JOURNAL OF BACTERIOLOGY, Mar. 2010, p. 1730–1734 Vol. 192, No. 60021-9193/10/$12.00 doi:10.1128/JB.00726-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

NOTES

F Plasmid TraF and TraH Are Components of an OuterMembrane Complex Involved in Conjugation�

Denis Arutyunov, Barbara Arenson, Jan Manchak, and Laura S. Frost*Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9

Received 4 June 2009/Accepted 4 January 2010

F plasmid TraF and TraH are required for F pilus assembly and F plasmid transfer. Using flotation sucrosedensity gradients, we found that TraF and TraH (as well as TraU and TraW) localized to the outer membranein the presence of the complete F transfer region, especially TraV, the putative anchor. Mutational analysis ofTraH revealed two domains that are important for its function and possible interaction with TrbI, which inturn has a role in stabilizing TraH.

The F plasmid (99,159 bp) of Escherichia coli is a modelsystem for the study of the horizontal gene transfer amongprokaryotes via conjugation (3, 10, 27). F encodes a 33.3-kbtransfer region that is responsible for the formation of matingjunctions between donor and recipient cells prior to DNAtransfer and establishment in the recipient. The hallmark of Fconjugation is the formation of extracellular filaments, F pili,that initiate contact between mating cells and retract, bringingthe donor and recipient cells together (5, 19). Synthesis of theF pilus is not well understood, despite the morphological sim-plicity of this organelle (7, 15, 28). The F transfer regionconsists of nearly 40 tra genes, with 18 being involved in con-struction of the transferosome, which is involved in pilus syn-thesis, mating pair stabilization, and DNA transfer (9). Eightof the encoded Tra proteins (TraA, -B, -C, -E, -G [the N-terminal domain], -K, -L, and -V) correspond to widely con-served members of type IV secretion systems (T4SS), whereasanother 9 (TraF, -G [C-terminal domain], -H, -N, -U, and -Wand TrbB, -C, and -I) are involved in the F-specific T4SS (4,18). Two other proteins (TraQ and -X) are specific to the Fplasmid itself. The roles of the F-specific proteins that areinvolved in pilus assembly and DNA transfer are intriguing,since other conjugative T4SS appear to function efficientlywithout them (18). These tra proteins do not affect F pilinlevels, and hence, they have been assigned functions in pilusassembly/retraction and mating pair stabilization, which arecharacteristics of F-like transfer systems (18). TraF, -H, and-W and TrbC are required for F pilus assembly (9), and mu-tations in traU reduce the number and the mean length of pilibut do not abolish pilus outgrowth (24). TraU is required forDNA transfer and has been tentatively grouped with TraN and-G as proteins involved in mating pair stabilization (18). TrbIis 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 aperiplasmic disulfide bond isomerase (6). Previously, we hy-pothesized that TrbB and TraF, the latter of which also has thethioredoxin-like domain but lacks the C-X-X-C motif, mighthave chaperone-like activity. These proteins might help F T4SSproteins such as TraH, -U, and -N, which have 6, 10, and 22conserved cysteines, respectively, achieve the correct confor-mation for assembly into the transferosome complex (6). In-terestingly, yeast two-hybrid (Y2H) analysis demonstrated thatTraF, -H, -U, and -W and TrbB and -I form an interactiongroup, with TraH directly linked to TraF, TraU, and TrbI (14).TraH is the only one of the three cysteine-rich proteins re-quired for pilus assembly; it is the largest protein (458 aminoacids [aa]; 50.2-kDa precursor, processed to 47.8 kDa) in theinteraction group and contains a C-terminal coiled-coil domainthat can contribute to its oligomerization and interaction withother T4SS proteins (18). Y2H analysis also showed that theC-terminal region of TraH is critical for its interaction withTraF (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 tipformation at the cell surface, since traH mutants are sensitiveto the M13K07 transducing phage, which binds to the pilus tip(1). Membrane fractionation studies of cells containing sub-clones of the F transfer region originally suggested that TraHfractionates with the inner membrane (IM) (22). TraH con-tains three N-terminal hydrophobic domains of approximately20 aa each, which supports this model. In contrast, Ham et al.predicted TraH to be a soluble periplasmic protein (12). Su-crose density gradient sedimentation studies suggested thatFLAG-tagged TraH, in the presence of F lac traH80, is in theouter membrane (OM) (23). Since TraH is extracted frommembrane preparations with guanidine-HCl or urea but notTriton X-100, Manwaring concluded that TraH is a peripher-ally associated outer membrane protein (23). By use of sub-clones of the F transfer region, TraF, -U, -W, and TrbB were

* Corresponding author. Mailing address: CW405 Biological Sci-ences Bldg., Department of Biological Sciences, University of Alberta,Edmonton, Alberta, Canada T6G 2E9. Phone: (780) 492-3248. Fax:(780) 492-9234. E-mail: laura.frost@ualberta.ca.

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localized to the periplasm, whereas TrbI was thought to be aninner membrane protein (21, 24, 29, 30). Using the F plasmidderivative pOX38-Tc (2), which carries the entire F transferregion, 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 (opticaldensity at 600 nm [OD600] of ca. 0.5) with appropriate antibi-otics at the following concentrations: 50 �g/ml ampicillin (Ap),20 �g/ml chloramphenicol (Cm), 25 �g/ml kanamycin (Km),200 �g/ml streptomycin (Sm), 100 �g/ml spectinomycin (Sp),and 10 �g/ml tetracycline (Tc). Sucrose density flotation stud-ies of cell membrane fractions and immunoblot analysis wereperformed as previously described (17). Cell pellets corre-sponding to 0.1 OD600 equivalents were used in all immuno-blot assays. Samples were boiled in sodium dodecyl sulfate(SDS) sample buffer for 5 min and were analyzed by resolvingSDS–15% polyacrylamide gel by using the Bio-Rad Minigelsystem. The positions of the inner and outer membrane frac-tions 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, an-ti-TraH, anti-TraU, anti-TraW, and anti-TrbB polyclonal an-tisera (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 blockingsolution and were incubated with the blots at room tempera-ture for 1 h. Anti-OmpA antibodies were used at a 10�5 dilu-

tion in 5% bovine serum albumin (BSA; Roche) to avoid heavybackground. Unfortunately, TrbI protein could not be over-produced and specific antibodies could not be raised.

Log-phase cultures of E. coli MC4100 (Smr) (17) containingpOX38-Tc (2) were separated into periplasmic, cytoplasmic,and membrane fractions according to a previously describedmethod (26). The fractions were tested for the presence ofTraH, TraF, TraU, and TraW by SDS-PAGE, followed byimmunoblot analysis. All four proteins were found associatedwith the membrane fraction and not the periplasmic fraction(Fig. 1A). TrbB was found in the periplasmic fraction, in agree-ment with its proposed role in disulfide bond isomerization (6;data not shown).

Sucrose density flotation gradients of the membrane prepa-rations of MC4100 (Smr) cells harboring pOX38-Tc (2),pOX38-Tc �traF::kan (6) and pOX38-Tc �traH::cat were per-formed to distinguish between OM and IM proteins accordingto reference 17. pOX38-Tc �traH::cat was constructed accord-ing to the method described by Elton et al. (6) by insertinga chloramphenicol acetyltransferase cassette into traH. Gradi-ents were fractionated, and a subset of the fractions (fractions26 to 54, renamed 1 to 29) that contained the proteins ofinterest were subjected to SDS-PAGE and immunoblot anal-yses (Fig. 2). OmpA and CpxA were controls for the outer andinner membrane fractions and helped define the subset offractions examined (Fig. 2, panels 1 and 2, respectively). TheTraE pilus assembly protein of the F plasmid was used as anIM marker for the F transfer system (Fig. 2, panel 9) (9). TraHfractionated as an OM protein in MC4100/pOX38-Tc (Fig. 2,panel 3), as did TraF, TraU, and TraW (Fig. 2, panels 5, 7, and8, respectively). TraH did not appear to be required for TraFlocalization, which was unaffected in a traH mutant (Fig. 2,

FIG. 1. (A) The F-specific proteins TraH, -F, -U, and -W weredetected in the membrane fraction when expressed from MC4100/pOX38-Tc. Proteins were detected by immunoblotting using antiseraspecific for each protein as described in the text. (B) TraF localizationwas tested in pOX38-Km and pOX38 �traV::cat. The cells were frac-tionated into cytoplasmic (C), periplasmic (P), and total membrane(M) fractions, and TraF was detected by immunoblotting with anti-TraF antibodies. TraV was complemented by pRS29 (pRS31 acted asa negative control). The following abbreviations are used: WT, wildtype; �V, pOX38 �traV::cat; �H, pOX38-Tc �traH::cat; and �F,pOX38 �traF::kan. The positions of the proteins are indicated byarrows on the right of each panel. The asterisk indicates a band thatreacts nonspecifically with anti-F antiserum.

FIG. 2. The cellular localizations of TraE, TraF, TraH, TraU, andTraW in subcellular fractions of E. coli MC4100/pOX38-Tc and itsderivatives. Flotation sucrose density gradients were performed withsubsequent immunodetection of tra proteins in a subset of gradientfractions (fractions 26 to 54, renumbered 1 to 29). The positions of theIM and OM fractions are shown above the gels, and the identities ofthe samples are indicated on the left. The panel numbers are indicatedon the right.

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panel 6). In addition, TraF did not appear to be required forTraH localization, although its absence caused a reduction inthe levels of TraH (Fig. 2, panel 4; see below).

TraF, -H, -U, and -W appear to be periplasmic proteins thatassociate with the outer membrane when in the context of thecomplete transfer apparatus. TrbC, which is fused to TraW inthe F-like R27 T4SS, might also be part of this complex (18).Therefore, an as yet unidentified transfer protein should act asan anchor in the outer membrane, directing these proteins tothis location. Of the 18 transferosome proteins, only TraV andTraN are known to be located in the OM, with TraV being theonly OM protein involved in pilus assembly. Preliminary local-ization studies using TraF as a test case and a traV insertionmutant, pOX38 �traV::cat (this study, constructed as describedabove for pOX38-Tc �traH::cat), demonstrated that the levelsof TraF decreased dramatically. However, the remaining TraFwas found in the periplasm (Fig. 1B). Complementation of thetraV mutation with pRS29, but not pRS31 (1), restored TraFlocalization to the outer membrane. Thus, TraV is probablythe anchor protein for both the F-specific transferosome pro-teins (TraF, -H, -U, and -W) as well as the TraV, -K, and -Bcomplex (13).

MC4100 (Smr) cells bearing pOX38-Tc (2) or insertionmutant pOX38-Tc �traH::cat, pOX38-Tc �traF::kan (6),pOX38-Tc �trbB::cat (6), pOX38-Tc �traW::cat (this study),pOX38 traU347 (Kmr) (24), or pOX38-trbI472 (Kmr) (21)were used in subsequent experiments. pOX38-Tc �traW::catwas constructed according to the method described by Elton etal. (6) by inserting a chloramphenicol acetyltransferase genewithin traW. Mating efficiencies of these mutants were deter-mined according to previously described methods using E. coliED24 (Spr) as the recipient (20). Transconjugants were se-lected based on double resistance toward chloramphenicol orkanamycin and spectinomycin (Fig. 2). Observed mating effi-ciencies were in agreement with the data obtained previously,as were the results of complementation assays using subclonescarrying the appropriate transfer gene (1, 6, 21, 24, 29, 30).These subclones were pK184TraH (Kmr) (this study), pFTraFand pFTrbB (Apr) (6), and pKI175 (Apr; traWU) (30) (Fig. 2).pK184TraH is based on the vector pK184 (Kmr) and containsthe traH gene plus its ribosome binding site cloned into theEcoRI and HindIII sites in pK184 (16). Immunoblot analysesrevealed that traF, traU, or trbB, but not traW, insertion mu-

tants had slightly reduced levels of TraH in MC4100 cellswhereas the trbI insertion mutant had undetectable levels ofTraH (Fig. 3). Since TraH interacts directly with TrbI, TraF,and TraU in Y2H assays (14), the absence of these proteinswould be expected to destabilize TraH. TraH is thought tointeract indirectly with TraW via TraU (14); its levels wereunaffected in a traW mutant. TraH was destabilized in a dsbAmutant and was undetectable by immunoblotting (data notshown) and decreased slightly in a trbB mutant, suggesting thatdisulfide bond formation (DsbA) and isomerization (TrbB) areimportant for TraH.

The absence of TrbI appeared to have the most profoundeffect on the level of TraH, although there was only a 20-folddecrease in mating efficiency, suggesting that enough TraH waspresent to support mating (Fig. 3). Complementation assaysperformed with pOX38-trbI472 and pBAD24TrbI plasmids(this study) restored the levels of TraH, possibly by stabilizingit (Fig. 3). pBAD24TrbI is based on the vector pBAD24 (Apr)and contains the trbI gene cloned into the EcoRI site inpBAD24 (11). However, complementation with pBAD24TrbIdid not restore mating efficiency to wild-type levels, confirmingthat the insertion mutation within pOX38-trbI472 has a weakpolar effect on downstream genes in the tra operon (21). Al-ternatively, overexpression of TrbI from pBAD24TrbI affectedmating efficiency.

Y2H analysis revealed two regions within TraH that ap-peared to be important for TraH-TrbI interactions (14). Thedeletion of 50 N-terminal amino acids (aa 25 to 75) from themature TraH gave a 40-fold increase in TraH-TrbI interactionin the Y2H assay (14). This region of TraH also contains thehighly conserved residues N31, T44, G60, and R65 (numera-tion includes the 25-aa signal peptide) (Fig. 4A). Site-directedmutagenesis was performed on plasmid pK184TraH by usingthe QuikChange kit (Stratagene). The mating abilities ofMC4100/pOX38-Tc �traH::cat/pK184TraH and derivativeswith amino acid substitutions N31A, T44A, G60A, and R65Awere determined according to previously described methodsusing ED24 (Spr) as the recipient (20). Transconjugants wereselected based on double resistance toward tetracycline andspectinomycin. TraH levels within the donor cells were moni-tored by immunoblot analysis. The N31A and T44A substitu-tions did not affect mating efficiency and did not change thelevel of TraH within donor cells (Fig. 5). The G60A and R65Asubstitutions decreased mating efficiency to undetectable lev-els. TraH levels remained unchanged in both mutants (Fig. 5).MC4100/pOX38-Tc �traH::cat cells with pK184TraHG60A orpK184TraHR65A were also resistant toward pilus-specificphage f1, suggesting that the pilus was not assembled.

Sequence analysis also showed the presence of conservedresidues N(L/I/Y)X(W/Y)XX(F/L) (N220IMWNAL226 in FTraH) within the putative TrbI interaction domain (aa 193 to226) (Fig. 4B) (14). Substitution of N220 with alanine (N220A)did not change the levels of TraH protein in pOX38-Tc�traH::cat/pK184TraHN220A but decreased the mating abilityto undetectable levels. The W223A mutation in TraH de-creased the level of TraH within donor cells and reduced themating efficiency 1,000-fold compared to the wild-type level(Fig. 5). The N220A and W223A mutants were resistant to f1phage and could not assemble functional pili. Thus, mutationsin N220 and W223 could affect TraH-TrbI interaction, or they

FIG. 3. Immunoblot analysis of the levels of TraH in the absence ofother members of Y2H interaction group by using pOX38-Tc and itsderivatives containing insertion mutations in traH, traF, trbB, traW,traU, or trbI. A loading control is shown in the lower panel, and themating efficiency (ME) expressed as a percentage of transconjugantsrelative to donor cells is given below the gels. n.d., not detected; n.a.,not applicable. The last line of data are the complementation data(percent complementation mating efficiency [CME]) obtained by useof clones as described in the text. Previously, TraH was found tointeract with TraF, TraU, and TrbI, and TraU interacts with TraW(14).

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may act independently to block TraH function. If TrbI is in theIM as previously reported (21), then the TrbI:TraH pair couldbe part of a second envelope-spanning structure analogous tothe TraV:TraK:TraB scaffold (8, 13).

Primary sequence analysis also revealed the presence ofa putative Walker A motif within aa 193 to 226 of TraH(G193CTVGGKS200) (9). Comparison of seven TraH orthologsrevealed that this motif is not conserved among TraH-likesequences (Fig. 4B). To confirm whether this sequence mightbe important in the F plasmid, a triple mutant (G193A/K199A/S200A) was constructed. It reduced mating efficiency 20-foldbut did not change the levels of TraH within donor cells (datanot shown). Single substitutions (G193A, K199A, or S200A)

did not change the mating efficiency or the level of TraH (datanot shown). Thus, TraH, a peripheral OM protein, is probablynot an NTPase, nor does it bind nucleotides.

Our data also revealed that several conserved amino acidresidues are critical for TraH function and structure and thatTraH stability is dependent on TrbI as well as DsbA and TrbB,which affect disulfide bond formation and isomerization, re-spectively. Thus, TrbI, in which mutations have only a minoreffect on mating ability, plays a more important role thanpreviously thought (21).

We thank Glen Armstrong, University of Calgary, for anti-OmpAantibodies and Tracy Raivio, University of Alberta, for anti-CpxAantibodies.

This work was supported by CIHR grant MT 62776 and NSERCgrant 139684.

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