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.     

Complete nucleotide sequence of pT181, a tetracycline-resistance plasmid from Staphylococcus aureus

pT181 is a naturally occurring Staphylococcus aureus plasmid, encoding inducible resistance to tetracycline. The plasmid has a copy number of about 20 per cell, and belongs to the incompatibility group inc3. The complete nucleotide sequence of pT181 has been determined and consists of 4437 bp. The nucleotide sequence contains 69.8% A-T and 30.2% G-C pairs. pT181 was found to contain four open reading frames capable of coding for polypeptides containing more than 50 amino acids. All the putative polypeptides are coded by one strand. The molecular weights of the four putative polypeptides are (in daltons): A, 37,500; B, 35,000; C, 23,000, and D, 18,000. Polypeptide A corresponds to the repC protein, earlier shown to be specifically required for pT181 replication. Polypeptide B (and possibly polypeptide D) are involved in tetracycline resistance. No role has yet been established for polypeptide C; deletion of the coding sequence for the C polypeptide has no detectable effect on any property of the pT181 plasmid. A region consisting of about 1200 bp contains information for the replication and copy number control of this plasmid. The sequencing results are discussed in relation to the replication properties and tetracycline resistance associated with the pT181 plasmid.     

Nucleotide sequence and functional properties of DNA encoding incompatibility in the broad host-range plasmid R1162

Summary A 370 base pair (bp) fragment of R1162 DNA encoding the incompatibility determinant has been cloned and sequenced. The DNA is located between 6.1 and 6.5 on the R1162 map, near the origin of replication. The sequence contains three perfectly conserved 20 bp direct repeats, with 11 bp of this sequence repeated a fourth time. The direct repeat unit shows some homology with that of another, unrelated broad host-range plasmid, RK2. The cloned DNA has two other properties: it lowers the copy number of R1162 when cloned into this plasmid, and it is required in cis for replication of R1162 satellite plasmids.     

Identification of the minimal essential region for the replication origin of miniF plasmid

Summary The minimal replication origin of miniF plasmid was found to lie within a region of 217 bp in length. This region contains an AT cluster and the four 19 bp direct repeats responsible for incompatibility, termed incB. Its location coincides with that of ori2 of plasmid F, previously inferred to be the replication starting point by electron microscopic analysis (Eichenlaub et al. 1981).Abbreviations kb kilobase(s) - bp base pairs - Ap ampicillin - Tc tetracycline     

The origin of transfer (oriT) of the conjugative plasmid R46: characterization by deletion analysis and DNA sequencing

Summary The origin of transfer (oriT) is the sequence within which conjugal transfer of plasmid DNA is initiated, and is absolutely required in cis for plasmid mobilization. We have cloned oriT from the 52 kb IncN plasmid R46 on a 600 bp fragment, and mapped the limits of the relevant sequence by deletion analysis and transposon mutagenesis. The nucleotide sequence of the oriT region contains 13 direct repeats of an 11 bp consensus sequence, 3 different pairs of 10 bp inverted repeats, and a segment that is extremely A-T rich. The direct repeats are within a region required for high frequency transfer and their sequence is such that their periodic alignment along the helix may induce curvature of the DNA. Analysis of Tn1725 insertions within the sequenced fragment of R46 revealed that, unlike most other transposons, transposition of Tn1725 can cause target sequence duplications of three different sizes.     

IncD, a genetic locus in F responsible for incompatibility with several plasmids of the IncFI group

Summary Cloning of mini-F DNA segments has led to the identification and mapping of a locus, incD, involved in incompatibility reactions with many IncFI plasmids. The cloned incD locus expressed incompatibility with F, R386, and six other IncFI plasmids but not with ColV3-K30 or pHH507 which lack sequence homology with the incD region. A sequence of 360 bp (48.66–49.02 FKB) was found to be sufficient for expression of incD incompatibility. Multicopy vectors containing incD are compatible with each other, but can be displaced by mini-F plasmids deleted for incD. These results indicate that incD-mediated incompatibility reactions require the presence of replication genes to which incD is normally linked. The degree of incompatibility exercised by incD is moderate compared with that of other inc loci in F, suggesting that incD is involved in an aspect of plasmid maintennce, such as partition, different from the functions of the other inc loci.     

Maintenance of the bacteriocinogenic plasmid Clo DF13 in Escherichia coli cells. I. Localisation and mutual interactions of four Clo DF13 incompatibility regions

Summary The incompatibility properties of the bacteriocinogenic plasmid Clo DF13 have been examined. By using Clo DF13, Clo DF13 deletion, and transposon insertion mutants as well as compatible R plasmids into which Clo DF13 fragments have been cloned, we could identify and localise four different incompatibility regions on the Clo DF13 genome. These regions, designated incA, incB, incC, and incD are located in the following positions: incA about incD between 1.8% and 9% of the Clo DF13 genome. We studied the contribution of each of the four inc regions, separately and/or in combination with each other, to the incompatibility between two plasmid replicons. Two types of incompatibility can be distinguished: Type I evoked by incD, that overlaps the replication control area of Clo DF13 and type II, caused by incA, B and C. From our observations we present a model for plasmid incompatibility based on a combination of the existing repressor dilution and membrane attachment models.     

The DNa-protein relaxation complex of the plasmid RK2: Location of the site-specific nick in the region of the proposed origin of transfer

Summary The broad host range plasmid, RK2, has been isolated as a DNA-protein relaxation complex. Nicking of the plasmid DNA in the relaxation complex occurs at a single specific site (rlx) located approximately 20 kb away from the origin of DNA replication. A cis-acting function required for plasmid transfer, the presumptive origin of transfer, maps in the same region as rlx. The region of RK2 encompassing rlx has been cloned onto pBR322 and shown to promote mobilization of the hybrid plasmid by an RK2 derivative. These results indicate that the RK2 relaxation complex nicks at or near the origin of transfer of the RK2 plasmid.     

Cloning of the replication and incompatibility regions of a plasmid derived from Rts1

A replication region, consisting of a 1.1-megadalton (Md) EcoRI/HindIII fragment, was isolated from an Rts1 derivative plasmid. This 1.1-Md fragment, designated as mini-Rts1, was ligated to either pBR322 or a nonreplicating DNA fragment specifying a drug resistance, and its replication properties were investigated. The mini-Rts1 plasmid was cured at a high frequency at 42 °C, while it was maintained stably at 37 °C despite it existed in low copy number. These behaviors are quite similar to those of Rts1. By dissecting the pBR322:mini-Rts1 chimeric plasmid with AccI endonuclease, an inc region of 0.34 Md in size was cloned, which expressed incompatibility toward Rts1. Proteins encoded on the mini-Rts1 genome were examined in the minicell system, and one specific product of 35,000 daltons in molecular weight was identified. Any polypeptides specific for the 0.34-Md inc⁺ region within mini-Rts1 were not detected.     

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.     

DNA segments of the IncX plasmid R485 determining replication and incompatibility with plasmid R6K

The expression of incompatibility properties between the IncX plasmids R6K and R485 of Escherichia coli was examined. For small autonomously replicating derivatives of both plasmid elements, the requirements for incompatibility expression include a functional R485 replicon and an active R6K beta-origin region. Functional R6K alpha and gamma origins are not directly involved in incompatibility expression between R6K and R485. A trans-acting replication system was constructed for plasmid R485. It consists of a 3.2-(kb) DNA fragment of R485 that specifies a product(s) in trans which supports replication from an R485 origin plasmid. A minimal R485 origin region of 591 bp was derived utilizing this trans-acting replication system and the nucleotide sequence of this origin region determined. The most striking feature of the sequence is the presence of six tandem 22-bp nucleotide sequence direct repeats.     

Sequence heterogeneity and differential expression of the α-Amy2 gene family in wheat

Summary Within plasmid pUB110 we have identified a 1.2 kb segment necessary and sufficient for driving autonomous replication in Rec⁺ cells at a wild-type copy number. This region can be divided into three functionally discrete segments: a 24 base pair (bp) region that acts as an origin, a 949 bp determinant of an essential replication protein, repU, and a 358 bp incompatibility region, incA, overlapping with the repU gene. The synthesis of the IncA determinant/s proceeds in the direction opposite to that of RepU. The positively (RepU) and negatively (IncA) trans-acting products seem to be involved in the control of plasmid replication. The RepU product has an M_r of 39 kDa, could be overproduced in Escherichia coli, and binds to the pUB110 origin region. Outside the minimal replicon a cis-acting, orientation dependent, 516 bp determinant is required (i) to compete with a coexisting incompatible plasmid and (ii) for segregational stability.     

Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904

To construct shuttle vectors based on an endogenous replicon, we isolated a small cryptic plasmid (pLP1) from Lactobacillus plantarum CCM 1904. The nucleotide sequence (2093 bp, 38.25 GC mol%) revealed one major open reading frame encoding for a 317 amino acid protein (Rep). Comparisons with proteins encoded by other Gram-positive bacteria plasmids strongly suggest that the protein encoded by pLP1 has a replicative role. The presence of a consensus sequence including a tyrosine residue known to be the replication protein binding site to the DNA (in phage φX174) strengthens this hypothesis. The DNA sequence contains also a sequence similar to the pC194 origin nick sequence, which initiates the plasmid replication at the plus origin, characteristic of plasmids which replicate following a rolling circle mechanism via single-stranded DNA intermediates. A set of 13 direct repeats of 17 bp could be involved in the expression of the incompatibility or in the copy number control as in the other plasmids. A promoter sequence located at the rep 5′ region has been identified and is functional in Bacillus subtilis.     

Mapping of RP4 plasmid using deletion mutants of pAS8 hybrid (RP4-ColE1)

Summary We used the hybrid plasmid pAS8 in order to conduct the genetic analysis of RP4 plasmid. The presence of two replicons in the hybrid plasmid permitted to expand the spectrum of deletion mutants of RP4 isolated, which are capable to autonomous replication. The shortening of the hybrid plasmid was achieved by P22 transduction, by induction of deletion mutants using mitomycin C, as well as by selection of Tra^- mutants on the basis of resistance of cells to P-specific phages. These techniques have lead to isolation of clones possessing different combinations of plasmid resistance determinants.Comparison of phenotypic characteristics of deletion plasmids pAS9, pAS10, pAS11, pAS12 and pAS10-2 permitted to propose the map for pAS8 plasmid with the following sequence of markers: trakan-ColE1-amp-tet...Heteroduplex analysis of deletion mutants of pAS8 permitted to construct a physical map and to elaborate in greater detail the functional map of RP4 plasmid. The correlation between the ability of mutants to replicate in polA (TS) strain at nonpermissive conditions and the length of the deleted segment permitted to map rep genes of RP4 on a region with coordinates 9.8–17.3 kb. A relationship between the manifestation of incompatibility of mutants with Inc P-1 plasmids and the length of deletions points out that inc genes are located on DNA region with coordinates 2.1–9.8 kb. The analysis of replication of deletion mutants and the manifestation of incompatibility just as of the data about the size of appropriate deletions permitted to make the conclusion about the functional and genetic independence of the replication control and incompatibility control in RP4 plasmid.     

The 20 bp, directly repeated DNA sequence of broad host range plasmid R1162 exerts incompatibility in vivo and inhibits R1162 DNA replication in vitro

Summary The broad host range plasmid R1162 contains a directly repeated, 20 bp DNA sequence in the region of the plasmid required in cis for replication and maintenance. This sequence has been chemically synthesized and cloned, and shown to be sufficient for expression of plasmid incompatibility. The sequence also inhibits replication of R1162 DNA in a cell-free system. The strengths of both these effects are determined by the number of direct repeats (DRs) present, and are also affected to similar degrees by different mutations within the repeated sequence. Several of the mutations were tested for their effect in cis on plasmid maintenance in the cell, and one was found to cause an increase in plasmid copy number. The results suggest that the direct repeats exert incompatibility by inhibiting DNA replication, presumably because they are the binding sites for a limiting essential protein.Abbreviations bp base pairs - Cb^r, Km^r, Sm^r resistance to carbenicillin, kanamycin, streptomycin, respectively - DR direct repeat     

Replication of ColE2 and ColE3 plasmids: Two ColE2 incompatibility functions

Summary We have identified and localized two incompatibility determinants (IncA and IncB) within a 1.3 kb segment of ColE2 sufficient for autonomous replication. The IncA determinant is localized in a region shorter than 250 bp and expresses incompatibility against both ColE2 and ColE3. The region which determines sensitivity to the IncA determinant seems to overlap with the region specifying the IncA determinant. The expression of the trans-acting factor(s) specifically required for replication of ColE2 interferes with expression of the IncA determinant against ColE2 but not against ColE3. The IncA determinant might be at least partly responsible for the copy number control of the plasmid. The IncB determinant is localized in a 50 bp region (origin) which is sufficient for initiation of replication in the presence of the trans-acting factor(s). The IncB determinant is specific for ColE2 and seems to be due to titration of the trans-acting essential replication factor(s) by binding.     

Separation of the minimal replication region of the F plasmid into a replication origin segment and a trans-acting segment

Summary An analysis was carried out on the replication functions within a 2.3 kilobase (kb) segment of the F plasmid which contains an origin (ori S) of replication and is capable of autonomous replication inEscherichia coli. Two separable regions were delineated for this segment: an origin region of approximately 1,140 bp in length and a segment of approximately 1,400 bp that functionsin trans to support replication of the origin region. The trans-acting segment is functional as part of an F replicon or when inserted into theE. coli chromosome. A prominent feature of the trans-acting segment is a coding sequence for a 29 K protein (Murotsu et al. 1981).     

Multiple mechanisms for expression of incompatibility by broad-host-range plasmid RK2

By cloning fragments of plasmid DNA, we have shown that RK2 expresses incompatibility by more than one mechanism. One previously identified (R. J. Meyer, Mol. Gen, Genet. 177:155--161, 1979; Thomas et al., Mol. Gen. Genet. 181:1--7, 1981) determinant for incompatibility is linked to the origin of plasmid DNA replication. When cloned into a plasmid vector, this determinant prevents the stable inheritance of a coresident RK2. However, susceptibility to this mechanism of incompatibility requires an active RK2 replicon and is abolished if another replicator is provided. We have also cloned a second incompatibility determinant, encoded within the 54.1- to 56.4-kilobase region of RK2 DNA, which we call IncP-1(II). An RK2 derivative remains sensitive to IncP-1(II), even when it is not replicating by means of the RK2 replicon. The 54.1- to 56.4-kilobase DNA does not confer susceptibility to the IncP-1(II) mechanism, nor does it encode a detectable system for efficient plasmid partitioning. The incompatibility may be related to the expression of genes mapping in the 54.1- to 56.4-kilobase region, which are required for plasmid maintenance and suppression of plasmid-encoded killing functions.     

Processes at the nick region link conjugation, T-DNA transfer and rolling circle replication

Data from prokaryotic replicative and conjugative systems, which interrelate DNA processing events initiated by a site-specific nick, are reviewed. While the replicative systems have been established in accordance with the rolling circle replication model, the mechanism of conjugative replication has not been elucidated experimentally. We summarize data involving random point mutagenesis of the RK2 transfer origin (oriT), which yielded relaxation-deficient and transfer-deficient derivatives having mutations exclusively in a 10bp region defined as the nick region. Features of the RK2 (IncP) nick region, including the DNA sequence, nick site position, and 5′ covalent attachment of the nicking protein, have striking parallels in other systems involving nicking and mobilization of single-stranded DNA from a supercoiled substrate. These other systems include T-DNA transfer occurring in Agrobacterium tumefaciens Ti plasmid-mediated tumorigenesis in plants, and the rolling circle replication of plasmids of Gram-positive bacteria and of φX174-like bacteriophage. The structural and functional similarities suggest that IncP conjugative replication, originating at the oriT, and T-DNA transfer replication, originating at the T-DNA border, produce continuous strands via a rolling circle-type replication.