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Journal of Bacteriology, October 2008, p. 6281-6289, Vol. 190, No. 19
0021-9193/08/$08.00+0     doi:10.1128/JB.00709-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of the traD Operon of Naphthalene-Catabolic Plasmid NAH7: a Host-Range Modifier in Conjugative Transfer ^,

Ryo Miyazaki, Yoshiyuki Ohtsubo, Yuji Nagata, and Masataka Tsuda^*

Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai 980-8577, Japan

Received 20 May 2008/ Accepted 21 July 2008

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Pseudomonas putida G7 carries a naphthalene-catabolic and self-transmissible plasmid, NAH7, which belongs to the IncP-9 incompatibility group. Adjacent to the putative origin of conjugative transfer (oriT) of NAH7 are three genes, traD, traE, and traF, whose functions and roles in conjugation were previously unclear. These three genes were transcribed monocistronically and thus were designated the traD operon. Mutation of the three genes in the traD operon resulted in 10- to 10⁵-fold decreases in the transfer frequencies of the plasmids from Pseudomonas to Pseudomonas and Escherichia coli and from E. coli to E. coli. On the other hand, the traD operon was essential for the transfer of NAH7 from E. coli to Pseudomonas strains. These results indicated that the traD operon is a host-range modifier in the conjugative transfer of NAH7. The TraD, TraE, and TraF proteins were localized in the cytoplasm, periplasm, and membrane, respectively, in strain G7 cells. Our use of a bacterial two-hybrid assay system showed that TraE interacted in vivo with other essential components for conjugative transfer, including TraB (coupling protein), TraC (relaxase), and MpfH (a channel subunit in the mating pair formation system).

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A number of bacterial plasmids are transmissible to other bacterial strains by conjugation, and plasmid-mediated horizontal gene transfer has contributed greatly to the plasticity of genomes and to the rapid acquisition of various genetic traits by the host cells. The mechanism of plasmid transfer in gram-negative bacteria has been well characterized, and the overall transfer is accomplished by two systems; the DNA transfer and replication (Dtr) system and the mating pair formation (Mpf) system. In addition, the membrane-bound coupling protein (CP) connects the two systems. First, the proteins of the Dtr system form a nucleoprotein complex called a relaxosome at the cognate origin of conjugative transfer (oriT) and prepare the plasmid DNA for transfer (23). The relaxase, a key enzyme of the relaxosome, catalyzes site- and strand-specific cleavage at oriT and covalently binds to the 5' end of the cleaved single-stranded DNA (ssDNA) (10). The relaxase-ssDNA complex next interacts with CP, which then recruits the complex to a cognate secretion pore formed by the Mpf proteins (31). Finally, the Mpf structure, which spans the cell envelope to form the channel, functions as a DNA transfer apparatus to translocate the relaxase-ssDNA substrate into recipient cells (2). The Mpf-CP system is a type IV secretion system (3).

The genes for the Dtr and Mpf systems and the CPs on self-transmissible plasmids are usually found in operons. Comparison of the transfer regions of various plasmids has revealed similar organizations and orders of transfer-related genes within the operons (28). Two broad-host-range plasmids, RP4 and R388, which belong to the IncP-1 and IncW groups, respectively, have been well characterized with respect to their transfer-related genes (6, 8, 10, 24, 31). The so-called Tra1 region in RP4 contains two operons that are transcribed divergently from a region containing oriT. One operon includes genes encoding relaxase and CP, and another operon, often termed the leader operon, includes three genes, traK, traL, and traM. Many other broad-host-range plasmids in the IncP-1, IncW, IncN, and IncQ groups, but not narrow-host-range plasmids such as F plasmid, also carry three-gene clusters corresponding to the RP4 traK-traL-traM cluster (Fig. 1). Previous studies of the IncP-1 and IncQ plasmids by other groups have suggested that the three-gene clusters are involved in conjugation (9, 17, 35) and that the role of the TraK protein is as a relaxosome component (36, 38). However, no further detailed characterization of the three-gene clusters has been reported.

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FIG. 1. Genetic organization of the transfer region of NAH7 and comparison with other plasmids. The pentagons indicate the sizes and transcriptional directions of genes, as follows: green, Dtr genes; yellow, Mpf genes; blue, CP genes; white, genes with unknown functions; and gray, other genes not apparently involved in transfer. Filled circles indicate the oriT regions. The traD, dtr, and mpf operons of NAH7 are labeled. The stb genes of R46 are required for stable plasmid inheritance in recombination-proficient strains (25), but the stb genes of R388 have not been analyzed experimentally. The value below each gene indicates the level of amino acid identity (expressed as a percentage) of the product to the product encoded by the gene in the NAH7 traD operon. Values less than 25% are not shown. The GenBank accession numbers of the nucleotide sequences are as follows: NAH7, AB237655; pWW0, NC_003350; R388, BR000038; R46, NC_003292; and RP4, L27758.

The 82-kb naphthalene-catabolic plasmid NAH7 is a self-transmissible plasmid from Pseudomonas putida G7 and belongs to the IncP-9 incompatibility group (7). Complete sequence determination of NAH7 has revealed the presence of essential genes for the Dtr and Mpf systems and for CP (30). NAH7 also has three genes, traD, traE (formerly orf16), and traF (formerly orf15), whose relative location and transcriptional direction are the same as those of the leader operon in RP4 (Fig. 1). In this study, we investigated the role of traD, traE, and traF in NAH7 transfer. We demonstrated that these three genes are expressed as one transcriptional unit and thus designated them the traD operon. The subcellular localization of their products and interactions with other transfer-related gene products were also investigated. Our comprehensive mating analyses further revealed that the traD operon functions as a host-range modifier in the conjugative transfer of NAH7.

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Bacterial strains, plasmids, and media. The bacterial strains and plasmids used in the present study are listed in Table 1. Escherichia coli cells were grown at 37°C in Luria-Bertani (LB) broth (18), and Pseudomonas cells were grown at 30°C in 1/3LB (0.33% tryptone, 0.16% yeast extract, 0.5% NaCl) broth. When the E. coli cells carried NAH7 derivatives, the growth temperature used was 30°C, since these plasmids cannot be maintained in E. coli at 37°C (34). The solid media were prepared by addition of 1.5% agar. Antibiotics were used at final concentrations of 50 µg/ml for ampicillin, 30 µg/ml for chloramphenicol, 25 µg/ml for kanamycin (Km), 20 µg/ml for gentamicin (Gm) and tetracycline, and 100 µg/ml for streptomycin.

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TABLE 1. Bacterial strains and plasmids used in this study

Methods used for DNA and RNA manipulation. Established methods were employed for preparation of plasmid DNAs, digestion with restriction endonucleases, ligation, agarose gel electrophoresis, and transformation of E. coli cells (18). Transformation of bacterial cells by electroporation was performed as described previously (14). PCR was performed with KOD-Plus DNA polymerase (Toyobo, Osaka, Japan) or ExTaq polymerase (Takara, Kyoto, Japan), and the primers used are listed in Table S1 in the supplemental material. Nucleotide sequencing was performed with an ABI Prism model 310 sequencer (Applied Biosystems, Foster City, CA). The nucleotide and protein sequences were analyzed by using Genetyx 13 software (SDC, Tokyo, Japan). Homology searches were performed using BLAST programs available at the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST/). The conserved domains and motifs were searched using the InterProScan (http://www.ebi.ac.uk/InterProScan/), COILS (http://www.ch.embnet.org/software/COILS_form.html), Jpred (http://www.compbio.dundee.ac.uk/www-jpred/index.html), and SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui/sosuiframe0.html) programs. Signal peptides were predicted using the SignalP server (http://www.cbs.dtu.dk/services/SignalP/).

Total RNA from P. putida G7 cells in mid-log phase was prepared by using ISOGEN (Nippongene, Toyama, Japan). Reverse transcription-PCR (RT-PCR) analysis using an RNA sample was performed with a TaKaRa RNA PCR kit (AMV) (version 3.0; Takara) and appropriate primer sets. To check whether there was contamination of total DNA, an RNA sample without DNase treatment was used for PCR amplification as a control.

Construction of KT2440-derived lacZ reporter strains. Three DNA fragments containing promoter regions of the dtr, mpf, and traD operons (designated Pdtr, Pmpf, and PtraD, respectively) were amplified by PCR using total DNA of strain G7 as the template and the following primers: EE_PtraA.F and PtraA_Sac.R for Pdtr, EE_Pmpf.F5 and Pmpf_Sac.R for Pmpf, and EE_PtraD.F and PtraD_Sac.R for PtraD. Each PCR product was ligated at its 5' terminus with a strong transcriptional terminator sequence, T0-T1, that was amplified by PCR using pUC18-mini-Tn7T-Gm as the template and primers Not_T0T1.F and T0T1_EcoT.R (4). Each ligated product was next fused at its 3' terminus with a pKLZ-W-derived promoterless lacZ gene to generate a terminator-promoter-lacZ cassette, and this cassette was subsequently inserted into a NotI site of mini-Tn7(Gm)P_A1/04/03ecfp-a. The resulting plasmids, pm7TPdtrLZT, pm7TPmpfLZT, and pm7TPtraDLZT, respectively (see Table S2 in the supplemental material), were introduced into KT2440 cells by electroporation with pTNS2, which encodes the specific TnsABC+D transposition pathway of Tn7 mutagenesis (4), to obtain Gm^r transformants. Each of the three transcriptional reporter strains obtained in this way (KTPdtr, KTPmpf, and KTPtraD) carried a single copy of the terminator-promoter-lacZ cassette at the chromosomal attTn7 site that is located downstream of the glmS gene.

Measurement of LacZ activity. The LacZ activity was measured using cell extract as described previously (21). The cell extract was mixed with an equal volume of an o-nitrophenyl-β-D-galactopyranoside solution in Z buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, 50 mM β-mercaptoethanol; pH 7.0). After incubation at 30°C, 0.5 volume of 1 M Na₂CO₃ was added to terminate the reaction. The amount of o-nitrophenol (ONP) produced was measured spectrophotometrically at an optical density at 415 nm. A molar extinction coefficient of 21,300 was used to calculate the ONP concentrations. One unit of LacZ activity was defined as the amount of LacZ generating 1 nmol of ONP per min, and the activity was normalized by using the amount of protein in the cell extract. The amount of protein was measured using a protein assay kit (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as a standard.

Mutagenesis of the traD operon of NAH7. Allelic exchange mutagenesis of NAH7 was carried out using pEX18Ap, which contains a sacB gene as a counterselective suicide marker (12). Approximately 800-bp regions located upstream and downstream of a target gene were amplified by PCR using the total DNA of strain G7 as the template, and the PCR-amplified Km^r gene from pUC4K was inserted between the two fragments. The ligated fragment was cloned into the multiple-cloning site of pEX18Ap. The resulting plasmid was introduced into G7 by electroporation, and Km^r transformants able to grow on 1/3LB agar containing 10% sucrose were selected. The expected double-crossover-mediated homologous recombination in the transformants was confirmed by PCR.

Construction of strains that contain chromosomal mini-Tn7 derivatives with the traD operon. The entire traD operon or part of it was amplified by PCR, and each amplified product was ligated with a 160-bp fragment containing a putative promoter and ribosome-binding site of traD. The fusion construct was inserted between the KpnI and StuI sites of pUC18-mini-Tn7T-Gm. The resulting plasmid (see Table S2 in the supplemental material) was used to transform Pseudomonas strains by electroporation with pTNS2, and Gm^r transformants that carried a single chromosomal copy of the fusion construct at the attTn7 site were selected. The Flp recombinase-mediated marker excision system (12) was used to remove the Gm^r gene in the chromosomal mini-Tn7 element. To insert the traD operon into the attTn7 site of the E. coli chromosome, the fusion construct and the pUCGM-derived Gm^r cassette were inserted into the KpnI and StuI sites of pUC18R6KT-mini-Tn7T, and the resulting plasmid was used to transform E. coli strains (see Table S2 in the supplemental material).

Measurement of cell growth and plasmid stability. The segregational stability of plasmids was measured as follows. Cells carrying the Km-resistant NAH7 derivatives were cultivated in Km-containing 1/3LB broth to the stationary phase. The culture was diluted 10⁴-fold, and a 50-µl aliquot was inoculated into 5 ml of fresh 1/3LB broth without Km. The culture was incubated until it reached the stationary phase (for approximately 20 generations) and was again diluted 10⁴-fold, and a 50-µl aliquot was inoculated into 5 ml of fresh 1/3LB broth for incubation to the stationary phase. This dilution-incubation procedure was carried out for an additional cycle (a total of approximately 60 generations). The turbidity (optical density at 660 nm) of the cell suspension during each cultivation was recorded by using a TVS062CA Bio-Photorecorder (Advantec MFS Inc., Tokyo, Japan), and the fraction of the Km^r cells in the stationary phase culture was determined by plating the appropriate amount of the cell suspension on 1/3LB agar plates with and without Km.

Mating assay. Donor and recipient cells separately grown overnight were harvested by centrifugation, washed with 1/3LB broth, and resuspended in fresh 1/3LB broth. They were then mixed and subsequently spotted on a sterile 0.45-µm-pore-size cellulose acetate filter (Advantec) placed on a 1/3LB agar plate. After incubation at 30°C for 24 h, the cells on the filter were suspended in 1/3LB broth, diluted, and plated on selective agar plates.

Protein localization analysis. To investigate the subcellular localization of the traD, traE, and traF products of NAH7, the 3' end of each gene in the mini-Tn7 transposon was fused with a FLAG tag-encoding DNA sequence. The resulting three mini-Tn7 derivatives were inserted into the chromosomal attTn7 site of the G7 derivatives (G7dD, G7dE, and G7dF, respectively) by using a mini-Tn7 transposon insertion mutagenesis system (see above). The cytoplasmic, periplasmic, and membrane fractions of the resulting strains were prepared as described previously (33). To evaluate successful fractionation, glucose-6-phosphate dehydrogenase, β-lactamase, and succinate dehydrogenase activities, respectively, were measured as fraction markers (13). After fractionation, cytoplasmic (6 µg), periplasmic (5 µg), and membrane (2 µg) proteins were separated by sodium dodecyl sulfate -polyacrylamide gel electrophoresis (15% acrylamide). The separated proteins were transferred to a polyvinylidene difluoride membrane (GE Healthcare UK Ltd., Buckinghamshire, England). The membrane was treated first with mouse anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO) and then with sheep anti-mouse antibody conjugated to horseradish peroxidase (GE Healthcare UK Ltd.), according to the manufacturer's instructions. Western blots were developed using the Immobilon Western chemiluminescent horseradish peroxidase substrate (Millipore, Billerica, MA) and a Lumi-Imager (FujiFilm, Tokyo, Japan).

Bacterial two-hybrid assay. A BacterioMatch II two-hybrid system (Stratagene, La Jolla, CA) was used to detect protein-protein interactions in vivo (19). The traD, traE, traF, traB, traC, and mpfH genes were amplified by PCR using the total DNA of G7 as the template, and each amplified product was cloned into pBT (bait) and pTRG (prey) vectors (see Table S2 in the supplemental material). The bacterial two-hybrid assay was performed according to the manufacturer's instructions. The strength of the interaction correlated with the ratio of the number of colonies obtained on the selective plates to the number of colonies on the nonselective plates.

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Transcriptional analysis of the traD operon. RT-PCR analysis using the RNA sample prepared from strain G7 in log phase revealed that the traD, traE, and traF genes of NAH7 were expressed as one transcriptional unit (Fig. 2), and this gene cluster was designated the traD operon. This analysis also suggested that the transcriptional start site(s) for the traD operon is within a 146-bp sequence that covers the region from nucleotide 162 to nucleotide 307 upstream of the putative start codon of traD. This 146-bp sequence contains the putative oriT (30). A transcriptional terminator motif was found at a position downstream of traF.

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FIG. 2. Transcriptional mapping of the NAH7 traD operon by RT-PCR. Each arrowhead indicates the position and direction of a primer used for RT-PCR. The number above each arrowhead indicates the nucleotide position of the primer when the translational start position of the traD gene was defined as position 1. The filled circle indicates the oriT region. The predicted transcriptional terminator (7-bp palindromic sequence) is indicated by the open circle with a vertical line. The cotranscribed regions are indicated by filled boxes, and the noncotranscribed regions are indicated by open boxes. +, RT-PCR product of the correct size detected by agarose gel electrophoresis; –, no detectable amplification product. RTase, reverse transcriptase.

To evaluate the expression level of the traD operon, we constructed a KT2440-derived reporter strain, KTPtraD, which carried at its chromosomal attTn7 site a promoterless lacZ gene fused with the promoter region of the traD operon (PtraD). The cells were cultivated overnight, and their LacZ activities were measured. The activity of PtraD was higher than the activities of promoter regions of the dtr and mpf operons (Pdtr and Pmpf, respectively) (Fig. 3). The presence of NAH7K2, an NAH7 derivative having a Km^r gene in a catabolic gene, nahAc (22), did not affect the PtraD or Pmpf activities but led to a decrease in the Pdtr activity. This result suggests that NAH7K2 carries a regulator gene(s) for Pdtr.

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FIG. 3. Transcriptional analysis of the dtr, traD, and mpf operons. The promoter regions of the operons, Pdtr, PtraD, and Pmpf, fused with the promoterless lacZ gene were introduced into the attTn7 site of the P. putida KT2440 chromosome. The LacZ activities of the strains were determined in the presence (+) and absence (–) of NAH7K2. The bars indicate the means of three independent experiments, and the error bars indicate standard deviations.

Host range of NAH7. NAH7K2, instead of NAH7, was used as the "wild-type" plasmid for the conjugal transfer experiments in this study since insertion of a Km^r gene into the nahAc gene did not affect the replication/maintenance or transfer frequency of NAH7 (data not shown). To investigate the host range of NAH7, donor strain G7K2(NAH7K2) was mated with Sphingomonas paucimobilis IAM12578G, Burkholderia multivorans ATCC 17616G, Acidovorax sp. strain KKS102G, P. putida KT2440G, Psedomonas fluorescens Pf-5G, and E. coli MV1190 (20). Km^r transconjugants of KT2440G, Pf-5G, and MV1190 were obtained at frequencies ranging from 10^–3 to 10^–6 per donor cell, supporting our previous finding that NAH7 can be conjugally transferred to and maintained in gammaproteobacterial strains (34). Our failure to obtain Km^r transconjugants of IAM12578G, ATCC 17616G, and KKS102G (<10^–10 per donor) suggests that NAH7 is not transferred to or maintained in alpha- or betaproteobacterial strains. Therefore, we used Pseudomonas and E. coli strains as the hosts for the remainder of the mating assays in this study.

Effect of the traD operon on plasmid transfer. To investigate whether the traD operon is involved in the NAH7 transfer, we constructed NAH7dD2, an NAH7 derivative completely lacking the traD operon. In the case of transfer from the G7 background to KT2440G, the transfer frequency of NAH7dD2 was approximately 3,000-fold lower than that of NAH7K2 (Fig. 4). To carry out the complementation analysis, the traD operon was inserted into the donor chromosome by using a mini-Tn7 transposition system, because our repeated attempts to introduce any traD operon-containing vector plasmids (e.g., pBBR1- and pVS1-based plasmids [11, 15]) into the donor cells were unsuccessful. When the traD operon was supplied from the chromosome of the donor strain, the transfer frequency of NAH7dD2 was the same as that of NAH7K, indicating that at least one product of the traD operon is required for efficient transfer of NAH7. It was possible that the decrease in the growth of donor cells or the segregational stability of NAH7dD2 led to the poorer conjugative transfer ability of this plasmid. However, this was not the case, since (i) the growth rates of the three donor strains in 1/3LB medium were indistinguishable from one another and (ii) the donor strains stably maintained their endogenous plasmids over at least 60 generations in the absence of any selective pressure (data not shown).

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FIG. 4. Conjugative transfer of NAH7 derivatives from the P. putida G7 background to P. putida KT2440G. Selection and counterselection were performed for resistance to Km and Gm, respectively. The transfer frequency was determined by dividing the number of transconjugants by the number of donors. The donor strains are indicated on the x axis, and their genetic characteristics are indicated in parentheses. The means and standard deviations of at least three independent assays are shown. WT, wild type.

Effect of traD, traE, and traF on plasmid transfer. To examine the roles of the three genes of the traD operon in NAH7 transfer, we constructed three NAH7 derivatives, NAH7dD, NAH7dE, and NAH7dF, which lacked traD, traE, and traF, respectively, and measured their transfer frequencies using Pseudomonas and E. coli strains. In the case of transfer from the G7 background to KT2440G (from Pseudomonas to Pseudomonas), NAH7dD, NAH7dE, and NAH7dF were transferred at frequencies that were approximately 24-, 6-, and 12,000-fold, respectively, lower than the frequency for NAH7K2 (Fig. 5A). Supplying the traD, traE, and traF genes from the donor chromosomes led to successful restoration of the transfer frequencies to the wild-type level. In the case of transfer from the G7 background to MV1190 (from Pseudomonas to E. coli), the transfer frequencies of the three NAH7 mutants were approximately 10-, 200-, and 2,700-fold lower, respectively, than that of NAH7K2 (Fig. 5B). The transfer frequency of the traD mutant plasmid was completely restored to that of NAH7K2 by supplying the traD gene in trans. Partial, but not complete, restoration of transfer frequencies was observed when the traE and traF genes were supplied in trans to the corresponding mutant plasmids. Use of another E. coli strain, XL1-Blue, as the recipient produced similar results, although the resulting transfer frequencies were higher than those obtained when MV1190 was used as the recipient (data not shown). This recipient-dependent difference in the transfer frequencies might have been due to a defect in the restriction system in XL1-Blue. The mutations in traD, traE, and traF of NAH7 led to reductions in the frequency of transfer from MV1190 to HB101 (from E. coli to E. coli) of approximately 6-, 40-, and 160-fold, respectively, and partial complementation was observed when the corresponding genes from the donor chromosomes were supplied in trans (Fig. 5C). It is remarkable that the mating experiments using the MV1190 derivative harboring NAH7dD, NAH7dE, or NAH7dF as the donor strain and P. putida KT2440G as the recipient strain (from E. coli to Pseudomonas) gave rise to no transconjugants (frequencies of <10^–9 per donor cell). This defect in the formation of transconjugants was restored by supplying each gene in trans from the donor chromosome (Fig. 5D). Similar results were obtained when P. fluorescens Pf-5G was used as the recipient strain (data not shown). These results indicated that all of the traD, traE, and traF products play essential roles in the conjugative transfer of NAH7 from E. coli to Pseudomonas strains.

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FIG. 5. Conjugative transfer of NAH7 derivatives using P. putida and E. coli strains, including conjugative transfer from P. putida G7 derivatives to P. putida KT2440G (A) and to E. coli MV1190 (B) and from E. coli MV1190 derivatives to E. coli HB101 (C) and to P. putida KT2440G (D). Selection was performed by using resistance to Km, and counterselection was performed by using resistance to Gm (for KT2440G), tetracycline (for MV1190), and streptomycin (for HB101). The transfer frequency was determined as described in the legend to Fig. 4. The donor strains are indicated on the x axis, and their genetic characteristics are indicated in parentheses. The means and standard deviations of at least three independent assays are shown. deriv., derivative; WT, wild type.

Supply of the traD operon in recipient Pseudomonas cells. To determine whether expression of the traD, traE, and traF genes in the recipient Pseudomonas strains allowed conjugative transfer of the corresponding mutant plasmids from E. coli, we constructed three KT2440-derived strains whose chromosomal attTn7 sites were occupied by the traD-, traE-, and traF-containing mini-Tn7 elements, respectively. Use of these recipient strains did not result in transfer of the three mutant plasmids from MV1190 (Fig. 6). This showed that the traD, traE, and traF products are required in the donor E. coli cells for transfer of NAH7 to recipient P. putida KT2440 cells.

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FIG. 6. Conjugative transfer of NAH7 derivatives from E. coli MV1190 to P. putida KT2440 derivatives carrying the genes of the traD operon. Selection and counterselection were performed by using resistance to Km and Gm, respectively. The transfer frequency was determined as described in the legend to Fig. 4. The donor and recipient strains are indicated on the x axis, and their genetic characteristics are indicated in parentheses. The means and standard deviations of at least three independent assays are shown. WT, wild type.

Subcellular localization of the TraD, TraE, and TraF proteins. We considered that the subcellular localizations of the TraD, TraE, and TraF proteins would be informative for predicting their functions. The TraD, TraE, and TraF derivatives having the FLAG tag at their C-terminal ends were expressed in the G7 derivatives harboring NAH7dD, NAH7dE, and NAH7dF, respectively, and we confirmed that each FLAG-tagged protein complemented the transfer frequency of the corresponding mutant at levels indistinguishable from that of the wild-type protein (data not shown). Subcellular fractionation and subsequent Western blot analysis revealed that FLAG-tagged TraD, TraE, and TraF proteins with the predicted sizes were located in the cytoplasmic, periplasmic, and membrane fractions, respectively (Fig. 7). However, the SignalP program did not predict that the TraE and TraF genes encode the signal peptide portions. Our computer analysis predicted that there is a transmembrane domain in the C-terminal portion of TraF.

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FIG. 7. Subcellular localization of TraD, TraE, and TraF in P. putida. Three G7 derivatives expressing the FLAG-tagged TraD, TraE, and TraF fusion proteins were fractionated into the cytoplasmic (C), periplasmic (P), and membrane (M) fractions by using the osmotic shock method (see Materials and Methods). Proteins were separated on 15% sodium dodecyl sulfate-polyacrylamide gels. All FLAG-tagged fusion proteins were detected by Western blotting with anti-FLAG antibody. The sizes of standards (in kDa) (Precision Plus protein standards; Bio-Rad Laboratories) are indicated on the left in each panel. The predicted molecular masses of the FLAG-tagged TraD, TraE, and TraF proteins are 17.9, 27.6, and 14.8 kDa, respectively. The values below the gels indicate the relative activities (expressed as percentages) of glucose-6-phosphate dehydrogenase (G6PD), β-lactamase, and succinate dehydrogenase (SDH) in the three fractions. ND, not detected.

Two-hybrid assay of the TraD, TraE, and TraF proteins. To obtain additional clues to help elucidate the functions of TraD, TraE, and TraF, the interactions of these proteins with one another and with other NAH7-specified Dtr/Mpf/CP proteins, such as TraB (coupling protein), TraC (relaxase in the Dtr system), and MpfH (VirB10-like channel subunit in the Mpf system), were investigated by using a bacterial two-hybrid assay (Table 2). The interactions previously reported for other plasmids were also detected in our assay of NAH7 (MpfH with TraB, TraC, and MpfH and TraB with TraC) (6, 28), confirming that the system used in this study worked well. The TraE protein interacted with itself, indicating its homomultimer formation. The TraE protein also weakly interacted with the TraB, TraC, and MpfH proteins, suggesting that TraE is a protein that is directly and pleiotropically involved in the Dtr and Mpf systems and their coupling. On the other hand, interactions of the TraD and TraF proteins with themselves and with other proteins were not detected by this assay.

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TABLE 2. Bacterial two-hybrid analysis of protein interactionsa

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Our mating assay in this study demonstrated that disruption of one of the traDEF genes in the traD operon of NAH7 led to an approximately 10- to 10⁵-fold decrease in the transfer frequencies, indicating that the traD operon is involved in efficient conjugative transfer. Furthermore, all of these genes were essential for NAH7 transfer from E. coli to Pseudomonas strains. These results indicated that the significance of the traD operon in plasmid transfer depends on the combination of donor and recipient strains, and the traD operon can be considered a host-range modifier in the conjugative transfer of NAH7.

Our transcriptional reporter assay revealed that the expression level of the traD operon, which is higher than the expression levels of the of the dtr and mpf operons, is not affected by the coresiding NAH7 plasmid (Fig. 3). The constitutive expression of the traD operon might be important to keep the host cells ready for conjugative transfer. In contrast, our failure to introduce the traD-, traE-, and/or traF-containing multicopy plasmids into P. putida cells suggests that high-level expression of these genes may have detrimental effect(s) in host cells. This may also be one plausible reason why the transfer frequencies of mutant plasmids were not sufficiently restored to the wild-type levels in some strains used for complementation. The optimal level of expression of the traD operon thus appears to be necessary for healthy conjugation.

The TraD protein of NAH7 shows low (26%) identity to a relaxosome component, TraK, of RP4, which wraps around an intrinsically bent region of oriT and is thought to alter the local DNA superhelicity and thus allow easier access of the relaxase to its target site (36, 38). While the RP4 TraK protein is essential for conjugative transfer between E. coli strains (9), the NAH7 TraD protein was not essential for transfer of NAH7 between two Pseudomonas strains, two E. coli strains, and from Pseudomonas to E. coli strains. Furthermore, the Jpred analysis predicted the presence of a helix-turn-helix DNA-binding motif in TraK but not in TraD. Because of these differences between the two proteins, at present it is not clear whether the NAH7 TraD protein has the same function as the RP4 TraK protein. The second genes in the traD operon and the RP4 leader operon are traE and traL, respectively (Fig. 1). However, the apparent roles of these genes in conjugation were also different since disruption of the traL gene of RP4 did not affect its transfer (17). The TraE protein shows 34% identity to the R46-encoded StbB protein, which is involved in stable inheritance of this IncN plasmid (25). The role of the TraE protein can be distinguished from that of the StbB protein since the traE mutation had no apparent effects on the segregational stability of NAH7 or the growth of host cells. Our computer analysis predicted that the TraE protein has a nucleoside triphosphatase activity and contains a coiled-coil motif in its C-terminal portion, and the latter prediction was consistent with the homomultimer formation of TraE. This protein was found to be located in the periplasm and also interact in vivo with TraB, TraC, and MpfH, which are presumed to be essential components of the transfer apparatus of NAH7. On the basis of these properties, TraE might function as an energy-supplying accessory component that promotes the transfer efficiency of the TraC-ssDNA complex through the type IV transfer apparatus. Among the three genes in the traD operon of NAH7, the traF gene is the most important since mutation of this gene resulted in the most drastic decreases in the transfer frequencies. The TraF protein showed no distinct identity to the third gene products, TraM and StbC, of the leader operons in RP4 and R46, respectively. Furthermore, the traF mutant of NAH7 was stably maintained, and its phenotype differs from the unstable maintenance phenotype of the R46 stbC mutant (25). More detailed biochemical analysis of TraF, as well as TraD and TraE, of NAH7 should reveal more definite roles of each protein in the conjugation of this plasmid.

The complete loss of transferability of traD, traE, and traF mutants of NAH7 from E. coli to P. putida was restored by supplying the corresponding genes in trans in the donor cells but not at all by supplying these genes in the recipient cells, indicating that the three genes function in donor cells. Furthermore, our observation that the NAH7 derivatives were transmissible from E. coli strains to E. coli strains raises the possibility that there is a P. putida-specific factor(s) directly or indirectly linked to the function of the traD operon. This is very likely since we indeed obtained chromosomal mutants of KT2440 that were able to receive the NAH7 mutants from the E. coli donor (our unpublished data). More detailed elucidation of each product of the traD operon, as well as characterization of P. putida-specified factor(s), should provide new insights into the host ranges of bacterial plasmid conjugation systems.

 
We are grateful to S. Molin (Technical University of Denmark) and H. P. Schweizer (Colorado State University) for generously providing vectors.

This work was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan. R.M. was supported by research fellowships from the Japan Society for the Promotion of Science for Young Scientists.

 
* Corresponding author. Mailing address: Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai 980-8577, Japan. Phone and fax: 81-22-217-5699. E-mail: mtsuda{at}ige.tohoku.ac.jp

Published ahead of print on 1 August 2008.

Supplemental material for this article may be found at http://jb.asm.org/.

Present address: Department of Fundamental Microbiology, University of Lausanne, 1015 Lausanne, Switzerland.

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Journal of Bacteriology, October 2008, p. 6281-6289, Vol. 190, No. 19
0021-9193/08/$08.00+0     doi:10.1128/JB.00709-08
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