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Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
Correspondence
J. Allan Downie
allan.downie{at}bbsrc.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: EMBL Outstation – Hinxton, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK.
Present address: Department of Biology and Biochemistry, University of Bath, BA2 7AY, UK.
Present address: Department of Biochemistry, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch Straße, D-35043 Marburg, Germany.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; d'Angelo-Picard et al., 2005). Such ‘quorum-sensing’ regulation involves the accumulation of the AHLs; as the concentration of AHLs rises, the levels of AHLs within the cells also rise, inducing gene expression by activating regulators that induce expression from specific promoters (Swift et al., 2001). Various cellular processes are regulated in this manner (Barnard et al., 2007; Swift et al., 2001). Many of the regulators that recognize AHLs belong to the LuxR class of DNA-binding proteins, so-named because LuxR regulates the production of light by bioluminescent bacteria (Fuqua et al., 2001). The AHLs recognized by LuxR are synthesized by the AHL synthase LuxI, and a large family of related bacterial AHL synthases has been identified (Gray & Garey, 2001).
One of the quorum-sensing regulatory systems controls the regulation of plasmid transfer by strains of Agrobacterium tumefaciens, which causes tumours in plants by directly transforming plant cells (Fuqua et al., 1994; Hwang et al., 1994; Piper et al., 1993). Such strains contain plasmids that carry genes required for plant pathogenicity, and the transfer of these plasmids between agrobacteria requires both an appropriate population density and the presence of a specialized carbon source (opines) (Fuqua & Winans, 1996; Piper et al., 1999). The DNA transferred to the plant includes genes for promoting cell proliferation and for synthesizing secondary metabolites called opines (Escobar & Dandekar, 2003). These plant-made opines are secreted from transformed plants and induce the expression of specialized uptake and catabolism genes in A. tumefaciens. One of the genes co-induced is traR, the product of which regulates the transfer of the pathogenesis plasmid. TraR dimers bind to the AHL 3-oxo-octanoyl-L-homoserine lactone (3-O-C8-HSL) and this induces the expression of plasmid-transfer genes, including the traI-trb operon. The traI gene product synthesizes 3-O-C8-HSL, resulting in positive autoregulation. The structure of TraR bound to 3-O-C8-HSL has been resolved (Vannini et al., 2002; Zhang et al., 2002) and a consensus TraR-binding sequence (the tra-box) has been defined (Zhu et al., 2000).
Rhizobium leguminosarum is closely related to A. tumefaciens and many of the genes required for the nitrogen-fixing symbioses between R. leguminosarum and legumes are present on plasmids (Downie et al., 1983; Young et al., 2006). The transfer of one of these plasmids (pRL1JI) between different rhizobia can be induced by potential recipient strains of Rhizobium in a population-density-dependent manner (Danino et al., 2003). As in A. tumefaciens, there is a TraR regulator that induces the expression of plasmid transfer genes, including a traI-trb operon; this requires the accumulation of 3-O-C8-HSL, which is synthesized by TraI in a population-density-dependent manner. Expression of traR, however, is under the direct control of a second LuxR-type regulator encoded by bisR, which is adjacent to traR on pRL1JI (Fig. 1). BisR induces traR expression in response to N-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone (3-OH-C14 : 1-HSL), which is produced by the chromosomally encoded cinI gene product (Lithgow et al., 2000). The cinI gene is so-called because in bacteriocin-type assays it is required for growth inhibition of some strains of R. leguminosarum (Lithgow et al., 2000); sensitive strains include those carrying pRL1JI and their sensitivity to CinI-made 3-OH-C14 : 1-HSL is related to the activation of TraR, because mutations affecting traR or traI expression cause a loss of the sensitivity phenotype. R. leguminosarum strains carrying pRL1JI do not make much 3-OH-C14 : 1-HSL, because BisR represses cinI expression (Wilkinson et al., 2002). Such strains carrying pRL1JI are poised to detect 3-OH-C14 : 1-HSL made by potential Rhizobium recipients, which lack bisR (and therefore pRL1JI) and so are not repressed for 3-OH-C14 : 1-HSL synthesis. Thus, when a strain of R. leguminosarum (donor) carrying pRL1JI is growing near a population of a strain lacking pRL1JI (potential recipient), 3-OH-C14 : 1-HSL made by the recipient strain activates BisR to induce traR expression in the donors (Fig. 1). Then, as the population density of the donors increases, TraR induces the plasmid-transfer genes and conjugal transfer of pRL1JI is induced (Danino et al., 2003). The sequenced R. leguminosarum bv. viciae strain 3841 lacks bisR (Young et al., 2006) and so this strain is one such potential recipient.
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; Fuqua et al., 2001), there is no obvious recognition motif upstream of the genes regulated by the LuxR-type regulators BisR and CinR (Danino et al., 2003; Lithgow et al., 2000; Wilkinson et al., 2002). In this work, we have mapped the transcript start sites of genes regulated by CinR, BisR and TraR and identified conserved regions of dyad symmetry that may represent the respective DNA-binding sites. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Antibiotics were added at the following final concentrations (µg ml–1): streptomycin, 400; kanamycin, 40; spectinomycin, 200; tetracycline, 5. Bacterial growth was monitored at 600 nm using a Perkin Elmer MBA2000 spectrophotometer. Green fluorescent protein (GFP) fluorescence was measured using a Tecan SAFIRE microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 510 nm, and for the GFP experiments bacterial growth was monitored at an absorbance of 600 nm in the same plate reader. Units of β-galactosidase activity were measured as described by Miller (1972) using a Titertek Multiscan spectrophotometer. Cultures were grown for 2 days in TY medium containing the appropriate antibiotics then diluted 1 : 100 in AMS (acid minimal salts) pyruvate minimal medium (Poole et al., 1994) for GFP assays or TY medium for lacZ assays. Where required, AHLs were added at the time of dilution to the final concentrations stated. Bacteriocin growth-inhibition tests were done as described previously (Wilkinson et al., 2002).
Bacterial strains and plasmids.
The strains and plasmids used in this study are listed in Table 1. A924 (cinR3 : : Tn5) was isolated from a Tn5 insertion library of strain 3841, and the Tn5 insertion was found by DNA sequencing to be located 205 bp from the translation start of cinR. Plasmid pIJ9718 was made by amplifying the cinI-cinR intergenic region from genomic DNA of strain 3841 (using Taq polymerase and the 5' primer 5'-TGCTCGTTTCAAACTCGGCTG-3' and the 3' primer 5'-GCGAGCGAATCGTAGCTGTC-3'), generating a 388 bp fragment which was ligated into pGEM-T Easy (Promega) following the manufacturer's instructions. All other plasmids were constructed using DNA from strain A34. The cinI–gfp transcriptional fusion plasmid pIJ9611 was made by amplifying a 450 bp HindIII DNA fragment (5' primer 5'-CTCGGCAAAGCTTACAAGGATATTTC-3'; 3' primer 5'-CAGAGCGTATCGGCGAAAAGCTTCTTGC-3') containing 136 bp of cinR coding region, the cinRI intergenic region and 70 bp of cinI coding region, followed by cloning the DNA fragment into the HindIII site of pRU1156. The traR–gfp transcriptional fusion plasmid pIJ9612 consists of a PCR-amplified 217 bp HindIII DNA fragment (5' primer 5'-GTACATTTGAAGCTTATCACTCCCCAC-3', 3' primer 5'-GAAGCTTTTCAGGGCACTCTTG-3') containing 26 bp of bisR coding region, the bisR-traR intergenic region and 82 bp of traR coding region cloned into the HindIII site of pRU1156. The repA–lacZ plasmid pIJ9753 was made by subcloning a 930 bp XbaI/KpnI fragment from pIJ9278 (Danino et al., 2003) into pMP220. The cinI–gfp plasmid, pIJ9884, was made by PCR amplification (5' primer 5'-CTCGGCAAAGCTTACAAGGATATTTC-3'; 3' primer 5'-CAGAGCGTATCGGCGAAAAGCTTCTTGC-3') followed by HindIII digestion and ligation into pRU1156, producing a plasmid containing 180 bp DNA fragment, which has the same 3' end as pIJ9611, but is shorter by 270 bp at the 5' end. The traR–gfp plasmid, pIJ9991, was made as pIJ9612 except the 5' primer was 5'-GAGTAACCCAAGCTTGGGTATCGGTTTG-3', generating an insert size of 188 bp.
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; Wexler et al., 2001). The oligonucleotides used in these studies are shown in the legends to the respective figures. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Primer extensions were performed (Fig. 2a) on RNA extracted from R. leguminosarum strain 8401 (lacking pRL1JI), which induces cinI, and from an isogenic cinR mutant (R. leguminosarum strain A741), which does not (Lithgow et al., 2000). Transcription of cinI initiated from a single site 27 bp upstream of the predicted cinI translational start. As expected (Lithgow et al., 2000), the appearance of this transcript required cinR (Fig. 2a). We also identified the transcription initiation site of cinI in R. leguminosarum strain 3841 (Fig. 2b), whose genome has been sequenced (Young et al., 2006). Strain 3841 lacks bisR and induces cinI expression, based on measurements of 3-OH-C14 : 1-HSL accumulation, which inhibits the growth of R. leguminosarum strain A34 (Fig. 3a). An isogenic cinR mutant (R. leguminosarum strain A924) did not produce 3-OH-C14 : 1-HSL (Fig. 3a). A cinR-dependent transcript was identified also, starting 27 bp upstream of the cinI translation initiation codon in this strain (Fig. 2b). In neither strain was there an obvious recognition sequence similar to a lux or tra box at the anticipated location, usually centred about 42–45 bp upstream of the transcription start. However, we did identify a region of dyad symmetry centred 60 bp upstream of the cinI transcription start site (Fig. 2b); a similar gap (65 bp) between a transcript start and quorum-sensing regulator (TraR) binding site has been observed previously (Pappas & Winans, 2003).
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) (Lithgow et al., 2000). The Tn5 is inserted 65 bp upstream of the 5' end of the cinI transcript and so there is not an intact promoter within 65 bp of the cinI transcript start (Fig. 2b). To define the 5' end of the cinI promoter, we amplified cinI promoter regions starting 79 bp and 360 bp upstream of the transcription start site and cloned the DNA products into a GFP transcriptional fusion vector to make pIJ9884 and pIJ9611, respectively. The results obtained with these promoter fusions (Fig. 2c) showed that an intact and functional CinR-dependent cinI promoter is within the 79 bp upstream of the transcription start site (Fig. 2c). We conclude that some of the 14 bp of the cinI upstream region between the 5' end of pIJ9884 (position –79) and the Tn5 insertion in A568 (position –65) must be required for normal cinI promoter activity. The Tn5 in A568 is within a region of dyad symmetry and it is possible that CinR may bind to this region interrupted by the Tn5 insertion in A568.
The expression of cinI is repressed by BisR (Wilkinson et al., 2002), although it is not known if this is due to repression by binding to the promoter or some other mechanism, such as the formation of inactive heterodimers with CinR. Cloned bisR repressed cinI expression in pIJ9611 and pIJ9884 (Fig. 2d). To identify whether BisR-mediated repression is retained in A568 (containing Tn5 in the promoter region of cinI), we made use of the observation that a weak promoter reads out from Tn5 (Berg et al., 1980). To determine if this could be observed with A568, we used a strain of R. leguminosarum whose growth is extremely sensitive to inhibition by the CinI-made 3-OH-C14 : 1-HSL (Wilkinson et al., 2002). A568 does cause some growth inhibition detectable by this strain (Fig. 3b) and we conclude that this is probably due to cinI expression from the weak Tn5 promoter. The growth inhibition by A568 was abolished by introducing the bisR gene, cloned on pIJ9581, into strain A568 (Fig. 3b), showing that BisR-mediated repression is retained even though normal cinI induction by CinR is lost in A568.
Transcription of traR
The next step in the induction of pRL1JI transfer is BisR-mediated induction of traR in response to 3-OH-C14 : 1-HSL (Fig. 1) (Danino et al., 2003). BisR shows 59 % overall identity to CinR (Wilkinson et al., 2002); in the predicted DNA contact regions (equivalent to residues 191–221 in TraR; Zhang et al., 2002) of BisR (residues 195–225) and CinR (residues 196–206) the identity is 68 % (21 identities in 31 residues). Database searches with residues 195–225 of BisR returned the highest significant score with CinR (with the exception of BisR itself and the likely orthologue of BisR in Rhizobium etli). This high similarity between the predicted DNA-binding domains of BisR and CinR from R. leguminosarum suggested that promoters regulated by BisR and CinR might share some sequence similarity. The transcription initiation site of traR was determined (Fig. 4a) using RNA isolated from R. leguminosarum A34 carrying pRL1JI and an isogenic bisR mutant (A549), both grown in the presence of 3-OH-C14 : 1-HSL to activate traR induction by BisR. A bisR-dependent transcript was observed starting 53 bp upstream from the predicted traR translation start. The region between bisR and traR is short and so the transcription start site of traR is only 52 bp downstream from the translation stop of bisR. No typical lux- or tra- box-like elements could be identified upstream of the transcription start site. We constructed two traR promoter fusion constructs (Fig. 4b), pIJ9612 starting 83 bp upstream of the transcription start site (within the end of bisR) and pIJ9991 starting 53 bp upstream of the traR transcription start site (at the translational stop of bisR). Induction of the traR promoter was assayed as described previously using strain A677 (Danino et al., 2003), which is a derivative of A. tumefaciens that makes no detectable AHLs (Vaudequin-Dransart et al., 1995). Normal induction of traR–gfp by 3-OH-C14 : 1-HSL was seen with pIJ9612, but there was no induction with pIJ9991 (Fig. 4c), demonstrating that the induction probably occurs following BisR binding to DNA included within the 83–53 bp region upstream of the traR transcript start. We compared the traR promoter region with the cinI promoter region to try to identify potential BisR- and CinR-binding motifs. Two regions of similarity were noted. The sequence TGAGGGAATTT, centred 41 bp upstream of the cinI transcript start (Fig. 2b), is similar to the sequence TGGGGGATTT, 41 bp upstream of the traR transcript start (Fig. 4b). However, these sequences are fully retained in the cinI2 : : Tn5 mutant A568 (Fig. 2b) and the traR promoter fusion plasmid pIJ9991 (Fig. 4b), neither of which allows expression of the downstream genes (Figs 3a and 4c). Therefore, these sequences alone seem very unlikely to be the CinR- and BisR-binding motifs. The other region of similarity seen was that the sequence CCCCACATGAG, starting 78 bp upstream of the traR transcription start site (Fig. 4b), which is present in the opposite orientation in the cinI promoter region (as CTCATCTGGGG, starting 64 bp upstream of the cinI transcription start site; Fig. 2b). This motif is fully retained in the inducible traR promoter fusion in pIJ9612 (Fig. 4b, c) and in the cinI : : Tn5 promoter mutant A568, in which BisR-mediated repression of cinI is retained (A568/pIJ9581 in Fig. 3b) and overlaps with the predicted CinR-binding site (Fig. 2b). Therefore the CTCATSTGGGG sequence might act as (part of) a BisR-binding site that could allow induction of traR, but prevent CinR binding to the cinI promoter. Clearly DNA footprinting experiments with BisR and CinR will be required to exactly define their DNA-binding sites.
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). The traI gene was transcribed from a single promoter, which required both TraR and 3-OH-C14 : 1-HSL (Fig. 5a and data not shown). In A. tumefaciens and Rhizobium sp. NGR234, transcription of traI requires a conserved TraR-binding site with dyad symmetry (tra-box) centred 42–45 bp upstream of the transcription start site (Fuqua et al., 2001; He et al., 2003). A predicted tra-box sequence, similar to the tra-box identified upstream of traI from pNGR234a (pSym) in Rhizobium NGR234, was identified centred 44 bp upstream of the traI transcript start on pRL1JI (Fig. 5b). This predicted TraR-binding site on pRL1JI shows some similarity (Fig. 5b) to the tra-box upstream of traI in A. tumefaciens and to other tra-boxes (Fuqua & Winans, 1996; Li & Farrand, 2000; Zhu et al., 2000).
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; Pappas & Winans, 2003). Using RNA from A34 and the traR mutant A627 grown with added 3-OH-C14 : 1-HSL to induce traR gene expression, the pRL1JI repA gene was found to be transcribed from two start sites, only one of which required traR (Fig. 6). The traR-independent promoter is likely to be constitutive because expression of repA will be necessary throughout growth.
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), and the increased expression observed when both traR and 3-O-C8-HSL are present is due to induction of the downstream TraR-dependent promoter. There is a potential tra-box centred 63 bp upstream of the TraR-dependent transcription start site of repA (Fig. 5), but it does not show strong dyad symmetry, typical of many other lux- and tra-boxes. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The reason for this complex cascade of regulation, compared with the relatively simple TraR-mediated gene induction in other rhizobia and agrobacteria (He & Fuqua, 2006), is presumably related to the unusual mechanism required to regulate recipient-induced plasmid transfer. This involves the induction of traR gene expression by BisR in donors in response to 3-OH-C14 : 1-HSL made by potential recipients. The consequent induction of traR expression in donors leads to the induction of the traI-trb operon expression, and presumably the traAFBH and traCDG operons (Farrand, 1998; Zhu et al., 2000), as in agrobacteria and other rhizobia.
Upstream of the traI-trbBCDEJKLFGHI operon on pRL1JI are two conserved tra-boxes similar to those identified in rhizobia and agrobacteria (Zhu et al., 2000). One is centred 43 bp upstream of the transcription start site of traI and the other is centred 63 bp upstream of the inducible transcription start site of the divergently transcribed repABC genes. There are two transcript starts upstream of repA; one, presumably constitutively expressed, is 12 bp upstream of the predicted translation initiation codon and the other, 5 bp upstream of the translation start, is regulated by TraR in response to AHLs. Thus, as has been observed in other rhizobia and agrobacteria, induction of conjugal transfer may be coupled to an increase in plasmid copy number.
Expression of traR is induced by BisR in response to 3-OH-C14 : 1-HSL, and this AHL is also recognized by CinR. BisR and CinR are 59 % identical at the amino acid sequence level (Wilkinson et al., 2002) and therefore BisR may have arisen as a duplication of CinR, retaining similar AHL-binding characteristics. However, despite significant conservation in the DNA-binding domains, their promoter specificities must be different, because BisR cannot induce expression of cinI and CinR cannot directly induce expression of traR (Danino et al., 2003; Lithgow et al., 2000; Wilkinson et al., 2002). In fact, BisR represses cinI expression (Wilkinson et al., 2002). Within the traR promoter region, we identified a possible BisR-binding site that is conserved in the opposite orientation in the cinI promoter region and so this is a possible site of repression, possibly by BisR preventing access of CinR to the cinI promoter. Evidence of direct binding of BisR to the cinI promoter came from the inhibition by BisR of the low levels of cinI expression in the mutant A568, which has Tn5 inserted in the cinI promoter. This mutant has greatly reduced cinI expression, probably associated with a weak promoter within Tn5. The bisR gene could repress this expression, and in this situation the simplest explanation is that the BisR-binding site is retained in this mutant. Therefore, a BisR-binding motif is predicted to be present in the cinI promoter downstream of the site of Tn5 insertion. The most likely location seems to be the conserved sequence overlapping with the predicted CinR-binding site, but we cannot exclude the possibility that BisR may bind to the conserved sequence centred about 40 bp upstream of the cinI transcript start. The precise location of BisR binding will require DNA footprinting experiments with purified BisR.
The mechanism by which BisR can act as an inducer of traR but a repressor of cinI expression is not clear. It seems likely that the BisR repression function can occur in the absence of 3-OH-C14 : 1-HSL, because BisR prevents the formation of this AHL. However, BisR-dependent induction of traR absolutely requires 3-OH-C14 : 1-HSL (Danino et al., 2003). Possibly the binding of 3-OH-C14 : 1-HSL can change the affinity of BisR for the traR promoter, such that in the presence of 3-OH-C14 : 1-HSL BisR binds more strongly to the traR-type promoter.
Although the precise promoter-binding regions for BisR and CinR have yet to be defined, our data have allowed us to delimit the regions at which they are likely to interact and provide new insights into the complex mechanisms controlling plasmid replication and maintenance in rhizobia.
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ACKNOWLEDGEMENTS |
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Edited by: C. W. Ronson
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Received 20 February 2007;
revised 5 April 2007;
accepted 11 April 2007.
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