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.     

Bacterial mutation affecting plasmid maintenance in Pseudomonas aeruginosa.

A bacterial mutation, risA, in Pseudomonas aeruginosa caused growth inhibition at 43 degrees C of risA strains containing P2 plasmids. Incubation at 43 degrees C resulted in selection for clones that had lost P2 plasmids.     

A specific region in the N terminus of a replication initiation protein of plasmid RK2 is required for recruitment of Pseudomonas aeruginosa DnaB helicase to the plasmid origin

Broad host range plasmid RK2 encodes two versions of its essential replication initiation protein, TrfA, using in-frame translational starts spaced 97 amino acids apart. The smaller protein, TrfA-33, is sufficient for plasmid replication in many bacterial hosts. Efficient replication in Pseudomonas aeruginosa, however, specifically requires the larger TrfA-44 protein. With the aim of identifying sequences of TrfA-44 required for stable replication of RK2 in P. aeruginosa, specific deletions and a substitution mutant within the N terminus sequence unique to TrfA-44 were constructed, and the mutant proteins were tested for activity. Deletion mutants were targeted to three of the four predicted helical regions in the first 97 amino acids of TrfA-44. Deletion of TrfA-44 amino acids 21-32 yielded a mutant protein, TrfA-44Delta2, that had lost the ability to bind and load the DnaB helicase of P. aeruginosa or Pseudomonas putida onto the RK2 origin in vitro and did not support stable replication of an RK2 mini-replicon in P. aeruginosa in vivo. A substitution of amino acid 22 within this essential region resulted in a protein, TrfA-44E22A, with reduced activity in vitro, particularly with the P. putida helicase. Deletion of amino acids 37-55 (TrfA-44Delta3) slightly affected protein activity in vitro with the P. aeruginosa helicase and significantly with the P. putida helicase, whereas deletion of amino acids 71-88 (TrfA-44Delta4) had no effect on TrfA activity in vitro with either helicase. These results identify regions of the TrfA-44 protein that are required for recruitment of the Pseudomonas DnaB helicases in the initiation of RK2 replication.     

Characterization of the stable maintenance properties of the par region of broad-host-range plasmid RK2.

A 3.2-kb fragment encoding five genes, parCBA/DE, in two divergently transcribed operons promotes stable maintenance of the replicon of the broad-host-range plasmid RK2 in a vector-independent manner in Escherichia coli. The parDE operon has been shown to contribute to stabilization through the postsegregational killing of plasmid-free daughter cells, while the parCBA operon encodes a resolvase, ParA, that mediates the resolution of plasmid multimers through site-specific recombination. To date, evidence indicates that multimer resolution alone does not play a significant role in RK2 stable maintenance by the parCBA operon in E. coli. It has been proposed, instead, that the parCBA region encodes an additional stability mechanism, a partition system, that ensures that each daughter cell receives a plasmid copy at cell division. However, studies carried out to date have not directly determined the plasmid stabilization activity of the parCBA operon alone. An assessment was made of the relative contributions of postsegregational killing (parDE) and the putative partitioning system (parCBA) to the stabilization of mini-RK2 replicons in E. coli. Mini-RK2 replicons carrying either the entire 3.2-kb (parCBA/DE) fragment or the 2.3-kb parCBA region alone were found to be stably maintained in two E. coli strains tested. The stabilization found is not due to resolution of multimers. The stabilizing effectiveness of parCBA was substantially reduced when the plasmid copy number was lowered, as in the case of E. coli cells carrying a temperature-sensitive mini-RK2 replicon grown at a nonpermissive temperature. The presence of the entire 3.2-kb region effectively stabilized the replicon, however, under both low- and high-copy-number-conditions. In those instances of decreased plasmid copy number, the postsegregational killing activity, encoded by parDE, either as part of the 3.2-kb fragment or alone played the major role in the stabilization of mini-RK2 replicons within the growing bacterial population. Our findings indicate that the parCBA operon functions to stabilize by a mechanism other than cell killing and resolution of plasmid multimers, while the parDE operon functions solely to stabilize plasmids by cell killing. The relative contribution of each system to stabilization depends on plasmid copy number and the particular E. coli host.     

The Tra2 core of the IncP(alpha) plasmid RP4 is required for intergeneric mating between Escherichia coli and Streptomyces lividans.

Escherichia coli cells and Streptomyces mycelia are able to form close contacts in the absence of a conjugative system which might facilitate intergeneric plasmid transfer without the genes required for mating pair formation (Tra2) of the RP4 plasmid. The same Tra2 genes found to be essential for RP4 plasmid transfer, RSF1010 mobilization, and donor-specific phage propagation in E. coli were also required for intergeneric transfer between E. coli and Streptomyces lividans.     

Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria.

The replication and maintenance properties of the broad-host-range plasmid RK2 and its derivatives were examined in nine gram-negative bacterial species. Two regions of RK2, the origin of replication (oriV) and a segment that encodes for a replication protein (trfA delta kilD, designated trfA*), are sufficient for replication in all nine species tested. However, stable maintenance of this minimal replicon (less than 0.3% loss per generation under nonselection conditions) is observed only in Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, and Azotobacter vinelandii. Maintenance of this minimal replicon is unstable in Rhizobium meliloti, Agrobacterium tumefaciens, Caulobacter crescentus, Acinetobacter calcoaceticus, and Rhodopseudomonas sphaeroides. A maintenance function has been localized to a 3.1-kilobase (kb) region of RK2 encoding three previously described functions: korA (trfB korB1 korD), incP1-(II), and korB. The 3.1-kb maintenance region can increase or decrease the stability of maintenance of RK2 derivatives dependent on the host species and the presence or absence of the RK2 origin of conjugal transfer (oriT). In the case of A. calcoaceticus, stable maintenance requires an RK2 segment that includes the promoter and the kilD (kilB1) functions of the trfA operon in addition to the 3.1-kb maintenance region. The broad-host-range maintenance requirements of plasmid RK2, therefore, are encoded by multiple functions, and the requirement for one or more of these functions varies among gram-negative bacterial species.     

Contribution of different segments of the par region to stable maintenance of the broad-host-range plasmid RK2.

A 3.2-kb region of the broad-host-range plasmid RK2 has been shown to encode a highly efficient plasmid maintenance system that functions in a vector-independent manner. This region, designated par, consists of two divergently arranged operons: parCBA and parDE. The 0.7-kb parDE operon promotes plasmid stability by a postsegregational killing mechanism that ensures that plasmid-free daughter cells do not survive after cell division. The 2.3-kb parCBA operon encodes a site-specific resolvase protein (ParA) and its multimer resolution site (res) and two proteins (ParB and ParC) whose functions are as yet unknown. It has been proposed that the parCBA operon encodes a plasmid partitioning system (M. Gerlitz, O. Hrabak, and H. Schwabb, J. Bacteriol. 172:6194-6203, 1990; R. C. Roberts, R. Burioni, and D. R. Helinski, J. Bacteriol. 172:6204-6216, 1990). To further define the role of this region in promoting the stable maintenance of plasmid RK2, the parCBA and parDE operons separately and the intact (parCBA/DE) par region (3.2 kb) were reintroduced into an RK2 plasmid deleted for par and assayed for plasmid stability in two Escherichia coli strains (MC1061K and MV10delta lac). The intact 3.2-kb region provided the highest degree of stability in the two strains tested. The ability of the parCBA or parDE region alone to promote stable maintenance in the E. coli strains was dependent on the particular strain and the growth temperature. Furthermore, the insertion of the ColE1 cer site into the RK2 plasmid deleted for the par region failed to stabilize the plasmid in the MC1061K strain, indicating that the multimer resolution activity encoded by parCBA is not by itself responsible for the stabilization activity observed for this operon. To examine the relative contributions of postsegregational cell killing and a possible partitioning function encoded by the intact 3.2-kb par region, stability assays were carried out with ParD provided in trans by a compatible (R6K) minireplicon to prevent postsegregational killing. In E. coli MV10delta lac, postsegregational killing appeared to be the predominant mechanism for stabilization since the presence of ParD substantially reduced the stability of plasmids carrying either the 3.2- or 0.7-kb region. However, in the case of E. coli MC1061K, the presence of ParD in trans did not result in a significant loss of stabilization by the 3.2-kb region, indicating that the putative partitioning function was largely responsible for RK2 maintenance. To examine the basis for the apparent differences in postsegregational killing between the two E. coli strains, transformation assays were carried out to determine the relative sensitivities of the strains to the ParE toxin protein. Consistent with the relatively small contribution of the postsegregational killing to plasmid stabilization in MC1061K, we found that this strain was substantially more resistant to killing by ParE in comparison to E. coli MV10delta lac. A transfer-deficient mutant of thepar-deleted plasmid was constructed for the stable maintenance studies. This plasmid was found to be lost from E. coli MV10delta lac at a rate three times greater than the rate for the transfer-proficient plasmid, suggesting that conjugation can also play a significant role in the maintenance of plasmid RK2.     

Evidence for stable transformation of Pseudomonas aeruginosa with a pUC-based plasmid

Pseudomonas aeruginosa was transformed with pUC8:16, a pUC-based plasmid bearing the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). Transformation was initially indicated by an increase in ampicillin resistance from 1500 to 2500 mg l^–1. Presence of the plasmid in P. aeruginosa was confirmed by amplification of a portion of vgb from and detection of VHb in the transformant but not the untransformed host. Southern blot analysis further indicated that pUC8:16 existed as an autonomous plasmid rather than integrated into the chromosome of the P. aeruginosa transformant.     

Regions of broad-host-range plasmid RK2 which are essential for replication and maintenance.

The sites of cleavage on the map of the broad-host-range plasmid RK2 (56 kilobases) were determined for the BglII, PstI, and SmaI restriction enzymes, and the determinants for tetracycline and ampicillin resistance were localized. The cleavage sites were clustered at or near the drug resistance genes. To localize regions required for plasmid replication and maintenance in Escherichia coli, we deleted nonessential regions of RK2 by partial digestion with the restriction endonuclease HaeII to produce small derivatives. The smallest stable replicon obtained contained five HaeII fragments of RK2 which total 5.4 kilobases. These fragments were derived from three regions of RK2 that are separated from each other by antibiotic resistance genes. One of these HaeII fragments (0.75 kilobases) has the properties expected of the origin of replication. The outer four fragments, located in two separate regions of RK2, were found to provide, in trans, functions that permit the replication of the HaeII fragment carrying the origin of the replication. These results indicate that at least two plasmid-encoded genes, capable of acting in trans, and a replication origin are required for RK2 replication and maintenance.     

Different relative importances of the par operons and the effect of conjugal transfer on the maintenance of intact promiscuous plasmid RK2.

The par region of the broad-host-range, IncP alpha plasmid RK2 has been implicated as a stability determinant by its ability to enhance the maintenance of mini-RK2 plasmids or heterologous replicons in a growing population of host cells. The region consists of two operons: parCBA, which encodes a multimer resolution system, and parDE, which specifies a postsegregational response mechanism that is toxic to plasmidless segregants. To assess the importance of this region to the stable maintenance of the complete RK2 plasmid in different hosts, we used the vector-mediated excision (VEX) deletion system to specifically remove the entire par region or each operon separately from an otherwise intact RK2 plasmid carrying a lacZ marker. The par region was found to be important to stable maintenance of RK2lac (pRK2526) in Escherichia coli and five other gram-negative hosts (Agrobacterium tumefaciens, Azotobacter vinelandii, Acinetobacter calcoaceticus, Caulobacter crescentus, and Pseudomonas aeruginosa). However, the relative importance of the parCBA and parDE operons varied from host to host. Deletion of parDE had no effect on the maintenance of pRK2526 in A. calcoaceticus, but it severely reduced pRK2526 maintenance in A. vinelandii and resulted in significant instability in the other hosts. Deletion of parCBA did not alter pRK2526 stability in E. coli, A. tumefaciens, or A. vinelandii but severely reduced plasmid maintenance in A. calcoaceticus and P. aeruginosa. In the latter two hosts and C. crescentus, the delta parCBA mutant caused a notable reduction in growth rate in the absence of selection for the plasmid, indicating that instability resulting from the absence of parCBA may trigger the postsegregational response mediated by parDE. We also examined the effect of the conjugal transfer system on RK2 maintenance in E. coli. Transfer-defective traJ and traG mutants of pRK2526 were stably maintained in rapidly growing broth cultures. On solid medium, which should be optimal for IncP-mediated conjugation, colonies from cells containing the pRK2526 tra mutants displayed significant numbers of white (Lac-) sectors on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates, whereas sectors appeared rarely in colonies from tra+ plasmid-containing cells. Both the traJ and traG mutations further reduced the maintenance of the already unstable deltapar derivative. Thus, these experiments with defined mutations in an intact RK2 plasmid have revealed (i) that the par region allows RK2 to adapt to the different requirements for stable maintenance in various hosts and (ii) that conjugal transfer can contribute to the maintenance of RK2 in a growing population, particularly under conditions that are favorable to RK2 transfer.     

DnaA protein is required for replication of the minimal replicon of the broad-host-range plasmid RK2 in Escherichia coli.

The minimal origin of replication of the broad-host-range plasmid RK2 has two potential recognition sequences for the DnaA protein of Escherichia coli. DNA transfer by transformation into a dnaA-null mutant of E. coli showed that DnaA protein is needed for replication or maintenance of mini-RK2. We isolated and purified DnaA protein as a chimeric protein, covalently attached to a piece of collagen and beta-galactosidase. The hybrid protein specifically bound to restriction fragments from the oriV region of RK2, which contained the two dnaA boxes. Deletion of the second dnaA box inactivated the origin and abolished the binding of the hybrid protein to the DNA fragment that had suffered the deletion. When the second dnaA box was replaced with an EcoRI linker of identical length, origin activity was restored. Binding experiments showed that the linker provided a weak dnaA box. An alternative explanation was that the linker restored proper spacing between sequences on either side of the deleted box, thus restoring origin activity.     

FP2 plasmid curing in Pseudomonas aeruginosa

A procedure for the elimination of the IncP-8 plasmid FP2 from Pseudomonas aeruginosa strain 1 was developed. The procedure consists of freezing cells, competent for transformation, in 15% glycerol at -70 degrees C for at least 48 h and screening survivors for loss of mercuric chloride resistance. Curing frequencies of 0.5% were achieved only in host cells carrying a dht mutation (unable to convert thymine to dihydrothymine).     

Instability of a high-copy-number mutant of a miniplasmid derived from broad host range IncP plasmid RK2

Mini-RK2 plasmids pCT460 and pCT461 which contain the oriVRK2, trfA and trfB regions of RK2 in addition to tetracycline and kanamycin resistance determinants, have copy numbers of 17 and 35 copies per chromosome equivalent, respectively. The difference in copy number is due to a 56-bp deletion in oriVRK2 in pCT461. In Escherichia coli only pCT461 is markedly unstable in batch culture while both are unstable (although pCT461 is more so) in bacteria on stock plates. The instability of pCT461 in bacteria on stock plates is recA+ dependent and appears to involve loss of plasmid DNA from bacteria rather than selective cell death. After storage of recA+ bacteria carrying pCT461 for a few weeks the remaining antibiotic-resistant bacteria carry a mixture of plasmid DNA species including parental pCT461, transposable element insertion derivatives, and, by far the majority, deletion derivatives. It appears that one particular plasmid region, which includes the kilD gene (which inhibits plasmid maintenance in the absence of korD which, however, is present on pCT460 and pCT461), is responsible for this instability in a gene dosage-dependent way. Most of these deletion derivatives are dependent on pCT461-specified trfA gene (essential for replication) so that they do not displace pCT461 entirely. Their presence reduces the copy number of pCT461, thus reducing the instability, and is probably ultimately responsible for pCT461 survival on stock plates. In many bacteria the same process which gives rise to deletion derivatives may result in degradation of plasmid DNA extensive enough to cause loss of pCT461.     

Conserved C-terminal region of global repressor KorA of broad-host-range plasmid RK2 is required for co-operativity between KorA and a second RK2 global regulator, KorB.

KorA and KorB proteins of IncP1 plasmid RK2 are encoded in the central control region (ccr) of the plasmid and act as global regulators of plasmid genes for replication, transfer and stable inheritance. KorA represses seven promoters on RK2, by binding to a defined operator site, OA, which always occurs in promoter regions. KorB recognises another operator, OB, which is found 12 times on the RK2 genome, but not always in promoter regions. At five of the KorA-regulated promoters, an OBsequence is also present. The presence of both KorA and KorB leads to severely decreased promoter activity. By measuring repression at different levels of KorA and KorB alone and in combination, we showed that there is at least 3. 4-fold co-operativity between them at korApin vivo. Testing the ability of previously isolated KorA mutants to act in a co-operative way in the presence of KorB in vivo or in vitro showed that the C-terminal part of KorA between amino acid positions 68 and 83 is required for this co-operativity. This region is part of a segment that is highly conserved between KorA and two other RK2 proteins, TrbA and KlcB. We propose that this conserved region may provide the basis for co-operativity with KorB either indirectly, by modulating DNA structure near the KorB binding site, or directly by serving as the "recognition" patch of each protein by KorB. It may thus serve as a key domain in allowing a sensitive response of the global circuits to changes in repressor concentration and thus modulation of replication, transfer and maintenance.