Plasmid RK2 Research Articles

Construction of a series of pSAM2-based integrative vectors for use in actinomycetes

We have developed vectors which allowed integration of cloned DNA at a single site in the chromosome of Streptomyces lividans 66. These vectors made use of (1) an Escherichia coli replicon, (2) a thiostrepton (Th)- and a streptomycin/spectinomycin-resistance gene for selection in Streptomyces, (3) a 3.5-kb fragment of the Streptomyces integrative plasmid pSAM2 containing its xis and int genes as well as its attachment site, attP, to direct the integration of the vectors at the chromosomal pSAM2 attachment site attB, (4) the origin of transfer of the IncP broad-host-range plasmid RK2 which allowed the mobilization of the vectors from E. coli to S. lividans, and (5) the Th-inducible tipA promoter to permit regulated transcription of cloned genes. We demonstrated that pPM927, a plasmid which contained all of these elements, was able to transfer cloned fragments from E. coli to S. lividans by conjugation, stably integrate into the chromosome, and express cloned genes from the tipA promoter. Furthermore, since pPM927 contained the pBR322 replicon, cloned fragments could be conveniently recovered from the S. lividans chromosome for analysis in E. coli by cleavage of genomic DNA isolated from transformed strains, intramolecular ligation and transformation. Since we have shown that the pSAM2 attB site forms part of a conserved prokaryotic tRNA gene, these integrative vectors are potentially useful tools for analysis and expression of genes in diverse bacteria.

A family of ATPases involved in active partitioning of diverse bacterial plasmids

Low copy-number bacterial plasmids F (the classical Escherichia coli sex factor) and prophage P1 encode partitioning functions which may provide fundamental insights into the active processes which ensure that bacterial genomes are segregated to both daughter cells prior to cell division. These partitioning systems involve two proteins: ParA and ParB. We report that incC from the broad host-range plasmid RK2 is a member of the family of ParA partitioning proteins and that these proteins (as well as related proteins encoded by plasmids from Agrobacterium tumefaciens and Chlamydia trachomatis) contain type I nucleotide-binding motifs. Also, we show that the cell division inhibitor MinD is homologous to members of the ParA family. Sequence comparisons of ParB proteins suggest that they may contain sites for phosphorylation. We propose that ATP hydrolysis by the ParA protein may result in phosphorylation of the ParB protein, thereby causing a conformational shift necessary to separate paired plasmid molecules at the cell division plane.

Location of a P1 plasmid replication inhibitor determinant within the initiator gene

The P1 plasmid replication initiator protein, RepA, binds to its own promoter and represses transcription efficiently. There are only about 20 RepA dimers present per repA gene. A possible reason for this highly restrained expression became evident when repA expression was increased by using foreign promoters: with fivefold overexpression, the replication rate was diminished, and with 40-fold overexpression, replication was not detectable. The inhibition was P1 specific: growth of Escherichia coli and replication of pSC101, R6K, and mini-F plasmids were not affected. The activity is apparently not from RepA itself. Excess purified RepA did not inhibit replication in vitro. Mutations of the repA translation initiation codon reduced synthesis of the initiator but not the inhibitory activity. Deletion from either the N- or C-terminal ends of repA (28 and 69 codons, respectively, out of the 286-codon open reading frame) affected the initiator but not the inhibitory activity. Further deletions affected both the activities. These results demonstrate that the integrity of the initiator is not required for inhibition, but involvement of an unstable initiator fragment or of initiator mRNA cannot be ruled out.

Replication of the broad-host-range plasmid RK2: direct measurement of intracellular concentrations of the essential TrfA replication proteins and their effect on plasmid copy number

The trfA gene of the broad-host-range plasmid RK2 is essential for initiation of plasmid replication. Two related TrfA proteins of 43 and 32 kilodaltons (kDa) are produced by independent translation initiation at two start codons within the trfA open reading frame. These proteins were o overproduced in Escherichia coli and partially purified. Rabbit antisera raised against the 32-kDa TrfA protein (TrfA-32) and cross-reacting with the 43-kDa protein (TrfA-43) were used in Western blotting (immunoblotting) assays to measure intracellular TrfA levels. In logarithmically growing E. coli HB101, RK2 produced 4.6 +/- 0.6 ng of TrfA-32 and 1.8 +/- 0.2 ng of TrfA-43 per unit of optical density at 600 nm (mean +/- standard deviation). On the basis of determinations of the number of cells per unit of optical density at 600 nm, this corresponds to about 220 molecules of TrfA-32 and 80 molecules of TrfA-43 per cell. Dot blot hybridizations showed that plasmid RK2 is present in about 15 copies per E. coli cell under these conditions. Using plasmid constructs that produce different levels of TrfA proteins, the effect of excess TrfA on RK2 replication was tested. A two- to threefold excess of total TrfA increased the copy number of RK2 by about 30%. Additional increases in TrfA protein concentration had no further effect on copy number, even at levels 170-fold above normal. An RK2 minimal origin plasmid showed a similar response to intracellular TrfA concentration. These results demonstrate that TrfA protein concentration is not strictly rate limiting for RK2 replication and that a mechanism that is independent of TrfA concentration functions to limit RK2 copy number in the presence of excess TrfA.

Mutations in the trfA replication gene of the broad-host-range plasmid RK2 result in elevated plasmid copy numbers

Mutated forms of trfA, the replication protein gene of plasmid RK2, that support a minimal RK2 origin plasmid in Escherichia coli at copy numbers up to 23-fold higher than normal have been isolated. Six such high-copy-number (copy-up) mutations were mapped and sequenced. In each case, a single base transition led to an amino acid substitution in the TrfA protein primary sequence. The six mutations affected different residues of the protein and were located within a 69-base-pair region encoding 24 amino acids. Dominance tests showed that each of the mutants can be suppressed by wild-type trfA in trans, but suppression is highly dependent on the amount of wild-type protein produced. Excess mutant TrfA protein provided in trans significantly increased the copy number of RK2 and other self-replicating derivatives of RK2 that contain a wild-type trfA gene. These observations suggest that the mutations affect a regulatory activity of the TrfA replication protein that is a key factor in the control of initiation of RK2 replication.

The kil-kor regulon of broad-host-range plasmid RK2: nucleotide sequence, polypeptide product, and expression of regulatory gene korC

Broad-host-range plasmid RK2 encodes several kil operons (kilA, kilB, kilC, kilE) whose expression is potentially lethal to Escherichia coli host cells. The kil operons and the RK2 replication initiator gene (trfA) are coregulated by various combinations of kor genes (korA, korB, korC, korE). This regulatory network is called the kil-kor regulon. Presented here are studies on the structure, product, and expression of korC. Genetic mapping revealed the precise location of korC in a region near transposon Tn1. We determined the nucleotide sequence of this region and identified the korC structural gene by analysis of korC mutants. Sequence analysis predicts the korC product to be a polypeptide of 85 amino acids with a molecular mass of 9,150 daltons. The KorC polypeptide was identified in vivo by expressing wild-type and mutant korC alleles from a bacteriophage T7 RNA polymerase-dependent promoter. The predicted structure of KorC polypeptide has a net positive charge and a helix-turn-helix region similar to those of known DNA-binding proteins. These properties are consistent with the repressorlike function of KorC protein, and we discuss the evidence that KorA and KorC proteins act as corepressors in the control of the kilC and kilE operons. Finally, we show that korC is expressed from the bla promoters within the upstream transposon Tn1, suggesting that insertion of Tn1 interrupted a plasmid operon that may have originally included korC and kilC.

Identification of a seventh operon on plasmid RK2 regulated by the korA gene product

Broad-host-range IncP plasmids possess a series of operons involved in plasmid maintenance, whose expression is coordinated by a series of regulators, most of which are encoded in a central regulatory operon. The nucleotide sequence of a new monocistronic operon located between coordinates 55.0 and 56.0 kb on the genome of the IncPα plasmids RK2 and RP4 is presented. The operon encodes a 34 kDa protein which has a net negative charge. Transcription of the operon, designated by us kfrA ( korF-regulated), is repressed not only by the product of the previously described korA gene but also by the product of a gene which we have designated korF and which has not been described previously. The korF gene is encoded downstream from KorB within the key korA/korB regulatory operon. We propose that KorF binds to a novel inverted repeat overlapping the promoter for the kfrA operon.

Broad host range plasm id RK2 encodes a polypeptide related to single-stranded DNA binding protein (SSB) ofEscherichia coli

Broad host range plasm id RK2 encodes a polypeptide related to single-stranded DNA binding protein (SSB) of<i>Escherichia coli</i>

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.

Tus and the terminators: The arrest of replication in prokaryotes

Bidirectional replication of prokaryotic circular chromosomes concludes when the two replication forks converge opposite the origin of replication in a region called the terminus. The terminus regions of several replicons contain sites referred to as terminators that arrest DNA replication and prevent replication forks from exiting the terminus. Function of the terminators is absolutely dependent upon a DNA binding protein that specifically binds the terminator sites and impedes replication forks. Studies on prokaryotic termination systems have now progressed to the point where termination sites have been sequenced and DNA binding proteins have been purified to near homogeneity. Very recently, several laboratories have completed the initial biochemical characterization of the mechanism of replication fork arrest, and one of these papers appears in this issue of cell. The studies that elucidated the components of the termination systems in Escherichia coli, Bacillus subtiiis, and the R6K plasmid will be reviewed here, as well as the more recent mechanistic studies. Tsrmlnator Sites The terminus of the E. coli chromosome is a large region (~360 kb) flanked on both sides by terminator sites, WA, Ter8, TerC, and TerD, that arrest replication forks (see figure; nomenclature for these sites is presented in the footnote). Before proceeding to the details of this inhibition of replication, the role of these sites in the replication cycle should be considered. inhibition is polar, and the important concept was developed that the terminus region functions as a replication-fork trap (Hill et al., 1987; deMassy et al., 1987). Specifically, replication forks from either direction are not prevented from entering the terminus region, but they are inhibited when they progress across the terminus and reach the other side. With respect to bidirectional replication of the chromosome, this means that if the two forks

Negative control of plasmid R6K replication: possible role of intermolecular coupling of replication origins.

The gamma origin binding sites of the replication initiator pi protein, composed of seven 22-base-pair (bp) direct repeats and previously shown to be essential for replication of plasmid R6K, can also act as an inhibitor of R6K replication in Escherichia coli cells if provided in trans. Inhibition is dependent upon the ability of these repeats to bind the R6K-encoded pi protein but is not overcome by increasing the intracellular pi level. The insertion of a second repeat cluster in close proximity to the gamma origin also can markedly inhibit replication. The severity of this effect is dependent upon the position, orientation, and number of repeats present in the extra cluster. As few as six extra repeats can result in a completely nonfunctional gamma origin. However, this inactive gamma origin plasmid containing the six extra repeats is functional when placed in a strain that underproduces the wild-type pi protein or when placed in the presence of any of several copy-up mutant pi proteins. On the basis of these observations, we propose that the nucleoprotein structures formed by the binding of pi protein to the seven 22-bp direct repeats at the gamma origin are capable of coupling with each other in vivo and that replication initiation is prevented at such coupled origins. In support of this model of replication control, we demonstrate by electron microscopy analysis that the pi protein has the ability to associate two DNA molecules containing gamma origin sequences and also show that pi enhances the DNA ligase-catalyzed multimerization of a DNA fragment containing the gamma origin.

Evidence of a ter specific binding protein essential for the termination reaction of DNA replication in Escherichia coli.

Activity binding specifically to the 22 bp of the DNA replication terminus (ter) sequence on plasmid R6K and the Escherichia coli genome was detected in the crude extract of E. coli cells. This activity was inactivated by heat or by protease but not by RNase treatments. Overproduction of the ter binding activity was observed when the extract was prepared from the cell carrying a plasmid with a chromosomal-derived 5.0 kb EcoRI fragment, on which one of the four terC sites, terC2, was also located. By mutagenesis of the 5.0 kb fragment on the plasmid with transposon Tn3 and subsequent replacement of the corresponding chromosomal region with the resulting mutant alleles, we isolated tau- mutants completely defective in ter binding activity. These mutants simultaneously lost the activity to block the progress of the DNA replication fork at any ter site, on the genome or the plasmid. It would thus appear that the ter binding protein plays an essential role in the termination reaction, at the ter sites.

In vivo cloning of Pseudomonas aeruginosa genes with mini-D3112 transposable bacteriophage

The transposition properties of the Pseudomonas aeruginosa mutator bacteriophage D3112 were exploited to develop an in vivo cloning system. Mini-D replicon derivatives of D3112 were constructed by incorporating broad host range plasmid replicons between short terminal D3112 sequences. These elements were made with small replication regions from the RK2, Sa, and pVS1 plasmids and selectable genes for tetracycline, carbenicillin, kanamycin, and gentamicin resistance. Some of the mini-D replicons also contain the RK2 oriT origin-of-transfer sequence, which allows them to be mobilized by conjugation to many different species of gram-negative bacteria. These elements were used to clone DNA by preparing lysates from P. aeruginosa cells harboring an inducible D3112 cts prophage and a mini-D replicon plasmid. These lysates were used to infect sensitive P. aeruginosa recipients and select recombinant plasmids as drug-resistant transductant colonies. These transductants form a gene library from which particular clones can be selected, such as by their ability to complement specific mutations. This system was used to clone nine different genes from the PAO chromosome. The ability of this system to precisely identify a gene was demonstrated by isolating clones of the argF+ and cys-59+ genes. Restriction maps of clones of these genes, which have different amounts of flanking DNA, located the positions of these genes. The sizes of the chromosomal DNA segments from 10 individual clones examined ranged from 6 to 21 kilobases (kb), with an average of about 10 kb. This is consistent with the approximately 40-kb DNA-packaging size of the D3112 phage.

A host-encoded DNA-binding protein promotes termination of plasmid replication at a sequence-specific replication terminus.

We have purified approximately 6600-fold an approximately 40-kDa protein (Ter protein) encoded by Escherichia coli that specifically binds to two sites at the 216-base-pair replication terminus (tau) of the plasmid R6K. Chemical footprinting experiments have shown that the Ter protein binds to two 14- to 16-base-pair sequences that exist as inverted repeats in the tau fragment. Site-directed mutagenesis of one of the terminus sequences (tau R) resulted in a mutant tau R that failed to bind to the Ter protein. The same mutant terminus also failed to terminate DNA replication in vivo. These experiments strongly suggest that the interaction of the Ter protein with tau sequences plays an essential role in the termination of DNA replication, specifically at tau.

Replication of an RK2 miniplasmid derivative in vitro by a DNA/membrane complex extracted from Escherichia coli: Involvement of the dnaA but not dnaK host proteins and association of these and plasmid-encoded proteins with the inner membrane

A DNA/membrane complex extracted from a miniplasmid derivative of the broad host range plasmid RK2 cultured in Escherichia coli capable of synthesizing new plasmid supercoiled DNA in vitro was treated with antibodies that were made against or reacted with the dnaA and dnaK host-encoded proteins, respectively. Anti-dnaA protein antibody inhibited total plasmid DNA synthesis significantly and the synthesis of supercoil plasmid DNA almost completely. In contrast, anti-dnaK protein antibody and nonimmune serum had little or no effect on total plasmid DNA synthesis. Both proteins were found to be present in the inner but not outer membrane fraction of E. coli. A variety of miniplasmid-encoded proteins which had previously been found in the DNA/membrane complex have also been localized to the inner but not outer membrane fraction. These include an essential initiation protein of 32 kDa (and an overlapping protein of 43 kDa coded for by the same gene), as well as a 30-kDa protein that may be linked to incompatibility functions. Various extraction methods were used to distinguish between the associated and the integral nature of the plasmid-encoded proteins. The results demonstrated that the essential replication proteins (32 and 43 kDa) as well as the 30-kDa protein was tightly bound to the inner membrane. Computer analysis of the amino acid sequence of the 32 (and 43)-kDa protein revealed a hydrophobic region that is only half that normally required to span the membrane. Other interactions are discussed with respect to attaching this protein to the membrane.

Analysis of nonpolar insertion mutations in the trfA gene of IncP plasmid RK2 which affect its broad-host-range property

Replication of broad-host-range plasmid RK2 requires the protein product(s) of the plasmid-encoded trfA gene to initiate replication at oriV, the vegetative replication origin. The trfA gene contains two translational starts which direct translation of two polypeptides, of 382 and 285 amino acids, which differ by the 97 amino acids at their N-terminus. Nonpolar insertions which abolish expression of the larger TrfA polypeptide but otherwise retain the trfA gene's normal expression signals severely reduce plasmid replication efficiency in Pseudomonas aeruginosa and to a lesser extent in Pseudomonas putida, but have very little effect in Escherichia coli. This indicates that the organization of the trfA gene, producing two polypeptides products, plays an important part in the broad-host-range of plasmid RK2 by providing a degree of flexibility in the way the plasmid's replication system interacts with host biochemistry.

Host-specific effects of the korA-korB operon and oriT region on the maintenance of miniplasmid derivatives of broad host-range plasmid RK2.

Two genetic determinants are sufficient for small derivatives of broad host-range plasmid RK2 to replicate in different Gram-negative bacteria: trfA, which encodes a replication initiator, and oriV, the origin of replication. In this study, nonessential RK2 determinants in the region encoding oriT, the origin of conjugative transfer, and the korA-korB operon, whose products regulate trfA expression, were tested for their effects on the stability of mini-RK2 plasmids in eight different hosts. We found that determinants of both regions can substantially alter plasmid stability, but the effects are not uniform in all hosts. The results also indicate that the effects of the korA-korB operon extend beyond that of the regulation of trfA transcription. This study further illustrates the different requirements for stable plasmid maintenance in diverse bacteria and the ability of wild-type RK2 to adapt to a variety of intracellular environments. The data also provide further evidence for the involvement of different regions of RK2 for stable maintenance in various hosts.

Termination sites T1 and T2 from the Escherichia coli chromosome inhibit DNA replication in ColE1-derived plasmids.

Inhibition sites T1 and T2 from the Escherichia coli terminus functioned with the same characteristics in ColE1-derived plasmids and in the chromosome. These characteristics included polarity and dependence on tus, a trans-acting factor required for inhibition. Inhibition in the terminus region of the R6K plasmid was also tus dependent.

Biological characteristics of plasmid carrying a repeated deoxyribonucleic acid sequence

It was found that a plasmid which had a foreign deoxyribonucleic acid (DNA) between two repeated sequences did not multiply in E. coli recBCsbcB, even if it multiplied in wild-type E. coli, E. coli recBC or E. coli recBCsbcBrecF when the insert was longer than 351 base pair. The multiplication of these plasmids were, however, inhibited when a plasmid expressing recF gene was introduced into E. coli recBCsbcBrecF. The inviability of the plasmid carrying the repeated sequence in E. coli recBCsbcB was discussed by the mechanism of recombination, and the functions of recF, recBC and sbcB were speculated. When E. coli recBC was transformed with pDR1 which was a derivative of pBR322 carrying a directly repeated sequence between which a DNA fragment derived from plasmid R6K with its origin was inserted, the intramolecular recombinant appeared. The recombinant recovered was, however, only the plasmid which had the replication origin of pBR322. The result suggests that pBR322 is compatible with pDR1 but R6K is not. The replication origin of R6K seems to be preferrentially used by pDR1.

Purification and characterization of RepA, a protein involved in the copy number control of plasmid pLS1

The promiscuous streptococcal plasmid pLS1 encodes for the 5.1 kDa RepA protein, involved in the regulation of the plasmid copy number. Synthesis of RepA was observed both in Bacillus subtilis minicells and in an Escherichia coli expression system. From this system, the protein has been purified and it appears to be a dimer of identical subunits. The amino acid sequence of RepA has been determined. RepA shows the alpha helix-turn-alpha helix motif typical of many DNA-binding proteins and it shares homology with a number of repressors, specially with the TrfB repressor encoded by the broad-host-range plasmid RK2. DNase I footprinting revealed that the RepA target is located in the region of the promoter for the repA and repB genes. Trans-complementation analysis showed that in vivo, RepA behaves as a repressor by regulating the plasmid copy number. We propose that the regulatory role of RepA is by limitation of the synthesis of the initiator protein RepB.