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.

A critical step in the initiation of DNA replication is the recruitment, loading, and activation of the replicative helicase. Helicase activity is not only necessary for progression of the replication fork but, as studies with the Escherichia coli chromosomal origin oriC have revealed, the DnaB helicase makes essential contacts with other proteins in the replication complex. Recruitment of the helicase in E. coli requires association of the DnaB hexamer with the E. coli DnaC accessory protein (1)(2)(3). The DnaB-DnaC complex interacts with the host initiation protein, DnaA, bound to specific sequences (DnaA boxes) at oriC. This association results in the loading of the helicase onto unwound single-stranded DNA in the origin and the release of DnaC (4 -7). Once loaded onto the single-stranded DNA, DnaB interacts directly with DnaG to facilitate primase loading onto the single-stranded DNA at the replication fork (8,9). This interaction becomes the primary regulator of Okazaki fragment synthesis (10) and also ensures the proper placement of primers for leading strand synthesis (11). The subsequent association of DnaB with the Tau subunit of the DNA polymerase III holoenzyme results in rapid movement of the replication fork (12,13).
Studies on plasmids and phages that replicate in E. coli have revealed additional strategies for recruiting DnaB to a replication origin. The replication initiation proteins encoded by the narrow host range plasmids pSC101 and R6K, RepA and , respectively, have been shown to interact with DnaB in vitro (14 -16). For plasmid pSC101, the RepA-DnaB interaction is essential for loading the helicase onto the origin in a process that also requires the DnaC and DnaA proteins (14). Bacteriophage utilizes the phage-encoded P protein instead of the host-encoded DnaC protein to recruit DnaB to the initiation complex (17,18). With phage P2, the phage-encoded B protein appears to be required for DnaB recruitment for lytic replication, although not replication of P2 as a plasmid (19). Broad host range plasmids are able to replicate in diverse bacteria. As such, they provide the means to examine replication of a specific replicon in different host backgrounds. RK2 is a promiscuous plasmid belonging to the IncP group. It is noted for its ability to transfer into and be stably maintained in a wide variety of Gram-negative bacteria. Only two regions of an IncP plasmid are essential for broad host range replication, the cis acting origin for DNA replication (oriV) and the trfA gene that encodes two forms of a trans-acting replication initiation protein.
The host bacterium provides all other proteins essential for replication. The simplicity of this system has allowed for a direct comparison of the mechanism for DNA replication initiation in different bacterial species (20,21).
IncP plasmids can be divided into two subgroups based on sequence differences: IncP␣, to which RK2 belongs, and IncP␤, which is represented by the plasmid R751. Both RK2 and R751 can replicate in E. coli and Pseudomonas aeruginosa. Earlier studies had shown that the smaller form of the RK2 initiation protein, TrfA-33, was sufficient for stable replication of an RK2 mini-replicon in E. coli or Pseudomonas putida but that the larger form of the protein, TrfA-44, was required for plasmid replication in P. aeruginosa (22)(23)(24). Recently, it has been shown that TrfA-44 is unique among plasmid initiation proteins in that it can load and activate the DnaB helicase of P. aeruginosa or P. putida on the RK2 origin in vitro in the absence of the DnaA protein (25). By contrast, the TrfA-33 protein requires DnaA protein to load and activate the helicase of P. putida and requires DnaA plus DnaC to load the helicase of E. coli. Consistent with the earlier in vivo studies, TrfA-33 did not function in vitro with the DnaB helicase of P. aeruginosa either in the presence or in the absence of P. aeruginosa DnaA (25).
Since it has been shown that the open complex formed on a supercoiled oriV template is indistinguishable when either TrfA-33 or TrfA-44 proteins are used with the DnaA proteins of E. coli, P. putida, or P. aeruginosa (20), the observed differences in activity of the two forms of the TrfA initiation protein in P. aeruginosa are likely due to a difference in their ability to interact with the P. aeruginosa DnaB helicase. This suggested a role for the N-terminal 97 amino acids, which are unique to TrfA-44, in DnaB recruitment in Pseudomonas. We therefore undertook a mutational analysis of the first 97 amino acids of this protein in an attempt to identify those regions within the N terminus that are important in the loading of the Pseudomonas helicases at the RK2 replication origin.

EXPERIMENTAL PROCEDURES
Construction of trfA Mutations-Plasmid pGC1 (20) was used to express His6TrfA-44(M98L/G254D/S267L), a variant of TrfA-44 that has six histidine residues inserted between the first amino acid (Met) and the second amino acid (Asn) of the native protein to allow for ease of purification, the M98L amino acid substitution that replaces the native methionine start of TrfA-33 with a leucine and thus eliminates TrfA-33 expression, and the G254D and S267L changes that result in a primarily monomeric form of the protein. A previous study had shown that although wild type TrfA protein is primarily a dimer in solution, it was the monomeric form that was essential for replication initiation activity (26). Plasmid pGC1 served as the template for the construction of three specific N-terminal deletions and one point mutant as follows.
Amino acids 21-32, 37-55, or 71-88 (numbering based on the native non-His-tagged protein) were deleted by using a PCR-based strategy. For each deletion, two pairs of primers were used to synthesize fragments that flanked either side of the desired deletion in the trfA gene. Both PCR fragments included restriction enzyme sites such that after digestion with the appropriate enzymes, ligation of the two fragments in the presence of a vector backbone resulted in the construction of a pGC1 derivative. These derivatives expressed the desired TrfA-44 deletion protein, which contained either one or two additional amino acids at the site of the deletion. The resulting plasmids were pZZ28, for expression of TrfA-44⌬2, pZZ25, for expression of TrfA-44⌬3, and pZZ23, for expression of TrfA-44⌬4.
To change amino acid residue 22 in the trfA gene of pGC1 from Glu (GAG) to Ala (GCG), and coincidentally introduce a SacII site, primer 5Ј-GGGTTTTCCGCCGCGGATGCCGAAAC-3Ј and its complement were used with pGC1 template and the PCR-based QuikChange sitedirected mutagenesis kit (Stratagene) to introduce the underlined base change. The EcoRI-SfiI fragment of one resulting mutant was sequenced to confirm the presence of the desired mutation, and this fragment was then used to replace the matching fragment in nonmutagenized pGC1 resulting in pZZ29.
C-terminal His 6 -tagged DnaA proteins of E. coli, P. putida, and P. aeruginosa (21) and C-terminal His 6 -tagged DnaB proteins of E. coli, P. putida, and P. aeruginosa (20) were purified as described. The modified proteins have been found to behave similarly to the native E. coli proteins in several in vitro assays (28,29). E. coli DnaC and E. coli DNA gyrase were purified from strains RSC680 and AN1459(pPS562), respectively, kindly provided along with purification protocols by Dr. Nick Dixon. HU was a generous gift from Dr. Roger McMacken. Commercially available proteins were SSB 1 (Promega), creatine kinase and bovine serum albumin (Fraction V) (Sigma), and DNA restriction and modification enzymes from various commercial sources.
The five pRR10 derivatives were then introduced separately into P. aeruginosa by electroporation (34) using 100 g/ml carbenicillin for selection. A single colony for each of these five strains was picked from a fresh overnight LB ϩ 100 g/ml carbenicillin plate, resuspended in 5 ml of LB, and then incubated for 24 h with shaking at 37°C. At subsequent time points, 50 l of the stationary phase culture was transferred to 5 ml of LB broth, and incubation at 37°C with shaking was continued. In addition, serial dilutions of the initial suspension and the subsequent overnight cultures were prepared, and aliquots were plated onto LB agar without antibiotics. After overnight incubation at 37°C, 100 colonies from these plates were patched to plates with LB ϩ 100 g/ml carbenicillin and then onto LB plates to determine the percentage of cells still maintaining the plasmid.

RESULTS
Rationale for the Construction of Specific Mutants-Plasmids RK2 (IncP␣ replicon) and R751 (IncP␤ replicon) can replicate in E. coli and P. aeruginosa, and both plasmids express two forms of the essential plasmid-encoded replication initiation protein. The larger form of the RK2 initiation protein, TrfA-44, is required for replication of the plasmid in P. aeruginosa, whereas the smaller form is sufficient for replication in E. coli (22)(23)(24). The smaller forms of the RK2 and the R751 initiation proteins, TrfA-33 and TrfA2, respectively, are each 285 amino acids in length, and when aligned using ClustalW (35), 91% of the residues are either identical or strongly similar (data not shown). The larger forms of the initiation protein of the two plasmids differ in size and are less related in the N-terminal regions. Alignment of the 382-amino-acid TrfA-44 protein of RK2 and the 407-amino-acid TrfA1 protein of R751 using ClustalW identified 78% residues that were identical or strongly similar. The alignment result for the N terminus of the larger proteins is shown in Fig. 1. Given the increased sequence variation of the two larger proteins as compared with the two smaller proteins, it seemed likely that those residues conserved in the N terminus of TrfA-44 and TrfA1 would be important for protein activity in P. aeruginosa.
Analysis of TrfA-44 using the secondary structure prediction program PredictProtein (36) identified four possible helical regions in the first 97 amino acids of the protein (Fig. 1). The fourth helical region overlapped with a region of striking similarity between TrfA-44 and TrfA1, comprised of amino acid residues 70 -90 of TrfA-44, that had been noted in an earlier study (37). We therefore decided to delete amino acids 71-88 of TrfA-44, which encompassed this helical region. The analysis of the in vitro activity of this deletion mutant, designated as TrfA-44⌬4, led to the construction of deletions TrfA-44⌬3 (amino acid residues 37-55 inclusive deleted) and TrfA-44⌬2 (amino acid residues 21-32 inclusive deleted), which removed, separately, two of the three remaining predicted helical regions.
In Vitro Helicase Loading Activity of TrfA-44 Deletion Mutants-Loading and activation of DnaB helicase at a replication origin on a supercoiled plasmid template can be examined in vitro using the FI* assay (2,29). The basis of this assay is that helicase unwinding of a supercoiled template in the presence of DNA gyrase and SSB produces a highly unwound form of the DNA, termed FI*, that can be distinguished electrophoretically from the template FI form.
The FI* assay was used to test the functionality of the mutant TrfA proteins in the recruitment, loading, and activation of DnaB at the RK2 origin. To ensure that slight differences in the activity of the mutants would be seen, the amount of DnaB helicase added to the standard assay was titrated to determine the lowest level of DnaB protein needed to get full activity with wild type TrfA-44 protein. The level of E. coli DnaB necessary for full activity, 1600 ng, was similar to that reported previously (29), and the addition of both E. coli DnaA and DnaC proteins was essential (data not shown). The level of Pseudomonas DnaB required for full activity with wild type TrfA-44 protein, 50 ng for DnaB from P. aeruginosa and 100 ng for P. putida DnaB (Fig. 2), was less then reported previously (20). As expected from previous work (25) . We then confirmed that TrfA-33 was able to load this lower level of P. putida helicase, but only in the presence of P. putida DnaA (Fig. 2, lanes 7-9), and that even with DnaA present, P. aeruginosa DnaB, at this lower concentration, could not be loaded by TrfA-33 (Fig. 2, lanes 2 and 3).
The ability of the three TrfA-44 deletion mutant proteins to load P. aeruginosa or P. putida helicase onto RK2 oriV was then tested. As shown in Fig. 3, TrfA-44⌬4 (lanes 5) was fully functional with DnaB from P. aeruginosa (A) or P. putida (B). TrfA-44⌬3 (lanes 4) had reduced activity, particularly with P. putida DnaB, whereas TrfA-44⌬2 (lanes 3) was not functional with either helicase. Activity with TrfA-44⌬2 was not restored by the addition of P. aeruginosa DnaA to reactions containing P. aeruginosa DnaB but was restored by the addition of P. putida DnaA to reactions containing P. putida DnaB protein (data not shown).
All three mutant proteins functioned as well as the wild type protein in the loading and activation of E. coli DnaB in the presence of E. coli DnaA and DnaC (Fig. 4). These results showed that the observed defects in the TrfA-44⌬2 and TrfA-44⌬3 mutants were specific to the recruitment and loading of DnaB from Pseudomonas in the absence of DnaA.
Point Mutation in TrfA-44 with Altered Helicase Loading Activity-The TrfA-44⌬2 mutant protein was deleted for 12    with the N terminus of TrfA1 of plasmid R751 (amino acid residues 1-123) using ClustalW (*, identical residues; :, strongly similar residues; ., weakly similar residues) is as shown. The methionine residue that is the start of the smaller TrfA proteins is shown in bold. The predicted secondary structure for the N terminus of TrfA-44, using PHD Predict (h, helical region; e, extended strand), is shown above the alignment. The three boxed-in sequences of TrfA-44 mark the residues deleted in TrfA-44⌬2, TrfA-44⌬3, and TrfA-44⌬4, respectively. The position of the TrfA-44E22A mutant is indicated by the lowercase e in the TrfA-44 sequence. amino acids. To determine whether a specific amino acid within the deleted region was required for helicase loading, we constructed a specific point mutant. Reasoning that a charged and/or polar amino acid residue might be involved in the stabilization of a DnaB/TrfA-44 interaction, we replaced the glutamate residue at position 22 with alanine, yielding TrfA-44E22A. This change alters the charge and polarity at this position but not the helical nature of this region as predicted using PredictProtein.
The TrfA-44E22A mutant protein was tested for helicase loading activity. This mutant protein, when added at 300 ng/ assay, had slightly reduced activity when compared with wild type TrfA-44 protein with P. aeruginosa or P. putida DnaB (data not shown). This reduction in activity was more pronounced, particularly with P. putida DnaB, when the amount of TrfA protein added to each reaction was decreased to 200 ng/assay (Fig. 5), suggesting that the point mutant was also altered in helicase recruitment or loading. The TrfA-44E22A mutant protein behaved as the wild type protein at all concentrations tested in assays with E. coli DnaA, DnaB, and DnaC (data not shown).
A filter binding assay was used to confirm that the reduced activity of the TrfA-44E22A point mutant was not due to reduced DNA binding activity of the mutant as compared with the wild type protein (data not shown). The three TrfA-44 deletion mutants were also not defective in binding to a DNA fragment containing RK2 oriV (data not shown).
Association of P. aeruginosa DnaB with Wild Type and Mutant TrfA-44 Proteins-Gel exclusion chromatography was used to confirm that the differences observed in the conversion of the RK2 mini-plasmid from the FI to the FI* form was due to differences in the ability of the DnaB helicase of P. aeruginosa to physically associate with certain of the TrfA-44 mutant proteins. Wild type and mutant TrfA-44 proteins were incu-bated with P. aeruginosa DnaB and the RK2 mini-replicon pTJS42. The reaction was then run through a Sepharose CL4-B column, and the eluted fractions were analyzed for the presence of DnaB protein. On this column, the template DNA is present in the void, as are any proteins that are stably associated either with the DNA or with a protein that is bound to the DNA. As shown in Fig. 6, P. aeruginosa DnaB interacted with TrfA-44 wild type protein bound to the template DNA and was therefore present in the void fractions (B) but did not form a stable complex with the TrfA-33 wild type protein bound to the template (A) as has been shown previously (25). P. aeruginosa DnaB also did not stably associate with the TrfA-44⌬2 mutant (C) and showed reduced association with the TrfA-44E22A mutant protein (D) and, to a lesser extent, the TrfA-44⌬3 mutant (E). Under the conditions used, the TrfA-44 wild type or mutant proteins could not be detected after Western blotting. However, TrfA protein could be detected if column fractions were directly applied to a nitrocellulose membrane that was then processed for detection as described under "Experimental Procedures." In all cases, the peak of TrfA protein was in the void fractions (10 and 11) (data not shown).
The stability of these plasmids in P. aeruginosa was then examined. As shown in Fig. 7, plasmids pRR10-98His⌬3, pRR10-98His⌬4, and pRR10-98HisE22A were almost as stable as pRR10-98His with less then 20% loss after ϳ84 generations of growth. Plasmid pRR10-98His⌬2, however, was extremely unstable. Only 6% of the P. aeruginosa cells maintained the plasmid after ϳ28 generations of growth. DISCUSSION Plasmid RK2 can replicate in a wide range of Gram-negative bacteria, and recent studies indicate that it does so by employing a variety of strategies to recruit the necessary host proteins. Early studies showed that RK2 mini-replicons expressing only the smaller replication initiation protein, TrfA-33, were unstable in P. aeruginosa (22)(23)(24). Recently, in vitro studies revealed that TrfA-33 was unable to load the DnaB helicase of P. aeruginosa onto the RK2 origin in the presence or absence of DnaA, although this form of the replication initiation protein was fully functional when the DnaA and DnaB proteins from P. putida or the DnaA, DnaB, and DnaC proteins from E. coli were provided (20). The experiments presented here demonstrate that specific regions within the first 97 amino acids of TrfA-44 are required for the recruitment of the P. aeruginosa DnaB helicase to the RK2 replication origin.
The deletion of amino acid residues 21-32 in TrfA-44 results in a mutant protein, TrfA-44⌬2, which fails to stably interact with P. aeruginosa DnaB in vitro (Fig. 6C) and is not able to recruit and activate the helicase at the RK2 origin as detected by the FI* assay (Fig. 3A). That this effect is due to the loss of the specific amino acid residues in the second helix predicted in the N terminus ( Fig. 1) and not simply due to a shortening in the length of the protein is clear from the observations that deletion of the 18 amino acid residues in the fourth predicted helix (positions 71-88) had no discernable effect on helicase recruitment and loading in vitro (Fig. 3) or protein activity in vivo (Fig. 7). Furthermore, the TrfA-44⌬2 protein was fully functional in FI* assays in which the TrfA-33 version of the protein is active, i.e. with E. coli DnaA, DnaB and DnaC proteins (Fig. 4) or with P. putida DnaA and DnaB proteins (data not shown).
The defect of TrfA-44⌬2 is also apparent in vivo (Fig. 7). The unstable replication of a plasmid expressing this mutant protein in P. aeruginosa is similar to that of an RK2 replicon expressing only TrfA-33 (22)(23)(24). The results presented in this study support the conclusion that RK2 mini-replicons which express only TrfA-33 are unstable in P. aeruginosa because this protein is incapable of loading the DnaB helicase, even in the presence of DnaA protein. In fact, it is not clear that the DnaA protein plays any role in RK2 replication in P. aeruginosa as a mini-replicon deleted for all four DnaA bindings sites in oriV was fully functional in P. aeruginosa (38).
Helicase loading in E. coli requires DnaA and DnaC and utilizes either form of the TrfA protein. In this bacterium, it is likely that the additional 97 amino acids on the N terminus of TrfA-44 play no role in the recruitment and activation of DnaB. Indeed, all four TrfA-44 mutant proteins tested in this study were as active as wild type protein in the FI* assay using the E. coli replication proteins ( Fig. 4 and data not shown). Inter-estingly, an earlier study found that a mini-replicon expressing only TrfA-33 was more stable in E. coli than a mini-replicon expressing only TrfA-44 (23).
P. putida helicase can be loaded in vitro by either of two mechanisms (25). One utilizes TrfA-33 and requires DnaA, similar to E. coli, and the other utilizes TrfA-44 and is DnaAindependent, similar to P. aeruginosa. It is clear from the results shown in Fig. 2, lanes 4 -6, that when both DnaA and TrfA-44 are present, the various possible protein interactions can interfere with helicase loading.
As shown in this study, the two Pseudomonas helicases differ somewhat in their interaction with the N terminus of TrfA-44. Although TrfA-44⌬2 was defective in vitro with both Pseudomonas helicases, the TrfA-44E22A substitution mutant and the TrfA-44⌬3 deletion mutant showed significantly reduced activity with P. putida DnaB (Figs. 3B and 5). In vivo, P. putida, like E. coli, is able to use TrfA-33 and DnaA to recruit DnaB, and as such, RK2 plasmid derivatives carrying trfA-44⌬2, trfA-44⌬3, or trfA-44E22A were able to stably replicate in this host. 2 The interaction of helicase with a replication initiation protein is not unique to TrfA-44. The replication initiation proteins of plasmids R6K ( protein) and pSC101 (RepA) as well as the chromosomal initiator protein DnaA have all been shown to interact with E. coli DnaB (5,14,16). The regions of DnaB that interact with these three proteins are not identical, although there is some overlap. Unlike the TrfA-44 protein, however, both the and RepA proteins require the E. coli DnaA protein for loading and activation of E. coli DnaB helicase. The role of the TrfA-44 replication initiation protein as being solely responsible for helicase loading and activation at the RK2 origin, therefore, is unique among plasmid replicons. This is possibly because most of the well studied replication systems are of narrow host range plasmids of E. coli and DnaA/DnaC interactions with DnaB may predominate in this organism but not in other bacteria. In the case of plasmid RK2, the fact that expression of the larger TrfA-44 initiation protein in Pseudomonas species obviates the need for DnaA and DnaC proteins in the recruitment and translocation of DnaB to the replication origin may be an important factor in extending the host-range of this plasmid.