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.     

The plasmid RK2 replication initiator protein (TrfA) binds to the sliding clamp beta subunit of DNA polymerase III: implication for the toxicity of a peptide derived from the amino-terminal portion of 33-kilodalton TrfA

The broad-host-range plasmid RK2 is capable of replication and stable maintenance within a wide range of gram-negative bacterial hosts. It encodes the essential replication initiation protein TrfA, which binds to the host initiation protein, DnaA, at the plasmid origin of replication (oriV). There are two versions of the TrfA protein, 44 and 33 kDa, resulting from alternate in-frame translational starts. We have shown that the smaller protein, TrfA-33, and its 64-residue amino-terminal peptide (designated T1) physically interact with the Escherichia coli beta sliding clamp (beta(2)). This interaction appears to be mediated through a QLSLF peptide motif located near the amino-terminal end of TrfA-33 and T1, which is identical to the previously described eubacterial clamp-binding consensus motif. T1 forms a stable complex with beta(2) and was found to inhibit plasmid RK2 replication in vitro. This specific interaction between T1 and beta(2) and the ability of T1 to block DNA replication have implications for the previously reported cell lethality caused by overproduction of T1. The toxicity of T1 was suppressed when wild-type T1 was replaced with mutant T1, carrying an LF deletion in the beta-binding motif. Previously, T1 toxicity has been shown to be suppressed by Hda, an intermediate regulatory protein which helps prevent over-initiation in E. coli through its interaction with the initiator protein, DnaA, and beta(2). Our results support a model in which T1 toxicity is caused by T1 binding to beta(2), especially when T1 is overexpressed, preventing beta(2) from interacting with host replication proteins such as Hda during the early events of chromosome replication.     

A broad host range replicon with different requirements for replication initiation in three bacterial species.

Plasmid RK2 is unusual in its ability to replicate stably in a wide range of Gram-negative bacteria. The replication origin (oriV) and a plasmid-encoded initiation protein (TrfA; expressed as 33 and 44 kDa forms) are essential for RK2 replication. To examine initiation events in bacteria unrelated to Escherichia coli, the genes encoding the replicative helicase, DnaB, of Pseudomonas putida and Pseudomonas aeruginosa were isolated and used to construct protein expression vectors. The purified proteins were tested for activity along with E.coli DnaB at RK2 oriV. Each helicase could be recruited and activated at the RK2 origin in the presence of the host-specific DnaA protein and the TrfA protein. Escherichia coli or P.putida DnaB was active with either TrfA-33 or TrfA-44, while P.aeruginosa DnaB required TrfA-44 for activation. Moreover, unlike the E.coli DnaB helicase, both Pseudomonas helicases could be delivered and activated at oriV in the absence of an ATPase accessory protein. Thus, a DnaC-like accessory ATPase is not universally required for loading the essential replicative helicase at a replication origin.     

Replication of mini RK2 plasmid in extracts of Escherichia coli requires plasmid-encoded protein TrfA and host-encoded proteins DnaA, B, G DNA gyrase and DNA polymerase III

Soluble extracts of Escherichia coli capable of carrying out replication of the mini-RK2 derivative pCT461 have been prepared from cells carrying this plasmid or from plasmid-free bacteria. The latter are dependent upon exogenously added plasmid-encoded replication protein (TrfA) and require additional DnaA protein for optimum activity. This dependence upon DnaA was confirmed by the failure of DnaA-deficient cell extracts to support replication of pCT461 in the absence of added DnaA protein. Replication is unidirectional and begins at or near oriV, the vegetative replication origin of RK2. DNase I protection studies with purified TrfA indicate that this protein acts by binding to short (17 base-pairs) directly repeated DNA sequences present in oriV. The in vitro replication is resistant to rifampicin but can be abolished by antibodies against DnaG protein (E. coli primase) or DnaB protein (helicase) and by DNA gyrase inhibitors. Inhibition by arabinosyl-CTP suggests that DNA polymerase III is responsible for elongation of nascent DNA strands. These results are discussed in relation to the mechanism of RK2 replication and in the context of the host range of the plasmid.     

Isolation of an inner membrane-derived subfraction that supports in vitro replication of a mini-RK2 plasmid in Escherichia coli

Previous results have demonstrated that the inner, but not the outer, membrane fraction of Escherichia coli is the site of membrane-associated DNA replication of plasmid RK2, a broad-host-range plasmid capable of replication in a wide variety of gram-negative hosts (K. Michaels, J. Mei, and W. Firshein, Plasmid 32:19-31, 1994). To resolve the inner membrane replication site further, the procedure of Ishidate et al. (K. Ishidate, E. S. Creeger, J. Zrike, S. Deb, G. Glauner, T. J. MacAlister, and L. I. Rothfield, J. Biol. Chem. 261:428-443, 1986) was used to separate the inner membrane into a number of subfractions, of which only one, a small subfraction containing only 10% of the entire membrane, was found to synthesize DNA inhibited by antibody prepared against the plasmid-encoded initiation protein TrfA. This is the same subfraction that was also found to bind oriV and TrfA to the greatest extent in filter binding assays (J. Mei, S. Benashski, and W. Firshein, J. Bacteriol. 177:6766-6772, 1995).     

Host-dependent requirement for specific DnaA boxes for plasmid RK2 replication

The replication origin of the broad-host-range plasmid RK2, oriV, contains four DnaA boxes, which bind the DnaA protein isolated from Escherichia coli. Using a transformation assay, mutational analysis of these boxes showed a differential requirement for replication in different Gram-negative bacteria. DnaA boxes 3 and 4 were required in E. coli and Pseudomonas putidabut not as strictly in Azotobacter vinelandii and not at all in P. aeruginosa. In vitro replication results using an extract prepared from E. coli demonstrated that the activity of origin derivatives containing mutations in boxes 3 or 4 or a deletion of all four DnaA boxes could be restored by the addition of increasing amounts of purified DnaA protein. High levels of DnaA protein in the presence of the TrfA protein also resulted in the stimulation of open complex formation and DnaB helicase loading on oriV, even in the absence of the four DnaA boxes. These observations at least raise the possibility that an alternative mechanism of initiation of oriV is being used in the absence of the four DnaA boxes and that this mechanism may be similar to that used in P. aeruginosa, which does not require these four DnaA boxes for replication.     

Identification of a potential membrane-targeting region of the replication initiator protein (TrfA) of broad-host-range plasmid RK2

Plasmid RK2 codes for two species of the replication initiator protein TrfA (33 and 44 kDa). Both polypeptides are strongly associated with membrane fractions of Escherichia coli host cells (W. Firshein and P. Kim, Mol. Microbiol. 23, 1-10, 1997). We investigated the role of a 12-amino-acid hydrophobic region (HR) in the membrane association of TrfA. Epitope-tagged polypeptide fragments of TrfA that contained HR were expressed and found to be associated with membrane fractions. Site-directed mutagenesis of trfA revealed that changes of specific amino acids in HR can affect both TrfA association with the membrane and its ability to support replication of an RK2 oriV plasmid in vivo. These results are consistent with the hypothesis that membrane association of TrfA is functionally relevant and that the HR region of TrfA is involved in membrane association and DNA replication in vivo.     

TrfA-dependent inner membrane-associated plasmid RK2 DNA synthesis and association of TrfA with membranes of different gram-negative hosts

TrfA, the replication initiator protein of broad-host-range plasmid RK2, was tested for its ability to bind to the membrane of four different gram-negative hosts in addition to Escherichia coli: Pseudomonas aeruginosa, Pseudomonas putida, Salmonella enterica serovar Typhimurium, and Rhodobacter sphaeroides. Cells harboring TrfA-encoding plasmids were fractionated into soluble, inner membrane, and outer membrane fractions. The fractions were subjected to Western blotting, and the blots were probed with antibody to the TrfA proteins. TrfA was found to fractionate with the cell membranes of all species tested. When the two membrane fractions of these species were tested for their ability to synthesize plasmid DNA endogenously (i.e., without added template or enzymes), only the inner membrane fraction was capable of extensive synthesis that was inhibited by anti-TrfA antibody in a manner similar to that of the original host species, E. coli. In addition, although DNA synthesis did occur in the outer membrane fraction, it was much less extensive than that exhibited by the inner membrane fraction and only slightly affected by anti-TrfA antibody. Plasmid DNA synthesized by the inner membrane fraction of one representative species, P. aeruginosa, was characteristic of supercoil and intermediate forms of the plasmid. Extensive DNA synthesis was observed in the soluble fraction of another representative species, R. sphaeroides, but it was completely unaffected by anti-TrfA antibody, suggesting that such synthesis was due to repair and/or nonspecific chain extension of plasmid DNA fragments.     

Analysis of mutations in trfA, the replication initiation gene of the broad-host-range plasmid RK2.

Plasmids with mutations in trfA, the gene encoding the replication initiation protein of the broad-host-range plasmid RK2, were isolated and characterized. Mutants identified from a nitrosoguanidine bank were defective in supporting the replication of a wild-type RK2 origin in Escherichia coli. Most of the mutations were clustered in a region of trfA corresponding to the carboxy-terminal quarter of the TrfA protein. 5' and 3' deletion mutants of trfA were also constructed. A C-terminal deletion of three amino acids of the Tr A protein was completely nonfunctional for RK2 replication. However, a deletion of 25 amino acids from the start of the 33-kDa TrfA protein was still competent for replication. Further characterization of the point and deletion trfA mutants in vivo revealed that a subset was capable of supporting RK2 replication in other gram-negative bacteria, including Pseudomonas putida, Agrobacterium tumefaciens, and Azotobacter vinelandii. Selected mutant TrfA proteins were partially purified and characterized in vitro. Velocity sedimentation analysis of these partially purified TrfA proteins indicated that the wild-type protein and all mutant TrfA proteins examined exist as dimers in solution. Results from in vitro replication assays corroborated the experimental findings in vivo. Gel retardation results clearly indicated that the point mutant TrfA-33:151S, which was completely defective in replication of an RK2 origin in all of the bacterial hosts tested in vivo, and a carboxy-terminal deletion mutant, TrfA-33:C delta 305, were not able to bind iterons in vitro. In addition to the partially defective or could not be distinguished from the wild-type protein in binding to the origin region. The mutant proteins with apparently normal DNA-binding activity in vitro either were inactive in all four gram-negative bacteria tested or exhibited differences in functionality depending on the host organism. These mutant TrfA proteins may be altered in the ability to interact with the replication proteins of the specific host bacterium.     

A critical DnaA box directs the cooperative binding of the Escherichia coli DnaA protein to the plasmid RK2 replication origin.

The requirement of DnaA protein binding for plasmid RK2 replication initiation the Escherichia coli was investigated by constructing mutations in the plasmid replication origin that scrambled or deleted each of the four upstream DnaA boxes. Altered origins were analyzed for replication activity in vivo and in vitro and for binding to the E. coli DnaA protein using a gel mobility shift assay and DNase I footprinting. Most strikingly, a mutation in one of the boxes, box 4, abolished replication activity and eliminated stable DnaA protein binding to all four boxes. Unlike DnaA binding to the E. coli origin, oriC, DnaA binding to two of the boxes (boxes 4 and 3) in the RK2 origin, oriV, is cooperative with box 4 acting as the "organizer" for the formation of the DnaA-oriV nucleoprotein complex. Interestingly, the inversion of box 4 also abolished replication activity, but did not result in a loss of binding to the other boxes. However, DnaA binding to this mutant origin was no longer cooperative. These results demonstrate that the sequence, position, and orientation of box 4 are crucial for cooperative DnaA binding and the formation of a nucleoprotein structure that is functional for the initiation of replication.     

Cooperative action of Escherichia coli ClpB protein and DnaK chaperone in the activation of a replication initiation protein

The Escherichia coli molecular chaperone protein ClpB is a member of the highly conserved Hsp100/Clp protein family. Previous studies have shown that the ClpB protein is needed for bacterial thermotolerance. Purified ClpB protein has been shown to reactivate chemically and heat-denatured proteins. In this work we demonstrate that the combined action of ClpB and the DnaK, DnaJ, and GrpE chaperones leads to the activation of DNA replication of the broad-host-range plasmid RK2. In contrast, ClpB is not needed for the activation of the oriC-dependent replication of E. coli. Using purified protein components we show that the ClpB/DnaK/DnaJ/GrpE synergistic action activates the plasmid RK2 replication initiation protein TrfA by converting inactive dimers to an active monomer form. In contrast, Hsp78/Ssc1/Mdj1/Mge1, the corresponding protein system from yeast mitochondria, cannot activate the TrfA replication protein. Our results demonstrate for the first time that the ClpB/DnaK/DnaJ/GrpE system is involved in protein monomerization and in the activation of a DNA replication factor.     

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.     

Positioning and the specific sequence of each 13-mer motif are critical for activity of the plasmid RK2 replication origin

The minimal replication origin of the broad-host-range plasmid RK2, oriV, contains five iterons which are binding sites for the plasmid-encoded replication initiation protein TrfA, four DnaA boxes, which bind the host DnaA protein, and an AT-rich region containing four 13-mer sequences. In this study, 26 mutants with altered sequence and/or spacing of 13-mer motifs have been constructed and analysed for replication activity in vivo and in vitro. The data show that the replacement of oriV 13-mers by similar but not identical 13-mer sequences from Escherichia coli oriC inactivates the origin. In addition, interchanging the positions of the oriV 13-mers results in greatly reduced activity. Mutants with T/A substitutions are also inactive. Furthermore, introduction of single-nucleotide substitutions demonstrates very restricted sequence requirements depending on the 13-mer position. Only two of the mutants are host specific, functional in Pseudomonas aeruginosa but not in E. coli. Our experiments demonstrate considerable complexity in the plasmid AT-rich region architecture required for functionality. It is evident that low internal stability of this region is not the only feature contributing to origin activity. Our studies suggest a requirement for sequence-specific protein interactions within the 13-mers during assembly of replication complexes at the plasmid origin.     

Interactions of plasmid-encoded replication initiation proteins with the origin of DNA replication in the broad host range plasmid RK2

The TrfA proteins, encoded by the broad host range plasmid RK2, are required for replication of this plasmid in a variety of Gram-negative bacteria. Two TrfA proteins, 33 and 44 kDa in molecular mass (designated TrfA-33 and TrfA-44, respectively), are expressed from the trfA gene of RK2 through the use of two alternative in-frame start codons within the same open reading frame. The two proteins have been purified from Escherichia coli to near homogeneity as a mixture of wild-type TrfA-44/33, as TrfA-33 alone and as a functional variant form of TrfA-44, designated TrfA-44(98L), which contains a leucine in place of the TrfA-33 methionine start codon. Cross-linking experiments demonstrated that TrfA-33 can multimerize in solution. By using gel mobility shift and DNase I footprinting techniques the binding properties of TrfA-33, TrfA-44(98L), and TrfA-44/33 to the origin of replication of plasmid RK2 were analyzed. All three protein preparations were able to bind very specifically to the cluster of five direct repeats (iterons) contained in the minimal origin of replication. Each protein preparation produced a ladder of TrfA/minimal oriV complexes of decreasing electrophoretic mobility. The DNase I protection pattern on the five iterons was identical for all three protein preparations and extended from the beginning of the first iteron to 5 base pairs upstream of the fifth iteron. Studies on the affinity of the proteins for DNA fragments containing one, two, or all five iterons of the origin revealed a strong preference of TrfA protein for DNA containing at least two iterons. To study the stability of TrfA.DNA complexes, association and dissociation rates of TrfA-33 and DNA fragments with one, two, or five iterons were measured. This analysis showed that unlike complexes involving two or five iterons the TrfA/one iteron complexes were highly unstable, suggesting some form of cooperativity between proteins or iterons in the formation of stable complexes and/or the requirement of specific sequences bordering the iterons at the RK2 origin of replication for the stabilization of TrfA/DNA complexes.     

The sequence encoding the 43-kilodalton trfA protein is required for efficient replication or maintenance of minimal RK2 replicons in Pseudomonas aeruginosa

The trfA gene of the broad-host-range plasmid RK2 encodes two proteins of 43- and 32-kDa by initiating translation at either of two in-phase AUG codons in a single open reading frame. At least one of these proteins is essential for replication of RK2 derivatives. In order to study the role of the 43-kDa protein, Bal31 deletions into the 5' end of the trfA gene were constructed and incorporated into minimal RK2 replicons. When examined in Escherichia coli, replication and maintenance properties of plasmids encoding only the 32-kDa protein were indistinguishable from those of plasmids encoding both the 43- and the 32-kDa proteins. In four other gram-negative hosts deletion of sequences encoding only the 43-kDa protein did not have a substantial effect on plasmid establishment or stable maintenance. However, in Pseudomonas aeruginosa, deletion of 43-kDa coding sequences greatly reduced the efficiency of plasmid maintenance, suggesting a host-specific role for the 43-kDa TrfA protein in RK2 replication.     

DnaA box sequences as the site for helicase delivery during plasmid RK2 replication initiation in Escherichia coli

DnaA box sequences are a common motif present within the replication origin region of a diverse group of bacteria and prokaryotic extrachromosomal genetic elements. Although the origin opening caused by binding of the host DnaA protein has been shown to be critical for the loading of the DnaB helicase, to date there has been no direct evidence presented for the formation of the DnaB complex at the DnaA box site. For these studies, we used the replication origin of plasmid RK2 (oriV), containing a cluster of four DnaA boxes that bind DnaA proteins isolated from different bacterial species (Caspi, R., Helinski, D. R., Pacek, M., and Konieczny, I. (2000) J. Biol. Chem. 275, 18454-18461). Size exclusion chromatography, surface plasmon resonance, and electron microscopy experiments demonstrated that the DnaB helicase is delivered to the DnaA box region, which is localized approximately 200 base pairs upstream from the region of origin opening and a potential site for helicase entry. The DnaABC complex was formed on both double-stranded superhelical and linear RK2 templates. A strict DnaA box sequence requirement for stable formation of that nucleoprotein structure was confirmed. In addition, our experiments provide evidence for interaction between the plasmid initiation protein TrfA and the DnaABC prepriming complex, formed at DnaA box region. This interaction is facilitated via direct contact between TrfA and DnaB proteins.     

Broad-host-range properties of plasmid RK2: importance of overlapping genes encoding the plasmid replication initiation protein TrfA.

The trfA gene, encoding the essential replication initiation protein of the broad-host-range plasmid RK2, possesses an in-frame overlapping arrangement. This results in the production of TrfA proteins of 33 and 44 kDa, respectively. Utilizing deletion and site-specific mutagenesis to alter the trfA operon, we compared the replication of an RK2-origin plasmid in several distantly related gram-negative bacteria when supported by both TrfA-44 and TrfA-33, TrfA-33 alone, or TrfA-44/98L (a mutant form of the TrfA-44 protein) alone. TrfA-44/98L is identical to wild-type TrfA-44 with the exception of a single conservative amino acid alteration from methionine to leucine at codon 98; this alteration removes the translational start codon for the TrfA-33 protein. Copy number and stability were virtually identical for plasmids containing both TrfA-44 and TrfA-33 proteins or TrfA-44/98L alone in Pseudomonas aeruginosa and Agrobacterium tumefaciens, two unrelated bacteria in which TrfA-33 is poorly functional. This, along with recent in vitro studies comparing TrfA-44, TrfA-33, and TrfA-44/98L, suggests that the functional activity of TrfA-44 is not significantly affected by the 98L mutation. Analysis of minimal RK2 derivatives in certain gram-negative bacterial hosts suggests a role of the overlapping arrangement of trfA in facilitating the broad host range of RK2. RK2 derivatives encoding TrfA-44/98L alone demonstrated decreased copy number and stability in Escherichia coli and Azotobacter vinelandii when compared with derivatives specifying both TrfA-44 and TrfA-33. A strategy employing the trfA-44/98L mutant gene and in vivo homologous recombination was used to eliminate the internal translational start codon of trfA in the intact RK2 plasmid. The mutant intact RK2 plasmid produced only TrfA-44/98L. A small reduction in copy number and beta-lactamase expression resulted in E. coli, suggesting that overlapping trfA genes also enhance the efficiency of replication of the intact RK2 plasmid.     

Conformation of a plasmid replication initiator protein affects its proteolysis by ClpXP system

Proteins from the Rep family of DNA replication initiators exist mainly as dimers, but only monomers can initiate DNA replication by interaction with the replication origin (ori). In this study, we investigated both the activation (monomerization) and the degradation of the broad‐host‐range plasmid RK2 replication initiation protein TrfA, which we found to be a member of a class of DNA replication initiators containing winged helix (WH) domains. Our in vivo and in vitro experiments demonstrated that the ClpX‐dependent activation of TrfA leading to replicationally active protein monomers and mutations affecting TrfA dimer formation, result in the inhibition of TrfA protein degradation by the ClpXP proteolytic system. These data revealed that the TrfA monomers and dimers are degraded at substantially different rates. Our data also show that the plasmid replication initiator activity and stability in E. coli cells are affected by ClpXP system only when the protein sustains dimeric form.     

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.     

Interactions of the origin of replication (oriV) and initiation proteins (TrfA) of plasmid RK2 with submembrane domains of Escherichia coli.

It has been possible to locate a submembrane domain representing less than 10% of the total membrane that appears to be responsible for sequestering some essential components required for plasmid RK2 DNA replication. This subfraction, whose cellular location in the membrane prior to extraction is still unknown, is derived from the inner membrane fraction, since it possesses enzyme marker activity (NADH oxidase) exclusively associated with the inner membrane. The subfraction was detected by a modification of the methods of Ishidate et al. (K. Ishidate, E. S. Kreeger, J. Zrike, S. Deb, B. Glauner, T. MacAlister, and L. I. Rothfield, J. Biol. Chem. 261:428-443, 1986) in which low pressure in a French pressure cell and lysozyme were used to preserve the supercoil plasmid DNA template during cell disruption. This was followed by successive cycles of sucrose gradient sedimentation and flotation density gradient centrifugation to reveal a number of subfractions, including the one of interest. The characteristics of plasmid interaction with the subfraction include the presence of supercoil DNA after extraction, the binding of the origin of plasmid replication (oriV) in vitro, and the association of the two plasmid-encoded initiation (TrfA) proteins (encoded by overlapping genes). However, another peak, the outer membrane fraction, also binds oriV in vitro, contains plasmid DNA in vivo, and associates with the TrfA initiation proteins. Nevertheless, it contains much less of the initiation proteins, and the specific activity of binding oriV is also much reduced compared with the other subfraction. There is a strong correlation between the association of the TrfA initiation proteins with a particular membrane fraction and the binding of oriV in vitro or plasmid DNA in vivo. Since the proteins are known to bind to repeated sequences in oriV (S. Perri, D. R. Helinski, and A. Toukdarian, J. Biol. Chem. 266:12536-1254, 1991; M. Pinkney, R. Diaz, E. Lanka, and C. M. Thomas, J. Mol. Biol. 203: 927-938, 1988), it appears that the initiation proteins themselves could be responsible, at least in part, for the association of plasmid DNA to the membrane.