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Journal of Bacteriology, November 2008, p. 7164-7169, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00843-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The Carboxyl-Terminal Domain of TraR, a Streptomyces HutC Family Repressor, Functions in Oligomerization

Masakazu Kataoka,^1* Takeshi Tanaka,² Toshiyuki Kohno,² and Yusuke Kajiyama¹

Department of Environmental Science and Technology, Faculty of Engineering, Shinshu University, Wakasato 4-17-1, Nagano-shi, Nagano 380-8553, Japan,¹ Mitsubishi Kagaku Institute of Life Sciences (MITILS), Minami-ooya 11, Machida-shi, Tokyo 194-8511, Japan²

Received 19 June 2008/ Accepted 12 August 2008

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Efficient conjugative transfer of the Streptomyces plasmid pSN22 is accomplished by regulated expression of the tra operon genes, traA, traB, and spdB. The TraR protein is the central transcriptional repressor regulating the expression of the tra operon and itself and is classified as a member of the HutC subfamily in the helix-turn-helix (HTH) GntR protein family. Sequence information predicts that the N-terminal domain (NTD) of TraR, containing an HTH motif, functions in binding of DNA to the cis element; however, the function of the C-terminal region remains obscure, like that for many other GntR family proteins. Here we demonstrate the domain structure of the TraR protein and explain the role of the C-terminal domain (CTD). The TraR protein can be divided into two structural domains, the NTD of M1 to R95 and the CTD of Y96 to E246, revealed by limited proteolysis. Domain expression experiments revealed that both domains retained their function. An in vitro pull-down assay using recombinant TraR proteins revealed that TraR oligomerization depended on the CTD. A bacterial two-hybrid system interaction assay revealed that the minimum region necessary for this binding is R95 to P151. A mutant TraR protein in which Leu121 was replaced by His exhibited a loss of both oligomerization ability and repressor function. An in vitro cross-linking assay revealed preferential tetramer formation by TraR and the minimum CTD. These results indicate that the C-terminal R95-to-P151 region of TraR functions to form an oligomer, preferentially a tetramer, that is essential for the repressor function of TraR.

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Gene transfer depending on conjugative plasmids has an important role in horizontal gene transfer to vary genetic traits in bacteria. Many plasmids have been isolated from gram-positive filamentous bacteria of the genus Streptomyces, and most of them are self-transmissible and able to mobilize chromosomal markers (7, 10, 18, 19). Unlike conjugation in gram-negative bacteria, conjugation in Streptomyces usually depends on a small number of gene products, designated Tra proteins. The "central repressor," designated TraR or KorA, usually exerts strict regulation of the expression of Tra proteins from Streptomyces plasmids. Mutations in regulatory genes can result in overexpression of tra genes, leading to lethal phenotypes (8, 13, 17). The TraR/KorA family proteins have a representative helix-turn-helix (HTH) motif in their N-terminal region and are classified as GntR family proteins (5, 9). Interestingly, the TraR/KorA family proteins show little similarity in their primary sequence but have similar secondary structures, and they have been classified into the HutC subfamily based on their secondary structure in the C-terminal region (23). Although recent computer-assisted studies have demonstrated the roles of the N-terminal domains (NTD) and C-terminal domains (CTD) of bacterial repressors, there have been fewer experimental studies on the roles of the CTD of GntR family proteins than on the distinctive N-terminal HTH DNA-binding domains. In TraR/KorA family proteins, DNA-binding activities and their target sites in the NTD have been studied previously (15, 26, 27), but the CTD functions remain obscure, like those for many other GntR proteins.

One of the most studied repressors of the TraR/KorA family is TraR from Streptomyces nigrifaciens plasmid pSN22. TraR binds specifically to four DNA regions containing similar 12-bp sequences in the bidirectional tra-traR promoter region designated TRE (transfer regulating element) and turns off the transcription of itself and the tra operon, carrying the traA, traB, and spdB genes (15). We have reported several characteristics of TraR (12-16, 28), but its domain and functional structures remain as obscure as those of other TraR/KorA family proteins. In the present study, we reveal the domains and functional structures of TraR at the protein level. The CTD plays an important role in forming a tetramer, and the formation of the tetramer depends on a region consisting of 55 amino acid residues. These results might be applicable not only to other Streptomyces TraR/KorA family proteins but also to the HutC subfamily proteins that are similar to the TraR/KorA family proteins in structure and function.

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Bacterial strains, plasmids, culture, and cloning experiments. Streptomyces lividans TK21 (19) was used as the general Streptomyces host strain. Escherichia coli JM109 (29) was used as the host for plasmid construction in E. coli. Streptomyces plasmid pSN2218K or -2219K (Fig. 1) was used for in vivo promoter analysis. E. coli plasmids pUC18 (29) and pBluescript SK (Stratagene) were used for routine cloning. Plasmids pGEX-4T1 (GE Healthcare) and pTrcHis (Invitrogen) were used to express TraR-derived proteins. Bacterial handling and in vitro DNA handling were performed according to standard protocols (19, 25). PCR for the construction of modified genes was performed using Vent DNA polymerase (NEB). DNA sequences were determined using an ALF Express sequencer (GE Healthcare). Enzymes for DNA manipulation were purchased from Toyobo and were used according to the manufacturer's instructions. The in vivo binding assay was conducted using the BacterioMatch two-hybrid system (Stratagene) according to the manufacturer's instructions.

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FIG. 1. Map of promoter-probe shuttle plasmid pSN2218K. All functional units were generated by PCR. Details of the generation strategy are available upon request. pUC ori, replication region of pUC19; bla, ampicillin resistance gene; tsr, thiostrepton resistance gene; Rep22, replication region of pSN22; Kan, promoterless kanamycin resistance gene; Fd, Fd terminator; MCS, multiple cloning site from pUC plasmid; M13 (–47) and M13 (RV), complementary sequences for the M13 universal primers. Note that pSN2219K is the same as pSN2218K except for the presence of a reverse-directed multiple cloning site.

Vector construction. DNA fragments corresponding to the entire TraR, TraR NTD (M1 to R95), and TraR CTD (R95 to E246) sequences, without the termination codon, were amplified by PCR with appropriate primers containing the BamHI recognition sequence for the N terminus and the XhoI recognition sequence for the C terminus. After digestion with BamHI and XhoI, the fragments were cloned into pBS digested with BamHI and XhoI, and the resulting plasmids were verified by their DNA sequences and used as base plasmids. For expression of glutathione S-transferase (GST) fusion proteins, the DNA fragments of TraR and the CTD were cloned into pGEX-4T1 by use of BamHI and XhoI. For in vitro immunodetection, the myc tag was added to the C termini of the TraR derivatives by PCR.

Pull-down assay. The GST-fused TraR derivatives were immobilized on 0.5-ml glutathione columns (GE Healthcare) and washed, and then 2 ml of total bacterial lysate, containing myc-tagged TraR or CTD, was added. After incubation at 4°C for 2 h, GST-TraR and GST-CTD were eluted, and the GST-TraR-enriched fractions were used for Western blot analysis with anti-myc-tag monoclonal antibody 5D4.

Isolation of mutant TraR. Loss-of-function TraR mutants were isolated as described previously (16), with minor revision. The DNA fragment of the traR TRE region on pUC18 was amplified by error-prone PCR, using Taq DNA polymerase and M13 primers in a buffer containing 2 mM MgSO₄, 2 mM Tris-HCl (pH 8.8), 10 mM (NH₄)₂SO₄, 0.1% Triton X-100, 1 mM MnCl₂, and a 4 mM concentration of each deoxynucleoside triphosphates. The amplified fragments were cloned into pSN2218K and transformed into S. lividans by the polyethylene glycol-assisted method. Derepressed mutants were selected by being overlaid with kanamycin (Km) (200 µg/ml) and thiostrepton (Tsr) (50 µg/ml), followed by replica plating on minimal medium (MM) containing Km (20 µg/ml) and Tsr (10 µg/ml).

Secondary structure prediction. Secondary structures of TraR and its derivatives were predicted by calculation using the Protein Structure Prediction Server (PSIPRED) (11, 21) or the PredictProtein Server (3, 24).

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TraR has two structural and functional domains. Previously, we indicated that TraR was an HTH DNA-binding regulator protein of the GntR family on the basis of its amino acid sequence and DNA-binding activity (14, 15). However, knowledge about the TraR domain structure at the protein level has remained unclear. To clarify this structure, TraR was subjected to limited proteolysis. TraR was expressed as a fusion protein with GST linked to the PreScission protease (GE Healthcare) recognition sequence in E. coli and was purified using a glutathione-Sepharose 4B column. GST was then extracted by digestion with PreScission protease. The purified TraR protein was subjected to limited proteolysis with trypsin at molecular ratios of 1:100 to 1:12,800 and was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The TraR protein was cleaved into two structural domains, of 10 kDa and 20 kDa, at molecular ratios of 1:400 and 1:1,600 (Fig. 2A). The N-terminal amino acid sequence of the 20-kDa domain was YAPGETSS, which corresponds to the sequence from Y96 to S103 of TraR (Fig. 2B), while that of the 10-kDa peptide was GPMYKA, which corresponds to the sequence of the N terminus of TraR and the PreScission protease cleavage adduct. Thus, TraR is composed of two structural domains, M1 to R95 and Y96 to E246.

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FIG. 2. Domain structure of TraR. (A) TraR expressed in E. coli was purified and subjected to partial tryptic digestion. Two peptide fragments, of 20 kDa and 10 kDa, were obtained at trypsin-to-TraR molecular ratios of 1:400 and 1:1,600. (B) Schematic representation of TraR domain structure. Amino acid sequencing of the 10-kDa and 20-kDa peptides in panel A showed that the former was the NTD (M1 to R95) and the latter was the CTD (Y96 to E246). The secondary structure of TraR is indicated along with the amino acid sequences adjacent to the trypsin digestion site and the N-terminal sequence.

To confirm whether the NTD and CTD were functional, the inhibitory effect of each peptide on TraR function was tested. The proteins corresponding to the whole TraR peptide, the NTD (M1 to R95), and the CTD (R95 to E246) were expressed from the tra promoter (p-tra) under TraR control (Fig. 3A). In a previous study, we found that TraR regulates both divergent promoters p-tra and p-traR and that the promoter strength of p-tra was higher than that of p-traR (13, 15). If the expressed peptide had an inhibitory effect on TraR repression, the effect would be enhanced by the negative loop effect due to the preferential expression of the peptide. The p-tra activity was estimated as the survival rate of S. lividans on MMT (MM containing Tsr) containing 2.5, 5, 10, or 20 µg/ml Km originating from expression of the downstream promoterless kan gene. The CFU of S. lividans harboring a plasmid in which the additional whole TraR protein was expressed was markedly decreased in accordance with the increase in the Km concentration, as well as that for cells harboring the control plasmid without the artificial TraR protein (Fig. 3B). The survival rate of S. lividans harboring the TraR expression plasmid on MMT containing 10 µg/ml Km was under 0.01%. In contrast, the survival rate of S. lividans harboring the NTD or CTD expression plasmid decreased gradually, and over 1% of colonies grew even on MMT containing 20 µg/ml Km (Fig. 3B). Thus, overexpression of the NTD or CTD peptide, but not TraR, led to a loss of TraR repressor function, suggesting that both structural NTD and CTD are essential for repressor function.

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FIG. 3. Inhibition of TraR repression of p-tra by domain expression. (A) Schematic structures of domain expression plasmids. The myc-tagged wild-type TraR expression plasmid was controlled by the traR promoter (p-traR) and TraR. NTD (M1-R95) and CTD (R95-E246) construct expression was controlled by p-tra. Transcription from p-tra was monitored by expression of the promoterless Km resistance gene in pSN2219K. The three artificial genes were expressed by following the initiation codon for the traA gene. (B) Effect of domain expression on TraR repression of p-tra. Expression of the promoterless Km gene of each construct was monitored as CFU on MM containing Tsr (20 µg/ml) and Km (0, 2.5, 5, 10, and 20 µg/ml). The survival rate of S. lividans harboring each plasmid is shown as CFU on each medium with respect to CFU on medium lacking Km.

TraR forms a homo-oligomer through its CTD. Based on its primary structure, the function of the NTD was expected to be DNA binding. Indeed, nuclear magnetic resonance analysis revealed a secondary structure that is characteristic of the winged HTH motif, the general structure of DNA-binding domains (28). However, the function of the CTD could not be predicted from the analysis of its primary structure or the predicted secondary structure. The CTD of GntR family proteins are generally thought to function in oligomerization and/or effector binding, but no experimental evidence has been produced to indicate whether the CTD of TraR and other central repressors of Streptomyces conjugation factors function in oligomerization or have a binding function. To clarify the function of the CTD, oligomerization was studied in an in vitro protein binding assay. GST-TraR and GST-CTD were expressed and immobilized on glutathione beads. A bacterial lysate in which myc-tagged TraR (TraRmyc) or CTD (CTDmyc) was expressed was then mixed with the immobilized beads. After washing and elution steps, the eluate was used for immunoblotting with anti-myc antibody. As shown in Fig. 4, TraRmyc was detected in eluates of GST-TraR and GST-CTD but not in control eluates. Significant CTDmyc signals were also detected in eluates of GST-TraR and GST-CTD. Although a faint signal was detected in the control, the signal strength was under 1% of the significant signal. Thus, TraR is able to form oligomers in vitro through the CTD, consisting of the R95-to-E246 region.

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FIG. 4. Binding of TraR and CTD in vitro. TraR, CTD, and complexin II (CPX, a presynaptic protein; used as a control) were expressed in E. coli as GST fusion proteins. After immobilization of the fusion proteins on glutathione-Sepharose beads, the beads were incubated with E. coli lysate in which myc-tagged TraR (TraRmyc) or CTD (CTDmyc) was expressed. The beads were then washed, and the immobilized proteins were eluted with glutathione and subjected to SDS-PAGE followed by immunoblotting with 5D4 anti-myc-tag monoclonal antibody.

Oligomerization depends on the R95-to-P152 region of the TraR CTD. To confirm TraR oligomerization, further binding analysis was performed using a bacterial two-hybrid (BTH) system. TraR, CTD, and NTD were used as bait and target peptides. In this system, binding capability was estimated as β-galactosidase activity. As shown in Fig. 5, when CTD was used as the bait, significant β-galactosidase activity was detected in the case of the TraR target (29.6 ± 3.9; n = 10) and the CTD target (74.7 ± 15.2; n = 10) but not in the case of the NTD target (20.3 ± 3.9; n = 3). When TraR was used as bait, similar results were obtained, but activities were lower than those obtained when CTD was used as bait (26.1 ± 1.8 for TraR, 41.8 ± 12.5 for CTD, and 21.0 ± 2.9 for NTD). NTD interaction with the target molecules was not detected in this assay. Thus, oligomerization of TraR through the CTD was reconfirmed by the BTH assay.

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FIG. 5. TraR-TraR binding in BTH assay. TraR-TraR and TraR-CTD binding was observed as significant β-galactosidase activity. CTD-CTD binding affinity was significantly stronger than that of CTD-TraR or TraR-TraR. The positive control (PC) and negative control (NC) were those supplied with the assay kit. Data are means ± standard deviations (SD) (n = 3 to 10).

The minimum region necessary for TraR oligomerization was then determined in the BTH assay with CTD and various CTD derivatives (Fig. 6A). The β-galactosidase activity for each target molecule is shown in Fig. 6B. Among the deletion mutants, the R95-P208 and R95-P151 mutants showed significant β-galactosidase activity with CTD. The deletion within the R95-to-P151 region (R95-P125 mutant) and any other region outside it (P151-Q197, Q197-E246, and P151-E246 mutants) did not show significant β-galactosidase activity. Thus, the essential region for TraR oligomerization was limited to the R95-to-P151 region.

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FIG. 6. Minimum region required for TraR-TraR binding. In the two-hybrid assay, CTD was used as the bait fragment and various deleted fragments were used as target molecules. (A) Peptides used as target molecules. (B) Average β-galactosidase activity for each set. Data are mean ± SD (n = 5-10).

Oligomerization is essential for TraR repression. The pull-down and BTH assays revealed that formation of oligomers by TraR depends on the R95-to-P151 region, both in vivo and in vitro. To find out whether TraR oligomerization is essential for repressor function, we isolated a series of TraR mutants that had lost repressor function as a result of random mutagenesis. Among the mutants, we found one in which Leu121 was replaced by His (TraR^L121H) within the R95-to-P151 region. S. lividans harboring traR^L121H-TRE/pSN2218K showed resistance to 20 µg/ml Km on MM, whereas S. lividans harboring traR-TRE/pSN2218K was unable to grow on MM containing 2.5 µg/ml Km. The growth of S. lividans harboring traR-TRE/pSN2218K and traR^L121H-TRE/pSN2218K as well as traR (C118-E246)-TRE/pSN2218K as a loss-of-function control is illustrated in Fig. 7A. To test the effect of the L121H mutation on oligomerization ability, the CTD of TraR^L121H (CTD^L121H) was used in the BTH assay as the target molecule. As shown in Fig. 7B, the CTD^L121H target did not show significant binding activity with either TraR bait or CTD bait. This result indicates that the L121H mutation abolished both the oligomerization activity of TraR and the repressor function, suggesting that the oligomerization of TraR is essential for repressor function.

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FIG. 7. Relationship between function and oligomerization of TraR. (A) Growth of S. lividans harboring traR-TRE/pSN2218K, traR(C118-E246)-TRE/pSN2218K, and traRL121H-TRE/pSN2218K on MM containing Tsr (plasmid marker) or Tsr and Km (10 µg/ml; promoter marker). (B) Results of the two-hybrid assay. The significant loss of β-galactosidase activity of CTDL121H indicates a loss of binding activity to TraR or CTD. Data are means ± SD (n = 6 to 10).

TraR preferentially forms a tetramer. We performed an in vitro cross-linking experiment to elucidate the number of TraR monomers utilized in the formation of oligomers. TraRmyc and CTDmyc (R95-P151 and R95-E246 mutants) were expressed in E. coli from the pTrc vector as His-tagged proteins. The molecular masses of the purified proteins on SDS-PAGE were 33 kDa, 24 kDa, and 13 kDa for TraR, CTD (R95-P151 mutant), and CTD (R95-E246 mutant), respectively. After purification, the purified proteins were cross-linked using formaldehyde at various concentrations. The cross-linked samples were subjected to SDS-PAGE without prior boiling, followed by Coomassie brilliant blue staining. As shown in Fig. 8A, a shifted band appeared for the cross-linked TraR sample. The shifted band at about 130 kDa was estimated to be a tetramer, indicating that TraR mainly forms homotetramers in vitro. To detect oligomer formation more precisely, we performed immunodetection using the myc tag attached at the C terminus of each construct (Fig. 8B). Cross-linking of TraR revealed multiple bands, corresponding to trimers, tetramers, and higher multimers, but a tetramer was the main product. The CTD (R95-P151 mutant) produced the same multimer pattern as TraR in the cross-linked sample, although a preferential formation of tetramers was not observed. The cross-linking of CTD (R95-E246 mutant) showed preferential trimer formation, although dimers and tetramers were also observed, as in the case of the CTD R95-P151 mutant. In addition, bands corresponding to dimers were observed in both boiled and nonboiled samples. These results indicate that TraR mainly forms tetramers, and the formation process requires the R95-to-P151 region, although the domain deletions affected the oligomerization pattern.

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FIG. 8. In vitro oligomerization of TraR. TraR (33 kDa), CTD (R95-P151) (24 kDa), and CTD (R95-E246) (13 kDa) were expressed in E. coli from the pTrc vector and purified using a Ni-nitrilotriacetic acid column (Qiagen). A cross-linking reaction was performed in Bicine buffer (20 mM Bicine, pH 7.2, 300 mM NaCl, 1 mM dithiothreitol). (A) Cross-linking of TraR with various concentrations of formaldehyde (0%, 0.5%, 1%, 2%, and 4% [vol/vol]). TraR (1 µg in 10 µl of buffer) was cross-linked by the addition of formaldehyde and incubation for 10 min at room temperature. The reaction was stopped by the addition of SDS sample buffer. The cross-linked samples were subjected to SDS-PAGE without prior boiling and stained with Coomassie brilliant blue. (B) Results of immunodetection of cross-linked TraR and CTDs with anti-myc antibody. TraR and CTD (50 ng in 10 µl of buffer) were cross-linked by the addition of formaldehyde (2% [vol/vol]) and subjected to immunoblotting. CL, cross-linked sample. Arrowheads indicate bands with molecular masses corresponding to a tetramer.

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In the present study, we have demonstrated the domain structure of TraR and the oligomerization function of the CTD. Oligomerization activity is indispensable for TraR repressor function. The TraR/KorA repressors show low homology in their primary structures but share similar secondary structures (data not shown). In addition, a recent comparative study based on the predicted secondary structure suggested that the TraR/KorA family should be classified within the HutC subfamily of the GntR family (23). Thus, our results might be applicable not only to other TraR/KorA proteins in Streptomyces but also to other HutC subfamily proteins owing to their similarity in structure and function.

TraR was divided into two structural domains, the DNA-binding NTD, consisting of 95 amino acid residues, and the CTD, consisting of 150 residues, by tryptic limited proteolysis. This suggests that a domain consisting of 1-2-3-β1-β2-4, a winged HTH motif followed by a helix, forms the DNA-binding domain in HutC subfamily proteins (2, 28). Indeed, preliminary results indicated that the TraR peptide of M1 to E100 was bound to the TRE region, although the dissociation constant was higher than that of the whole TraR protein (T. Tanaka and T. Kohno, unpublished results). Thus, the CTD would be essential for the stabilization of the TRE-TraR complex through oligomerization. Results of the BTH assay indicated that the R95-to-P151 region of TraR is essential for oligomerization. The TraR^L121H mutation led to impairment of repressor function and oligomerization activity. Computer-assisted structure prediction indicated that no secondary structural change would be caused by the L121H mutation. Leu121 is highly conserved among HutC subfamily repressors, suggesting that the leucine residue is essential for repressor function, in particular through oligomerization.

What is the role of oligomerization in repressor function? Our in vitro cross-linking experiment indicated that TraR preferentially formed tetramers, but dimers and trimers were also observed. In GntR family proteins, it is generally thought that the repressors act as dimers, and in silico analysis also suggested that GntR family repressors bind to their target sites as dimers (22). Certainly, in a preliminary experiment, we observed that two molecules of TraR bound to one TRE box (Tanaka et al., unpublished result). Like Cro and LacI (4, 20), the TraR-TRE complex might be stabilized by dimer formation on a TRE. In addition, allosteric regulation of each TRE dimer TraR subunit by oligomerization might be essential for strict repression by TraR. This model is supported by the inhibition experiment shown in Fig. 3. The inhibitory effects of the NTD and CTD might be due to competition for TraR binding to the TRE and destabilization of the TraR-TRE complex by inhibition of TraR oligomerization, respectively.

This is the first experimental report on the C-terminal function of the TraR/KorA protein. For the HutC subfamily, the CTD structure was estimated by molecular modeling based on the UbiC structure (1) and by X-ray analysis of PhnF (6). Our results are inconsistent with the results for the PhnF CTD, which indicated that PhnF forms a dimer through the β8 structure. To confirm the possibility of TraR oligomerization through β8, we performed in vitro cross-linking of the TraR mutant in which β8 was deleted. The deletion of β8 did not affect tetramer formation (data not shown). It is possible that TraR and PhnF have different oligomerization modes, although the difference between the mechanisms remains obscure.

 
We thank M. Nakane and Y. Kohori for technical assistance.

This work was supported in part by the Hokuto Biological Science Foundation and by a grant from the Research Foundation for the Electrotechnology of Chubu to M.K.

 
* Corresponding author. Mailing address: Department of Environmental Science and Technology, Faculty of Engineering, Shinshu University, Wakasato 4-17-1, Nagano-shi, Nagano 380-8553, Japan. Phone: 81-26-269-5538. Fax: 81-26-269-5550. E-mail: mars{at}shinshu-u.ac.jp

Published ahead of print on 22 August 2008.

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Journal of Bacteriology, November 2008, p. 7164-7169, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00843-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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