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.     

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.     

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.     

Host range of conjugation and replication functions of the Escherichia coli sex plasmid Flac. Comparison with the broad host-range plasmid RK2

The Escherichia coli conjugative plasmid Flac has a restricted host range, in that transfer to Pseudomonas aeruginosa is not detectable. The molecular basis for this host-range restriction was studied by a separate comparison of the replication and conjugation systems of Flac with those of the broad host-range plasmid RK2. The origin of transfer of Flac (oriT_F) was cloned onto a small RK2 replicon. The hybrid plasmid, pDG2906, could be transferred efficiently by both the Flac and RK2 conjugation systems to an E. coli recipient. The Flac conjugation system was able to transfer pDG2906 to P. aeruginosa, but only at a frequency of 10^?4 of that of the RK2 conjugation system. A second hybrid plasmid, containing the replication region of Flac with the transfer region of RK2, could not be established in P. aeruginosa. These results show that Flac is able to mediate low frequency transfer to P. aeruginosa, and that the lack of replication in Pseudomonas is ultimately responsible for the restricted host range.     

Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2

Summary A DNA sequence cosisting of 617 base pairs (bp) from the region of the origin of replication of the broad-host range plasmid RK2 has been determined. Included within this sequence is a 393 bp HpaII restriction fragment that provides a functional origin or replication when other essential RK2 specified functions are provided in trans. Also contained in this sequence is a region, distinguished functionally from the replication origin, which is involved in the expression of inc ₂ incompatibility, i.e., the ability of derivatives of RK2 to eliminate a resident RK2 plasmid. The 617 bp sequence includes eight 17 base pair direct repeats with 5 located within the region required for a functional replication origin and 3 within the region involved in inc ₂ incompatibility. In addition, a 40 bp region rich in A-T followed by a 60 bp stretch having a high G+C content is present. Deletion evidence indicates that the A-T rich and possibly the G+C regions are required for a functional replication origin. Based on the evidence contained in this and the preceding paper (Thomas et al. 1980 b) a model will be presented for the involvement of these specific sequences in the initiation of RK2 DNA replication, plasmid maintenance and plasmid incompatibility.     

Species-Dependent Phenotypes of Replication-Temperature-Sensitive trfA Mutants of Plasmid RK2: a Codon-Neutral Base Substitution Stimulates Temperature Sensitivity by Leading to Reduced Levels of trfA Expression

TrfA is the only plasmid-encoded protein required for initiation of replication of the broad-host-range plasmid RK2. Here we describe the isolation of four trfA mutants temperature sensitive for replication in Pseudomonas aeruginosa. One of the mutations led to substitution of arginine 247 with cysteine. This mutant has been previously described to be temperature sensitive for replication, but poorly functional, in Escherichia coli. The remaining three mutants were identical, and each of them carried two mutations, one leading to substitution of arginine 163 with cysteine (mutation 163C) and the other a codon-neutral mutation changing the codon for glycine 235 from GGC to GGU (mutation 235). Neither of the two mutations caused a temperature-sensitive phenotype alone in P. aeruginosa, and the effect of the neutral mutation was caused by its ability to strongly reduce the trfA expression level. The double mutant and mutant 163C could not be stably maintained in E. coli, but mutant 235 could be established and, surprisingly, displayed a temperature-sensitive phenotype in this host. Mutation 235 strongly reduced the trfA expression level also in E. coli. The glycine 85 codon in trfA mRNA is GGU, and a change of this to GGC did not significantly affect expression. In addition, we found that wild-type trfA was expressed at much lower levels in E. coli than in P. aeruginosa, indicating that this level is a key parameter in the determination of the temperature-sensitive phenotypes in different species. The E. coli lacZ gene was translationally fused at the 3′ end and internally in trfA, in both cases leading to elimination of the effect of mutation 235 on expression. We therefore propose that this mutation acts through an effect on mRNA structure or stability.     

Straight core structure of DNA replication origins in the mammalian AMPD2 locus

Identification of the nucleotide consensus sequence in mammalian replication origins is a difficult and controversial problem. The hypothesis that local DNA topology could be involved in recognition by replication proteins is an exciting possibility. Secondary DNA structures, including intrinsically bent DNA, can be easily detected, and they may indicate a specific pattern in or near mammalian replication origins. This work presents the entire mapping of the intrinsically bent DNA sites (IBDSs), using in silico analysis and a circular permutation assay, of the DNA replication origins oriGNAI3, oriC, oriB, and oriA in the mammalian amplified AMPD2 gene domain. The results show that each origin presents an IBDS that flanks the straight core of these DNA replication sites. In addition, the in silico prediction of the nucleosome positioning reveals a strong indication that the center of an IBDS is localized in a nucleosome-free region (NFR). The structure of each of these curved sites is presented together with their helical parameters and topology. Together, the data that we present here indicate that the oriGNAI3 origin where preferential firing to the replication initiation events in the amplified AMPD2 domain occurs is the only origin that presents a straight, narrow region that is flanked on both sides by two intrinsically bent DNA sites within a short distance (～300 bp); however, all of the origins present at least one IBDS, which is localized in the NFR region. These results indicate that structural features could be implicated in the mammalian DNA replication origin and support the possibility of detecting and characterizing these segments.     

Comparison of the organisation of the genomes of phenotypically diverse plasmids of incompatibility group P: members of the IncP beta sub-group are closely related

Summary Comparison of physical maps of the broad host range plasmids R751, R906 and R772, belonging to the IncP sub-group of the Escherichia coli incompatibility group P, reveals two large regions of similarity, separated by dissimilar regions which contain the majority of the cleavage sites for restriction endonucleases with hexanucleotide recognition sites. Mapping of the regions of these plasmids which show homology to probes specific for genetically characterised segments of the distantly related IncP plasmid RK2, involved in plasmid maintenance or conjugal transfer, reveals that all four plasmids share a similar genetic organisation. In each case the homologous plasmid back-bone is interrupted by heterologous segments both between the essential replication loci oriV and trfA, and between the conjugal transfer regions tra1 and tra2, although in the case of R772 the segment of the backbone carrying the trfA and tra2 regions is inverted relative to that of the other plasmids. However, in the case of pJP4, shown to be a fourth member of the IncP sub-group, the back-bone is interrupted only by a single large segment adjacent to the trfA region. Mapping of the regions of the four IncP plasmids which show homology to Tn501 and nucleotide sequence determination at the ends of the homologous regions reveals that R906, R772 and pJP4 share a common mercury resistance region. This region, which appears to have been inactivated in R772, was probably inserted into a common ancestor of these plasmids by the transposition of an element related to an ancestor of Tn501. R751 shows no trace of the mercury resistance region, but contains a short relict of Tn501, derived from an independent insertion event.     

The Two Cis-Acting Sites,parS1 and oriC1, Contribute to the Longitudinal Organisation of Vibrio cholerae Chromosome I

The segregation of bacterial chromosomes follows a precise choreography of spatial organisation. It is initiated by the bipolar migration of the sister copies of the replication origin (ori). Most bacterial chromosomes contain a partition system (Par) with parS sites in close proximity to ori that contribute to the active mobilisation of the ori region towards the old pole. This is thought to result in a longitudinal chromosomal arrangement within the cell. In this study, we followed the duplication frequency and the cellular position of 19 Vibrio cholerae genome loci as a function of cell length. The genome of V. cholerae is divided between two chromosomes, chromosome I and II, which both contain a Par system. The ori region of chromosome I (ori_I) is tethered to the old pole, whereas the ori region of chromosome II is found at midcell. Nevertheless, we found that both chromosomes adopted a longitudinal organisation. Chromosome I extended over the entire cell while chromosome II extended over the younger cell half. We further demonstrate that displacing parS sites away from the ori_I region rotates the bulk of chromosome I. The only exception was the region where replication terminates, which still localised to the septum. However, the longitudinal arrangement of chromosome I persisted in Par mutants and, as was reported earlier, the ori region still localised towards the old pole. Finally, we show that the Par-independent longitudinal organisation and ori_I polarity were perturbed by the introduction of a second origin. Taken together, these results suggest that the Par system is the major contributor to the longitudinal organisation of chromosome I but that the replication program also influences the arrangement of bacterial chromosomes.

Author summary

Proper chromosome organisation within the cell is crucial for cellular proliferation. However, the mechanisms driving bacterial chromosome segregation are still strongly debated, partly due to their redundancy. Two patterns of chromosomal organisation can be distinguished in bacteria: a transversal chromosomal arrangement, such as in E. coli, where the origin of replication (ori) is positioned at midcell and flanked by the two halves of the chromosome (replichores), and a longitudinal arrangement, such as in C. crescentus, where ori is recruited to the pole and the replichores extend side by side along the long axis of the cell. Here, we present the first detailed characterization of the arrangement of the genetic material in a multipartite genome bacterium. To this end, we visualised the position of 19 loci scattered along the two V. cholerae chromosomes. We demonstrate that the two chromosomes, which both harbour a Par system, are longitudinally organised. However, the smaller one only extended over the younger cell half. In addition, we found that disruption of the Par system of chromosome I released its origin from the pole but preserved its longitudinal arrangement. Finally, we show that the addition of an ectopic ori perturbed this arrangement, suggesting that the replication program contributes to chromosomal organisation.     

Molecular cloning of replication and incompatibility regions from the R-plasmid R6K.

The construction of a physical map of R6K DNA on the basis of specific cleavage of R6K DNA by HindIII and EcoRI restriction endonucleases allowed us to determine the location of the R6K replication and drug resistance regions. Molecular cloning techniques were used to dissect the replication and incompatibility functions of R6K. This R-plasmid possesses two origins of replication, α and β, separated by a stretch of 3900 nucleotides. A region close to ori α. controls the copy number of the composite replicon. Inverted duplications which are 100 to 200 nucleotides long are found at the positions of ori α and ori β, respectively. A 1400-nucleotide long sequence within the region bounded by the inverted duplications and separate from the origins and the control region is involved in the R6K self-replication and replication under conditions of polymerase I deprivation. This region also contains some of the incompatibility genes of R6K. The sequentially asymmetrically bidirectional mode of R6K replication is due to the existence of a replication termination site. This terminator is located outside the sequences bounded by the inverted duplications and is not essential for plasmid DNA replication.     

Conditional lethal mutants of the kilB determinant of broad host range plasmid RK2

We have examined the relationship of kilB to the other known determinants which map in the 14'-22' region of RK2. These are trfA, which encodes a diffusible replication function, and tra3, which specifies a function required for plasmid transmissibility. We found that, in addition to kilB, both tra3 and trfA functions are expressed by the cloned 14'-22' region of RK2. Four temperature-sensitive mutants of kilB were isolated by in vitro mutagenesis of the cloned segment. At 42 degrees C these mutant plasmids can be maintained in Escherichia coli cells which lack a korB+ helper plasmid. At 30 degrees C the helper plasmid is required. Our analysis of these mutants revealed that kilB function is distinct from those of trfA and tra3. One mutant plasmid was temperature-sensitive for maintenance of an RK2 ori plasmid, but this phenotype was shown to be independent of the KilB(ts) phenotype. Thus, kilB appears to be a separate new locus in this portion of the RK2 genome. In addition, these mutants allowed us to test for the existence of an essential replication determinant (trfB) in the 50.4'-56.4' region of RK2. Our results demonstrate that this region is non-essential for replication from the RK2 ori in E. coli. We propose an alternative hypothesis to explain the role of the RK2 trfB region for plasmid maintenance in E. coli.     

Characterization of the Holliday Junction Resolving Enzyme Encoded by the Bacillus subtilis Bacteriophage SPP1

Recombination-dependent DNA replication, which is a central component of viral replication restart, is poorly understood in Firmicutes bacteriophages. Phage SPP1 initiates unidirectional theta DNA replication from a discrete replication origin (oriL), and when replication progresses, the fork might stall by the binding of the origin binding protein G38P to the late replication origin (oriR). Replication restart is dependent on viral recombination proteins to synthesize a linear head-to-tail concatemer, which is the substrate for viral DNA packaging. To identify new functions involved in this process, uncharacterized genes from phage SPP1 were analyzed. Immediately after infection, SPP1 transcribes a number of genes involved in recombination and replication from P _E2 and P _E3 promoters. Resequencing the region corresponding to the last two hypothetical genes transcribed from the P _E2 operon (genes 44 and 45) showed that they are in fact a single gene, re-annotated here as gene 44, that encodes a single polypeptide, named gene 44 product (G44P, 27.5 kDa). G44P shares a low but significant degree of identity in its C-terminal region with virus-encoded RusA-like resolvases. The data presented here demonstrate that G44P, which is a dimer in solution, binds with high affinity but without sequence specificity to several double-stranded DNA recombination intermediates. G44P preferentially cleaves Holliday junctions, but also, with lower efficiency, replicated D-loops. It also partially complemented the loss of RecU resolvase activity in B. subtilis cells. These in vitro and in vivo data suggest a role for G44P in replication restart during the transition to concatemeric viral replication.     

Genetic studies of F plasmid maintenance genes involved in copy number control,incompatability, and partitioning

We have identified a 2.1-kilobase (kb) region (44.1 to 46.19 kb) in F that is necessary and sufficient to form low copy number minireplicons. Within this region we have mapped (i) mutations (cop) inducing 4.4- to 28-fold increases in copy number and (ii) two separate regions that determine incompatability (incB and incC). The 2.1-kilobase region has also been shown by others to contain (i) an origin of replication, ori (ii) a locus (aos) necessary for sensitivity to the plasmid replication inhibitor, acridine orange, and (iii) nine, 19- to 22-base-pair direct repeat sequences organized in two clusters. In the present work we more accurately locate the aos locus and show that it, as well as ori, incB, incC, and some cop mutations, map within or overlap the direct repeat regions. Analysis of other cop mutations indicates that they reduce or destroy the incompatability reaction associated with the 2.1-kb region; however, these cop mutations do not map within the incB or incC determinants. A 2-fold copy number elevation and unstable plasmid maintenance also results from deletion of the 46.19- to 49.2-kb region. Results described here and elsewhere suggest that the instability of the deletion mutant reflects the loss of partitioning gene, a gene that is probably identical to an inc locus, incD, that had been identified in this region in prior work. Whether or not the incD locus has anything to do with the slight copy number elevation is unknown.     

Gene encoding a replication initiator protein and replication origin of conjugative plasmid pSA1.1 of Streptomyces cyaneus ATCC 14921

pSA1.1 is a 9.1-kb multicopy plasmid originally isolated from Streptomyces cyaneus (formerly S. azureus) ATCC 14921. This plasmid accumulates single-stranded DNA in S. lividans and is therefore considered to replicate by a rolling-circle replication. In the present work, the rep gene encoding the replication initiator protein and the replication origin ori of pSA1.1 were determined. The rep and ori are located on separate regions. The Rep protein of pSA1.1 belongs to superfamily I which includes A proteins of phages. Nucleotide sequence of the surrounding putative nicking site of pSA1.1 shows good agreement with those of the pC194 group plasmids and phages. The direction of replication was also determined.     

Genetic analysis of a lactococcal plasmid replicon

Summary The sequence and genetic organization was determined of the 2508 by lactococcal portion of pFX2, which was derived from a crypticLactococcus lactis subsp.lactis plasmid and used as the basis for construction of a series of lactococcal vectors. A lactococcal plasmid plus origin and two replication protein-coding regions (repA andrepB) were located. RepA has a helix-turn-helix motif, a geometry typical of DNA-binding proteins. RepB shows a high degree of homology to the plasmid replication initiation proteins from other gram-positive bacteria andMycoplasma. The transcribed inverted repeat sequence betweenrepA andrepB could form an attenuator to regulate pFX2 replication. Upstream of theori site, and in a region which was non-essential for replication, a 215 by sequence identical to the staphylococcal plasmid pE194 and carrying the RS_A site was identified. The genetic organization of this lactococcal plasmid replicon shares significant similarity with pE194 group plasmids.     

Distribution of 2-kb miniplasmid pBMB2062 from Bacillus thuringiensis kurstaki YBT-1520 strain in Bacillus species

A multi-copy and small plasmid pBMB2062 from Bacillus thuringiensis kurstaki YBT-1520 strain was cloned and characterized and its distribution was analyzed using dot-blot analysis with the ORF1 fragment as a probe. Bacillus species of 84 serotypes were evaluated. The pBMB2062 plasmid was found to be present in commercial B. thuringiensis kurstaki (H3abc) and aizawai (H7) insecticides of various serotypes, and one Bacillus cereus UW85 strain (produced Zwittermicin fungicide and Cry toxin synergist). The sequences of 7 pBMB2062-like plasmids from randomly selected Bacillus species (positive signal in the dot-blot analysis) were highly conserved. Two open reading frames (ORFs), ORF1 and ORF2, were present in this plasmid. ORF1 was found to be necessary for plasmid replication, whereas ORF2 did not play a role in replication or stability. Based on its sequence homology, ORF2 was a putative solitary antitoxin. Furthermore, the copy number of the replicon of pBMB2062 was higher than those of ori1030 and ori44 based on the thermogenic data, and ori2062 could drive the stable replication of a recombinant plasmid (11 kb total size) in B. thuringiensis.     

Tn7 insertion mutations affecting the host range of the promiscuous IncP-1 plasmid R18

The genetic basis of plasmid host range has been investigated by Tn7 insertion mutagenesis of the promiscuous plasmid R18 in Pseudomonas aeruginosa. Six mutants have been isolated on the basis of greatly reduced transferability into Escherichia coli C while retaining normal transferability within P. aeruginosa. Their physical mapping shows that two of them map at coordinate 11.72 ± 0.14 kb, in the region of the origin of plasmid replication (oriV) and one at 18.0 ± 0.3 kb, in the trans-acting gene essential for initiation of replication at oriV (trfA). Three map at 48.4 ± 0.5 kb in the region of the origin of plasmid transfer (oriT) and the site at which a single-strand nick is introduced in the plasmid DNA-protein relaxation complex (rlx). Consistent with the postulated defective replication of the oriV and trfA mutants was their inability to transform E. coli C or K12 while being able to transform P. aeruginosa. As expected the oriT/rlx mutants transformed both hosts as effectively as R18. Furthermore the trfA mutant was readily curable by mitomycin C in a DNA polymerase I-proficient P. aeruginosa and spontaneously lost from a polymerase-deficient mutant of P. aeruginosa suggesting a role of this polymerase in the replication of R18. Extensive transfer tests from P. aeruginosa into a range of enteric bacteria, other Pseudomonas species and into other Gram-negative bacteria indicated a complex host range pattern for these mutants. It appears that both plasmid replication and conjugation genes are responsible for host range in addition to the involvement of host gene products.