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F plasmid TraF and TraH are required for F pilus assembly and F plasmid transfer. Using flotation sucrose density gradients, we found that TraF and TraH (as well as TraU and TraW) localized to the outer membrane in the presence of the complete F transfer region, especially TraV, the putative anchor. Mutational analysis of TraH revealed two domains that are important for its function and possible interaction with TrbI, which in turn has a role in stabilizing TraH.
The F plasmid (99,159 bp) of Escherichia coli is a model system for the study of the horizontal gene transfer among prokaryotes via conjugation (3, 10, 27). F encodes a 33.3-kb transfer region that is responsible for the formation of mating junctions between donor and recipient cells prior to DNA transfer and establishment in the recipient. The hallmark of F conjugation is the formation of extracellular filaments, F pili, that initiate contact between mating cells and retract, bringing the donor and recipient cells together (5, 19). Synthesis of the F pilus is not well understood, despite the morphological simplicity of this organelle (7, 15, 28). The F transfer region consists of nearly 40 tra genes, with 18 being involved in construction of the transferosome, which is involved in pilus synthesis, mating pair stabilization, and DNA transfer (9). Eight of the encoded Tra proteins (TraA, -B, -C, -E, -G [the N-terminal domain], -K, -L, and -V) correspond to widely conserved members of type IV secretion systems (T4SS), whereas another 9 (TraF, -G [C-terminal domain], -H, -N, -U, and -W and TrbB, -C, and -I) are involved in the F-specific T4SS (4, 18). Two other proteins (TraQ and -X) are specific to the F plasmid itself. The roles of the F-specific proteins that are involved in pilus assembly and DNA transfer are intriguing, since other conjugative T4SS appear to function efficiently without them (18). These tra proteins do not affect F pilin levels, and hence, they have been assigned functions in pilus assembly/retraction and mating pair stabilization, which are characteristics of F-like transfer systems (18). TraF, -H, and -W and TrbC are required for F pilus assembly (9), and mutations in traU reduce the number and the mean length of pili but do not abolish pilus outgrowth (24). TraU is required for DNA transfer and has been tentatively grouped with TraN and -G as proteins involved in mating pair stabilization (18). TrbI is thought to play a role in pilus retraction, since trbI mutants have unusually long pili (21). TrbB contains the thioredoxin-like domain with a C-X-X-C motif and appears to be a periplasmic disulfide bond isomerase (6). Previously, we hypothesized that TrbB and TraF, the latter of which also has the thioredoxin-like domain but lacks the C-X-X-C motif, might have chaperone-like activity. These proteins might help F T4SS proteins such as TraH, -U, and -N, which have 6, 10, and 22 conserved cysteines, respectively, achieve the correct conformation for assembly into the transferosome complex (6). Interestingly, yeast two-hybrid (Y2H) analysis demonstrated that TraF, -H, -U, and -W and TrbB and -I form an interaction group, with TraH directly linked to TraF, TraU, and TrbI (14). TraH is the only one of the three cysteine-rich proteins required for pilus assembly; it is the largest protein (458 amino acids [aa]; 50.2-kDa precursor, processed to 47.8 kDa) in the interaction group and contains a C-terminal coiled-coil domain that can contribute to its oligomerization and interaction with other T4SS proteins (18). Y2H analysis also showed that the C-terminal region of TraH is critical for its interaction with TraF (28 kDa, processed to 25.9 kDa) and TraU (36.8 kDa, processed to 34.3 kDa) and that a deletion within the N-terminal region of TraH enhanced its interaction with TrbI (14.1 kDa) (14).
Mutations in traH affect pilus outgrowth but not pilus tip formation at the cell surface, since traH mutants are sensitive to the M13K07 transducing phage, which binds to the pilus tip (1). Membrane fractionation studies of cells containing subclones of the F transfer region originally suggested that TraH fractionates with the inner membrane (IM) (22). TraH contains three N-terminal hydrophobic domains of approximately 20 aa each, which supports this model. In contrast, Ham et al. predicted TraH to be a soluble periplasmic protein (12). Sucrose density gradient sedimentation studies suggested that FLAG-tagged TraH, in the presence of F lac traH80, is in the outer membrane (OM) (23). Since TraH is extracted from membrane preparations with guanidine-HCl or urea but not Triton X-100, Manwaring concluded that TraH is a peripherally associated outer membrane protein (23). By use of subclones of the F transfer region, TraF, -U, -W, and TrbB were localized to the periplasm, whereas TrbI was thought to be an inner membrane protein (21, 24, 29, 30). Using the F plasmid derivative pOX38-Tc (2), which carries the entire F transfer region, we reassessed the localization of TraH as well as TraF, TraU, and TraW (23.6 kDa, processed to 21.7 kDa).
E. coli strains were grown at 37°C in Luria-Bertani (LB) broth (1% tryptone [Difco], 0.5% yeast extract [Difco], 1% NaCl [BDH]) with shaking to mid-exponential phase (optical density at 600 nm [OD600] of ca. 0.5) with appropriate antibiotics at the following concentrations: 50 μg/ml ampicillin (Ap), 20 μg/ml chloramphenicol (Cm), 25 μg/ml kanamycin (Km), 200 μg/ml streptomycin (Sm), 100 μg/ml spectinomycin (Sp), and 10 μg/ml tetracycline (Tc). Sucrose density flotation studies of cell membrane fractions and immunoblot analysis were performed as previously described (17). Cell pellets corresponding to 0.1 OD600 equivalents were used in all immunoblot assays. Samples were boiled in sodium dodecyl sulfate (SDS) sample buffer for 5 min and were analyzed by resolving SDS-15% polyacrylamide gel by using the Bio-Rad Minigel system. The positions of the inner and outer membrane fractions were determined using polyclonal antibodies to the C-terminal region of OmpA, the major outer membrane porin, and CpxA, the inner membrane sensor of the CpxAR two-component system (25). Anti-CpxA, anti-TraE, anti-TraF, anti-TraH, anti-TraU, anti-TraW, and anti-TrbB polyclonal antisera (raised in rabbits) were diluted 1:7,000, 1:5,000, 1:2,000, 1:1,000; 1:500, 1:20,000 and 1:10,000, respectively, in blocking solution and were incubated with the blots at room temperature for 1 h. Anti-OmpA antibodies were used at a 10−5 dilution in 5% bovine serum albumin (BSA; Roche) to avoid heavy background. Unfortunately, TrbI protein could not be overproduced and specific antibodies could not be raised.
Log-phase cultures of E. coli MC4100 (Smr) (17) containing pOX38-Tc (2) were separated into periplasmic, cytoplasmic, and membrane fractions according to a previously described method (26). The fractions were tested for the presence of TraH, TraF, TraU, and TraW by SDS-PAGE, followed by immunoblot analysis. All four proteins were found associated with the membrane fraction and not the periplasmic fraction (Fig. (Fig.1A).1A). TrbB was found in the periplasmic fraction, in agreement with its proposed role in disulfide bond isomerization (6; data not shown).
Sucrose density flotation gradients of the membrane preparations of MC4100 (Smr) cells harboring pOX38-Tc (2), pOX38-Tc ΔtraF::kan (6) and pOX38-Tc ΔtraH::cat were performed to distinguish between OM and IM proteins according to reference 17. pOX38-Tc ΔtraH::cat was constructed according to the method described by Elton et al. (6) by inserting a chloramphenicol acetyltransferase cassette into traH. Gradients were fractionated, and a subset of the fractions (fractions 26 to 54, renamed 1 to 29) that contained the proteins of interest were subjected to SDS-PAGE and immunoblot analyses (Fig. (Fig.2).2). OmpA and CpxA were controls for the outer and inner membrane fractions and helped define the subset of fractions examined (Fig. (Fig.2,2, panels 1 and 2, respectively). The TraE pilus assembly protein of the F plasmid was used as an IM marker for the F transfer system (Fig. (Fig.2,2, panel 9) (9). TraH fractionated as an OM protein in MC4100/pOX38-Tc (Fig. (Fig.2,2, panel 3), as did TraF, TraU, and TraW (Fig. (Fig.2,2, panels 5, 7, and 8, respectively). TraH did not appear to be required for TraF localization, which was unaffected in a traH mutant (Fig. (Fig.2,2, panel 6). In addition, TraF did not appear to be required for TraH localization, although its absence caused a reduction in the levels of TraH (Fig. (Fig.2,2, panel 4; see below).
TraF, -H, -U, and -W appear to be periplasmic proteins that associate with the outer membrane when in the context of the complete transfer apparatus. TrbC, which is fused to TraW in the F-like R27 T4SS, might also be part of this complex (18). Therefore, an as yet unidentified transfer protein should act as an anchor in the outer membrane, directing these proteins to this location. Of the 18 transferosome proteins, only TraV and TraN are known to be located in the OM, with TraV being the only OM protein involved in pilus assembly. Preliminary localization studies using TraF as a test case and a traV insertion mutant, pOX38 ΔtraV::cat (this study, constructed as described above for pOX38-Tc ΔtraH::cat), demonstrated that the levels of TraF decreased dramatically. However, the remaining TraF was found in the periplasm (Fig. (Fig.1B).1B). Complementation of the traV mutation with pRS29, but not pRS31 (1), restored TraF localization to the outer membrane. Thus, TraV is probably the anchor protein for both the F-specific transferosome proteins (TraF, -H, -U, and -W) as well as the TraV, -K, and -B complex (13).
MC4100 (Smr) cells bearing pOX38-Tc (2) or insertion mutant pOX38-Tc ΔtraH::cat, pOX38-Tc ΔtraF::kan (6), pOX38-Tc ΔtrbB::cat (6), pOX38-Tc ΔtraW::cat (this study), pOX38 traU347 (Kmr) (24), or pOX38-trbI472 (Kmr) (21) were used in subsequent experiments. pOX38-Tc ΔtraW::cat was constructed according to the method described by Elton et al. (6) by inserting a chloramphenicol acetyltransferase gene within traW. Mating efficiencies of these mutants were determined according to previously described methods using E. coli ED24 (Spr) as the recipient (20). Transconjugants were selected based on double resistance toward chloramphenicol or kanamycin and spectinomycin (Fig. (Fig.2).2). Observed mating efficiencies were in agreement with the data obtained previously, as were the results of complementation assays using subclones carrying the appropriate transfer gene (1, 6, 21, 24, 29, 30). These subclones were pK184TraH (Kmr) (this study), pFTraF and pFTrbB (Apr) (6), and pKI175 (Apr; traWU) (30) (Fig. (Fig.2).2). pK184TraH is based on the vector pK184 (Kmr) and contains the traH gene plus its ribosome binding site cloned into the EcoRI and HindIII sites in pK184 (16). Immunoblot analyses revealed that traF, traU, or trbB, but not traW, insertion mutants had slightly reduced levels of TraH in MC4100 cells whereas the trbI insertion mutant had undetectable levels of TraH (Fig. (Fig.3).3). Since TraH interacts directly with TrbI, TraF, and TraU in Y2H assays (14), the absence of these proteins would be expected to destabilize TraH. TraH is thought to interact indirectly with TraW via TraU (14); its levels were unaffected in a traW mutant. TraH was destabilized in a dsbA mutant and was undetectable by immunoblotting (data not shown) and decreased slightly in a trbB mutant, suggesting that disulfide bond formation (DsbA) and isomerization (TrbB) are important for TraH.
The absence of TrbI appeared to have the most profound effect on the level of TraH, although there was only a 20-fold decrease in mating efficiency, suggesting that enough TraH was present to support mating (Fig. (Fig.3).3). Complementation assays performed with pOX38-trbI472 and pBAD24TrbI plasmids (this study) restored the levels of TraH, possibly by stabilizing it (Fig. (Fig.3).3). pBAD24TrbI is based on the vector pBAD24 (Apr) and contains the trbI gene cloned into the EcoRI site in pBAD24 (11). However, complementation with pBAD24TrbI did not restore mating efficiency to wild-type levels, confirming that the insertion mutation within pOX38-trbI472 has a weak polar effect on downstream genes in the tra operon (21). Alternatively, overexpression of TrbI from pBAD24TrbI affected mating efficiency.
Y2H analysis revealed two regions within TraH that appeared to be important for TraH-TrbI interactions (14). The deletion of 50 N-terminal amino acids (aa 25 to 75) from the mature TraH gave a 40-fold increase in TraH-TrbI interaction in the Y2H assay (14). This region of TraH also contains the highly conserved residues N31, T44, G60, and R65 (numeration includes the 25-aa signal peptide) (Fig. (Fig.4A).4A). Site-directed mutagenesis was performed on plasmid pK184TraH by using the QuikChange kit (Stratagene). The mating abilities of MC4100/pOX38-Tc ΔtraH::cat/pK184TraH and derivatives with amino acid substitutions N31A, T44A, G60A, and R65A were determined according to previously described methods using ED24 (Spr) as the recipient (20). Transconjugants were selected based on double resistance toward tetracycline and spectinomycin. TraH levels within the donor cells were monitored by immunoblot analysis. The N31A and T44A substitutions did not affect mating efficiency and did not change the level of TraH within donor cells (Fig. (Fig.5).5). The G60A and R65A substitutions decreased mating efficiency to undetectable levels. TraH levels remained unchanged in both mutants (Fig. (Fig.5).5). MC4100/pOX38-Tc ΔtraH::cat cells with pK184TraHG60A or pK184TraHR65A were also resistant toward pilus-specific phage f1, suggesting that the pilus was not assembled.
Sequence analysis also showed the presence of conserved residues N(L/I/Y)X(W/Y)XX(F/L) (N220IMWNAL226 in F TraH) within the putative TrbI interaction domain (aa 193 to 226) (Fig. (Fig.4B)4B) (14). Substitution of N220 with alanine (N220A) did not change the levels of TraH protein in pOX38-Tc ΔtraH::cat/pK184TraHN220A but decreased the mating ability to undetectable levels. The W223A mutation in TraH decreased the level of TraH within donor cells and reduced the mating efficiency 1,000-fold compared to the wild-type level (Fig. (Fig.5).5). The N220A and W223A mutants were resistant to f1 phage and could not assemble functional pili. Thus, mutations in N220 and W223 could affect TraH-TrbI interaction, or they may act independently to block TraH function. If TrbI is in the IM as previously reported (21), then the TrbI:TraH pair could be part of a second envelope-spanning structure analogous to the TraV:TraK:TraB scaffold (8, 13).
Primary sequence analysis also revealed the presence of a putative Walker A motif within aa 193 to 226 of TraH (G193CTVGGKS200) (9). Comparison of seven TraH orthologs revealed that this motif is not conserved among TraH-like sequences (Fig. (Fig.4B).4B). To confirm whether this sequence might be important in the F plasmid, a triple mutant (G193A/K199A/S200A) was constructed. It reduced mating efficiency 20-fold but did not change the levels of TraH within donor cells (data not shown). Single substitutions (G193A, K199A, or S200A) did not change the mating efficiency or the level of TraH (data not shown). Thus, TraH, a peripheral OM protein, is probably not an NTPase, nor does it bind nucleotides.
Our data also revealed that several conserved amino acid residues are critical for TraH function and structure and that TraH stability is dependent on TrbI as well as DsbA and TrbB, which affect disulfide bond formation and isomerization, respectively. Thus, TrbI, in which mutations have only a minor effect on mating ability, plays a more important role than previously thought (21).
We thank Glen Armstrong, University of Calgary, for anti-OmpA antibodies and Tracy Raivio, University of Alberta, for anti-CpxA antibodies.
This work was supported by CIHR grant MT 62776 and NSERC grant 139684.
Published ahead of print on 15 January 2010.