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J Bacteriol. Nov 2005; 187(21): 7341–7350.
PMCID: PMC1272987

Identification of the rctA Gene, Which Is Required for Repression of Conjugative Transfer of Rhizobial Symbiotic Megaplasmids

Abstract

An analysis of the conjugative transfer of pRetCFN42d, the symbiotic plasmid (pSym) of Rhizobium etli, has revealed a novel gene, rctA, as an essential element of a regulatory system for silencing the conjugative transfer of R. etli pSym by repressing the transcription of conjugal transfer genes in standard laboratory media. The rctA gene product lacks sequence conservation with other proteins of known function but may belong to the winged-helix DNA-binding subfamily of transcriptional regulators. Similar to that of many transcriptional repressors, rctA transcription seems to be positively autoregulated. rctA expression is greatly reduced upon overexpression of another gene, rctB, previously identified as a putative activator of R. etli pSym conjugal transfer. Thus, rctB seems to counteract the repressive action of rctA. rctA homologs are present in at least three other bacterial genomes within the order Rhizobiales, where they are invariably located adjacent to and divergently transcribed from putative virB-like operons. We show that similar to that of R. etli pSym, conjugative transfer of the 1.35-Mb symbiotic megaplasmid A of Sinorhizobium meliloti is also subjected to the inhibitory action of rctA. Our data provide strong evidence that the R. etli and S. meliloti pSym plasmids are indeed self-conjugative plasmids and that this property would only be expressed under optimal, as yet unknown conditions that entail inactivation of the rctA function. The rctA gene seems to represent novel but probably widespread regulatory systems controlling the transfer of conjugative elements within the order Rhizobiales.

Rhizobia are gram-negative soil bacteria that are able to establish nitrogen-fixing symbiotic associations with leguminous plants. Besides the chromosome, their genomes are usually constituted of large plasmids which carry genetic material relevant for diverse functions. In some species, the plasmid contribution to the total genome size is certainly astonishing (from 25 to 50% of the genome size [20, 42]). Most of the genes for nodulation and nitrogen fixation are usually located in one of these large replicons, known as symbiotic plasmids (pSyms), or clustered in “symbiosis islands” within the chromosome. In other cases, the participation of another plasmid in symbiosis is considered important enough to justify the denomination of pSym. For example, Sinorhizobium meliloti carries two megaplasmids: pSymA (1.35 Mb), the “true” pSym, contains most of the genes needed for nodulation and nitrogen fixation, whereas pSymB (1.68 Mb) harbors exopolysaccharide biosynthetic genes (19), which are also required for the establishment of symbiosis.

Advances in the knowledge of the transfer systems of these pSyms have been hindered by some particular characteristics of these replicons, such as their large size and the difficulty of detecting their transfer under laboratory conditions. However, there is abundant evidence of symbiotic plasmid transfer among these bacteria in soil (11, 26, 50, 56, 58). In addition, genome sequencing has revealed that many rhizobial pSyms carry genes potentially involved in conjugal transfer (19, 22, 46). For example, putative DNA transfer and replication genes (dtr) involved in the processing of plasmid DNA during conjugative transfer have been identified in several rhizobial pSyms, including pSymA of S. meliloti (2), pRetCFN42d (pSym of Rhizobium etli [22]), and pNGR234a (pSym of the broad-host-range strain Rhizobium sp. strain NGR234 [46]). These Dtr functions are commonly encoded by two divergently transcribed operons, traA and traCDG, with a hypothetical origin of conjugative transfer (oriT) in the intergenic regions (reviewed in reference 52).

Other genes likely involved in the conjugal transfer of pSym plasmids have also been identified, such as complete trb-like systems (in pNGR234a [46] and pRL1JI, the pSym of Rhizobium leguminosarum bv. viciae [59]) or virB-like (2, 22) type IV secretion systems (T4SS), which potentially code for mating pair formation (Mpf) systems required for the transfer of DNA across the membranes of donor and recipient cells during conjugation (52).

Despite the evidence indicating the potential of these plasmids for conjugation, their transfer under laboratory conditions is usually undetectable or occurs at negligible frequencies. Uncovering the regulatory networks that likely control the conjugative transfer of most of these plasmids is one of the keys to identifying the environmental conditions promoting the transfer of these symbiotic megaplasmids and to better understanding the role of pSym lateral spread in rhizobial ecology.

In Agrobacterium, a genus of the Rhizobiaceae family, conjugal transfer of the tumor-inducing plasmid (pTi) is a quorum sensing-dependent highly regulated process (5, 31). Opines produced by the plant during infection control transcription from at least five promoters in gene operons involved in plasmid transfer (38, 47). Some rhizobial symbiotic and nonsymbiotic plasmids with quorum-sensing conjugal transfer regulation systems have been reported as well (15, 25, 27, 28, 40, 57). However, other rhizobial pSyms, such as pRetCFN42d of R. etli CFN42 and pSymA of S. meliloti 1021, seem to have no quorum sensing-like genes carried in their genomes (2, 22). This suggests that these plasmids either have different regulatory systems controlling their transfer or, as suggested previously (22), may have lost the capacity for efficient lateral spread.

The presence of S. meliloti pSymA has been described as an ancient event of lateral transfer mediated by import of pSymA from an unknown bacterium, as indicated by its distinctive G+C content and codon usage (19), but this plasmid has never been described as self-transmissible under laboratory conditions.

In R. etli, the ability of the pSym pRetCFN42d to cointegrate with the accompanying conjugative plasmid pRetCFN42a provides an alternative means for lateral spread (10, 12, 57). However, Pérez-Mendoza and coworkers have recently identified a gene (yp028) involved in the promotion of R. etli pSym transfer at relatively high frequencies in standard media in the absence of pRetCFN42a (45). The identification of this gene opened the possibility that conjugal transfer functions encoded by the R. etli pSym might be silent under laboratory conditions and that transfer could be activated with the participation of yp028 under as yet unknown conditions.

Here we report the identification of the rctA gene present in the symbiotic plasmids of R. etli and S. meliloti. rctA prevents the transfer of these two megaplasmids in standard media and is required for the repression of conjugal transfer genes under these conditions. The existence of this novel regulatory system to actively limit pSym transfer under certain conditions further supports our interpretation that conjugative transfer of these megaplasmids may be a highly regulated process responding to as yet unknown signals.

MATERIALS AND METHODS

Bacterial strains and plasmids.

All bacteria and plasmids used for this work are listed in Table Table1.1. R. etli and S. meliloti strains were grown at 30°C on TY (tryptone-yeast extract-CaCl2 [6]). Escherichia coli and Agrobacterium tumefaciens were grown on Luria-Bertani (LB) medium (54). When required, antibiotics were added at the following concentrations: nalidixic acid (Nx), 20 μg/ml; spectinomycin (Spc), 100 μg/ml for R. etli and 200 μg/ml for S. meliloti; kanamycin (Km), 50 μg/ml for R. etli and A. tumefaciens and 200 μg/ml for S. meliloti; gentamicin (Gm), 10 μg/ml; rifampin, 50 μg/ml; streptomycin (Sm), 100 μg/ml for R. etli and A. tumefaciens, 200 μg/ml for S. meliloti, and 25 μg/ml for E. coli; and tetracycline (Tc), 2 μg/ml for Rhizobium and 10 μg/ml for E. coli.

TABLE 1.
Bacterial strains and plasmids used for this study

Bacterial matings.

Donor strains, grown to an approximate optical density at 600 nm (OD600) of 0.2, and recipient strains, grown to late exponential phase, were washed and mixed at a 1:1 ratio of donor to recipient. Mating mixtures were resuspended in 50 μl of TY medium and loaded onto a sterile nitrocellulose filter with a 0.45-μm pore size. Filter mating mixtures were deposited on TY-agar plates and incubated overnight at 30°C. Cells were resuspended by vigorous vortexing and diluted in liquid medium. To calculate transfer frequencies, donor, recipient, and transconjugant CFU were counted after mating disruption and plating of serial dilutions. Transconjugants were selected on plates supplemented with appropriate antibiotics. The transfer frequency was expressed as the number of transconjugants per output recipient. Donor and recipient levels of spontaneous resistance to selective antibiotics were also determined.

Cell transformations.

Bacterial transformation was carried out by electroporation using an electrocell manipulator apparatus (BTX 600). Electrocompetent cells were prepared according to the instructions of the manufacturer and then stored at −80°C. For electroporation, cells were thawed on ice, mixed with plasmid DNA (0.3 to 0.5 μg/ml of cell suspension), and then transferred to a 0.2-cm-electrode-gap chilled cuvette. A pulse with a 2.5-kV/cm field strength, a 6.8-ms length, and a 129-Ω set resistance was applied, and cells were immediately suspended in 1 ml of TY or LB medium and then incubated at 30°C for 15 h (R. etli) or at 37°C for 1 h (E. coli). Appropriate dilutions were plated on selective media.

Plasmid profiles.

Plasmid profiles were visualized by the Eckhardt procedure (16), as modified by Hynes and McGregor (32).

DNA hybridizations.

Total genomic DNAs were isolated by standard procedures (41), digested with the endonucleases EcoRI and BamHI, electrophoresed in 0.8% agarose gels, and then transferred to positively charged nylon membranes by the method of Southern (54). DNA hybridization probes were labeled with digoxigenin according to the manufacturer's instructions (Roche, Barcelona, Spain). Hybridization and membrane washes were carried out under high-stringency conditions. Membranes were prepared for chemiluminescence detection (Roche) and exposed to Kodak X-Omat film (Sigma).

PCR, cloning, and sequencing.

Standard DNA techniques were used as described previously by Sambrook et al. (54). The Tn5 insertions of the different mutants were subcloned into pUC18 as EcoRI fragments. Transposon arms were subcloned, and insertion sites were mapped by DNA sequencing using an IS50-specific primer. For each insertion, sequence stretches of between 600 nucleotides (nt) and >1,000 nt were obtained. Sequencing was performed with an ABI 373 automated sequencer. DNA sequence editing, translation, and analysis were performed with the Vector NTI 5.5 software package (Oxford Molecular) and the program BLAST from the network service at NCBI (1). The RctA winged-helix DNA-binding domain was identified using the program Superfamily 1.65 (23; http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/). Three-dimensional prediction structures of proteins were prepared using the 3D-JIGSAW program (version 2.0) (3, 4, 14; http://www.bmm.icnet.uk/servers/3djigsaw/).

All oligonucleotides used for constructions are listed in Table S1 in the supplemental material. Different gene fusions to the β-glucuronidase reporter gene gusA were constructed by cloning the pRetCFN42d traA-traC and yp037-rctA DNA regions into the vector p53gus. Primers UptraAE and LwtraCE were used to amplify a 1,394-bp DNA fragment containing the traC promoter, and primers UptraAXb and LwtraCK were used to amplify a 1,363-bp DNA fragment containing the traA promoter. These two PCR fragments were cloned into a dephosphorylated blunt-ended pMOSBlue vector. Among the two possible orientations, the correct one was identified by PCR using UptraA− (E or Xb) and the vector-specific primer T7 and then cloned as a SpeI/KpnI (in the p53RetraC::gus construct) or XbaI/KpnI (in the p53RetraA::gus construct) fragment into p53gus, which was previously digested with the corresponding restriction enzymes. p53Reyp037::gus and p53Reyp038::gus were constructed by cloning the PCR fragments containing the yp037 promoter (amplified with yp037up and yp037lw) and the yp038 promoter (amplified with yp038up and yp038lw) into p53gus, which was previously digested with XhoI/XbaI (restriction enzyme sites are underlined in the primer sequences).

S. meliloti gene fusion plasmids were constructed by cloning a traA1-traC fragment of 480 bp, previously amplified with primers SmtraACup and SmtraAClw, into the pGEM-T Easy cloning vector. Using the XbaI, EcoRI, and SalI restriction enzyme sites at the 5′ ends of the primers, different fragment orientations were obtained with respect to the gusA reading frame in p53gus. All p53gus derivatives were introduced into rhizobial strains by conjugation using the S17-1 mobilizing strain.

For complementation experiments, different plasmids derived from pTE3 were constructed. The different rctA alleles were amplified using specific primers with restriction sites at their 5′ ends to facilitate subsequent cloning in front of the trp promoter in pTE3. pTEYp038 contains the rctA gene of pRetCFN42d as a 750-bp fragment previously amplified with primers RerctA-F and RetrctA-R. pTEAtu5160 contains the rctA gene of pAtC58 as a 450-bp fragment that was amplified with the primers AtrctA-F and AtrctA-R. pTESMa1323 contains the rctA gene of pSymA as a 430-bp fragment that was obtained after amplification with primers SmrctA-F and SmrctA-R.

Construction of an S. meliloti pSymA SMa1323 mutant.

The oligonucleotides used to construct the pSymA SMa1323 mutant are listed in Table S1 in the supplemental material. Two pairs of primers, SmrctA1-SmrctA2 and SmrctA3-SmrctA4, were used to amplify two DNA fragments which flanked the S. meliloti 1021 SMa1323 gene. Each PCR product was cloned into pUC18, the R fragment was cloned as a SmaI/XbaI fragment, generating pUC18-R, and the L fragment was cloned as a BamHI/HindIII fragment, generating pUC18-L. A triple ligation reaction included an Sm/Spc resistance gene cassette (a BamHI fragment purified from the pHP45Ω vector), the R fragment (as a BamHI/HindIII fragment purified from pUC18-R), and pUC18-L (previously linearized with BamHI/HindIII). The resulting construct (Ap, Sm, and Spc resistant; pUC18LCR) was restricted with SmaI/EcoRV, and the corresponding LCR fragment (containing the fragment L plus the resistance Cassette plus R) was cloned into suicide plasmid pSUP202 and introduced by conjugation into strain 1021 of S. meliloti. Allele replacement events were selected as described previously (55), and rctA mutants were verified after hybridization with a labeled plasmid, pUC18LCR, as a probe.

Gene expression assays.

Rhizobial strains containing reporter gene fusion plasmids were grown in TY selective medium to an approximate OD600 of 0.8. Cultures were diluted (1/200 to 1/500) in TY medium containing 20 μg/ml Gm to an approximate OD600 of 0.4 to 0.6. Cells in 1.5 ml of medium were pelleted, washed three times with sterile distilled water to remove any traces of TY, and finally resuspended in 1.5 ml of assay buffer (dithiothreitol, 5 mM; EDTA, 1 mM; Na2HPO4-NaH2PO4, 50 mM). Three hundred microliters of this sample was stored at −20°C for the Bradford Bio-Rad protein assay. Another 200 μl was used to determine the β-glucuronidase activity by mixing with 740 μl of assay buffer, 50 μl of 0.1% sodium dodecyl sulfate, and 100 μl of chloroform and vortexing twice for 15 seconds each time. Samples were incubated at 37°C for 10 min, and then 10 μl of 100 mM NPG (4-nitrophenyl-β-d-glucuronide), preheated at 37°C, was added. Samples were incubated at 37°C until they turned yellow. The reactions were ended by adding 200 μl of 1 M Na2CO3. Samples were centrifuged for 5 min (12,000 rpm), and the absorbance of the upper phase of each sample was measured at 405 nm with a spectrophotometer. Values of β-glucuronidase activity were expressed as specific β-glucuronidase activities (nmol/min/mg of protein). Mean values and standard deviations were calculated from at least three independent experiments.

RESULTS

Isolation and genetic characterization of conjugative transfer regulatory elements in the R. etli symbiotic plasmid.

The symbiotic plasmid of R. etli CFN42, pRetCFN42d, has never been shown to be self-transmissible. However, data from a previous work indicated that conjugative transfer of this pSym may be silent under laboratory conditions, suggesting the possibility of an active repression system in this plasmid (45). In order to identify putative genes participating in the repression of R. etli pSym conjugal transfer, strain CFNX182 of R. etli (pRetCFN42a) was subjected to random Tn5 mutagenesis using the suicide vector pSUP2021 (55). About 2.2 × 106 transposants were obtained from 10 independent matings, where each CFNX182 Kmr transconjugant should harbor a single Tn5 insertion in its genome. The pool of CFNX182 transposants was used as a donor en masse in conjugation with the plasmidless A. tumefaciens GMI9023 strain as the recipient. Transposon insertions causing a loss of functionality of a hypothetical repressor of conjugative plasmid functions would result in a relief of repression and therefore would allow plasmid transfer. Tn5 insertions able to promote the transfer of any plasmid in strain CFNX182 could be easily identified among A. tumefaciens GMI9023 Kmr transconjugants. Some 400 GMI9023 transconjugants were obtained from such mating. Plasmid profiles of 11 randomly chosen transconjugants showed that they all carried a plasmid with a similar size to that of the R. etli symbiotic plasmid pRetCFN42d. Southern hybridization analysis of digested genomic DNAs revealed that each transconjugant harbored a single copy of Tn5 in its genome (Fig. (Fig.1).1). Transposon insertions could be grouped into five different types, which were named Tn5.1, Tn5.2, Tn5.6, Tn5.8, and Tn5.13. These results suggested that different Tn5 insertions were producing the hypothetical derepression of plasmid transfer and that most, if not all, such insertions seemed to have occurred on the pSym plasmid pRetCFN42d but not on any other of the four additional plasmids carried by R. etli CFNX182.

FIG. 1.
Southern blots of EcoRI- and BamHI-digested genomic DNAs of 11 A. tumefaciens GMI9023 Kmr transconjugants, with digoxigenin-labeled Tn5 as a probe. Lanes: 1 and 14, digoxigenin-labeled DNA molecular weight marker; 2 to 12, transconjugants harboring a ...

To verify that the mutant pRetCFN42d plasmids had indeed gained self-transmissibility, the five different types of A. tumefaciens GMI9023 derivatives were then individually used as donors for conjugation with R. etli CFNX218Spc. All the Tn5 insertions promoted transfer of the Tn5-encoded Km resistance from A. tumefaciens to R. etli, but at three different frequencies (Table (Table2).2). Insertions Tn5.1 and Tn5.2 promoted transfer at relative high frequencies under laboratory conditions (about 10−3 transconjugants per recipient cell), whereas the Tn5.6 mutant plasmid was transferred at a ninefold lower frequency than the Tn5.1 or Tn5.2 insertion (about 10−4); insertions Tn5.8 and Tn5.13 also promoted the transfer of pRetCFN42d, but at much lower frequencies (Table (Table2).2). Plasmid profiles of the various transconjugant types confirmed the transfer of the Tn5-tagged pRetCFN42d mutants in all cases. Individual R. etli transconjugants arising from each of the later matings were again used as donors in new matings with A. tumefaciens GMI9023 as the receptor strain. The conjugal transfer frequencies from the individual R. etli CFNX218Spc donors were similar to those previously obtained from the A. tumefaciens GMI9023 donors (data no shown). These results confirmed that Tn5 insertions harbored by pRetCFN42d and selected with our strategy promoted conjugative transfer of this plasmid in standard laboratory media without the help of any other R. etli plasmid, in contrast to the wild-type plasmid, which shows no detectable self-transfer under these conditions (10, 45).

TABLE 2.
Transfer frequencies of different pRetCFN42d Tn5 mutant derivatives from a plasmidless A. tumefaciens strain to R. etli CFNX218Spc

The EcoRI fragments from the mutant plasmids containing the transposon insertions promoting high-frequency transfer of pRetCFN42d, i.e., Tn5.1, Tn5.2, and Tn5.6, were cloned, and the Tn5 flanking DNAs were sequenced. Sequence comparison with the pRetCFN42d genome sequence allowed the identification of the DNA regions interrupted by Tn5. Insertions Tn5.1 and Tn5.2 were located within the same EcoRI fragment but were 67 bp apart. In both mutants, the transposon had interrupted the coding sequence of the yp038 gene (Fig. (Fig.2),2), located adjacent to a hypothetical virB-like operon present in pRetCFN42d. In contrast, insertion Tn5.6 was mapped upstream of the yp028 open reading frame (Fig. (Fig.2).2). yp028 is located downstream of traA, the hypothetical plasmid relaxase gene. Recently, yp028 was described as a gene which may act in the activation of conjugal transfer of the R. etli symbiotic plasmid (45).

FIG. 2.
Locations of different Tn5 insertions in the pRetCFN42d genome. Transposon positions are shown as black triangles.

yp038 represses conjugal transfer of the R. etli symbiotic plasmid.

yp038 encodes a hypothetical protein of 149 amino acids that lacks significant similarity to any protein sequence of known function described in the databases. Nevertheless, this gene displays sequence conservation with several open reading frames of unknown function present in genomes of different bacteria within the order Rhizobiales (see below; Fig. Fig.3A).3A). Using the software program Superfamily 1.65 (23, 39), a “winged-helix” DNA-binding domain was identified in the hypothetical products of yp038 and its homologues (Fig. (Fig.3A).3A). The winged-helix DNA-binding proteins constitute a subfamily within the large ensemble of helix-turn-helix proteins (18). The three-dimensional structure predicted by the 3D-JIGSAW (version 2.0) comparative modeling program (3, 4, 14) showed for Yp038 a similar α/β topology (Fig. (Fig.3B)3B) to that of the winged-helix domains of several crystallized transcriptional regulators (35, 62).

FIG. 3.
(A) Genomic locations of rctA copies in pRetCFN42d from R. etli, pSymA from S. meliloti, pAtC58 from A. tumefaciens, and the chromosome of Mesorhizobium sp. strain BNC1. RctA open reading frames are shown as striped arrows, and virB-like genes are shown ...

The high pRetCFN42d transfer frequencies observed upon interruption of yp038 (insertions Tn5.1 and Tn5.2) suggested that this gene could act by repressing conjugal transfer of the R. etli symbiotic plasmid. To confirm this hypothesis, R. etli cells harboring Tn5.1 and Tn5.2 pSym mutants were complemented in trans with a yp038 gene under the control of the trp promoter (pTEyp038). Conjugal transfer of the mutant plasmids was then tested in matings with A. tumefaciens GMI9023 as the recipient. In all cases, the presence of the cloned yp038 gene resulted in a complete inhibition of transfer of the mutant pSyms, confirming the important role of yp038 in silencing the conjugative transfer of R. etli pSym. In contrast, complementation of the pRetCFN42d Tn5.1 mutant derivative with a yp038 gene under the control of the trp promoter in the A. tumefaciens GMI9023 background resulted in a reduction of only 2 to 3 log of the transfer frequency of the mutant plasmid but not in the complete inhibition of pSym transfer (data not shown). This result suggested that the product of the yp038 gene expressed in trans was not able to totally repress plasmid conjugation in the A. tumefaciens genetic background.

We also analyzed the effect of a yp038 mutation on the expression of pRetCFN42d conjugal transfer genes by using transcriptional reporter gene fusions. The expression of the putative Dtr (p53RetraA::gus and p53RetraC::gus) and Mpf (p53yp037::gus) genes was elevated (between 4.7- and 6.7-fold) in a yp038 mutant (pRetCFN42d::Tn5.1) with regard to the wild-type plasmid (Table (Table3).3). Furthermore, the expression of all of these genes returned to the wild-type levels when the yp038 mutation was complemented in trans with a cloned yp038 gene (pTEyp038) (Table (Table3).3). In contrast to the case for conjugal transfer genes, we observed that rctA expression (yp038::gus fusion) was greatly reduced in a RctA background and returned to wild-type levels when a cloned rctA gene was provided in trans (Table (Table3).3). These data suggest that rctA expression is positively autoregulated.

TABLE 3.
Influence of rctA genotype on expression of R. etli pSym conjugal transfer genes

These results clearly show that yp038 actively participates in keeping low expression levels of genes likely involved in the conjugal transfer of pRetCFN42d. In view of the role of yp038 in the regulation of conjugal transfer of pRetCFN42d, we have renamed this gene rctA (regulation of conjugal transfer).

Overexpression of yp028 activates the expression of pRetCFN42d conjugal transfer genes.

In a previous work, we demonstrated that the overexpression of yp028 promotes the conjugal transfer of pRetCFN42d (45). Here we have found that the Tn5.6 insertion, located upstream of the yp028 coding sequence (Fig. (Fig.2),2), promotes transfer of the R. etli symbiotic plasmid at similar frequencies to those observed when yp028 is overexpressed (Table (Table2)2) (45).

To determine whether the overexpression of yp028 or the Tn5.6 transposon insertion could affect the transcription of pRetCFN42d conjugal transfer genes, we analyzed the expression of the traA and traC genes, as well as that of the yp037 gene, the hypothetical first gene of the virB-like operon coding for the putative Mpf of pRetCFN42d. The expression of yp028 from the trp promoter led to enhanced expression of hypothetical transfer genes (between 4.1- and 6.1-fold) (Table (Table4).4). Likewise, the Tn5.6 insertion increased the expression of the hypothetical transfer genes between four- and fivefold (data not shown). Thus, this mutation had a similar effect to that observed when the yp028 gene was overexpressed.

TABLE 4.
Influence of cloned yp028 gene on expression of pRetCFN42d conjugal transfer genes

The location of the Tn5 insertion upstream of the yp028 gene coding region, the observed plasmid pSym transfer frequencies, and the effect on the expression of conjugal transfer genes from pRetCFN42d allow us to propose that the Tn5.6 insertion may have the effect of deregulating the yp028 gene. Altogether, these results support the idea that yp028 acts as a positive element in the regulation of pRetCFN42d conjugal transfer. Therefore, we have renamed this gene rctB.

rctB has a role in reducing rctA expression.

As shown above, the conjugal transfer of the R. etli symbiotic plasmid seems to be promoted by the action of the rctB gene and repressed by the rctA gene. Therefore, we wondered whether the functions of these conjugal transfer regulators were somewhat related. Using a transcriptional fusion of the rctA gene to the gus reporter gene (p53yp038::gus), we analyzed the effect of rctB on the expression of rctA. As shown in Table Table4,4, overexpression of the rctB gene cloned under the control of the trp promoter led to a significant reduction of rctA expression (Table (Table4),4), indicating that the rctB product interferes with rctA expression. A similar effect on rctA expression was observed in the pRetCFN42d::Tn5.6 mutant (data not shown). These results suggest that the role of rctB in enhancing the expression of conjugal transfer genes might be an indirect phenomenon due to a negative effect of the rctB product on rctA expression or activity.

The rctA gene is present in several bacteria within Rhizobiales.

Although the predicted rctA gene product has no significant sequence conservation with proteins of known function, it displays sequence conservation (between 45% and 57% sequence identity; Fig. Fig.3B)3B) with at least three other open reading frames of unknown function present in the sequenced genomes of different bacterial species within the Rhizobiales order. The hypothetical products of SMa1323 located in pSymA of S. meliloti 1021 and of Atu5160 (from pAtC58, the cryptic plasmid of A. tumefaciens C58) not only show very significant sequence conservation with RctA but are also located in similar genomic contexts, divergently transcribed from virB-like operons carrying the hypothetical plasmid T4SS (Fig. (Fig.3A).3A). Indeed, for R. etli and S. meliloti these genes were annotated virB1 to virB11 (19, 22).

A third rctA homolog has been identified within the unfinished genome sequence of a gram-negative bacterium that was isolated by its ability to degrade chelating compounds such as EDTA, nitrilotriacetate, and diethylenetriaminepentaacetate (34, 43, 44). This strain was initially classified as Agrobacterium sp. strain BNC1 (7) but was recently renamed Mesorhizobium sp. strain BNC1, and its genome is being sequenced by the DOE Joint Genome Institute (NZ_AAED00000000). The shotgun sequence shows that the genome of this bacterium is composed of a unique 4,922,255-bp replicon. Similar to the rhizobium and A. tumefaciens counterparts, the rctA gene homolog from this bacterium seems to be located adjacent to a hypothetical virB-like operon (Fig. (Fig.3A3A).

Recently, Chen and coworkers (13) have described the functionality of the pAtC58 T4SS, named AvhB. This system is essential for pAtC58 plasmid transfer, suggesting that AvhB products comprise an Mpf system (13). Although pAtC58 carries a rctA homolog (60), this plasmid displays high-frequency conjugative transfer under laboratory conditions, in contrast to the R. etli and S. meliloti pSyms (13, 53). This suggests that either the pAtC58 rctA copy is not functional or the genetic background may influence the activity of rctA.

To test whether the rctA homologs from S. meliloti pSymA and pAtC58 are functional, the putative S. meliloti and A. tumefaciens rctA homologs were placed under the control of the heterologous trp promoter (pTESMa1323 and pTEAtu5160) and then introduced into a pRetCFN42d RctA mutant (pRetCFN42d::Tn5.1). Using A. tumefaciens GMI9023 as the recipient, the transfer of pRetCFN42d::Tn5.1 from R. etli CFNX218Spc became undetectable in the presence of either the pSymA or pAtC58 rctA homolog, showing that both of these genes were able to replace the R. etli rctA gene in silencing the conjugal transfer of the pRetCFN42d plasmid.

S. meliloti rctA gene represses conjugal transfer of the symbiotic plasmid pSymA.

As shown above, pSymA of S. meliloti also seems to contain a functional rctA copy, the SMa1323 gene. To test whether this gene has any influence in the repression of conjugal transfer of S. meliloti pSymA, an SMa1323 mutant derivative of strain 1021 was constructed, and pSymA transfer was tested in a mating with A. tumefaciens GMI9023 as the recipient. An S. meliloti 1021 derivative carrying pSymA tagged with an Sm/Spc cassette in the otsA gene was used as the donor in a control mating. The rctA mutation generated increments of S. meliloti pSymA transfer frequencies under laboratory conditions from undetectable to 1 × 10−5, suggesting strong similarities in the regulation of pSym conjugal transfer between S. meliloti and R. etli. Furthermore, we also tested whether the rctA mutation in pSymA affects the expression of the conjugal transfer genes traA1 and traC. Similar to the case for R. etli, a rctA mutation in S. meliloti resulted in enhanced expression of both Dtr genes (Table (Table5),5), which also correlates with the effect of this mutation on pSym transfer. Thus, the symbiotic megaplasmids from both species, R. etli and S. meliloti, share not only similar conjugal transfer genetic organizations but also similar genetic regulatory systems that limit their transfer under standard laboratory conditions.

TABLE 5.
Effect of rctA mutation on expression of S. meliloti pSymA conjugal transfer genes

DISCUSSION

The ability to nodulate leguminous plants provides rhizobia with the capacity to exploit an exclusive ecological niche and therefore with significant advantages over a strictly saprophytic lifestyle. Thus, it seems reasonable to think that the gain of the genetic information necessary to nodulate a specific host should be a very important event in the evolution of these soil bacteria. Genetic and ecological studies have shown that symbiotic plasmids do transfer in soil, as evidenced by the presence of similar pSyms in different genomic backgrounds (21, 36). These studies suggest the occurrence of horizontal transfer during the diversification of natural populations of rhizobia. Indeed, typical conjugal transfer genes have been identified in most pSym genomes sequenced so far.

Several symbiotic plasmids of R. leguminosarum, such as pRL1JI (33) and pRL5JI (8) of R. leguminosarum bv. viciae, pSym5 of R. leguminosarum bv. trifolii (29), and pRP2JI (37) of R. leguminosarum bv. phaseoli, have been shown to be self-transmissible. However, for most rhizobial symbiotic plasmids, significant conjugal transfer does not occur under laboratory conditions, with transfer frequencies ranging from very low to undetectable.

In the particular case of R. etli, the transfer of pRetCFN42d has always been regarded as a cointegration phenomenon with the resident self-transmissible plasmid pRetCFN42a (10, 12, 57). Based on this, some authors suggested that the putative oriT and tra genes identified in the pRetCFN42d genome could be nonfunctional and viewed as evolutionary relics (22).

The demonstration of the functionality of the pRetCFN42d mob region and the identification of the yp028 gene as a possible activator of pRetCFN42d conjugative transfer provided the first strong evidence that this plasmid is self-transmissible (45). These findings led us to hypothesize the existence of a transfer regulation system that might repress the conjugal transfer functions under laboratory conditions. Here we have identified the rctA gene as an essential element of a regulatory system silencing the conjugative transfer of the R. etli symbiotic plasmid in standard laboratory media. Correlating with this, rctA is also required to maintain low expression levels of conjugal transfer genes in this plasmid. In contrast to that of transfer genes, the expression of rctA under laboratory conditions is high and seems to be positively autoregulated, as deduced from the low rctA expression levels observed in a rctA mutant. The putative rctA gene product shows no sequence homology to other proteins of known function. However, an analysis of the secondary and tertiary structures indicated that the predicted rctA gene product belongs to the winged-helix DNA-binding protein subfamily, which includes a number of both activators and repressors of gene transcription (18, 51). Altogether, the data suggest that the rctA gene product directly represses the transcription of transfer genes to prevent plasmid conjugation under nonfavorable conditions. The fact that the pRetCFN42d rctA mutant derivatives show self-transmissibility at high frequencies indicates that pSym conjugation can only be achieved under conditions leading to inactivation of the rctA gene product or to a reduction of rctA gene expression. This seems to be a requirement to obtain enhanced expression of the conjugal transfer genes traA and traCDG and the putative virB-like genes encoding a likely Mpf system. The rctB gene plays an important role in this regulatory process, as rctA expression is greatly reduced under conditions where rctB expression is enhanced, thereby leading to enhanced expression of transfer genes. Since rctA seems to autoregulate its own expression, it is not possible to anticipate whether the rctB product directly represses rctA transcription or interferes with the activity of the rctA gene product. There are two plausible mechanisms to explain the operation of this system. One of these entails binding of the RctA protein to its respective operators; this binding might be antagonized by protein-protein interactions with RctB. Alternatively, RctB might also bind to DNA, hindering the repression mediated by RctA. Resolution between these alternatives must await the purification of these proteins for use in binding assays in vitro.

Nonetheless, the data accumulated allow us to propose a working model for the regulation of R. etli pSym conjugative transfer (Fig. (Fig.4).4). We hypothesize that under unfavorable conditions for conjugation, like those present in the laboratory, the high expression of rctA allows the cell to maintain low expression levels of conjugal transfer genes so that efficient plasmid conjugation cannot proceed. Under favorable yet unknown conditions, the expression of rctB would be enhanced through an unknown mechanism, thereby leading to a reduction of rctA expression and/or activity which would result in the relief of expression of conjugal transfer functions and the transfer of pSym.

FIG. 4.
Proposed model for conjugal transfer regulation of the R. etli symbiotic plasmid. +, activation; −, repression or inhibition. The lower part shows the normal status, i.e., the status under unfavorable conditions for conjugation (laboratory ...

The regulatory network controlling pRetCFN42d conjugal transfer is probably very different from those described for other symbiotic and nonsymbiotic rhizobial plasmids, where plasmid transfer seems to be a quorum sensing-dependent phenomenon (15, 30, 57). This assumption is supported by the novel and important roles of the rctA and rctB genes in the control of pRetCFN42d conjugation, as described in this work, together with the absence of putative quorum-sensing gene homologs in pRetCFN42d (22).

We have also shown that conjugative transfer of the S. meliloti 1.35-Mbp pSymA is subject to rctA-dependent repression under laboratory conditions, by a mechanism likely similar to that found for pRetCFN42d. As for the R. etli pSym plasmid, our data provide strong evidence that the S. meliloti megaplasmid pSymA is self-conjugative but that this property is only expressed under certain conditions.

Since rctA is functional in both plasmids, it seems reasonable to think that both pSyms may share other elements of the conjugation regulatory network. However, we have been unable to identify an rctB homolog within the pSymA sequence, which suggests that the derepression of conjugal transfer genes in both plasmids may be achieved through different regulatory cascades or in response to different environmental conditions.

The rctA gene does not seem to be exclusive to rhizobial Sym plasmids. The A. tumefaciens pAtC58 plasmid also contains an rctA gene, and we have shown that this gene, when expressed from a heterologous promoter, is able to replace the R. etli allele in inhibiting pRetCFN42d conjugative transfer. However, unlike the R. etli and S. meliloti pSyms, pAtC58 was reported to conjugate under laboratory conditions (13, 53), in spite of harboring a rctA gene. It is possible that rctA is not expressed in A. tumefaciens, or more likely, the RctA proteins are not able to cause a complete repression of plasmid conjugation in the Agrobacterium background. Evidence favoring the latter possibility comes from the facts that R. etli RctA was not fully able to repress the conjugation of the pRetCFN42d Tn5.1 derivative in the GMI9023 background, whereas the product of the A. tumefaciens rctA allele was able to totally repress the conjugation of pRetCFN42d rctA mutants in R. etli cells.

We have also identified a homolog of rctA within the genome of the biodegradative bacterium Mesorhizobium sp. strain BNC1, where it is also located divergently transcribed from a hypothetical virB-like operon. According to the unfinished genome sequence, this bacterium carries a single replicon and no extrachromosomal elements. Thus, it is possible that the rctA and virB-like genes found in this genome belong to a conjugative element integrated in the chromosome.

The presence of a system for the repression of conjugal transfer genes might represent a widespread regulatory system for conjugative elements within the Rhizobiales order as an alternative to the better-known quorum sensing-dependent regulation described for several rhizobial and agrobacterial plasmids. The existence of an active system to silence the expression of plasmid conjugal transfer genes suggests the importance of achieving lateral spread only under the most favorable conditions. Uncovering additional elements in this regulatory network will probably be the best way to identify the optimal conditions for pSym transfer in nature, a key aspect to fully understand the ecology and evolution of this group of symbiotic bacteria.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was supported by grant BIO99-0904 from MCyT to J. Sanjuan, by grant IN226802 from DGAPA, UNAM, to S. Brom, and by a CSIC-CONACYT cooperation grant to J. Sanjuan and S. Brom. The support of predoctoral fellowships from MCYT and CSIC (to D. Pérez-Mendoza) and from CONACYT (to E. Sepúlveda) is gratefully acknowledged. J. A. Herrera-Cervera and M. J. Soto were supported by MEC postdoctoral contracts.

We thank A. Domínguez-Ferreras for providing strain Sm 10OTSS and L. Girard for providing plasmid p53Gus.

Footnotes

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

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