. cynodontis

A 50-kilobase (kb) cryptic plasmid was found in three geographic isolates of Clavibacter xyli subsp. cynodontis (Cxc). The DNA region essential for replication of the plasmid was cloned in an Escherichia coli vector. The resulting vector, which functions as a shuttle vector between E. coli and Cxc, was characterized further with respect to its stability and copy number.     

Enhanced production of single copy backbone-free transgenic plants in multiple crop species using binary vectors with a pRi replication origin in <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Single transgene copy, vector backbone-free transgenic crop plants are highly desired for functional genomics and many biotechnological applications. We demonstrate that binary vectors that use a replication origin derived from the Ri plasmid of Agrobacterium rhizogenes (oriRi) increase the frequency of single copy, backbone-free transgenic plants in Agrobacterium tumefaciens mediated transformation of soybean, canola, and corn, compared to RK2-derived binary vectors (RK2 oriV). In large scale soybean transformation experiments, the frequency of single copy, backbone-free transgenic plants was nearly doubled in two versions of the oriRi vectors compared to the RK2 oriV control vector. In canola transformation experiments, the oriRi vector produced more single copy, backbone-free transgenic plants than did the RK2 oriV vector. In corn transformation experiments, the frequency of single copy backbone-free transgenic plants was also significantly increased when using the oriRi vector, although the transformation frequency dropped. These results, derived from transformation experiments using three crops, indicate the advantage of oriRi vectors over RK2 oriV binary vectors for the production of single copy, backbone-free transgenic plants using Agrobacterium-mediated transformation.     

The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector

Summary A plant gene vector cassette to be used in combination with various Escherichia coli gene-cloning vectors was constructed. This cassette contains a replication and mobilization unit which allows it to be maintained and to be transferred back and forth between E. coli and Agrobacterium tumefaciens hosts provided these hosts contain plasmid RK2 replication and mobilization helper functions. The cassette also harbors a transferable DNA unit with plant selectable marker genes and cloning sites which can be combined with different bacterial replicons, thus facilitating the reisolation of transferred DNA from transformed plants in E. coli. The vector cassette contains two different promoters derived from the T-DNA-encoded genes 5 and nopaline synthase (NOS). By comparing the levels of expression of the marker enzymes linked to each of these promoter sequences, it was found that the gene 5 promoter is active in a tissue-specific fashion whereas this is not the case for the NOS promoter. This observation provides the first documented instance of a gene derived from a procaryotic host the expression of which is apparently regulated by plant growth factors.Abbreviations OCS octopine synthase (gene) - NOS nopaline synthase (gene) - NPT-II neomycin phosphotransferase (gene) of transposon Tn5 - vir Ti-plasmid region encoding virulence functions - Cb carbenicillin - Gm gentamycin - Km kanamycin - Cm chloramphenicol - Sm streptomycin - Sp spectinomycin - Rif rifampicin - Ery erythromycin - bom basis of mobilization - ori _r origin of conjugational plasmid transfer - Tra, Mob functions required for conjugational transfer of plasmids - BAP N⁶-benzylaminopurine - NAA -naphthaleneacetic acid - CTAB N-cetyl-N,N,N-trimethyl-ammonium bromide     

Behavior of Inc-Q plasmids in Agrobacterium tumefaciens

Inc-Q plasmids were introduced into Agrobacterium tumefaciens, by mobilization from Escherichia coli with an Inc-P plasmid, or by transformation with purified plasmid DNA. It was found that they were stably maintained. The presence of an Inc-Q plasmid did not influence tumorigenicity. These results suggest that these plasmids may be used in genetic complementation studies of Ti plasmid mutants in A. tumefaciens.     

Cloning and characterisation of the recA gene of Agrobacterium tumefaciens C58

Summary A gene library of Agrobacterium tumefaciens C58 has been constructed in the plasmid vector pACYC184. A recombinant plasmid was isolated from the library by interspecific complementation in E. coli, which contained the A. tumefaciens recA gene. Heterologous Southern blotting and DNA sequence analysis have demonstrated the existence of considerable homology between the recA genes of A. tumefaciens, E. coli and R. meliloti.Abbreviations MMS methyl methanesulfonate - UV ultraviolet light - bp base pairs - kbp kilo base pairs - dATP deoxyadenosine 5-triphosphate - dNTP deoxynucleoside triphosphate - Ap ampicillin - Cm chloramphenicol - Km kanamycin - Tet tetracycline     

OriC plasmids do not affect timing of chromosome replication in Escherichia coli K12

Summary The variability of the time interval between successive rounds of chromosome replication was estimated by density-shift experiments, by measuring the conversion of heavy DNA to hybrid density and light DNAs upon transfer of a steady-state culture growing in medium with [¹³C]glucose and ¹⁵NH₄Cl to medium with light isotopes. The coefficient of variation (CV%) for the interreplication time of the Escherichia coli K12 chromosome was found to be 17%, i.e. similar to that for interdivision time. The presence of additional copies of oriC in the cell on a high copy number plasmid did not increase the CV of interreplication time. It is concluded that a single rate-limiting event is unlikely to time the initiation of chromosome replication. The regulation of initiation at oriC and the coordination with cell division is discussed.     

The region essential for efficient autonomous replication of pSa in Escherichia coli

Summary Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.     

Role of DnaA protein during replication of plasmid pBR322 in Escherichia coli

Summary The in vivo role of the Escherichia coli protein DnaA in the replication of plasmid pBR322 was investigated, using a plasmid derivative carrying an inducible dnaA ⁺ gene. In LB medium without inducer, the replication of this plasmid, like that of pBR322, was inhibited by heat inactivation of chromosomal DnaA46 protein so that plasmid accumulation ceased 1 to 2 h after the temperature shift. This inhibition did not occur when the plasmid dnaA ⁺ gene was expressed in the presence of the inducer isopropyl-1-thin--d-galactopyranoside (IPTG). Inhibition was also not observed in glycerol minimal medium or in the presence of low concentrations of rifampicin or chloramphenicol. Deletion of the DnaA binding site and the primosome assembly sites (pas, rri) downstream of the replication origin did not affect the plasmid copy number during exponential growth at 30° C, or after inactivation of DnaA by a shift to 42° C in a dnaA46 host, or after oversupply of DnaA, indicating that these sites are not involved in a rate-limiting step for replication in vivo. The accumulation of the replication inhibitor, RNAI, was independent of DnaA activity, ruling out the possibility that DnaA acts as a repressor of RNAI synthesis, as has been suggested in the literature. Changes in the rate of plasmid replication in response to changes in DnaA activity (in LB medium) could be resolved into an early, rom-dependent, and a late, rom-independent component. Rom ^– plasmids show only the late effect. After heat inactivation of DnaC, plasmid replication ceased immediately. These results, together with previously published reports, suggest that DnaA plays no specific role during in vivo replication of ColE1 plasmids and that the gradual cessation of plasmid replication after heat inactivation of DnaA in LB medium results from indirect effects of the inhibition of chromosome replication and the ensuing saturation of promoters with RNA polymerase under nonpermissive growth conditions.     

Complementation analysis of replication and maintenance functions of broad host range plasmids RK2 and RP1

It has previously been concluded that regions tentatively designated trfA and trfB, located at 16–18.7 and 54–56 kb, respectively, on the genome of broad host range plasmid RK2 provide trans-acting functions involved in plasmid replication and maintenance in Escherichia coli (Thomas et al., 1980). A third region, the replication origin, ori_RK2, located at 12 kb on the genome, is also required. A segment of DNA containing ori_RK2 can be linked to a nonreplicating selective marker and can replicate as an autonomous plasmid so long as DNA of RK2 carrying the gene for one or more trans-acting replication functions is present in the same cell on an independent plasmid or integrated into the chromosome. It is demonstrated here that the trfA region alone can provide the trans-acting functions necessary for replication from ori_RK2. Deletion of the trfB region in trans to an ori_RK2 plasmid does not correlate with alteration in copy number or stability of the ori_RK2 plasmid. Temperature-sensitive mutants defective in plasmid maintenance can apparently arise from mutations in both the trfA and trfB regions as indicated by complementation analysis of three different mutants. The trfA and trfB regions from two mutant plasmids have been cloned and used to allow a physically separate but functionally dependent ori_RK2 plasmid to replicate at 30 °C. When the source of trfA and trfB is a trfB mutant the ori_RK2 plasmid is temperature stable but is temperature sensitive when the source is a trfA mutant. This confirms that only trfA is essential for initiation at and elongation from ori_RK2 which is probably the primary event in RK2 replication and suggests that the trfB region plays some other role in plasmid maintenance in plasmids carrying all three regions, ori_RK2, trfA, and trfB.     

Site-specific mutagenesis of the Ti plasmid by transformation of Agrobacterium tumefaciens with mutagenized T-DNA fragments cloned in E. coli plasmids

Summary A DNA fragment covering the complete T-region of the Ti plasmid from Agrobacterium tumefaciens strain C58 was cloned in the Escherichia coli cosmid pHC79. This fragment was mutagenized by insertion of transposon Tn5. The isolated DNA from hybrid plasmids was used to transform cells of A. tumefaciens strain C58 applying the freeze-thaw method. Although the E. coli plasmids with the mutagenized Ti plasmid fragment cannot replicate in these cells, they can be rescued by recombination with the homologous region of the Ti plasmid. The cointegrates formed were resolved in a second recobination event, which was detected by loss of the drug resistance marker of the E. coli plasmid. Subcloning of the Ti plasmid fragments labeled with Tn5 showed that the frequency of rescue of the hybrid plasmid as a cointegrate and its segregation in agrobacteria depend on the degree of homology with the Ti plasmid. We also applied the strategy for site-directed Tn5 mutagenesis to insert specifically the replication origin of bacteriophage fd and the thymidine kinase gene from Herpes virus into the T-DNA of Ti plasmid-C58.     

Plasmid R1 in Salmonella typhimurium: Molecular instability and gene dosage effects

Plasmid R1 drd-19 and two of its copy mutants (pKN102 and pKN103) were transferred from Escherichia coli to Salmonella typhimurium, where the expression of the copy mutations was studied further. The copy number (ratio of plasmid DNA to chromosomal DNA) was the same in S. typhimurium and in E. coli. The activities of the plasmid-coded antibiotic-metabolizing enzymes β-lactamase, chloramphenicol acetyltransferase, and streptomycin adenylyltransferase as well as the resistances to ampicillin and streptomycin were proportional to the gene dosage up to at least a threefold increase in the steady state plasmid copy number, whereas resistance to chloramphenicol showed no increase with increased number of plasmid copies per chromosome equivalent. Also the resistance to rifampicin was affected since S. typhimurium cells became more sensitive the higher the copy number of the resident plasmid. Furthermore, plasmid R1 showed molecular instability in S. typhimurium cells since there was a tendency to dissociate into resistance transfer factors and resistance determinants and also to form miniplasmids. This tendency to instability was more pronounced the higher the plasmid copy number.     

dam methylation and the initiation of DNA replication on oriC plasmids

Summary Plasmid DNA containing the replication origin of the Escherichia coli chromosome (oriC) has been shown to be inefficient as a template for DNA synthesis in vitro when isolated from dam mutants. here, we extend this study to hemimethylated oriC plasmids and to replication in dam-3 mutant enzyme extracts. The results show that: (1) hemimethylated oriC plasmids replicate with the same low efficiency as nonmethylated DNA; (2) DNA synthesis starts at oriC regardless of the methylated state of the template; (3) replication in dam-3 enzyme extracts is inefficient because this strain is deficient in DnaA protein; and (4) consistent with this observation, the copy number of the oriC plasmid pFH271 is reduced in the dam-3 mutant. However, we have found that low DnaA protein levels in dam-3 mutants are not sufficient to explain the reduced transformation efficiency of oriC plasmids. We suggest that there must exist in vivo inhibitory factors not present or present in low quantities in vitro which specifically recognize the hemimethylated or nonmethylated forms of the oric region.     

Studies on the alteration of chromosome copy number and cell division potential in a dnaA mutant of Escherichia coli

Summary The dnaA167 mutant of Escherichia coli, N167, maintains, on the average, two replicating chromosomes per cell at the perimissive growth temperature of 30°C and only one per cell at the higher permissive growth temperature of 38°C. When the growth temperature of this mutant is changed from 30° to 38°C the cells rapidly readjust their chromosome copy number from two to one. I have examined the kinetics of this transition with reference to DNA replication and cell division. My results indicate that this mutant uncouples cell division from chromosome duplication to achieve the appropriate copy number, suggesting that the dnaA gene product may be involved in the coordination between these two cellular events.