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Journal of Bacteriology, February 2008, p. 861-871, Vol. 190, No. 3
0021-9193/08/$08.00+0     doi:10.1128/JB.01310-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Regulation of Motility by the ExpR/Sin Quorum-Sensing System in Sinorhizobium meliloti{triangledown} ,{dagger}

Hanh H. Hoang,{ddagger} Nataliya Gurich, and Juan E. González*

Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas 75083-0688

Received 12 August 2007/ Accepted 7 November 2007


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ABSTRACT
 
A successful symbiotic relationship between Sinorhizobium meliloti and its host Medicago sativa (alfalfa) depends on several signaling mechanisms, such as the biosynthesis of exopolysaccharides (EPS) by S. meliloti. Previous work in our laboratory has shown that a quorum-sensing mechanism controls the production of the symbiotically active EPS II. Recent microarray analysis of the whole-genome expression profile of S. meliloti reveals that the ExpR/Sin quorum-sensing system regulates additional physiological processes that include low-molecular-weight succinoglycan production, nitrogen utilization, metal transport, motility, and chemotaxis. Nearly half of the flagellar genes and their dependence on quorum sensing are prominently displayed in our microarray analyses. We extend those observations in this work and confirm the findings by real-time PCR expression analysis of selected genes, including the flaF, flbT, flaC, cheY1, and flgB genes, involved in motility and chemotaxis. These genes code for regulators of flagellum synthesis, the chemotactic response, or parts of the flagellar apparatus. Gene expression analyses and visualization of flagella by electron microscopy performed at different points in the growth phase support our proposed model in which quorum sensing downregulates motility in S. meliloti. We demonstrate that the ExpR/Sin quorum-sensing system controls motility gene expression through the VisN/VisR/Rem relay. We also show that the ExoS-dependent two-component system suppresses motility gene expression through VisN and Rem in parallel to quorum sensing. This study contributes to our understanding of the mechanisms that govern motility in S. meliloti.


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INTRODUCTION
 
An organism's ability to sense and move toward a favorable environment or away from a hazardous one provides it with a substantial and often crucial advantage over nonmotile competitors. More than 70% of microorganisms are motile and chemotactic, and much of the ground-breaking work in this field has been derived from the studies of such bacteria as Escherichia coli, Pseudomonas aeruginosa, and Serratia marcescens (12, 26, 29, 40, 47, 52, 57). Although equipped with an extremely useful ability, motile organisms must expend large amounts of energy to synthesize, assemble, and activate the machinery for motility and chemotaxis. As much as 2% of the energy expenditure in the cell can be attributed to the maintenance of such activities; therefore, it is often in the bacterium's best interest to strictly regulate its ability to move toward those conditions most favorable for survival (35, 52). Many factors can influence an organism's movement including, but not limited to, temperature, pH, ion concentration, and the availability of metabolites (52). There is increasing evidence that connects the mechanism of quorum sensing, a population-density-dependent mode of gene regulation, to the control of motility. In S. marcescens, Vibrio cholerae, and E. coli, production of the motility machinery is upregulated as the population density increases (3, 14, 53, 54, 61). In these organisms, activation of motility at a high population density probably serves to disperse the bacteria from a nutrient-depleted environment or to initiate the search for a new host.

In addition to quorum sensing, studies in several organisms have established a connection between the production of bacterial polysaccharides and motility. For example, the Burkholderia solanacearum phcA gene is necessary for the production of a virulence-associated exopolysaccharide (EPS), and a mutant lacking this function also exhibits increased motility (7). Boles and McCarter showed that the presence of the Vibrio parahaemolyticus scrABC gene operon activates swarming motility and decreases the production of capsular polysaccharides, both of which are important for colonization of the host (6). In addition, the igaA gene product from Salmonella enterica positively controls motility while demonstrating a negative effect on the wca genes involved in capsule synthesis (9). Furthermore, the ahlI, ahlR, and aefR genes in the plant pathogen Pseudomonas syringae positively regulate EPS production but inhibit swarming motility (44).

Sinorhizobium meliloti, a soil bacterium from the {alpha}-proteobacteria family, is capable of fixing atmospheric nitrogen for its leguminous alfalfa host (Medicago sativa) under nitrogen-limiting conditions (27, 34, 56). This symbiotic interaction involves multiple complex signaling mechanisms between both partners. Studies from several laboratories, in addition to the completed S. meliloti genome, have substantially contributed to our understanding of the genetic components of this bacterium's flagellar system. The S. meliloti genome contains at least 41 motility- and chemotaxis-related genes that are chromosomally located in a 45-kb region commonly referred to as the "flagellar regulon" (51). These genes are categorized into three main classes and are expressed in a hierarchical manner. Class I genes (visN and visR) lie at the apex and consequently control the expression of the genes in class II and class III (49). Class II is subdivided into class IIA (fliM and orf38) and class IIB (mot) genes, which participate in flagellar assembly and motor functions, respectively. Only the class IIA genes exert control over the expression of the genes in class III. The third group of genes (fla and che) includes those encoding the flagellin subunits and chemotactic proteins. The flagellar system in S. meliloti differs from that of E. coli in several ways. S. meliloti harbors the visN/visR operon instead of the master regulatory operon, flhDC (33, 49). The visN and visR genes are independently transcribed, and their gene products are believed to form heterodimers that contain the N-terminal ligand-binding and C-terminal DNA-binding motifs typically seen in the LuxR-family of transcriptional regulators (49). The mot genes in S. meliloti are placed in the second class rather than in the third class as they are in E. coli. Furthermore, the rotation of the four to eight peritrichous flagella present in S. meliloti occurs strictly in a clockwise direction, and the direction of movement is modulated by the speed of the rotary motors (25, 43, 50). In E. coli, the switch between clockwise and counterclockwise rotation of the flagellar bundles changes the direction of movement (15).

Previous work has shed some light on the mechanisms that control motility in S. meliloti. Sourjik et al. (49) showed that a mutation in either visN or visR is sufficient to dramatically decrease the expression of representative flagellar and chemotaxis genes in class II and class III, which, in turn, abolishes motility in the S. meliloti strain RU10. The regulatory mechanism utilized by the visN/visR operon remains unclear. More recently, work by Rotter et al. demonstrated that an OmpR-like regulator, which the authors named Rem, controls the expression of several classes of motility genes in S. meliloti strain RU11 (46). Rem seems to directly regulate motility by binding to the promoters of flgB, fliF, orf38, and rem, and its activation depends on the VisN/VisR regulators. Yao et al. (60) and more recently Wells et al. (59) revealed that the expression of several flagellar genes and the production of flagella are downregulated in the S. meliloti strain Rm1021 that contains either a mutation in the gene exoS, which is part of a two-component sensor-kinase system, or exoR, a gene with no known homology (10, 45, 59, 60). Work from our laboratory has shown that the process of quorum sensing may also play a role in the expression of various motility related genes. The Sin quorum-sensing system in S. meliloti is comprised of the SinR transcriptional regulator and the SinI autoinducer synthase, which specifies the synthesis of at least five different N-acyl homoserine lactone (AHL) signaling molecules (38). In conjunction with the ExpR transcriptional regulator, the sin AHLs control numerous cell functions that play important roles in the bacteria's ability to form a successful symbiotic association with its alfalfa host plant (36). Through the use of DNA microarray expression profiling, we showed that motility genes are among those that rely on the ExpR/Sin quorum-sensing system (28). These genes encode many of the components of the motility apparatus, including the basal body, hook, and filament structures, and two transcriptional regulators. In the present study, we demonstrate that the ExpR/Sin quorum-sensing system and the ExoR/ExoS/ChvI pathway both control motility gene expression via the visN/visR operon. Furthermore, in contrast to well-characterized systems such as that of E. coli and S. marcescens, the motility system in S. meliloti is downregulated with the increase in population density.


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MATERIALS AND METHODS
 
Bacterial strains and media conditions. Table 1 lists the bacterial strains used in this study. Starter cultures were grown in 2 ml of TYC broth (5 g of tryptone, 3 g of yeast extract, and 0.4 g of CaCl2/liter) with streptomycin (500 µg/ml) for 2 days at 30°C. The strains were subcultured (1:100) in 20 ml of minimal glutamate mannitol (MGM) low-phosphate medium (50 mM morpholinepropanesulfonic acid, 19 mM sodium glutamate, 55 mM mannitol, 0.1 mM K2HPO4-KH2PO4, 1 mM MgSO4, 0.25 mM CaCl2, 0.004 mM biotin [pH 7]) and grown at 30°C with constant shaking. Luria-Bertani (LB) medium is supplemented with 2.5 mM MgSO4 and 2.5 mM CaCl2. When necessary, tetracycline (10 µg/ml), chloramphenicol (20 µg/ml), gentamicin (75 µg/ml), hygromycin (75 to 100 µg/ml), or neomycin (200 µg/ml) was added.


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  		TABLE 1. Bacterial strains used in this study

Construction of S. meliloti strains. To construct the visN mutant, we first PCR amplified and cloned a fragment (using primers 1 and 2, Table 2) containing the visN open reading frame into the SpeI site of pJQ200SmSp. In parallel, the hygromycin cassette containing visN-specific 5'-linkers was amplified with primers 3 and 4 and then used as a primer for insertion/deletion PCR (KOD polymerase; Novagen) using a modified version of the protocol described by Geiser et al. (19). The PCR conditions were as follows: 95°C for 5 min; 30 cycles of 95°C for 30 s, 55°C for 30 s, and 68°C for 12 min; a final extension at 68°C for 10 min. The template for the insertion/deletion PCR was pVisN/sacB. The plasmid carrying the visN mutation was transferred into Rm8530 and its derivatives by triparental mating and selected on medium containing 5% sucrose and the appropriate antibiotics as described previously (22). The SMc03046 (rem) mutant was constructed by a similar strategy. In this case, a PCR fragment consisting of the SMc03046 open reading frame was amplified with primers 5 and 6 and then cloned into the NotI site of pPCR-Script Amp SK(+). The gentamicin cassette containing SMc03046-specific 5' linkers was amplified with primers 7 and 8, and this PCR product was inserted within the SMc03046 sequence of the SMc03046/pPCR-Script Amp SK(+) clone. The insertion/deletion PCR conditions consisted of the following: 95°C for 5 min; 30 cycles of 95°C for 30 s, 55°C for 30 s, 68°C for 8 min; a final extension at 68°C for 10 min. Next, the Gm-SMc03046 fragment was amplified with primers 5 and 6 and cloned into the NotI site of pJQ200SmSp. After homogenization, the transconjugates were selected on medium containing 5% sucrose with antibiotics. Mutations were transferred back into the Rm8530 wild-type strain or its derivatives using {phi}M12h1 as described elsewhere (22). Likewise, strain derivatives that carry the exoS mutation were constructed by phage transduction with {phi}M12h1 grown on Rm1021 exoS.


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  		TABLE 2. Primers used for gene cloning and disruption

Complementation assays. Cultures of Rm8530 were grown in 20 ml of MGM low-phosphate medium to late log phase. The AHLs from bacterial cells and culture supernatant were extracted with an equal volume of dichloromethane, dried, and resuspended in 200 µl of 70% methanol. These AHLs were added to a growing culture of Rm8530 sinI at optical densities at 600 nm (OD600) of 0.4, 0.7, and 1.0. Cells were then collected at an OD600 of 1.2, and gene expression analysis was performed as described below.

RNA purification. Bacterial cultures were grown to an OD600 of 0.2, 0.8, or 1.2 in MGM low-phosphate medium (0.1 mM phosphate) supplemented with 500 µg of streptomycin/ml. Cells were harvested by centrifugation (10,000 x g for 1 min at 4°C), and cell pellets were immediately frozen in liquid nitrogen. Total RNA was purified by using the RNeasy MiniKit (Qiagen, Hilden, Germany). Cells were resuspended in 10 mM Tris-HCl (pH 8.0) and disrupted in RLT buffer (supplemented with β-mercaptoethanol) provided with the RNeasy kit in Fast Protein tubes (MP Biomedicals, Solon, OH) using the Ribolyser (Hybaid, Heidelberg, Germany) (30 s, level 6.5) prior to spin column purification according to the RNeasy MiniKit RNA purification protocol. The RNA samples were treated with the Qiagen on-column RNase-free DNase kit and further purified. Samples were DNase-treated a second time with the Turbo RNase-free DNase (Ambion) according to the manufacturer's instructions. An additional RNA clean-up step was performed, and the concentrations of the samples were determined with a spectrophotometer at A260 for cDNA synthesis.

Affymetrix GeneChip microarray hybridization and expression analysis. The cDNA synthesis, labeling, and hybridization to the S. meliloti/M. truncatula Affymetrix GeneChip (Santa Clara, CA) were performed according to a previously described protocol (5) by the Core Microarray facility at UT Southwestern Medical Center (Dallas, TX). For each experiment, 10 µg of total RNA were prepared from two independent biological replicates per strain that were grown to mid- and late-logarithmic phases (OD600 values of 0.8 and 1.2, respectively). The GeneChip Scanner 3000 was used to measure the signal intensity of each array, and all probe sets were scaled to the target signal value of 500. The .CEL files that were generated by Affymetrix GeneChip Operating Software (GCOS version 1.4) were used for data analysis by the GeneSifter (VizX Labs) software. The arrays for gene expression in Rm8530 (considered here as the wild-type strain) were used as the baseline, or reference sample, to compare expression in the sinI mutant strain. The fold change in gene expression was calculated as the log2 X/Y, where X is the signal intensity for the sinI mutant, and Y is the signal intensity for Rm8530. Genes were considered differentially expressed if the fold change in expression was ≥2 or ≤–2 and the P value was ≤0.05.

Real-time PCR analysis. The first-strand cDNA mixture for each strain was prepared with the RETROscript kit from Ambion using 1.0 µg of total RNA per reaction, and 1 µl of the cDNA reaction was used as a template for the real-time PCR setup. The probe and primer sequences, designed by using Beacon Designer 2.1, are listed in Table 3. The SMc00128 gene (30) was one of several used as a control for equal loading in each real-time PCR. Each reaction mixture contains 0.3 µM sense oligonucleotide, 0.3 µM antisense oligonucleotide, 0.2 µM TaqMan probe, 0.5 of an Omni Mix HS PCR beads (each PCR bead contains 1.5 U of Taq DNA polymerase, 10 mM Tris-HCl [H 9.0], 50 mM KCl, 1.5 mM MgCl2, 200 µM deoxynucleoside triphosphate, and stabilizers including bovine serum albumin) in a 25 µl-reaction volume. The experiment was performed with the Cepheid Smart Cycler v2.0c programmed as follows: stage 1, 95°C for 120 s; and stage 2 (two-temperature cycle repeat for 40 times), 95°C for 15 s, 60°C for 30 s. A difference of one threshold cycle (Cy) value equals a twofold change in gene expression. The fold change was calculated as 2{Delta}Ct, where Ct indicates the level of gene expression in the specified strain.


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  		TABLE 3. Probe and primer sequences used in real-time PCR analysis

Examination of flagella by electron microscopy. Cultures were grown in minimal low phosphate medium supplemented with streptomycin to OD600s of 0.2 and 1.2 for early and late logarithmic growth, respectively. Three biological replicas for each strain and condition were evaluated. The samples were prepared by placing 10 µl of the culture on a carbon/Formvar-coated grid (400 mesh; Electron Microscopy Sciences) for 2 min and then removing excess fluids with the edge of a damp sheet of filter paper. Samples were stained with 10 µl of 1% phosphotungstic acid for 10 s, and again the excess fluids were removed. After a wash with 10 µl of double-distilled H2O for 20 s and removal of the remaining fluids, the grids were air dried for 10 min. The samples were viewed at x4,000 magnification with the Zeiss 906E transmission electron microscope at 60 kV. Between 600 and 1,000 bacteria were examined in each sample, and the total number of flagella was divided by the number of cells to determine the number of flagella per cell.


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RESULTS
 
Quorum sensing downregulates flagellar gene expression in S. meliloti. We previously demonstrated, by DNA microarray expression profiling, that the ExpR quorum-sensing regulator in conjunction with the sin AHLs control numerous cell functions, including the biosynthesis of EPS II, a polymer that plays an important role in the bacteria's ability to form a successful symbiotic association with its alfalfa host plant (36). In that work, we observed that the expression of numerous genes involved in motility and chemotaxis is dramatically elevated in the absence of the sin-specified autoinducer signaling molecules. At least half of the flagellar genes are quorum sensing regulated, including representatives from the flg (class II), fli (class II), and fla (class III) gene clusters (28). These experiments have all been performed in low-phosphate minimal media (see Materials and Methods) in an attempt to replicate the conditions in the soil where the levels of nutrients and phosphate are scarce. Our laboratory and others have shown that S. meliloti grown in this medium produces large amounts of EPSs (23, 39), is highly motile under low-cell-population-density conditions (this study), and efficiently invades its host plant (24, 28, 36, 39).

We have confirmed our earlier findings here by utilization of the Affymetrix GeneChip, which comprises the complete genome of S. meliloti Rm1021 and Medicago truncatula. In our previous work, we had suggested that perhaps the number of differentially regulated genes extracted from that study was actually an underestimation. Indeed, although the Affymetrix data correlate well with our previous spotted microarray data, many more motility-related genes appear as upregulated in the quorum-sensing mutant in the Affymetrix data set (see additional information in the supplemental material). Several factors may account for this observation, including the difference in sensitivity between the two microarray technologies, the sequence specificity and length, and data analysis. Interestingly, visN appears on the Affymetrix data list as quorum sensing dependent, and this is the first report that links quorum sensing to the control of this regulator of motility. Also included in this list is SMc03046, whose product has homology to transcriptional regulators that contain phosphorylation domains like those of two-component systems. The SMc03046 gene lies within the flagellar gene cluster, directly upstream of the flgE operon. During the preparation of this manuscript, a report by Rotter et al. showed that this protein (designated by the authors as Rem) is a direct activator of motility in S. meliloti (46).

High population density downregulates motility gene expression in S. meliloti. Our microarray data indicated that flagellar gene expression is elevated in a strain that contains the ExpR regulator but cannot synthesize sin AHLs (referred to here as the quorum-sensing mutant [QS mutant]). To further confirm these observations, we analyzed the expression of selected motility and chemotaxis genes by real-time PCR in various S. meliloti strains (Fig. 1A). The flaF, flbT, and cheY1 genes code for regulators of motility and chemotaxis, whereas the flaC gene product constitutes one of the four flagellin subunits of the motility apparatus. The products of flgB and flgF likely comprise components of the basal body rod, and motA encodes a transmembrane motor protein involved in flagellar motor rotation. To determine the effect of population density, we compared the expression of these genes in the wild-type strain (Rm8530) and its sinI derivative at optical densities (OD600) of 0.2, 0.8, and 1.2, which correspond to the onsets of the logarithmic phase (early log), mid-logarithmic phase, and stationary phase (late log), respectively. All of the genes chosen for analysis were upregulated in the mutant that is unable to make any sin AHLs, suggesting that motility gene regulation somehow depends on these signaling molecules (Fig. 1A). Furthermore, we showed that the difference in gene expression between the wild-type and QS mutant strains increased from essentially no change at early log phase to as much as 70-fold at late log phase, as in the case of flgB expression. As the cell population density increased, we observed a concomitant decrease in the expression of motility-related genes in the wild-type strain, while their expression remained high in the QS mutant. This decrease in expression is directly related to the production of the sinI AHLs, since their exogenous addition to a QS mutant brought the expression of the motility-related genes to wild-type levels (Fig. 1B). These results are consistent with the hypothesis that quorum sensing downregulates the expression of the motility genes in S. meliloti.


Figure 1
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  		FIG. 1. High population density downregulates motility and flagellum production. (A) Expression of several motility and chemotaxis genes in Rm8530 versus Rm8530 sinI mutant at different growth phases. The relative expression is calculated as the fold change in gene expression between these two strains. (B) In trans complementation of the sinI phenotype by addition of crude AHLs. Expression of flaF, visN, and rem in an Rm8530 sinI quorum-sensing mutant was examined in the presence or absence of exogenously added AHLs obtained from the wild-type strain. Gene expression was measured at late log phase. (C) Electron microscopy to examine the presence of flagella in Rm8530 (wild type) and Rm8530 sinI (QS mutant) grown in minimal low phosphate to early (I and III) or late (II and IV) logarithmic phase. Between 600 and 1,000 cells were observed for each strain and condition in order to determine the average number of flagella per cell. Scale bars, 1 µm.

The quorum-sensing-proficient strain loses the ability to synthesize flagella at a high population density. We prepared samples for electron microscopy using bacterial cultures that were grown to early and late logarithmic phase as described in Materials and Methods. The Rm8530 strain and its QS mutant derivative showed flagella associated with the bacteria when the population density was low (OD600 of 0.2), whereas at late logarithmic phase there were fewer than 0.015 flagella per wild-type cell, and the QS mutant shows numbers consistent with more than one flagella per cell (Fig. 1C). The lack of flagella in the Rm8530 strain is not due to the high viscosity environment created by the synthesis of EPS II. Motility gene expression still exhibited a population-density-dependent pattern even after the introduction of an expA mutation, which renders the bacterium unable to produce EPS II (data not shown). These data correlate well with our gene expression studies and suggest that the production of flagella is suppressed at a high population density in S. meliloti.

Motility gene expression in S. meliloti under low-phosphate conditions requires the ExpR regulator. Our laboratory has previously shown that the activation of EPS II production by the sin AHLs requires the presence of the ExpR transcriptional regulator (36). These findings, coupled with our observations in the present study, suggest that the regulatory effects of the Sin quorum-sensing system on flagellar gene expression may likewise be mediated through the ExpR regulator (28, 36). To determine any requirement for ExpR in the regulation of motility, we also examined expression of 10 motility- and chemotaxis-related genes (flaF and flaC are representative) at the early-, mid-, and late-logarithmic growth phases in the wild type (Rm8530), the sinI mutant, and a strain that contains a disruption in the expR gene (Rm1021) (Fig. 2). The expression of these genes remains high throughout the growth cycle in a strain unable to produce sin AHLs (Rm8530 sinI), decreases in the wild-type strain as a function of population density, and remains low in the strain lacking the ExpR regulator (Rm1021) at all population densities (Fig. 2). To various degrees, Rm1021 exhibited extremely low transcriptional levels similar to that seen in the nonmotile mutant Rm8530 visN (see below). These results suggest that under the low-phosphate conditions of these studies, the expression of genes involved in motility seems to require the presence of ExpR.


Figure 2
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  		FIG. 2. Motility gene expression requires the presence of ExpR. The expression of representative motility genes (A) flaF and (B) flaC in the indicated strains is represented by CT values obtained from quantitative real-time PCR analysis. The CT value is defined as the PCR cycle at which the fluorescence intensity from each gene amplification event crosses the background level threshold. A smaller CT value corresponds to a higher level of gene expression, and 2{Delta}CT equals the fold change in expression.

Quorum-sensing controls motility gene expression through the regulators VisN/VisR and Rem. Sourjik and coworkers previously showed that in S. meliloti RU10 a visN or visR mutant strain is unable to activate motility gene expression and is thus completely defective in swimming motility (49). However, the manner in which visN/visR is controlled, as well as the mechanism of action for VisN/VisR, remains unclear. To address the first point, we examined our microarray data for any hints that would suggest a regulation of visN/visR by quorum sensing since these players are involved in controlling the same motility phenotype. In fact, visN appears to be downregulated in the wild-type strain compared to the QS mutant (Rm8530 sinI). We further examined the expression of visN and visR at different population densities (early, mid, and late log) and observed that they exhibit an expression pattern similar to those of the representative motility genes examined in Fig. 1A. As the population density increases, the transcription of these two genes become more downregulated by 4- to 6-fold at mid-log phase and by 8- to 16-fold at late log phase depending on the gene examined (Fig. 3). This effect is directly related to the production of AHLs, since the addition of crude AHLs to a Rm8530 sinI mutant led to a visN expression pattern similar to the one shown by the wild type (Fig. 1B). On the other hand, VisN/VisR do not seem to control quorum sensing since we observed no change in the expression of either sinI or expR (Fig. 4A). In addition, the expression of the EPS II biosynthetic gene expE2, previously shown to be quorum sensing controlled, is not affected by VisN/VisR (Fig. 4A). Our results suggest that quorum sensing controls motility by acting through the visN/visR operon.


Figure 3
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  		FIG. 3. Quorum sensing controls motility through the regulators VisN/VisR and Rem. Gene expression analysis was examined by using real-time PCR as described in Materials and Methods. The relative gene expression is represented for the Rm8530 strain (wild type) and is the fold change in gene expression compared to the Rm8530 sinI mutant. Expression of the indicated motility-related genes (visN, visR, and rem) decreases in the wild-type strain as the population increases from the early-, mid-, and late-logarithmic phases.


Figure 4
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  		FIG. 4. VisN/VisR and Rem control motility in a population-density-dependent manner. (A) The change in expression for a panel of motility and chemotaxis related genes between Rm8530 and a visN mutant. Measurements were obtained at early-, mid-, and late-logarithmic growth phases. The effects of a visN mutation on the expression of the autoinducer synthase gene sinI, the quorum sensing regulator gene expR, and the EPS II biosynthetic gene expE2 are also shown. (B) Expression of motility and chemotaxis genes at the different growth phases in Rm8530 compared to that of the rem mutant.

As discussed above, rem appeared in our microarray data as significantly downregulated in a quorum-sensing-proficient strain. That observation was further expanded by the gene expression analyses at various growth phases (Fig. 3). The expression of rem displayed characteristics similar to those of visN/visR. At early logarithmic phase, rem is highly expressed in both the wild-type and the QS mutant strains, but as the population density increases during the mid-logarithmic and late logarithmic phases, rem expression is dramatically suppressed by 16- and 47-fold, respectively (Fig. 3). As with visN, complementation of the Rm8530 sinI strain with AHLs in trans leads to expression levels of rem comparable to those seen in the wild-type strain (Fig. 1B).

VisN/VisR and Rem control motility gene expression in a population-density-dependent manner. The work regarding visN/visR by Sourjik et al. was performed in the S. meliloti RU10 strain (49); therefore, we sought to determine whether a similar effect of VisN/VisR would be observed in our Rm8530 strain and whether the regulation is dependent on the quorum-sensing mechanism. To address this issue, we first constructed an Rm8530 visN mutant derivative by introducing within the visN coding sequence an antibiotic gene cassette that confers resistance to hygromycin. We confirmed, by using the swimming motility assay, that this strain does have a dramatic motility defect and does not move from the point of inoculation in swimming agar (data not shown). The expression of various motility genes in this mutant was analyzed and compared to the wild-type strain at different growth phases (Fig. 4A). The motility defect of a visN mutation can be viewed most obviously at the early log phase, when motility gene expression in the wild-type strain is at its highest level. At this point, differences in expression between these two strains range from 3- to 49-fold. During mid-logarithmic growth, the wild-type strain experiences a repression in motility; therefore, the difference in gene expression compared to the visN mutant is significantly reduced. We believe that the downregulation of motility gene expression in a quorum-sensing-proficient strain (Rm8530) causes it to behave similarly to the Rm8530 visN mutant as the population increases. No discernible differences can be seen at the late log phase because suppression of motility gene expression in the wild-type strain has essentially reached the level of the visN motility mutant (Fig. 4A).

Since rem appeared as quorum sensing regulated in our microarray analyses, we set out to construct a rem mutant in the Rm8530 strain and analyzed motility gene expression under various growth phases. The rem mutant is also unable to move in swimming agar (data not shown) and lacked expression of several motility genes. Among the genes that we examined, flgB, flgF, and motA displayed the most dramatic differences ranging from 59-, 34-, and 26-fold, respectively (Fig. 4B). An important point to note is that the control of motility by Rem is also quorum sensing dependent because the differential gene expression can only be seen at the early-logarithmic-growth phase when the Rm8530 wild-type strain exhibits its highest level of motility. Our data are similar to the work by Rotter and coworkers that showed the OmpR-like protein Rem exerting control over motility in strain RU11 (46). However, we did not observe a clear effect of Rem on the expression of several genes, such as flaF, flbT, and flaC, which had displayed obvious dependency on quorum sensing and VisN. At least in the S. meliloti Rm8530 strain, Rem appears to regulate the expression of only a subset of the motility genes controlled by VisN/VisR.

The ExoS/ChvI two-component system potentially suppresses motility gene expression through VisN/VisR and Rem. Recent work by Yao et al. and Wells et al. demonstrated the dependence of motility on the ExoR and ExoS/ChvI regulatory relay in the S. meliloti strain Rm1021 that lacks the ExpR regulator (59, 60). To see whether the same holds true for an expR+ derivative under the conditions (low phosphate at the indicated growth phases) used here, we transduced the exoS96 mutation into the expR+ strain and its sinI derivative. The exoS96 mutation results in the production of a constitutively active ExoS sensor kinase that suppresses motility gene expression (10). We reasoned that motility gene expression might be downregulated by ExoS/ChvI through VisN/VisR and/or Rem. We addressed this possibility by examining the level of visN and rem expression in the sinI mutant, an exoS mutant or a sinI exoS double mutant. The relative expression for each gene is calculated as the fold change in expression in Rm8530 compared to the indicated mutant strains (Fig. 5). In the case of visN, we observed a 12-fold-higher level of expression in the wild-type strain Rm8530 versus the exoS mutant grown to mid-logarithmic phase (Fig. 5). This result indicates that expression of visN is low in the exoS mutant. On the other hand, a sinI exoS mutant exhibited similar expression to the Rm8530 strain. This suggests that the presence of the sinI mutation can partially negate the low level of visN expression due to the exoS mutation. The data support the theory that the quorum-sensing system and ExoS-related pathway both may control motility by acting on the visN/visR operon. The effect of ExoS on rem expression differs slightly from that of visN. As predicted, when we compared the wild-type with the sinI mutant, rem expression dropped by 16-fold as the population increased to mid-log phase (Fig. 5). Under the same conditions, rem expression is 18-fold higher in the wild type compared to the exoS mutant. When we introduced the sinI mutation into the exoS strain, expression of rem was still about 17-fold lower compared to Rm8530 at mid-log phase. Unlike the case of visN expression, the presence of sinI did not seem to compensate, to any degree, for the low level of rem expression in the exoS mutant during mid-log growth. The reason for this difference remains to be elucidated.


Figure 5
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  		FIG. 5. The ExoS/ChvI two-component system suppresses motility through VisN/VisR and Rem. The relative expression (i.e., the fold change in gene expression) of visN and rem at mid-logarithmic growth phase in Rm8530 compared to the indicated mutant strain is charted.

The ExoR and ExoS/ChvI products are also known for their involvement in the regulation of succinoglycan, another EPS made by S. meliloti, as well as motility (10, 41). We hypothesized that not only is the control of EPS production closely linked to that of motility, but the quorum-sensing system may interact in an unknown manner with the ExoR and ExoS/ChvI relay to regulate motility and chemotaxis or vice versa. One possibility may include the regulation of the exoR and/or exoS genes by the ExpR/Sin quorum-sensing system. Therefore, we analyzed the expression of the exoR and exoS genes in the relevant strains at mid-log phase and found that the expression of exoR and exoS does not depend on the ExpR/Sin system, at least at the level of transcription (data not shown). We also considered the notion that ExoR or ExoS/ChvI may control expression of expR; however, when we analyzed the exoS mutants, no such effect on the transcript level of expR was observed. Independent of quorum sensing, we observed a three- to fivefold increase in the expression of exoR in strains that carried an exoS mutant allele, which produces a constitutively active protein (10). The cumulative results suggest that, in response to different environmental signals, motility gene expression is controlled by the ExoS/ChvI pathway in parallel with the ExpR/Sin quorum-sensing system. The convergence point is likely VisN/VisR or a factor that controls their expression.


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DISCUSSION
 
Bacteria must constantly move in their quest for nutritional sources or the proper host for survival. Most bacterial species have the ability to sense changes in the environment and rapidly direct their movement accordingly. However, this useful ability comes at a great cost since ca. 2% of the cell's energy expenditure is applied toward the assembly and maintenance of the motility apparatus (52). Quorum sensing, or population density-dependent gene regulation, is among the many strategies used by bacteria to control the process of motility and chemotaxis (14, 16, 17, 53, 61). Our laboratory has reported, through a series of microarray analyses, that the expression of many motility- and chemotaxis-related genes is regulated by the ExpR/Sin quorum sensing system (Fig. 1A; see also the supplemental material) (28). These results support the idea that the Sin quorum-sensing system, in combination with the ExpR regulator, suppresses the expression of the motility genes in S. meliloti at high population densities. The necessity for both the Sin system and the ExpR regulator in the control of motility is analogous to the regulation of EPS II biosynthesis by quorum sensing (36). Marketon et al. reported that the activation of EPS II production by the sin AHLs requires the ExpR regulator (36). In fact, our laboratory also demonstrated that most of the control exerted by the Sin system on S. meliloti gene expression depends on the presence of ExpR (28). These observations support the role of the ExpR/Sin quorum-sensing system as the common regulator of multiple cellular processes.

An expR+ strain that is unable to make the sin AHLs not only fails to suppress motility but also lacks the ability to make EPS II (36). One possible explanation for our results is that the change in the medium viscosity resulting from the lack of EPS II synthesis might be directly sensed as a trigger for the elevated expression of the flagellar genes in the sinI mutant. However, when we use strains that lack the ability to sense population density and that also carry a mutation in the expA gene necessary for EPS II production, a similar pattern of motility gene repression was observed. These findings support the theory that SinI through the synthesis of AHLs rather than the control of EPS II production, probably acts as the signal that regulates gene expression. In the present study, we show that quorum sensing controls motility via the regulators VisN/VisR and Rem in a population density-dependent manner. The products of VisN/VisR were previously reported to be essential for the activation of motility gene expression (49), and Rem was shown to positively activate motility gene expression by direct contact of the promoters of flgB, fliF, orf38, and rem (46). Quorum sensing acts upstream of these regulators to suppress motility when the bacteria reach the late logarithmic growth phase. We have further delineated the pathway by connecting quorum sensing with the ExoR/ExoS/ChvI relay, another previously reported mechanism that also controls motility and EPS biosynthesis (59, 60). Based on the accumulated evidence, we have compiled a working model for the control of motility and EPS production in S. meliloti. Our current model (depicted in Fig. 6) emphasizes several important features and observations made by our laboratory and others. First, the regulation of EPS production and motility is intertwined in an inverse manner. The ExpR/Sin quorum-sensing system plays a role in the activation of EPS II production that is critical for a successful interaction with the host plant (36). As we have elaborated in this work, quorum sensing also suppresses motility gene expression and flagella synthesis at a high population density. The ExoR/ExoS/ChvI relay, known for its involvement in succinoglycan production, also suppresses motility (59, 60). Furthermore, these two systems seem to exert their control on motility in distinct, parallel pathways but converge at the VisN/VisR juncture. We postulate that the sin AHLs may form a complex with ExpR, which then directly suppresses the transcription of the visN/visR operon. Alternatively, the autoinducer-ExpR complex could exert control on an unknown repressor of VisN/VisR. Similarly, the ExoR/ExoS/ChvI pathway seems to suppress motility through VisN/VisR in response to an unknown signal. This seems likely to depend on the particular environment encountered by the bacteria (59). We postulate that the bacteria may use the quorum-sensing mechanism as a switch to fine-tune bacterial motility, in addition to other cellular processes, in response to bacterial population density.


Figure 6
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  		FIG. 6. Model for the control of motility and EPS production in S. meliloti. Quorum sensing activates the production of EPS II and downregulates the process of motility. The ExpR/Sin quorum-sensing (QS) system could regulate motility directly through VisN/VisR or by acting on a repressor of this operon. There is no evidence for any transcriptional control of ExoS and ExoR by the ExpR/Sin system. We also cannot disregard a quorum-sensing-dependent regulation of an undiscovered component that might interfere with the on/off state of the ExoS/ChvI two-component system (TCS). The ExoS/ChvI two-component system controls the biosynthesis of succinoglycan and also seems to suppress motility by acting through VisN/VisR and Rem. ExoR has been suggested to act as a repressor of the ExoS/ChvI system (59).

Recent work by Gibson et al. focuses on a regulator called CbrA, a potential two-component histidine kinase that is involved in the symbiosis between S. meliloti strain Rm1021 and its host plant alfalfa (20). These authors provide evidence that CbrA also controls motility, presumably through the regulators VisN/VisR and Rem. Interestingly, expression of the sinI gene was mildly reduced in a cbrA mutant, suggesting that CbrA may act upstream of the quorum sensing system. However, the strain Rm1021 used in the work of Gibson et al. lacks the ExpR regulator that is required for most quorum sensing-dependent activity in S. meliloti (20, 21). Therefore, it would be informative to see how CbrA behaves in the context of a quorum sensing-proficient strain background, with respect to regulation of motility.

The sinI quorum-sensing mutant resembles, in many ways, the fadD mutant in S. meliloti strain GR4 that was studied by Soto et al. (48). In that study, the authors showed that a disruption in fadD, encoding a long-chain fatty acyl coenzyme A ligase, resulted in a mutant that is hyperflagellated, exhibits swarming motility, and is less symbiotically efficient compared to the wild-type strain. These phenotypes are dependent on cell population density, culture medium, and the medium viscosity. The similarities between the fadD and sinI mutants raise the question of whether there is any connection between the pathways in which these proteins play a role.

Studies from the laboratory of Wolfgang D. Bauer have also focused on motility and chemotaxis in S. meliloti and, more recently, its control by the ExpR/Sin quorum-sensing system. Earlier work by Wei and Bauer has shown that motility is somehow suppressed when cells are transferred to starvation medium (58). These authors also demonstrated that motility was more rapidly lost than flagella integrity. It is interesting that one of the starvation conditions used in that particular study included no or low phosphate similar to that used in our current work. More recently, Gao et al. showed that the sinI and expR mutants exhibited less swarming motility than the wild-type strain (18). Why and if this is in any way connected to the quorum-sensing effects on motility that we report here warrants further investigation.

The present study provides new insights into the mechanisms that control motility in S. meliloti and how this regulation may be germane to its interaction with the alfalfa host. This is the first report that directly connects quorum sensing with the control of motility in S. meliloti and offers an example of a few cases in which quorum sensing decreases bacterial motility. One particularly noteworthy instance can be found in P. syringae. Elegant work by Quiñones et al. (44) showed that analogous to the ExpR/Sin quorum-sensing system in S. meliloti, the AhlI-AhlR quorum-sensing system of P. syringae positively regulates EPS synthesis and represses motility. AHL-deficient P. syringae mutants are hypermotile and show an enhanced ability to invade the host leaves compared to the wild type. These authors suggest that the increased motility enhances the pathogen's ability to explore and invade protected sites on the leaves (44). In contrast, S. meliloti quorum-sensing mutants do not invade nodules as efficiently as the wild-type strain.

In organisms such as E. coli and S. marcescens, motility is upregulated by quorum sensing (14, 32, 54, 55). Under normal circumstances, bacteria in search of food sources must disperse from an area of high population density since those environments are rapidly depleted of nutrients. However, consideration for the lifestyles of S. meliloti supports a different method for the regulation of motility by quorum sensing. This soil bacterium is able to enter a symbiotic relationship with alfalfa, where it can gain access to nutrient sources, but it first must be able to reach the plant host amid the soil milieu, an environment that typically contains very low levels of phosphate (in the range of 1 to 10 µM). We have shown that under the low-phosphate conditions used in this study, the ExpR regulator is necessary for high levels of motility gene expression. We speculate that perhaps the ExpR/Sin system not only refines the regulation of motility in conjunction with other mechanisms but may also offer the bacteria a selective advantage under physiological environments in response to particular cues (i.e., low phosphate and population density). Studies have shown that flagellum production is not necessary for nodulation or nitrogen fixation because nonmotile S. meliloti strains are still able to invade plants and form a symbiotic interaction (1, 2, 8). In fact, flagellin has been well characterized as an inducer of the plant defenses (11). Therefore, upon reaching its proper niche, the necessity for movement in S. meliloti is essentially superfluous and might be detrimental to the invasion process. This could explain the decreased ability of S. meliloti quorum-sensing mutants to invade nodules. We believe that the gathering of S. meliloti around the alfalfa roots and the achievement of a quorum provides an elegant strategy for the simultaneous activation of the invasion signal EPS II and the downregulation of motility. These parallel events contribute to the success of S. meliloti during its symbiosis with the plant host.

The widely used S. meliloti strain Rm1021 is frequently studied in optimal conditions that negate the need for a fine-tuning mechanism, such as quorum sensing. In these experiments the bacteria are often added directly or close to the site where the host-symbiotic interaction will take place, obviating the need for movement toward the host. In addition, cultures are grown in media likely to contain levels of nutrients and ions higher than those usually found in the soil. Under these conditions, where starvation, desiccation, and competition with other organisms are unlikely to occur, the need for EPSs such as EPS II, flagella, and other enhancing mechanisms is reduced. Quorum sensing, in our view, potentially plays a very important role in preparing the bacterium for the crucial change in lifestyle that ensues during its association with the plant host. It is therefore essential that we strive to study model organisms, such as S. meliloti, under conditions that aim to replicate, as faithfully as possible, those encountered by the bacteria in its natural environment.


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ACKNOWLEDGMENTS
 
We thank the members of our laboratory for their helpful discussions and critical reading of the manuscript. We are also very grateful to Kostia Bergman for strains and his valuable advice. Dave Muirhead was extremely generous with his suggestions and the use of his electron microscope. Jay Ingram was supportive and helpful with the electron microscopy work.

This study was supported by National Science Foundation grant MCB-9733532 to J.E.G. and National Institutes of Health grant 1R01GM069925 to J.E.G.


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FOOTNOTES
 
* Corresponding author. Mailing address: RL11, Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083-0688. Phone: (972) 883-2526. Fax: (972) 883-2409. E-mail: jgonzal{at}utdallas.edu Back

{triangledown} Published ahead of print on 16 November 2007. Back

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

{ddagger} Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115. Back


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Journal of Bacteriology, February 2008, p. 861-871, Vol. 190, No. 3
0021-9193/08/$08.00+0     doi:10.1128/JB.01310-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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