A LuxR/LuxI-Type Quorum-Sensing System in a Plant Bacterium, Mesorhizobium tianshanense, Controls Symbiotic Nodulation
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《细菌学杂志》
Department of Microbiology, MOA Key Lab of Microbiological Engineering of Agricultural Environment, Nanjing Agricultural University, Nanjing, China,Department of Microbiology, Chinese Agricultural University, Beijing, China,Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
ABSTRACT
The ability of rhizobia to symbiotically fix nitrogen from the atmosphere when forming nodules on their plant hosts requires various signal transduction pathways. LuxR-LuxI-type quorum-sensing systems have been shown to be one of the players in a number of rhizobium species. In this study, we found that Mesorhizobium tianshanense, a moderate-growth Rhizobium that forms nodules on a number of licorice plants, produces multiple N-acyl homoserine lactone (AHL)-like molecules. A simple screen for AHL synthase genes using an M. tianshanense genomic expression library in Escherichia coli, coupled with a sensitive AHL detector, uncovered a LuxI-type synthase, MrtI, and a LuxR-type regulator, MrtR, in M. tianshanense. Deletions of the mrtI or mrtR locus completely abolished AHL production in M. tianshanense. Using lacZ transcriptional fusions, we found that expression of the quorum-sensing regulators is autoinduced, as mrtI gene expression requires MrtR and cognate AHLs and mrtR expression is dependent on AHLs. Compared with the wild-type strains, quorum-sensing-deficient mutants showed a marked reduction in the efficiency of root hair adherence and, more importantly, were defective in nodule formation on their host plant, Glycyrrhiza uralensis. These data provide strong evidence that quorum sensing plays a critical role in the M. tianshanense symbiotic process.
INTRODUCTION
Bacteria exchange small, self-produced chemical molecules to monitor their population density and control a variety of physiological functions in a cell density-dependent manner, a process called quorum sensing (5, 8, 10). Many gram-negative bacteria use a set of diffusible N-acyl homoserine lactones (AHLs, also called autoinducers) that generally serve as cell-to-cell communication signals (39, 41). The key regulatory components of these signaling systems are LuxI-type proteins, which act as AHL synthases, and LuxR-type proteins, which serve as AHL receptors and AHL-dependent transcription factors. In general, the LuxR-like proteins are each responsible for binding a cognate AHL molecule, binding specific target gene promoters, and activating transcription when cognate AHLs have achieved a critical threshold concentration (7, 44, 46). The LuxI-like proteins are required to synthesize AHL molecules, which contain invariant homoserine lactone moieties and highly variable fatty acyl groups. The homoserine lactone moiety is derived from S-adenosylmethionine, while the acyl chains are derived from an acyl-acyl carrier protein (23, 24, 40).
Many important bacterial behaviors are regulated by quorum sensing, including virulence, antibiotic production, biofilm formation, and symbiosis (22, 39). Nitrogen fixation and endosymbiotic interactions between legume plants and the genera Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, and Azorhizobium, collectively termed rhizobia, have been intensively studied (18). Intricate signaling between the host and rhizobial symbiont is required for successful symbiotic interactions, which result in the reduction of atmospheric N2 to ammonia by the bacteroids (32). Quorum sensing has been implicated as a key player in the symbiotic process (11). For example, a few studies have reported that AHL signals produced by several species of Rhizobium and Sinorhizobium appear to control various functions, such as exopolysaccharide production (14, 19, 25), plasmid transfer (12, 36), and nodulation (3, 4, 29), all of which are related to symbiosis. In Rhizobium leguminosarum bv. viciae, four quorum-sensing systems (rai, rhi, cin, and tra) have been identified and are intertwined in a complex regulatory network. The cin locus is situated at the top of the quorum-sensing network. Mutations of cinI and cinR abolish the production of 3-OH-C14:1-HSL and cause a decrease in levels of all of the short-chain AHLs produced by the enzymes encoded by raiI, traI, and rhiI (17). Examination of mutations in either rhiA and rhiR for nodulation of beans showed a decreased number of nodules, but only in combination with a nodFE mutant, leading to the hypothesis that the rhi system seems to play a role in nodulation efficiency (3). The tra system is clearly shown to regulate the conjugal transfer of pRL1J1, a symbiotic plasmid, but the advantage of having plasmid transfer under the control of the cin system is still unknown (43).
To date there have been no detailed studies on quorum-sensing regulatory systems in the Mesorhizobium genus, a moderately growing rhizobium. However, genome sequences of Mesorhizobium loti predict the presence of several LuxI-LuxR family proteins. In previous reports, we described the detection of AHL-like quorum-sensing signals from Mesorhizobium huakuii (45) and studied the possible roles of quorum sensing in biofilm formation in this strain (38). In the present study, we detected AHL signals from an M. tianshanense strain, a moderately growing root nodule bacterium which was originally isolated from an arid saline desert soil in northwestern China in 1995 (2). M. tianshanense was later widely found in dry soils and acts as a nitrogen-fixing symbiont for at least eight different plant species, including Glycyrrhiza (licorice) (35), whose roots are one of the most important crude medicines in Asia and Europe. We have developed a novel method to identify the AHL synthase gene from M. tianshanense and found that quorum sensing in M. tianshanense plays a critical role in symbiosis.
MATERIALS AND METHODS
Bacterial strains, plasmids, and culture conditions. Bacterial strains and plasmids used in this study are listed in Table 1. Mesorhizobium strains, which have been deposited in the Culture Collection of Beijing Agricultural University (CCBAU; Beijing, China), were grown at 28°C in TY medium (37). Escherichia coli was grown at 37°C in LB medium (31), and Agrobacterium tumefaciens was grown at 28°C in AT medium (9). For M. tianshanense, a spontaneous streptomycin-resistant mutant of CCBAU 3306, HMZ0, was used as the parental strain in this study. Chromosomal mrtI-lacZ and mrtR-lacZ transcriptional reporter fusions were constructed by PCR, amplifying the internal fragment of mrtI and intact 5' mrtR (including its putative promoter region), and these fragments were cloned into pVIK112 (15). The resulting plasmids were then integrated into the chromosome at the mrtI and mrtR loci, respectively, by homologous recombination. In-frame deletions in the mrtI and mrtR genes were constructed by overlapping PCR of flanking regions of the target genes and cloning into the pWM91 suicide vector (20). The resulting plasmids were introduced into HMZ0, and double-crossover events were selected on sucrose plates (10%) after the first "cross-on" homologous recombination. A plasmid that constitutively expresses mrtR was constructed by cloning of the mrtR genes into the pBBR1-MCS5 vector (16) and introduced into M. tianshanense strains by electroporation. -Galactosidase activity assays were performed as previously described (21).
Screening of M. tianshanense AHL synthase genes. Two- to 10-kb fragments of HMZ0 genomic DNA partially digested with HincII were cloned into the pEZSeq-Kan vector using the pEZSeq cloning kit (Lucigen, Wisconsin). Approximately 50,000 transformants with insertions were pooled and saved in 20% glycerol at –70°C. The library was then inoculated into LB medium containing appropriate antibiotics in 96-well plates at a dilution of approximately 20 cells per well. After overnight incubation at 37°C, 20 μl of supernatant from each well was transferred to a new 96-well plate in which each well was overlaid with 200 μl AT medium (9) agar (0.5%) containing 107 cells/ml AHL bioassay strain cells (45) and 5-bromo-4-chloro-3-indolyl -D-galactoside (X-Gal; 20 μg/ml) in each well. Plates were incubated at 28°C for 12 to 16 h and examined for X-Gal hydrolysis (blue color). For each blue-colored well, bacteria were then streaked out to single colonies from the corresponding original wells and inoculated to LB medium in 96-well plates to repeat the above assays. Plasmid DNA was purified from E. coli transformants that were able to produce AHLs to activate AHL bioassay reporters and subjected to DNA sequencing.
AHL bioassays. Spent medium (10%, vol/vol) from Mesorhizobium strains collected from various time points as indicated was added into AT medium (9), and approximately 107 AHL bioassay strain cells per ml (45) were added. These cultures were incubated with aeration for 12 h and assayed for -galactosidase specific activity (21).
Thin-layer chromatography (TLC) analysis of AHLs released by Mesorhizobium strains was performed as previously described (33). Aliquots from ethyl acetate extracts of spent media were applied to C18 reversed-phase TLC plates (Whatman), chromatographed, and developed as previously described (38).
Nodulation assays. Glycyrrhiza uralensis (Asian licorice) seeds were treated with 75% ethanol and 0.1% HgCl2 and were surface sterilized. The treated seeds were then placed in petri dishes, germinated in the dark at 28°C for 2 days, and then planted in 200- by 30-mm test tubes (three seeds per tube) filled with autoclaved Vermiculite and Perlite (10:1) and a plant nutrient solution of Fahraeus medium (6) supplemented with 28 mg N/kg Vermiculite. Plants were grown in a plant growth chamber at 28°C with a 16-h-8-h day-night cycle. After 6 days, plants were inoculated with 300-μl cultures of wild-type or mutant M. tianshanense grown in TY medium (optical density at 600 nm [OD600] of 2.0). The plants were pulled out to count the number of nodules at the time indicated. There were at least 12 replicates for each inoculation.
Root hair attachment assay. Seeds of Glycyrrhiza uralensis were surface sterilized and germinated as described above. Fifteen seedlings roots, approximately 1.5 cm in length, were incubated for 4 h at 28°C in 30 ml of 25 mM phosphate buffer (pH 7.5) containing 300 μl of cell suspension (OD600, 1.5) in the absence or presence of 10% cell-free supernatants of wild-type cultures. After incubation, roots were washed with 2 ml phosphate buffer three times with gentle shaking. The number of attached bacteria was determined by vortexing roots with glass beads and plating the cell suspension on TY medium plates.
Root hair deformation assays. Root hair deformation assays were performed according to previous publications (13, 26) with a few modifications. Briefly, M. tianshanense strains (wild type and quorum-sensing mutants) were grown in yeast mannitol medium (37), and at an OD600 of 0.3, 10% seed extract of Glycyrrhiza uralensis was added. Cultures were incubated for an additional 36 h. Bacterium-free supernatant containing Nod factors was stored at –20°C. Glycyrrhiza uralensis seedlings were then incubated with 10% M. tianshanense supernatants prepared as described above in petri dishes for 2 days. Roots were microscopically examined, and the number of deformed root hairs in the susceptible zone was counted.
Nucleotide sequence accession number. The mrtR-mrtI DNA sequence reported here has been deposited in the GenBank database under accession number DQ123807.
RESULTS AND DISCUSSION
AHL production in various Mesorhizobium strains. Although quorum-sensing regulation has been studied extensively in a number of rhizobia, there are limited studies on this system in Mesorhizobium species. To investigate whether quorum sensing plays any role in Mesorhizobium biology, the spent culture supernatants from eight type strains of the genus Mesorhizobium were examined for AHL production. High-cell-density cultures were extracted with ethyl acetate and subjected to thin-layer chromatograph detection of AHL content. The AHL profiles of these eight strains were quite different (Fig. 1A). M. chaoconense, M. plurifarium, M. mediterraneum, and M. tianshanense produced multiple AHL-like molecules. A single spot was detected from the extracts of M. amorphae and M. huakuii supernatants, confirming our previous report (45). No AHL-like molecules were detected from supernatants of M. loti and M. ciceri under these conditions. Interestingly, at least four annotated AHL synthase genes exist, based on M. loti genome sequencing. Overexpressing one of these synthase genes in E. coli produced at least three different AHLs (data not shown). These results indicated that those AHL synthase genes in M. loti are not expressed under routine culture conditions.
M. tianshanense, which showed the greatest diversity of AHL signaling molecule production among the Mesorhizobium strains tested, was selected for further studies. AHL activities from supernatants collected during the growth of a spontaneous streptomycin-resistant mutant (HMZ0) of the type strain CCBAU 3306 were assayed by measuring the induction of a lacZ reporter of an AHL bioassay strain (45). As shown in Fig. 1B, accumulation of AHL activity in supernatants of HMZ0 displayed a typical cell density-dependent pattern, with low AHL activity at low cell density (OD600 below 1) and higher activities as the bacterial culture grew.
Identification of a LuxI/LuxR-type quorum-sensing regulatory system in M. tianshanense. In an effort to identify the genes involved in AHL production in M. tianshanense, we developed a novel, simple screening method. Briefly, a genomic library of M. tianshanense was constructed on a high-copy-number vector flanked by a Plac promoter, which may turn on transcription of synthase genes that might not be expressed in the nonnative host (for instance, a quorum regulator from Agrobacterium tumefaciens requires its own RpoA to function as an activator [27]). Supernatants from the genomic library expressed in E. coli were added to 96-well plates by adding AT medium agar containing the AHL bioassay cells and X-Gal and subsequently screening for AHL activity by identifying blue wells (hydrolysis of X-Gal). This method is much less labor-intensive than traditional transposon mutagenesis, since it is a "gain-of-function" screen without requiring pure culture during the initial screens (see Materials and Methods). In addition, it has a potential advantage of identifying multiple AHL synthase genes from a single genome, genes which otherwise could not be isolated via transposon mutagenesis.
Approximately 50,000 independent E. coli clones containing 2- to 10-kb M. tianshanense DNA fragments were screened, and supernatants from three clones clearly showed strong AHL activity. DNA sequencing (GenBank accession no. DQ123807) revealed that all three had the same 4,063-bp insertion, illustrated in Fig. 2. One 665-bp open reading frame (ORF) encoded a putative protein of 221 amino acids that is highly similar (98% identity at the protein level) to AHL synthase CinI (AAF89990) of R. leguminosarum, which is responsible for synthesizing 3OH-C14:1-HSL molecules (17). This ORF is thus annotated as mrtI (Mesorhizobium tianshanense I). MrtI is also similar to other Rhizobium proteins, such as CinI (AAL59596) (4) and RaiI (AAC38172) (30) from Rhizobium etli (95% and 39% identity, respectively) and TraI (U00090) (12) from Rhizobuim sp. strain NGR234 (28% identity). An ORF upstream of MrtI encodes a putative protein of 272 amino acids similar to several LuxR-type transcriptional activators, such as CinR (98% amino acid identity) of R. leguminosarum (AAF89989), BisR (58% identity) of R. leguminosarum bv. viciae (AA021111), RaiR (32% identity) of R. leguminosarum (CAD 20930), and TraR (28% identity) of R. leguminosarum bv. viciae. This ORF is therefore annotated as mrtR (Mesorhizobium tianshanense R). Of note, the overall nucleotide sequences of the mrtR-mrtI region are very similar to the cinR-cinI region (located on the chromosome) of R. leguminosarum with 90% homology at the DNA sequence level, suggesting a recent DNA horizontal transfer event between these two rhizobia. It would be interesting to determine whether the mrtR-mrtI locus of M. tianshanense is located on the chromosome or on an indigenous megaplasmid, if any such elements exist. In addition, we examined the presence of this mrtR-mrtI locus in other Mesorhizobium strains. A set of specific primers targeting the M. tianshanense mrtR-mrtI region was used to perform PCR amplification on the seven other Mesorhizobium strains shown in Fig. 1A. Even though many of these strains produced AHLs, no PCR product was detected (data not shown). This result suggests that mrtR/mrtI genes are unique to M. tianshanense.
Mutations in mrtI or mrtR completely abolish AHL production in M. tianshanense. To investigate whether LuxR/I homologs MrtR/I obtained from our screens in E. coli are involved in autoinducer production in M. tianshanense, null mutations of mrtI and mrtR were constructed by replacing each gene with a tetracycline-resistant cassette. Compared to the wild-type strain, both mrtI and mrtR null mutant strains have similar growth rates when cultured in TY medium (data not shown), indicating that, unlike in some cases of rhizobial systems (12, 42), mutations in these putative quorum-sensing regulators do not affect growth. Cell-free culture supernatants of these strains were then assayed for AHL activity and AHL content. As noted above, the wild-type supernatant contained high AHL activity (Fig. 3A) and produced at least seven different AHLs on a TLC analysis. Strikingly, no AHL was detected in the supernatant of the mrtI mutants from both liquid assays and TLC assays. Similarly, a deletion in the mrtR gene completely abolished AHL production (Fig. 3). To ensure that the loss of AHL production in the mrtR mutant was not due to a polar effect of the mrtR mutation on downstream mrtI expression, a plasmid containing a Plac-controlled mrtR fusion was introduced to provide the MrtR proteins in trans. Overexpression of MrtR in the mrtR null mutant recovered AHL production in the resulting strain (data not shown).
There are two possibilities for why a deletion in mrtI/mrtR abolishes all AHL production under our testing conditions. One is that MrtI is responsible for synthesizing all these AHLs. Alternatively, MrtI-MrtR are situated at the top of a cascade of other quorum-sensing regulators, similar to the case of CinR-CinI in R. leguminosarum bv. viciae (17). Analysis of the spent culture supernatant of the E. coli strain expressing mrtR and mrtI revealed that at least five different AHLs are made (Fig. 3B). The two minor spots on the TLC plate may not have been visible due to the lower concentration of AHLs produced in E. coli, even though mrtR and mrtI were on a high-copy-number plasmid (Fig. 3A). A twofold dilution of concentrated spent culture supernatant from the wild-type M. tianshanense applied on a TLC plate showed the same five-spot AHL pattern (data not shown). In addition, we have repeated our screen for AHL synthase genes in E. coli several times, and no new synthase genes were identified other than mrtI. These lines of evidence suggest that the mrtI gene may be involved in the synthesis of all AHLs in M. tianshanense. Structural properties of these AHLs produced by MrtI are currently under investigation. Intriguingly, the MrtI protein is virtually identical to the CinI protein from R. leguminosarum (98%), which is responsible for synthesis of 3-OH-C14:1-HSL molecules (17). Different AHL synthases produce AHLs that vary in acyl chain length, oxidation at the C-3 position, and saturation. It has been proposed that this variability is not only a function of the enzyme acyl chain specificity but may also be influenced by the available cellular pool of acyl-ACPs (40).
Autoregulation of mrtI and mrtR genes. In order to study the expression of the AHL synthase gene mrtI, an mrtI-lacZ transcriptional fusion was constructed in a wild-type and mrtR mutant strain of M. tianshanense. This construct also disrupted the mrtI locus (see Materials and Methods); thus, no AHL was produced in these strains (data not shown). Very low levels of -galactosidase activity were detected in the mrtR mutant background even in the presence of AHLs (10% spent culture supernatant of wild-type M. tianshanense) (Fig. 4A, left panel). However, in the mrtR+ background, mrtI was fully induced only when the supernatant prepared from the cell-free spent medium of the wild-type culture (containing the maximal amount of AHLs assayed by an AHL bioassay) was added (Fig. 4A, right panel). Addition of supernatants from the mrtI mutant culture did not induce mrtI-lacZ expression (data not shown). These data indicate that mrtI expression requires both a functional MrtR and autoinducers produced by MrtI.
To investigate mrtR expression, an mrtR-lacZ transcriptional fusion was created by cloning an intact 5'-end mrtR fragment (including the putative promoter region) into the suicide vector pVIK112 (15) and then transforming it into wild-type or the mrtI mutant M. tianshanense to select for Campbell-type integration by homologous recombination. This construct preserved a functional copy of the mrtR gene. Figure 4B shows that mrtR expression is dependent on AHLs. In the mrtI mutant background, mrtR was induced by adding the supernatant containing AHLs, while in the wild-type background, lacZ expression reached high levels at high cell density. Taken together, these data suggest that in M. tianshanense, the expression of both mrtR and mrtI genes is autoregulated, which is a hallmark of quorum-sensing regulation (7).
Influence of quorum sensing on nodulation and root hair attachment. Quorum sensing has been shown to be involved in symbiotic processes in certain rhizobia. For example, compared to wild-type nodules, nodules of cinI mutants of R. etli CNPAF512 exhibited a 60 to 70% reduction in nitrogen fixation efficiency (4). To test if quorum sensing in M. tianshanense plays a role in symbiosis, a nodulation assay on Asian licorice (Glycyrrhiza uralensis) was performed using wild-type and mrtI mutant strains. Seventeen days after inoculation, observations of nodule formation were started from plant roots inoculated with the wild-type culture (54% of plants developed 2.5 nodules on average). The number of wild-type nodules per plant reached a maximal level (all plants were nodulated, with an average of 14.6 nodules per plant) about 2 months after inoculation. In contrast, no nodules formed on the plant roots inoculated with either mrtI or mrtR mutant cultures 2 months after inoculation, indicating that these quorum-sensing mutants are profoundly defective in nodulation. These observations provide compelling evidence that quorum sensing in M. tianshanense is involved in symbiosis.
Nodule formation and symbiotic nitrogen fixation by rhizobia require a number of well-regulated steps. Rhizobium cells attach to legume root hairs before infection, which can be viewed as an early recognition event in the infection process. To examine whether quorum sensing in M. tianshanense regulates root hair adherence, the efficiency of root hair attachment of wild-type and mrtI mutant strains was compared. After a 4-hour root incubation, approximately 5 x 105 wild-type bacteria attached to five roots, but less than 2 x 105 mrtI mutants attached. Addition of spent culture supernatant of wild-type bacteria during the binding assay was able to restore partial attachment efficiency of mrtI mutants. These data suggest that quorum sensing may regulate the bacterial root hair adherence process, thus possibly affecting later nodulation stages. The most apparent selective mechanism of bacterial attachment to root hairs in rhizobium-legume associations involves the specific binding of bacterial hetero-polysaccharides to lectins on root hair surfaces (1, 34). However, the 60% reduction of root hair attachment efficiency of mrtI mutants may not result in the total absence of nodulation. We also performed root hair deformation assays to determine whether quorum sensing is involved in Nod factor production. Nod factors, which are produced by rhizobia upon induction of flavonoids secreted by plant roots and cause root hair deformation, are considered to be essential during the early stages of Rhizobium infection (28). Although the structure of Nod factors and genes involved in Nod factor production in M. tianshanense are currently unknown, we were able to induce root hair deformation with supernatants from M. tianshanense cultured with Asian licorice root extracts. However, both wild-type and quorum-sensing mutants had the same capacity to induce root hair deformation (data not shown), indicating that quorum sensing may not be involved in Nod factor production during M. tianshanense symbiosis. The exact mechanisms by which quorum sensing interacts with the symbiotic process are currently under investigation.
In this study, we identified the quorum-sensing regulators MrtR and MrtI, which are homologs of LuxR and LuxI family proteins in M. tianshanense. The MrtR-MrtI quorum-sensing system controls multiple AHL-like autoinducer production in an autoregulating fashion and is a critical component of the symbiotic colonization of M. tianshanense host plants. This is the first functional analysis of quorum-sensing regulation in the genus Mesorhizobium. M. tianshanense was recently discovered, and genes and gene regulation involved in symbiosis are largely unknown. However, quorum sensing is obviously a key player during the microbe-host plant interaction of this bacterium. Interestingly, M. tianshanense is able to nodulate and fix nitrogen on plants grown in an arid environment. Therefore, further exploration of cell-cell communications between M. tianshanense and bacteria as well as plant hosts is very important from both economic and environmental points of view.
ACKNOWLEDGMENTS
We thank Yunrong Chai, Adam Joelsson, and Ansel Hsiao for helpful discussion and critically reviewing the manuscript.
This study was supported by the NSFC Fund for Distinguished Young Scholars (30325004), NSFC 30570011, Fok Ying Tong Education Foundation (91023), and the 973 project grant (001CB1089).
H.Z. and Z.Z. contributed equally to this work.
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ABSTRACT
The ability of rhizobia to symbiotically fix nitrogen from the atmosphere when forming nodules on their plant hosts requires various signal transduction pathways. LuxR-LuxI-type quorum-sensing systems have been shown to be one of the players in a number of rhizobium species. In this study, we found that Mesorhizobium tianshanense, a moderate-growth Rhizobium that forms nodules on a number of licorice plants, produces multiple N-acyl homoserine lactone (AHL)-like molecules. A simple screen for AHL synthase genes using an M. tianshanense genomic expression library in Escherichia coli, coupled with a sensitive AHL detector, uncovered a LuxI-type synthase, MrtI, and a LuxR-type regulator, MrtR, in M. tianshanense. Deletions of the mrtI or mrtR locus completely abolished AHL production in M. tianshanense. Using lacZ transcriptional fusions, we found that expression of the quorum-sensing regulators is autoinduced, as mrtI gene expression requires MrtR and cognate AHLs and mrtR expression is dependent on AHLs. Compared with the wild-type strains, quorum-sensing-deficient mutants showed a marked reduction in the efficiency of root hair adherence and, more importantly, were defective in nodule formation on their host plant, Glycyrrhiza uralensis. These data provide strong evidence that quorum sensing plays a critical role in the M. tianshanense symbiotic process.
INTRODUCTION
Bacteria exchange small, self-produced chemical molecules to monitor their population density and control a variety of physiological functions in a cell density-dependent manner, a process called quorum sensing (5, 8, 10). Many gram-negative bacteria use a set of diffusible N-acyl homoserine lactones (AHLs, also called autoinducers) that generally serve as cell-to-cell communication signals (39, 41). The key regulatory components of these signaling systems are LuxI-type proteins, which act as AHL synthases, and LuxR-type proteins, which serve as AHL receptors and AHL-dependent transcription factors. In general, the LuxR-like proteins are each responsible for binding a cognate AHL molecule, binding specific target gene promoters, and activating transcription when cognate AHLs have achieved a critical threshold concentration (7, 44, 46). The LuxI-like proteins are required to synthesize AHL molecules, which contain invariant homoserine lactone moieties and highly variable fatty acyl groups. The homoserine lactone moiety is derived from S-adenosylmethionine, while the acyl chains are derived from an acyl-acyl carrier protein (23, 24, 40).
Many important bacterial behaviors are regulated by quorum sensing, including virulence, antibiotic production, biofilm formation, and symbiosis (22, 39). Nitrogen fixation and endosymbiotic interactions between legume plants and the genera Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, and Azorhizobium, collectively termed rhizobia, have been intensively studied (18). Intricate signaling between the host and rhizobial symbiont is required for successful symbiotic interactions, which result in the reduction of atmospheric N2 to ammonia by the bacteroids (32). Quorum sensing has been implicated as a key player in the symbiotic process (11). For example, a few studies have reported that AHL signals produced by several species of Rhizobium and Sinorhizobium appear to control various functions, such as exopolysaccharide production (14, 19, 25), plasmid transfer (12, 36), and nodulation (3, 4, 29), all of which are related to symbiosis. In Rhizobium leguminosarum bv. viciae, four quorum-sensing systems (rai, rhi, cin, and tra) have been identified and are intertwined in a complex regulatory network. The cin locus is situated at the top of the quorum-sensing network. Mutations of cinI and cinR abolish the production of 3-OH-C14:1-HSL and cause a decrease in levels of all of the short-chain AHLs produced by the enzymes encoded by raiI, traI, and rhiI (17). Examination of mutations in either rhiA and rhiR for nodulation of beans showed a decreased number of nodules, but only in combination with a nodFE mutant, leading to the hypothesis that the rhi system seems to play a role in nodulation efficiency (3). The tra system is clearly shown to regulate the conjugal transfer of pRL1J1, a symbiotic plasmid, but the advantage of having plasmid transfer under the control of the cin system is still unknown (43).
To date there have been no detailed studies on quorum-sensing regulatory systems in the Mesorhizobium genus, a moderately growing rhizobium. However, genome sequences of Mesorhizobium loti predict the presence of several LuxI-LuxR family proteins. In previous reports, we described the detection of AHL-like quorum-sensing signals from Mesorhizobium huakuii (45) and studied the possible roles of quorum sensing in biofilm formation in this strain (38). In the present study, we detected AHL signals from an M. tianshanense strain, a moderately growing root nodule bacterium which was originally isolated from an arid saline desert soil in northwestern China in 1995 (2). M. tianshanense was later widely found in dry soils and acts as a nitrogen-fixing symbiont for at least eight different plant species, including Glycyrrhiza (licorice) (35), whose roots are one of the most important crude medicines in Asia and Europe. We have developed a novel method to identify the AHL synthase gene from M. tianshanense and found that quorum sensing in M. tianshanense plays a critical role in symbiosis.
MATERIALS AND METHODS
Bacterial strains, plasmids, and culture conditions. Bacterial strains and plasmids used in this study are listed in Table 1. Mesorhizobium strains, which have been deposited in the Culture Collection of Beijing Agricultural University (CCBAU; Beijing, China), were grown at 28°C in TY medium (37). Escherichia coli was grown at 37°C in LB medium (31), and Agrobacterium tumefaciens was grown at 28°C in AT medium (9). For M. tianshanense, a spontaneous streptomycin-resistant mutant of CCBAU 3306, HMZ0, was used as the parental strain in this study. Chromosomal mrtI-lacZ and mrtR-lacZ transcriptional reporter fusions were constructed by PCR, amplifying the internal fragment of mrtI and intact 5' mrtR (including its putative promoter region), and these fragments were cloned into pVIK112 (15). The resulting plasmids were then integrated into the chromosome at the mrtI and mrtR loci, respectively, by homologous recombination. In-frame deletions in the mrtI and mrtR genes were constructed by overlapping PCR of flanking regions of the target genes and cloning into the pWM91 suicide vector (20). The resulting plasmids were introduced into HMZ0, and double-crossover events were selected on sucrose plates (10%) after the first "cross-on" homologous recombination. A plasmid that constitutively expresses mrtR was constructed by cloning of the mrtR genes into the pBBR1-MCS5 vector (16) and introduced into M. tianshanense strains by electroporation. -Galactosidase activity assays were performed as previously described (21).
Screening of M. tianshanense AHL synthase genes. Two- to 10-kb fragments of HMZ0 genomic DNA partially digested with HincII were cloned into the pEZSeq-Kan vector using the pEZSeq cloning kit (Lucigen, Wisconsin). Approximately 50,000 transformants with insertions were pooled and saved in 20% glycerol at –70°C. The library was then inoculated into LB medium containing appropriate antibiotics in 96-well plates at a dilution of approximately 20 cells per well. After overnight incubation at 37°C, 20 μl of supernatant from each well was transferred to a new 96-well plate in which each well was overlaid with 200 μl AT medium (9) agar (0.5%) containing 107 cells/ml AHL bioassay strain cells (45) and 5-bromo-4-chloro-3-indolyl -D-galactoside (X-Gal; 20 μg/ml) in each well. Plates were incubated at 28°C for 12 to 16 h and examined for X-Gal hydrolysis (blue color). For each blue-colored well, bacteria were then streaked out to single colonies from the corresponding original wells and inoculated to LB medium in 96-well plates to repeat the above assays. Plasmid DNA was purified from E. coli transformants that were able to produce AHLs to activate AHL bioassay reporters and subjected to DNA sequencing.
AHL bioassays. Spent medium (10%, vol/vol) from Mesorhizobium strains collected from various time points as indicated was added into AT medium (9), and approximately 107 AHL bioassay strain cells per ml (45) were added. These cultures were incubated with aeration for 12 h and assayed for -galactosidase specific activity (21).
Thin-layer chromatography (TLC) analysis of AHLs released by Mesorhizobium strains was performed as previously described (33). Aliquots from ethyl acetate extracts of spent media were applied to C18 reversed-phase TLC plates (Whatman), chromatographed, and developed as previously described (38).
Nodulation assays. Glycyrrhiza uralensis (Asian licorice) seeds were treated with 75% ethanol and 0.1% HgCl2 and were surface sterilized. The treated seeds were then placed in petri dishes, germinated in the dark at 28°C for 2 days, and then planted in 200- by 30-mm test tubes (three seeds per tube) filled with autoclaved Vermiculite and Perlite (10:1) and a plant nutrient solution of Fahraeus medium (6) supplemented with 28 mg N/kg Vermiculite. Plants were grown in a plant growth chamber at 28°C with a 16-h-8-h day-night cycle. After 6 days, plants were inoculated with 300-μl cultures of wild-type or mutant M. tianshanense grown in TY medium (optical density at 600 nm [OD600] of 2.0). The plants were pulled out to count the number of nodules at the time indicated. There were at least 12 replicates for each inoculation.
Root hair attachment assay. Seeds of Glycyrrhiza uralensis were surface sterilized and germinated as described above. Fifteen seedlings roots, approximately 1.5 cm in length, were incubated for 4 h at 28°C in 30 ml of 25 mM phosphate buffer (pH 7.5) containing 300 μl of cell suspension (OD600, 1.5) in the absence or presence of 10% cell-free supernatants of wild-type cultures. After incubation, roots were washed with 2 ml phosphate buffer three times with gentle shaking. The number of attached bacteria was determined by vortexing roots with glass beads and plating the cell suspension on TY medium plates.
Root hair deformation assays. Root hair deformation assays were performed according to previous publications (13, 26) with a few modifications. Briefly, M. tianshanense strains (wild type and quorum-sensing mutants) were grown in yeast mannitol medium (37), and at an OD600 of 0.3, 10% seed extract of Glycyrrhiza uralensis was added. Cultures were incubated for an additional 36 h. Bacterium-free supernatant containing Nod factors was stored at –20°C. Glycyrrhiza uralensis seedlings were then incubated with 10% M. tianshanense supernatants prepared as described above in petri dishes for 2 days. Roots were microscopically examined, and the number of deformed root hairs in the susceptible zone was counted.
Nucleotide sequence accession number. The mrtR-mrtI DNA sequence reported here has been deposited in the GenBank database under accession number DQ123807.
RESULTS AND DISCUSSION
AHL production in various Mesorhizobium strains. Although quorum-sensing regulation has been studied extensively in a number of rhizobia, there are limited studies on this system in Mesorhizobium species. To investigate whether quorum sensing plays any role in Mesorhizobium biology, the spent culture supernatants from eight type strains of the genus Mesorhizobium were examined for AHL production. High-cell-density cultures were extracted with ethyl acetate and subjected to thin-layer chromatograph detection of AHL content. The AHL profiles of these eight strains were quite different (Fig. 1A). M. chaoconense, M. plurifarium, M. mediterraneum, and M. tianshanense produced multiple AHL-like molecules. A single spot was detected from the extracts of M. amorphae and M. huakuii supernatants, confirming our previous report (45). No AHL-like molecules were detected from supernatants of M. loti and M. ciceri under these conditions. Interestingly, at least four annotated AHL synthase genes exist, based on M. loti genome sequencing. Overexpressing one of these synthase genes in E. coli produced at least three different AHLs (data not shown). These results indicated that those AHL synthase genes in M. loti are not expressed under routine culture conditions.
M. tianshanense, which showed the greatest diversity of AHL signaling molecule production among the Mesorhizobium strains tested, was selected for further studies. AHL activities from supernatants collected during the growth of a spontaneous streptomycin-resistant mutant (HMZ0) of the type strain CCBAU 3306 were assayed by measuring the induction of a lacZ reporter of an AHL bioassay strain (45). As shown in Fig. 1B, accumulation of AHL activity in supernatants of HMZ0 displayed a typical cell density-dependent pattern, with low AHL activity at low cell density (OD600 below 1) and higher activities as the bacterial culture grew.
Identification of a LuxI/LuxR-type quorum-sensing regulatory system in M. tianshanense. In an effort to identify the genes involved in AHL production in M. tianshanense, we developed a novel, simple screening method. Briefly, a genomic library of M. tianshanense was constructed on a high-copy-number vector flanked by a Plac promoter, which may turn on transcription of synthase genes that might not be expressed in the nonnative host (for instance, a quorum regulator from Agrobacterium tumefaciens requires its own RpoA to function as an activator [27]). Supernatants from the genomic library expressed in E. coli were added to 96-well plates by adding AT medium agar containing the AHL bioassay cells and X-Gal and subsequently screening for AHL activity by identifying blue wells (hydrolysis of X-Gal). This method is much less labor-intensive than traditional transposon mutagenesis, since it is a "gain-of-function" screen without requiring pure culture during the initial screens (see Materials and Methods). In addition, it has a potential advantage of identifying multiple AHL synthase genes from a single genome, genes which otherwise could not be isolated via transposon mutagenesis.
Approximately 50,000 independent E. coli clones containing 2- to 10-kb M. tianshanense DNA fragments were screened, and supernatants from three clones clearly showed strong AHL activity. DNA sequencing (GenBank accession no. DQ123807) revealed that all three had the same 4,063-bp insertion, illustrated in Fig. 2. One 665-bp open reading frame (ORF) encoded a putative protein of 221 amino acids that is highly similar (98% identity at the protein level) to AHL synthase CinI (AAF89990) of R. leguminosarum, which is responsible for synthesizing 3OH-C14:1-HSL molecules (17). This ORF is thus annotated as mrtI (Mesorhizobium tianshanense I). MrtI is also similar to other Rhizobium proteins, such as CinI (AAL59596) (4) and RaiI (AAC38172) (30) from Rhizobium etli (95% and 39% identity, respectively) and TraI (U00090) (12) from Rhizobuim sp. strain NGR234 (28% identity). An ORF upstream of MrtI encodes a putative protein of 272 amino acids similar to several LuxR-type transcriptional activators, such as CinR (98% amino acid identity) of R. leguminosarum (AAF89989), BisR (58% identity) of R. leguminosarum bv. viciae (AA021111), RaiR (32% identity) of R. leguminosarum (CAD 20930), and TraR (28% identity) of R. leguminosarum bv. viciae. This ORF is therefore annotated as mrtR (Mesorhizobium tianshanense R). Of note, the overall nucleotide sequences of the mrtR-mrtI region are very similar to the cinR-cinI region (located on the chromosome) of R. leguminosarum with 90% homology at the DNA sequence level, suggesting a recent DNA horizontal transfer event between these two rhizobia. It would be interesting to determine whether the mrtR-mrtI locus of M. tianshanense is located on the chromosome or on an indigenous megaplasmid, if any such elements exist. In addition, we examined the presence of this mrtR-mrtI locus in other Mesorhizobium strains. A set of specific primers targeting the M. tianshanense mrtR-mrtI region was used to perform PCR amplification on the seven other Mesorhizobium strains shown in Fig. 1A. Even though many of these strains produced AHLs, no PCR product was detected (data not shown). This result suggests that mrtR/mrtI genes are unique to M. tianshanense.
Mutations in mrtI or mrtR completely abolish AHL production in M. tianshanense. To investigate whether LuxR/I homologs MrtR/I obtained from our screens in E. coli are involved in autoinducer production in M. tianshanense, null mutations of mrtI and mrtR were constructed by replacing each gene with a tetracycline-resistant cassette. Compared to the wild-type strain, both mrtI and mrtR null mutant strains have similar growth rates when cultured in TY medium (data not shown), indicating that, unlike in some cases of rhizobial systems (12, 42), mutations in these putative quorum-sensing regulators do not affect growth. Cell-free culture supernatants of these strains were then assayed for AHL activity and AHL content. As noted above, the wild-type supernatant contained high AHL activity (Fig. 3A) and produced at least seven different AHLs on a TLC analysis. Strikingly, no AHL was detected in the supernatant of the mrtI mutants from both liquid assays and TLC assays. Similarly, a deletion in the mrtR gene completely abolished AHL production (Fig. 3). To ensure that the loss of AHL production in the mrtR mutant was not due to a polar effect of the mrtR mutation on downstream mrtI expression, a plasmid containing a Plac-controlled mrtR fusion was introduced to provide the MrtR proteins in trans. Overexpression of MrtR in the mrtR null mutant recovered AHL production in the resulting strain (data not shown).
There are two possibilities for why a deletion in mrtI/mrtR abolishes all AHL production under our testing conditions. One is that MrtI is responsible for synthesizing all these AHLs. Alternatively, MrtI-MrtR are situated at the top of a cascade of other quorum-sensing regulators, similar to the case of CinR-CinI in R. leguminosarum bv. viciae (17). Analysis of the spent culture supernatant of the E. coli strain expressing mrtR and mrtI revealed that at least five different AHLs are made (Fig. 3B). The two minor spots on the TLC plate may not have been visible due to the lower concentration of AHLs produced in E. coli, even though mrtR and mrtI were on a high-copy-number plasmid (Fig. 3A). A twofold dilution of concentrated spent culture supernatant from the wild-type M. tianshanense applied on a TLC plate showed the same five-spot AHL pattern (data not shown). In addition, we have repeated our screen for AHL synthase genes in E. coli several times, and no new synthase genes were identified other than mrtI. These lines of evidence suggest that the mrtI gene may be involved in the synthesis of all AHLs in M. tianshanense. Structural properties of these AHLs produced by MrtI are currently under investigation. Intriguingly, the MrtI protein is virtually identical to the CinI protein from R. leguminosarum (98%), which is responsible for synthesis of 3-OH-C14:1-HSL molecules (17). Different AHL synthases produce AHLs that vary in acyl chain length, oxidation at the C-3 position, and saturation. It has been proposed that this variability is not only a function of the enzyme acyl chain specificity but may also be influenced by the available cellular pool of acyl-ACPs (40).
Autoregulation of mrtI and mrtR genes. In order to study the expression of the AHL synthase gene mrtI, an mrtI-lacZ transcriptional fusion was constructed in a wild-type and mrtR mutant strain of M. tianshanense. This construct also disrupted the mrtI locus (see Materials and Methods); thus, no AHL was produced in these strains (data not shown). Very low levels of -galactosidase activity were detected in the mrtR mutant background even in the presence of AHLs (10% spent culture supernatant of wild-type M. tianshanense) (Fig. 4A, left panel). However, in the mrtR+ background, mrtI was fully induced only when the supernatant prepared from the cell-free spent medium of the wild-type culture (containing the maximal amount of AHLs assayed by an AHL bioassay) was added (Fig. 4A, right panel). Addition of supernatants from the mrtI mutant culture did not induce mrtI-lacZ expression (data not shown). These data indicate that mrtI expression requires both a functional MrtR and autoinducers produced by MrtI.
To investigate mrtR expression, an mrtR-lacZ transcriptional fusion was created by cloning an intact 5'-end mrtR fragment (including the putative promoter region) into the suicide vector pVIK112 (15) and then transforming it into wild-type or the mrtI mutant M. tianshanense to select for Campbell-type integration by homologous recombination. This construct preserved a functional copy of the mrtR gene. Figure 4B shows that mrtR expression is dependent on AHLs. In the mrtI mutant background, mrtR was induced by adding the supernatant containing AHLs, while in the wild-type background, lacZ expression reached high levels at high cell density. Taken together, these data suggest that in M. tianshanense, the expression of both mrtR and mrtI genes is autoregulated, which is a hallmark of quorum-sensing regulation (7).
Influence of quorum sensing on nodulation and root hair attachment. Quorum sensing has been shown to be involved in symbiotic processes in certain rhizobia. For example, compared to wild-type nodules, nodules of cinI mutants of R. etli CNPAF512 exhibited a 60 to 70% reduction in nitrogen fixation efficiency (4). To test if quorum sensing in M. tianshanense plays a role in symbiosis, a nodulation assay on Asian licorice (Glycyrrhiza uralensis) was performed using wild-type and mrtI mutant strains. Seventeen days after inoculation, observations of nodule formation were started from plant roots inoculated with the wild-type culture (54% of plants developed 2.5 nodules on average). The number of wild-type nodules per plant reached a maximal level (all plants were nodulated, with an average of 14.6 nodules per plant) about 2 months after inoculation. In contrast, no nodules formed on the plant roots inoculated with either mrtI or mrtR mutant cultures 2 months after inoculation, indicating that these quorum-sensing mutants are profoundly defective in nodulation. These observations provide compelling evidence that quorum sensing in M. tianshanense is involved in symbiosis.
Nodule formation and symbiotic nitrogen fixation by rhizobia require a number of well-regulated steps. Rhizobium cells attach to legume root hairs before infection, which can be viewed as an early recognition event in the infection process. To examine whether quorum sensing in M. tianshanense regulates root hair adherence, the efficiency of root hair attachment of wild-type and mrtI mutant strains was compared. After a 4-hour root incubation, approximately 5 x 105 wild-type bacteria attached to five roots, but less than 2 x 105 mrtI mutants attached. Addition of spent culture supernatant of wild-type bacteria during the binding assay was able to restore partial attachment efficiency of mrtI mutants. These data suggest that quorum sensing may regulate the bacterial root hair adherence process, thus possibly affecting later nodulation stages. The most apparent selective mechanism of bacterial attachment to root hairs in rhizobium-legume associations involves the specific binding of bacterial hetero-polysaccharides to lectins on root hair surfaces (1, 34). However, the 60% reduction of root hair attachment efficiency of mrtI mutants may not result in the total absence of nodulation. We also performed root hair deformation assays to determine whether quorum sensing is involved in Nod factor production. Nod factors, which are produced by rhizobia upon induction of flavonoids secreted by plant roots and cause root hair deformation, are considered to be essential during the early stages of Rhizobium infection (28). Although the structure of Nod factors and genes involved in Nod factor production in M. tianshanense are currently unknown, we were able to induce root hair deformation with supernatants from M. tianshanense cultured with Asian licorice root extracts. However, both wild-type and quorum-sensing mutants had the same capacity to induce root hair deformation (data not shown), indicating that quorum sensing may not be involved in Nod factor production during M. tianshanense symbiosis. The exact mechanisms by which quorum sensing interacts with the symbiotic process are currently under investigation.
In this study, we identified the quorum-sensing regulators MrtR and MrtI, which are homologs of LuxR and LuxI family proteins in M. tianshanense. The MrtR-MrtI quorum-sensing system controls multiple AHL-like autoinducer production in an autoregulating fashion and is a critical component of the symbiotic colonization of M. tianshanense host plants. This is the first functional analysis of quorum-sensing regulation in the genus Mesorhizobium. M. tianshanense was recently discovered, and genes and gene regulation involved in symbiosis are largely unknown. However, quorum sensing is obviously a key player during the microbe-host plant interaction of this bacterium. Interestingly, M. tianshanense is able to nodulate and fix nitrogen on plants grown in an arid environment. Therefore, further exploration of cell-cell communications between M. tianshanense and bacteria as well as plant hosts is very important from both economic and environmental points of view.
ACKNOWLEDGMENTS
We thank Yunrong Chai, Adam Joelsson, and Ansel Hsiao for helpful discussion and critically reviewing the manuscript.
This study was supported by the NSFC Fund for Distinguished Young Scholars (30325004), NSFC 30570011, Fok Ying Tong Education Foundation (91023), and the 973 project grant (001CB1089).
H.Z. and Z.Z. contributed equally to this work.
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