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Horizontal transfer of antibiotic resistance genes carried by conjugative plasmids poses a serious health problem. As conjugative relaxases are transported to recipient cells during bacterial conjugation, we investigated whether blocking relaxase activity in the recipient cell might inhibit conjugation. For that purpose, we used an intrabody approach generating a single-chain Fv antibody library against the relaxase TrwC of conjugative plasmid R388. Recombinant single-chain Fv antibodies were engineered for cytoplasmic expression in Escherichia coli cells and either selected in vitro for their specific binding to TrwC, or in vivo by their ability to interfere with conjugation using a high-throughput mating assay. Several intrabody clones were identified showing specific inhibition against R388 conjugation upon cytoplasmic expression in the recipient cell. The epitope recognized by one of these intrabodies was mapped to a region of TrwC containing Tyr-26 and involved in the conjugative DNA-processing termination reaction. These findings demonstrate that the transferred relaxase plays an important role in the recipient cell and open a new approach to identify specific inhibitors of bacterial conjugation.
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Conjugation is one of the main mechanisms through which horizontal gene transfer occurs in bacteria. Three main modules are responsible for conjugal DNA processing and transfer (Llosa et al., 2002): (i) the relaxosome, being the relaxase its principal protein component, responsible for DNA cleavage at the origin of transfer (oriT), (ii) the transport apparatus, a type IV secretion system (T4SS) through which the DNA and proteins are transported to a recipient cell and (iii) a coupling protein, which acts as a link between the other two modules. It is well established that T4SS of pathogenic bacteria are able to transport protein substrates (Christie and Vogel, 2000; Schulein et al., 2005). In recent years, the idea that T4SSs of conjugative plasmids have proteins as their main substrates (Llosa et al., 2002; Ding et al., 2003; Schulein et al., 2005) was validated by demonstration of T4SS-dependent transfer of several plasmid-coded proteins in the absence of DNA transport, i.e. Sog primase of conjugative plasmid ColIb-P9 to different bacterial cells (Wilkins and Thomas, 2000); relaxase MobA of mobilizable plasmid RSF1010 by Legionella pneumophila Dot+Icm to bacterial cells (Luo and Isberg, 2004) or by Agrobacterium tumefaciens VirB+VirD4 to plant cells (Vergunst et al., 2005); relaxase TraA of conjugative plasmid pATC58 to human endothelial cells by VirB+VirD4 of Bartonella henselae (Schulein et al., 2005); proteins VirE2, VirF and relaxase VirD2 of Agrobacterium Ti plasmids to plant cells (Vergunst et al., 2000; 2005) and, finally, relaxase TrwC of conjugative plasmid R388 to bacterial cells (Draper et al., 2005). A bipartite signal sequence present in the C-terminal region of many transferred proteins was correlated with transport proficiency by site-directed mutagenesis (Vergunst et al., 2005).
As all these proteins are specifically and efficiently transported, it is expected that they perform a function in the recipient cells. Several reports point to the implication of relaxase VirD2 in nuclear import of the T-strand and its integration into the plant genome (Herrera-Estrella et al., 1990; Howard et al., 1992; Tinland et al., 1995; Mysore et al., 1998). Other instances of T4SS-mediated protein transport were demonstrated using reporter screens for reactions catalysed by the transferred proteins, i.e. primase activity of the Sog protein (Wilkins and Thomas, 2000) and site-specific recombinase and integrase activities of relaxase TrwC (Draper et al., 2005). These reports emphasize that the transported proteins remain in a functional state within the recipient cells. Nevertheless, the physiological functions that relaxases could play in recipi ent cells during conjugative transfer have not been elucidated. In vitro studies on DNA cleavage and strand transfer reactions catalysed by relaxase TrwC indicated that its two catalytically active tyrosyl residues Tyr-18 and Tyr-26 play non-equivalent roles during conjugative DNA processing. Tyr-18 was proposed to be required for initiation while Tyr-26 would be used in the termination reaction (Grandoso et al., 2000). Thus, we reasoned that, if relaxases play a function after being transported to recipients, perhaps conjugation could be inhibited by blocking relaxase activity in recipient cells. This is the idea that founded this work.
Stable intracellular antibodies (intrabodies) have been used to inactivate proteins in the cytoplasm of yeast (Carlson, 1988; Visintin et al., 1999), plant (Tavladoraki et al., 1993; Jobling et al., 2003) and especially in mammalian cells (Biocca et al., 1993; Rondon and Marasco, 1997; Lobato and Rabbitts, 2004), but not in bacteria, due to the strongly reducing environment of their cytoplasm, which prevents the formation of the disulphide bonds conserved in the immunoglobulin domains and needed for their folding and stability (Wörn and Plückthun, 2001). Fortunately, production of functional single-chain Fv antibodies (scFvs) in the cytoplasm of Escherichia coli was found to be feasible in a strain (named FÅ113) carrying mutations in the major intracellular disulphide bond-reduction systems, thioredoxin reductase (trxB) and glutation oxidoreductase (gor) (Bessette et al., 1999; Ritz et al., 2001; Fernández, 2004). Cytoplasmic expression of scFvs in E. coli FÅ113 in the presence of protein chaperones (e.g. DsbC) (Jurado et al., 2002) or as N-terminal fusions to thioredoxin 1 (Trx1) (Jurado et al., 2006) allows their correct folding, thus enabling antigen recognition. In this work, we demonstrate that intracellular expression of scFvs raised against relaxase TrwC protects recipient E. coli cells against conjugative transfer of plasmid R388. Results show that immunization led to interference with R388 conjugation in a highly specific mode, indicating that important conjugative DNA-processing reactions occur in the recipient cell.
Anti-TrwC-N293 scFvs inhibit R388 conjugation
In order to obtain scFvs specific against conjugative plasmid R388 relaxase TrwC, we immunized mice with native TrwC-N293, a protein fragment comprising the N-terminal 293 amino acids of TrwC containing the relaxase domain. After immunization, immunoglobulin variable gene segments were amplified by RT-PCR from mice splenocytes, assembled into scFv genes and cloned in phagemid vectors (see Experimental procedures). A library of ∼105 independent clones was obtained in E. coli XL-1 Blue and rescued into phage antibody (Phab) particles. This Phab library was subsequently used in four rounds of panning selection against purified TrwC-N293 adsorbed onto enzyme-linked immunosorbent assay (ELISA) plates. The overall scheme is represented in Fig. 1. Clear enrichment in the number of Phabs with ability to bind TrwC-N293 was obtained along three successive rounds of panning (Fig. S1). To ensure that recovered Phabs adequately bound TrwC-N293, 96 clones from third and fourth rounds of panning were screened for TrwC-N293 binding by ELISA. The sequence of 24 clones showing higher binding signals in ELISA was determined. All sequenced clones coded for the same scFv, named P4.E7 (Fig. S2).
To test for the effect of scFv-P4.E7 on R388 conjugation we performed conventional mating experiments in which scFv-P4.E7 was expressed in the cytoplasm of recipient E. coli cells. In order to ensure its functional cytoplasmic expression, scFv-P4.E7 was produced as a Trx1- N-terminal fusion (Trx1-scFv-P4.E7) in the cytoplasm of E. coli strain FÅ113 (Jurado et al., 2006). A donor E. coli strain containing an R388 derivative plasmid (pSU2007::Tnlux) was mated with recipient E. coli FÅ113 cells expressing Trx1-scFv-P4.E7, or the control fusion Trx1-scFv-B7 (Table 1). The scFv-B7 used as a negative control recognizes a protein from Pseudomonas putida that is not present in E. coli (Fraile et al., 2001). The conjugation frequencies obtained from these experiments are shown in Table 2. As observed, the conjugation frequency decreased to 5% of controls when Trx1-scFv-P4.E7 was expressed in recipient cells, suggesting that scFv-P4.E7 could be acting in vivo as an intrabody inhibiting TrwC function (∼20-fold) during R388 conjugation.
Table 1. Plasmids used in this work.
pCANTAB5E derivative with 6xHis and E-tag Periplasmic expression of scFvs; phage display vector
Matings were carried out between donor strain CSH53 (pSU2007::lux + pUC8::lacIq) and recipient strain FÅ113 expressing the intrabodies indicated in column 1. Arbitrary units of light (measured with a Fluoroskan microplate luminometer) obtained at the moment of maximum light emission (around 9 h) for controls (FÅ113 recipients expressing no scFv or with control scFv-B7) are listed in column 2. Conjugation frequencies (column 3) were calculated as the number of transconjugants (GmRTcR) divided by the number of donors (GmRSmR). In columns 2 and 3, the first value corresponds to the mean value, while standard deviations (assuming a log-normal distribution) appear in parentheses.
Only donor cells present.
Recipient cells do not express any scFv.
In vivo screening of intrabodies inhibiting R388 conjugation
The results obtained with scFv-P4.E7 prompted us to look for more potent scFv inhibitors. To this end, we screened in vivo for intrabodies able to inhibit R388 conjugation using a pool of Trx1-scFv fusions cloned from sublibrary 1 (obtained after the first round of panning), which contains a higher diversity of intrabody sequences. The automated conjugation assay used to screen in vivo for scFv acting as conjugation inhibitors utilizes a donor strain containing pSU2007::Tnlux, a R388 plasmid derivative harbouring a lux operon under the control of the lac promoter, and a non-mobilizable high copy-number plasmid (pUC18::lacIq) overproducing the LacI repressor, which hinders light emission in the donor cell. Transfer of pSU2007::Tnlux to recipients can be followed by an increase in light emission due to transcription of the lux operon in the transconjugants, unless an inhibitor blocks transfer of pSU2007::Tnlux. The recipient cells in this screening were E. coli FÅ113 cells bearing a plasmid encoding an intrabody as a Trx1-scFv fusion. Neither Trx1 fusion nor intrabody expression were handicaps for detecting light emission because light units obtained with control intrabody scFv-B7 did not differ significantly from the values obtained for FÅ113 without intrabody (Table 2). One thousand independent Trx1-scFv clones were tested for their ability to inhibit pSU2007::Tnlux transfer using the light emission assay. Clones showing light emission values lower than 2% of controls (i.e. recipients with no intrabodies or with an unrelated intrabody) were selected, their amino acid sequence determined (Fig. S2) and used in conventional mating experiments to quantify the R388 conjugation frequency. All of them resulted in a significant reduction, ranging from 40- to over 10 000-fold, of R388 conjugation frequency (Table 2).
Binding of inhibitory scFvs to TrwC-N293 in vitro
The scFv clones selected for their inhibitory function on R388 conjugation in vivo (Table 2) were tested for binding to TrwC-N293 in vitro. Soluble protein extracts from E. coli FÅ113 cells expressing the corresponding Trx-scFv fusions were used in ELISA and Western blot to detect TrwC-N293. Fusions Trx-scFv-B7 and Trx-scFv-P4.E7 were used as negative and positive binding controls respectively. Binding to TrwC-N293 in vitro was detected only for clone scFv-P1.F2, and the positive control scFv-P4.E7, both in ELISA (Fig. 2A) and Western blots (Fig. 3A). Both scFv-P1.F2 and scFv-P4.E7 showed specificity for TrwC-N293 (Fig. 2A), because neither of them bound BSA or TraI-N293, the relaxase of plasmid pKM101 (Paterson et al., 1999) that shares 51% identity with TrwC-N293. As expected, control intrabody Trx-scFv-B7 did not bind to these antigens. Nonetheless, the difference in binding signals obtained in ELISA suggested a higher affinity of scFv-P4.E7, as could be anticipated for an antibody selected in vitro purely for its binding to TrwC-N293. This was confirmed by a quantitative estimation of the relative affinities of these scFvs for TrwC-N293 (Fig. 2B) using a competitive ELISA-based assay to determine, under equilibrium conditions, the amount of free antibodies in solution in the presence of increasing amounts of antigen (Friguet et al., 1985). This method allowed us to estimate a ∼13-fold difference in the dissociation constant (KD) of scFv-P1.F2 (KD∼8 μM) and scFv-P4.E7 (KD∼0.6 μM), demonstrating the higher affinity of the intrabody selected in vitro.
Specificity of intrabodies as inhibitors of R388 conjugation
To determine the range specificity of seven selected intrabodies, we tested their effect on transfer of conjugative plasmids pKM101 and F, whose relaxase domains are 51% and 37% identical to TrwC respectively. Although all seven intrabodies were active against R388 (Table 2), they did not reduce pKM101 or F conjugation (with the exception of intrabody scFv-P1.A2) (Table 3), indicating that they were specific for the R388 relaxase, and were not affecting any general property of the recipient cell. To rule out the possibility that the analysed scFv intrabodies were affecting the integrity and/or functionality of the T4SS channel used during R388 conjugation, we analysed the transmission of a second plasmid (CloDF13) whose relaxase (MobC) is not homologous to R388 relaxase. CloDF13 does not code for a T4SS, and employs the R388 T4SS for mobilization. As shown in Table 3, recipient cells expressing each of the seven analysed anti-TrwC scFvs displayed similar mobilization values for CloDF13 as those obtained by the control intrabody scFv-B7, while they inhibited R388 conjugation from the very same donor cells. Thus, we conclude that the T4SS transport channel is working adequately when recipient cells express the intrabodies, so conjugation inhibition should be occurring after TrwC has entered the recipient cytoplasm. Intrabody scFv-P1.A2 was particularly interesting because it was the only one exhibiting ‘broad range’ inhibition.
Table 3. Plasmid specificity of conjugation inhibition by scFvs.a
Control scFv-B7 (×10−2)
The experiment was carried out as described in Table 2. The first column shows the plasmids contained in donor cells. Columns 2–9 show the conjugation frequencies obtained when the intrabody indicated in the top row was expressed in recipient cells (FÅ113).
A R388 derivative (pSU2007::lux) was used. Frequencies are expressed as GmRTcR transconjugants per GmRSmR donor.
In order to have appropriate selective marker genes, donors contained the mobilizable plasmid pSU4280 (that contains pKM101 MOB region) and the helper plasmid pKM101Δmob (that provides the pKM101 T4SS). Transfer frequencies express the ratio of CmRTcR transconjugants per CmRSmR donor.
pOX38Cm, a CmR derivative of F, was used. Transfer frequencies are expressed as CmRTcR transconjugants per CmRSmR donor.
pSU4814, a CmR CloDF13 derivative, was mobilized by T4SS provided by R388. Transfer frequencies are expressed as CmRTcR transconjugants per CmRSmR donor.
Plasmid pSU1445, a trwC-deficient derivative of plasmid R388 was complemented by either a non-mobilizable plasmid harbouring trwC (pSU2621)f or the mutant relaxase trwC(Y26F) (pSU1659)g, or trwC(Y26A) (pSU1628)h in donor DH5α. Transfer frequencies are expressed as TpRTcR transconjugants per TpRNxR donor.
Identifying the epitopes recognized by intrabodies against R388 conjugation
Several truncated proteins derived from TrwC (Table 1) were used to delimit the TrwC segment recognized by scFv-P1.F2 and scFv-P4.E7 (Fig. 3A). Total protein extracts from E. coli cells overexpressing each of the truncated TrwC fragments were blotted to a membrane and probed with scFv-P1.F2 or scFv-P4.E7 antibodies. In both cases, the intrabodies bound to the protein region comprised between amino acids 1 and 192. Recognition was highly specific, as no additional proteins present in the extracts were distinguished. No TrwC bands were detected with the negative control antibody (scFv-B7), although it bound weakly to other proteins of the extract.
To map precisely the epitopes bound by these intrabodies, scFvs were hybridized with membranes containing immobilized overlapping peptides spanning the TrwC-N293 sequence (Fig. 3B and C). Three consecutive overlapping dodecapeptides were specifically recognized by scFv-P4.E7 (Fig. 3B), while they were not recognized by the control antibody scFv-B7. Such three spots have in common the sequence LAGNIGEG, that corresponds to amino acid residues 59–66 of TrwC, a variable segment in the MOBF protein family (see Fig. 3D). The result of a similar experiment, but using intrabody scFv-P1.F2, is shown in Fig. 3C. In this case, a different target was recognized. The common sequence in four consecutive overlapping hexadecapeptides recognized by scFv-P1.F2 was GADDYYA (residues 22–28). Interestingly, these residues are contained within the conserved motif I in the MOBF relaxase family (Francia et al., 2004) (see Fig. 3D). The position of the epitopes recognized by these intrabodies is labelled in the 3D structure of TrwC-N293 (Guasch et al., 2003; Boer et al., 2006) (Fig. 4). As shown, the two epitopes are exposed to the solvent in the 3D structure of TrwC-N293.
Biological significance of the TrwC epitope recognized by intrabody scFv-P1.F2
TrwC contains two catalytic tyrosines, Tyr-18 and Tyr-26. The epitope GADDYYA recognized by intrabody scFv-P1.F2 is contained in motif I of MOBF relaxases and contains the Tyr-26 that catalyses a transesterification reaction related to TrwC physiology (Grandoso et al., 2000). Various experiments indicated that Tyr-18 was responsible of the conjugation initiation reaction (that is, first cleavage on the plasmid nic site), while Tyr-26 was proposed to act in the termination reaction, by analogy to what happens in replication of bacteriophage phiX-174 (Hanai and Wang, 1993). Thus, we speculated that intrabody scFv-P1.F2 was impeding recircularization of the incoming ssDNA by sequestering the Y26-containing loop, presumably accessible to intrabody contact, and thus interfering with the termination reaction of conjugation. It is known that mutations Y26F or Y26A leave TrwC with a residual activity reducing about 20-fold the conjugation frequency of R388 (Grandoso et al., 2000). Therefore, in case scFv-P1.F2 was interfering specifically with the biological activity of Tyr-26, this intrabody should not have any additional effect in conjugation driven by TrwC mutants Y26F or Y26A. To investigate this possibility we tested the inhibition that scFv-P1.F2 exerts over the conjugation frequency of R388 with alleles trwC-Y26F or trwC-Y26A. In ELISA, scFv-P1.F2 bound these mutant proteins with identical affinity to TrwC-N293 (data not shown). By contrast, intrabody scFv-P1.F2 did not inhibit further the function of any of the TrwC-Y26 mutant proteins in vivo (Table 3, column 4). Taken together these data suggest that Tyr-26 is essential for the inhibitory function of this intrabody in vivo. Thus, by interfering with Tyr-26-catalysed termination reaction, intrabody scFv-P1.F2 reduced R388 conjugation frequency about 20-fold, the same effect produced by the Y26F mutation.
The above results strongly indicate that Tyr-26 function occurs in the recipient cell. If so, a possibility exists that conjugation could be rescued by supplying Tyr-26 in trans from within the recipient cell. In order to test for this possibility, complementation assays were carried out as shown in Table 4. Mating experiments used donors with mutant variant TrwC(Y26F) and recipients expressing wild-type TrwC or TrwC(Y18F) to provide the Tyr-26 missing in the incoming TrwC. A significant 10-fold recovery in the conjugation frequency occurred when TrwC(Y18F) was expressed in the recipient, but no increase was detected when wild-type TrwC protein was used for complementation (Table 4).
Table 4. TrwC complementation from the recipient cell.a
Donor strain was DH5α[F–endA1 recA1 gyrA96 thi-1 hsdR17 supE44 relA1Δ(argF-lacZYA)U169 Φ80dlacZΔM15] (Lloyd, 1991) harbouring pSU1445 (a trwC-deficient R388 derivative) plus a second plasmid containing either trwC or one of its mutant variants (listed in column 1). Each single mutant relaxase used in donor was complemented in the recipient strain UB1637 (F–λ–lys his trp rpsL recA56) (de la Cruz and Grinsted, 1982) by expressing either the intact relaxase or the alternative single mutant (listed in column 2), in such a way that the tyrosyl residue that is absent in the donor is provided by the recipient. Donors and recipients were grown on LB with appropriate antibiotics to stationary phase and mated for 1 h in LB agar plates at 37°C. Transfer frequencies are listed in column 3.
Transfer frequencies, calculated as the ratio of KmRSmR transconjugants per KmRNxR donor, are expressed as relative to the value obtained with wild-type TrwC and are the average of six independent experiments.
Complementation from the recipient with the same protein present in the donor [TrwC, TrwC(Y18F), TrwC(Y26F) respectively] produced the same frequencies obtained in the absence of protein in the recipient.
The expression levels of all proteins were checked as indicated in Supplementary material and resulted to be similar (data not shown).
What epitopes are recognized by the other selected intrabodies? Intrabody scFv-P4.E7 is recognizing a surface region (shown in Fig. 4) which is far from both Y26 and the catalytic centre. However, conjugation of the Tyr-26 mutants to recipients containing this intrabody (Table 3, column 3) occurs at the same frequencies as when using the native protein. Thus, we assume that scFv-P4.E7 is also affecting Tyr-26 access to the active site. This seems also to be the case for intrabodies B2 and D2 (Table 3, columns 7 and 8). Conversely, intrabodies H1, A2 and E2 (Table 3, columns 5, 6 and 9) do still show an additional reduction in the conjugation frequency when the Tyr-26 mutant relaxases are used, suggesting that these intrabodies are recognizing epitopes involved in other conjugation-related functions that occur in the recipient cell.
Conjugative T4SSs and related systems transport a series of proteins to recipient cells. Among them are conjugative relaxases. Their transport could be the consequence of relaxases merely functioning as pilot proteins in order to thread the DNA in the transport channel. However, they could also play one or more additional active roles within the recipient, perhaps contributing to the termination phase of conjugative DNA processing reactions, as originally envisioned by Llosa et al. (2002). Based on previous results demonstrating relaxase TrwC transport to recipient cells (Draper et al., 2005), we decided to analyse possible roles of TrwC within recipient cells. The method used consisted in expressing anti-TrwC intrabodies in the recipients and studying their inhibitory effects on conjugation. The finding that scFvs can be oxidized and folded properly in the cytoplasm of E. coli trxB gor mutants (strain FÅ113) as N-terminal fusions to Trx1 (Jurado et al., 2006), opened the possibility of screening directly a library of scFvs generated from immunized mice independently of their dependence on disulphide bonds for stability. After one or four rounds of in vitro selection for scFv binding to TrwC-N293 using phage-display technology, either individual clones or pools of scFvs were tested in vivo for their ability to inhibit R388 conjugation by their functional expression in the cytoplasm of E. coli trxB gor recipient cells as N-terminal fusions to Trx1. This strategy retrieved several intrabodies interfering R388 conjugation and two of them were shown to interact in vitro specifically with TrwC-N293.
Intrabody scFv-P4.E7, obtained after extensive in vitro panning, exhibited a higher affinity for TrwC-N293 in vitro (Fig. 2), and was capable of significant inhibition of R388 conjugation in vivo (∼20-fold) (Table 2). A direct in vivo search for intrabodies in sublibrary 1, using a high-throughput screening system based on the lux operon, retrieved even more potent inhibitors of R388 conjugation than scFv-P4.E7 (Table 2). Intrabodies suppressing R388 conjugation 40- to 10 000-fold were isolated from the in vivo screening, although only one of them (scFv-P1.F2) demonstrated sufficient affinity for TrwC-N293 in vitro to reveal a physical protein–protein interaction in ELISA and Western blot. Unfortunately, we were not able to detect a direct in vitro interaction to TrwC-N293 of those intrabodies having the highest inhibitory activity in vivo (e.g. scFv-P1.D2 or scFv-P1.A2) most likely due to low in vitro affinity. Nevertheless, their mere finding indicates that conjugation can be practically abolished by expressing specific intrabodies within recipient cells. In this respect, intrabody A2 was particularly interesting, because it inhibited conjugation of R388, pKM101 and F. It thus seems possible to find broad range intrabodies, capable of inhibiting conjugation of whole sets of plasmids like the MOBF plasmids cited above.
In vivo screening of the more diverse sublibrary 1 proved to be a better method to obtain functionally efficient intrabodies. From the basis of these experiments, it also became clear that in vitro affinity was not directly correlated with efficiency of the intrabody in vivo. The use of several rounds of panning implies that those antibodies showing the highest expression and affinity will be selected, without necessarily recognizing epitopes related to the functional properties of the antigen. In fact, intrabody scFv-P4.E7 recognizes a region of TrwC (Fig. 3) that is not known to be directly involved in catalysis, which could be the cause of its lower inhibitory effect in vivo despite its higher affinity in vitro. On the other hand, scFv P1.F2 recognizes the sequence GADDYYA that contains TrwC residue Tyr-26, which is involved in a transesterification reaction important in the conjugation cycle (Grandoso et al., 2000).
The fact that R388 conjugation could be inhibited by anti-TrwC intrabodies resident in recipient cells confirms previous genetic data demonstrating TrwC relaxase transport to recipients during conjugation (Draper et al., 2005). Results from this work also indicate that relaxase transport is important (if not essential) for conjugation to reach its final outcome. Thus, TrwC is carrying out an important function while in the recipient. Intrabody scFv-P1.F2 shed light on the nature of this function. TrwC contains two catalytic tyrosines, Tyr-18 involved in the initial cleavage on the plasmid nic site, and Tyr-26 that is proposed to act in the termination reaction. TrwC atomic structure revealed that Tyr-26 is located in an external mobile loop of the protein (Guasch et al., 2003; Boer et al., 2006). Thus, it is tempting to speculate that the scFv-P1.F2 intrabody is impeding recircularization of the incoming ssDNA by sequestering the Tyr-26-containing loop, presumably accessible to intrabody contact, and thus interfering with the termination reaction of conjugation. The remaining analysed intrabodies may also affect the termination reaction in a variety of ways through their binding to various surface locations on TrwC-N293 structure. These could include interfering with Tyr-26 positioning in the active site, with nic site DNA positioning, or with the conformational changes needed for these reactions to proceed. As intrabody scFv-P4.E7 did not interfere with transfer facilitated by TrwC-Y26F, we must assume that it also interferes with the movement of the Tyr-26 loop, while it does not affect access of Tyr-18 from a second relaxase molecule. The epitopes recognized by the remaining intrabodies could not be mapped, so they do not warrant further speculation at this level of detail.
Interestingly, the 20-fold reduction in conjugation frequency achieved by scFv-P1.F2 intrabody in vivo was similar to that produced when conjugation was catalysed by the TrwC(Y26F) mutant protein. The fact that TrwC-Y26 mutants still transfer at 5% of wild-type frequency was taken to imply that alternatives exist to the termination reaction catalysed by Tyr-26 (Grandoso et al., 2000). As relaxases of other MOB families contain just one active Tyr (Pansegrau and Lanka, 1996), we suggested (Grandoso et al., 2000) that Tyr-18 could also act at termination (as a reverse of the initiation reaction). If this were the case, conjugation of a TrwC(Y26F) mutant plasmid should not be further inhibited by scFv-P1.F2. As can be seen in Table 3, R388 conjugation from a TrwC(Y26F)-containing donor was not inhibited by scFv-P1.F2 intrabody even when the intrabody recognizes the mutant and native proteins with similar affinities. Thus, we interpret these results as indicating that the effect of scFv-P1.F2 is to abolish the reaction catalysed by Tyr-26, that this reaction occurs in the recipient cell, and that it is important, but not absolutely essential for R388 conjugation to occur.
An additional, most enticing result was that wild-type conjugation frequency from TrwC(Y26F) donors could be partially restored when TrwC(Y18F) (but not wild-type TrwC) was expressed in recipient cells. This result has an interesting and intriguing interpretation. Our present hypothesis for conjugative DNA processing during conjugation requires the existence of different conformations in which TrwC can attack phosphodiester bonds and different DNA orientations (Boer et al., 2006): in the initiation reaction, TrwC is in a conformation in which both Tyr-18 and Tyr-26 are free but only Tyr-18 can attack a supercoiled DNA containing the cleavage site in oriT. On the other hand, in the termination reaction, Tyr-18 is covalently linked to DNA while Tyr-26 is free [mutant TrwC(Y18F) would resemble such a situation]. It can be speculated that Tyr-26 can only attack a nic site positioned in the active site when TrwC is in the termination conformation. If we assume that mutant Y18F is locked in the termination conformation, Tyr-26 would be active, which would explain the increase in the conjugation frequency when TrwC(Y18F), but not wild-type TrwC, was present in the recipient cell. It seems logical that the transesterification reaction is more efficient when Tyr-26 lies in the same TrwC monomer that contains Tyr-18 covalently linked to ssDNA, so explaining the only partial recovery of conjugation frequency by ‘in trans’ Tyr-26. It should be remembered that TrwC protein purifies as a dimer (Grandoso et al., 1994), thus providing support for a physical interaction between relaxase monomers. The above interpretation was suggested to us by the curious observation that the atomic structures of relaxases TrwC_R388 (Boer et al., 2006) and TraI_F (Larkin et al., 2005) in complex with ssDNA containing their respective nic sites could be solved only when mutants Y18F and Y16F, respectively, were used. Boer et al. (2006) inferred, from the structure of their TrwC-DNA complex, the existence of two DNA exit pathways. One of them would be occupied in the initiation reaction while the second would be utilized in termination for the binding of the new nic site, because the first path is still occupied by the DNA covalently linked to Tyr-18. It implies that when the new nic site enters the recipient cell it would be cleaved by Tyr-26 only if Tyr-18 is not available, as our results from Table 4 support.
Inhibition of R388 conjugation by six of the seven selected intrabodies was highly specific because conjugation of other plasmids of the MOBF family (Fernández-López et al., 2006), such as pKM101 and F, was not inhibited. Specificity was particularly striking in the case of intrabody scFv-P1.F2, because it distinguished the TrwC sequence GADDYYA from the related sequences AKDDYYS and KDNYYV of relaxases TraI_pKM101 and TraI_F respectively. Besides, when R388 and CloDF13 reside both in the same donor, the presence of any of the seven intrabodies in the recipient cell reduced R388 conjugation but not CloDF13 mobilization, which uses its own relaxase. Thus, physical obstruction at the T4SS channel entry site as a cause for R388 conjugation inhibition can be ruled out because, this being the case, blocking of CloDF13 mobilization would also have taken place.
In summary, this work clarifies the mechanism used by MOBF plasmids (such as F and R388) to terminate conjugative DNA processing. It demonstrates that the relaxase second catalytic tyrosine (Y26 in TrwC) plays an important role in the termination reaction. Thus, the relaxase is more than just a pilot protein for DNA transport. The question arises of how does termination occur in plasmids containing a single catalytic Tyr, such as the MOBP plasmid RP4 or the MOBQ plasmid RSF1010. We suggest that, in these cases, a second protein monomer has to enter the recipient cell to be able to affect the termination reaction. Precedence for this reaction can be found in the termination of rolling circle replication carried out by protein RepA of plasmid pT181 (Novick, 1998).
Plasmids used are listed in Table 1. Standard molecular biology methods (Sambrook et al., 1989) were used to purify, analyse, manipulate and amplify DNA.
Production of a phage displays scFv library
Two Balb/c female mice were intraperitoneally injected with 40 μg native TrwC-N293 (TrwC relaxase domain, containing the first 293 amino acids of the protein, purified as in Guasch et al., 2003) with Ribi adjuvant (Monophosphoryl Lipid A + Synthetic Trehalose Dicorynomycolate; Sigma). The process was repeated at day 25. Mice were sacrificed at day 38 to obtain lymphocytes from their spleens. The following steps to construct the scFv library were carried out as described in Fraile et al. (2001). scFv genes were assembled in a VH-linker-VL arrangement and cloned between the SfiI and NotI sites of phagemid pCANTAB-5EHis (Fernández et al., 2000). The recombinant library was electroporated into E. coli XL-1 Blue strain [recA1 gyrA96 relA1 endA1 hsdR17 supE44 thi1 lac (F′proAB lacIqlacZΔM15 Tn10); Stratagene]. Around 105 transformant colonies were harvested, pooled and stored at −80°C. This library was infected with VCS-M13 (KmR) helper phage in order to produce (rescue) M13 phage-scFv particles (Phabs). Phabs were recovered from culture supernatants, checked for scFv-gp3 production by Western blot, titrated on XL-1 Blue cells, and panned on ELISA immunoplates coated with TrwC-N293 (10 μg ml−1) as described in Fraile et al. (2001). Bound Phabs were eluted using 0.1 M glycine (pH 2.5) and used to infect E. coli XL-1 Blue cells (sublibrary 1). Subsequent rounds of panning and elution of Phabs bound to TrwC-N293 were carried out in identical conditions, resulting in sublibraries 2–4.
Rapid screening for Phabs binding to TrwC-N293 coming from sublibraries 3 and 4 was carried out by small-scale phagemid rescue on single colonies of E. coli XL-1 Blue (Fraile et al., 2001). Supernatants (containing Phabs) were tested by ELISA, using anti-M13 monoclonal antibody (MAb)-peroxidase (POD) conjugate (Amersham Bioscience GE) as secondary antibody to determine their specific binding to TrwC-N293.
scFv cloning for expression in E. coli cytoplasm
Phagemid DNAs from E. coli XL-1 Blue sublibrary 1 (around 104 cfu) and from individually selected Phabs from sublibraries 3 and 4 were extracted using a Plasmid DNA Extraction Kit (Qiagen). DNA fragments encoding scFv were digested either with restriction enzymes NcoI+NotI or with BsaAI+NotI. In our system BsaAI site was placed in order to generate a cohesive end compatible with NcoI, thus allowing the cloning of full-length scFv clones having an internal NcoI in addition to that introduced by oligonucleotide primer during amplification. Digested fragments were cloned into the vector backbone of plasmid pTB7 (Jurado et al., 2006) digested NcoI+NotI. From this intracellular expression vector, scFvs are expressed under the control of a Ptrc promoter as N-terminal fusions to Trx1 and containing 6xHis and E-tags at their C-termini. Ligation products were electroporated into E. coli strain FÅ113 [DHB4 trxB::Km gor552 . . . Tn10 (TcR) aphC*] (Bessette et al., 1999; Ritz et al., 2001) and plated on Luria–Bertani (LB) agar plates supplemented with 2% glucose and ampicillin (100 μg ml−1) (LBGA).
Automated conjugation assay
A whole cell automated assay for conjugation, based on visible light emission, was carried out using a BIOMEK 2000 liquid handling robot (Beckman) as described in Fernández-López et al. (2005) with some modifications. Basically, donor cells contain a derivative of plasmid R388 carrying a lux operon under the control of an engineered lac promoter. Light emission in the donor is repressed by LacI. Upon conjugation, lux can be expressed, and light thus produced, in the recipient cells. E. coli FÅ113 cells expressing scFvs were used as recipients. The donor strain was an E. coli CSH53 [araΔ(lac–pro) strA thi (phi80ΔlacI)] derivative carrying conjugative plasmid pSU2007::Tnlux (a R388 derivative containing the lux operon of Photorhabdus luminiscens under the control of the lac promoter) and a non-mobilizable high copy-number plasmid (pUC18::lacIq) overproducing the lac repressor. A single colony of the donor strain, previously checked for non-luminescence, was grown at 30°C in LBGA + kanamycin (50 μg ml−1) overnight. Individual colonies of FÅ113 scFv sublibrary 1 were pre-inoculated in 96 deep well plates (Axigen) in 750 μl LBGA and grown overnight at 30°C without agitation. Pre-inoculated plates were centrifuged, pellets washed with LB and resuspended in 750 μl LB, from where 40 μl were used to inoculate 96 well plates with 750 ml LB-Ap without glucose. Inocula plates were incubated for 3 h with agitation at 30°C, induced with 0.1 mM IPTG, and allowed to grow for a further 16 h. Plates were centrifuged and donor cells were added to each well in a 1:1 donor : recipient ratio. Conjugative matings were carried out in 96 well black microtiter plates (Thermo Electron Corporation) containing 300 μl Muller–Hinton solid media over which the conjugation mix (10 μl) was laid. Mating plates were incubated at 30°C and light emission detected in a microplate luminometer (Fluoroskan Ascent; Thermolab Systems). Clones were selected by low light emission (< 2% of controls in three independent experiments).
Conventional conjugation assays
Escherichia coli strains used as donor and recipient in each mating experiment are specified in the corresponding table. Strains were grown at 30°C on LB with appropriate antibiotics depending on the harboured plasmids. In case of recipient strain FÅ113 carrying scFv clones, pre-inocula were statically cultured overnight in LBGA and inocula in LB-Ap with shaking until OD600 = 0.5, when IPTG was added to 0.1 mM final concentration. Donors and recipients (in proportion 1:1) were mated on the surface of LB agar plates for 1 h at 30°C (unless otherwise indicated). Suitable dilutions of the conjugative mix were plated onto selective medium. Transfer frequency is the number of transconjugants divided by the number of donor cells. As a log-normal distribution is expected for transfer frequencies, these were calculated by log conversion of frequencies to obtain the mean and standard deviation values, which were expressed as the anti-log of the calculated figures.
Enzyme-linked immunosorbent assay
Purified relaxase domains of R388 protein TrwC (TrwC-N293) and pKM101 protein (TraI-N293) were adsorbed overnight at 4°C to 96 well immunoplates (Maxisorb; Nunc) at 10 μg ml−1 in PBS 1×. Bovine serum albumin (Sigma) was used as a negative control. The following steps were carried out at room temperature, using the conditions described in Jurado et al. (2002; 2006). Phabs or protein extracts from FÅ113 cells expressing scFvs were added to coated wells. Secondary antibodies anti-M13 MAb-POD conjugate or anti-E-tag-MAb-POD (Amersham Bioscience GE) were used to detect the corresponding primary antibodies (Phabs or E-tagged scFvs). The colour developing reaction for the peroxidase was carried out as in Jurado et al. (2002) and the OD490 of plates determined (Benchmark microplate reader, Bio-Rad).
The affinity of scFv antibodies for TrwC-N293 was estimated by indirect ELISA (Friguet et al., 1985). Increasing amounts of the antigen were incubated with a fixed amount of scFv for 2 h at room temperature. The proportion of antibody that remains unsaturated at equilibrium was measured using the incubated mixtures TrwC-N293-scFv as primary antibody in an ELISA. In parallel, direct ELISA using several dilutions of the scFv extract was carried out as explained above. Klotz plots of the binding of scFv-P1.F2 and scFv-P4.E7 to TrwC-N293 were drawn from the formula A0/(A0 − A) = 1 + KD/a0, being A0 the absorbance measured for the antibody in the absence of antigen and A, the absorbance measured for the antibody at different antigen concentrations (a0). Dissociation constants (KD) were deduced from the slopes calculated from linear regressions.
Whole cell protein extracts and clarified extracts from E. coli were separated by standard SDS-PAGE using Miniprotean system (Bio-Rad) and transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore) using a semidry electrophoresis transfer apparatus (Bio-Rad), or to a nitrocellulose membrane (Trans-Blot Transfer Medium; Bio-Rad) using wet electrophoresis transfer device (Bio-Rad). After protein transfer, membranes were blocked with 30 ml of MBT-buffer and incubated 1 h with an appropriate dilution of the scFv extract. For immunodetection of E-tagged scFvs, membranes were incubated with a 1:5000 dilution of mAb anti-E-tag-POD conjugate for 1 h and developed by SuperSignal West Pico Chemiluminescent Substrate (Pierce), employing an image-capturing system (Chemidoc; Bio-Rad).
Overlapping dodeca- or hexadecapeptides from the TrwC-N293 sequence were prepared by automated spot synthesis (Abimed, Langerfeld, Germany) onto an amino-derivatized cellulose membrane, immobilized by their C-termini (Frank and Overwing, 1996). Membranes were blocked, incubated with scFv extracts and developed as described for Western blots above.
We are grateful to María Lucas for the gift of purified proteins TrwC-N293_R388 and TrwC-N293(Y26F)_R388, and to Fernando Roncal for peptide membrane construction. This work was supported by Grants BFU2005-03477/BMC (Spanish Ministry of Education) and LSHM-CT-2005-019023 (European VI Framework Program) to F.C and Grants BIO2005-03964 (Spanish Ministry of Education) and LSHB-CT-2005-512061 (European VI Framework Program) to L.A.F. M.P.G.-B. is a postdoctoral scientist supported by a fellowship from Fundación Marqués de Valdecilla, and later by Grant LSHM-CT-2005-019023. B.G.-P. and P.J. are recipients of PhD fellowships from Spanish Ministry of Education. G.M. is a Ramón y Cajal Research Associate.