INTRODUCTION

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Bacterial motility and chemotaxis are essential qualities for optimum adaptation to different environments. Flagellar synthesis and motility are maximal under nutritional stress and require 2 to 3% of the energy of a cell. Therefore, the expression of some 50 genes involved in this process is strictly regulated by a hierarchy of controls (19). The current paradigm of flagellar gene regulation was derived from studies of Escherichia coli and Salmonella enterica serovar Typhimurium (13, 15, 18, 20, 29). The E. coli chemotaxis, flagellar, and motility genes map in four separate clusters often referred to as the flagellar regulon. The genes of this regulon are organized in three classes that are expressed in hierarchial order, although their map positions do not necessarily reflect the assignments to these classes. Class I is represented by a master operon that encodes the transcription activators, FlhC and FlhD, which in turn regulate the expression of class II (18). This includes genes that determine the flagellar basal body and the flagellin-specific export apparatus and fliA, which encodes a ²⁸ (F) transcription factor for class III.

Sinorhizobium (Rhizobium) meliloti, a member of the alpha subgroup of proteobacteria, exhibits significant deviations from the enterobacterial (gamma subgroup) paradigm of chemotaxis in its flagellar structure and mode of rotation (23). The complex, rigid flagellar filaments of S. meliloti consist of four closely related flagellin subunits, FlaA, FlaB, FlaC, and FlaD, encoded by four linked but independently transcribed genes (24, 33). The right-handed flagellar helices rotate exclusively in the clockwise mode, and swimming cells respond to tactic stimuli by modulating their flagellar rotary speed (31, 32). Two novel motor proteins, MotC and MotD, are essential players in the control of flagellar rotary speed (23). The organization of the S. meliloti chemotaxis (che), flagellar (fla, flg, flh, and fli), and motility (mot) genes is distinctly different from that in enterobacteria, since all known 41 genes are clustered in one contiguous 45-kb chromosomal region (33). Among these are 10 genes of a che operon, four mot genes (23), 15 flg, flh, and fli genes encoding components of the basal body and the flagellar export apparatus, 4 flagellin (fla) genes, and 5 genes of hitherto unknown function. Notably, MotA is encoded in the same operon as FliM, FliN, and FliG, and a new gene, orf38, necessary for flagellum formation, is part of the motB-motC-motD operon (33). In keeping with established nomenclature (20), we refer to the entirety of these genes as the flagellar regulon.

We have identified in S. meliloti two related members of the LuxR family, VisN and VisR (for "vital for swimming"), formerly named Orf12 and Orf13, respectively (33), that function as global activators of the flagellar regulon. Transcriptional activators of the LuxR family have been found in various bacterial species, where they regulate such different processes as conjugation, cell division, and antibiotic production (6, 34). Typical members of this family are TraR, which regulates plant-pathogenic genes of Agrobacterium tumefaciens (5, 22), and RhiR and RaiR, which regulate the nodulation genes of Rhizobium leguminosarum and Rhizobium etli (3, 8, 26). LuxR is best known for its role in quorum sensing, i.e., the ability to monitor population density by sensing the external concentration of autoinducer molecules, notably homoserine lactones. All these transcription factors have similar structures with a ligand-binding domain and DNA-binding domains of the helix-turn-helix type, which place them in the LuxR-FixJ-UhpA-NarL superfamily of transcription factors (6, 9). The LuxR-like regulators, VisN and VisR, of S. meliloti described here act as master controls of a gene cascade that encodes flagellar, motor, and chemotaxis proteins.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. Derivates of E. coli K-12 and S. meliloti MVII-1 (12) and the plasmids used in this study are listed in Table 1.

Media and growth conditions. E. coli strains were grown in Luria broth (19) at 37°C. S. meliloti strains were grown in TYC at 30°C (23). Motile cells prepared for immunoblots and -galactosidase assays (see below) were grown overnight in TYC, diluted in 15 ml of RB (7) to an optical density at 600 nm (OD₆₀₀) = 0.05, layered on Bromfield plates (31), and grown on a slowly rotating platform at 30°C for 17 h to an OD₆₀₀ of ca. 0.1 to 0.5. Swarm plates containing Bromfield medium and 0.3% Bacto Agar were inoculated with 3-µl droplets of the test culture and incubated at 30°C for 2 days. Antibiotics were used at the following final concentrations: for E. coli, kanamycin, 50 mg/liter; gentamicin, 10 mg/liter; and tetracycline, 10 mg/liter; for S. meliloti, neomycin, 100 mg/liter; streptomycin, 600 mg/liter; gentamicin, 20 mg/liter; and tetracycline, 10 mg/liter.

DNA methods. S. meliloti chromosomal DNA was isolated and purified as previously described (31). Plasmid DNA was purified with Qiagen spin columns or Qiagen 100 tips, as described by the manufacturer (Qiagen, Hilden, Germany). DNA fragments or PCR products were purified from agarose gels by use of the QiaEX DNA purification kit (Qiagen). PCR amplification of chromosomal DNA (33) and Southern analyses followed previously published protocols (23, 33). DNA was sequenced by the method of Sanger et al. (27) with a Quick Denature Sequenase kit (Amersham Buchler, Braunschweig, Germany) or with an ABI 310 automatic sequencer (Applied Biosystems, Weiterstadt, Germany). Sequences were aligned and compared by using GCG sequence analysis software (4); similarities were calculated by the method of Henikoff and Henikoff (9).

Gene replacement and complementation. Deletions were generated in vitro by using two PCR steps as described by Higuchi (10). PCR products containing the desired deletions were cloned into the mobilizable suicide vector pK18 mob sacB (28) and used to transform E. coli S17-1. Clones were sequenced to ascertain the accuracy of PCR-generated portions. Filter crosses of E. coli S17-1 and S. meliloti and sequential selections on neomycin and on 10% sucrose were performed as previously described (31). Complementation with pML122-based recombinant genes (Table 1) followed established protocols (16).

Immunoblots. Aliquots (1 ml) of cell culture of wild-type S. meliloti RU11/001 and various deletion strains at an OD₆₀₀ of 0.3 were harvested by centrifugation at 20,000 × g for 6 min, resuspended in 20 µl of sodium dodecyl sulfate sample buffer, and heated to 100°C for 8 min. Samples were stored at 20°C. Fla, FliM, and MotC proteins were separated electrophoretically in a 10 to 15% linear acrylamide gradient and CheY1 was separated in a 12.5 to 20% linear gradient by the method of Laemmli (17). Electrophoretic transfer of proteins from gels to 0.45-µm-pore-size nitrocellulose (Hybond ECL; Amersham) was performed in a tank blot device (Biological Laboratories, Harvard University) for 1.5 h at 500 mA. The nitrocellulose blots were blocked overnight at room temperature in 80 mM Na₂HPO₄-20 mM NaH₂PO₄-100 mM NaCl-0.1% (vol/vol) Tween 20 (pH 7.5) (Bio-Rad)-5% instant nonfat dry milk on a shaking platform. The blots were probed with anti-FliM-MalE, anti-Fla, and anti-CheY1 polyclonal antibodies at a 1:1,000 dilution for 2 h. Anti-MotC polyclonal antibody was purified, as described by Platzer et al. (23), at a 1:250 dilution for 3 h. The blots were washed three times (10 min each), incubated with donkey anti-rabbit horseradish peroxidase-linked whole immunoglobulin antibody (Amersham) diluted 1:2,500, and washed four times (10 min each). Detection of bands by enhanced chemiluminescence (Amersham) was performed as specified by the manufacturer.

Construction of lacZ fusions and -galactosidase assay. The broad-host range vector pPHU234 and two derivates, pPHU235 and pPHU236 (Table 1), served as vehicles for translational fusions to a promoterless lacZ (11). Five lacZ fusions were constructed to test gene expression in S. meliloti: (i) pRU1770, a recombinant of a 217-bp XbaI-HindIII visN (9-bp) fragment and pPHU234; (ii) pPHU2250, a recombinant of a 1,969-bp EcoRI-PstI tlpA (165-bp) fragment and pPHU235; (iii) pRU2269, a recombinant of a 786-bp EcoRI-HindIII motA (15-bp) fragment and pPHU235; (iv) pRU2274, a recombinant of a 550-bp EcoRI-PstI flaA (62-bp) fragment and pPHU236; and (v) pRU2278, a recombinant of a 561-bp orf38 (23-bp) fragment and pPHU 234 (lengths of coding sequences are given in parentheses). The resulting lacZ fusion plasmids were used to transform E. coli S17-1 and then conjugally transferred to wild-type and mutant S. meliloti strains by a streptomycin-tetracycline double selection, as described by Labes et al. (16). Cultures of S. meliloti cells containing lacZ fusions were sampled, diluted in Z-buffer to an OD₆₀₀ of 0.2, permeabilized with 1 drop of toluene, and assayed for -galactosidase activity by the method of Miller (21).

	RESULTS

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VisN and VisR are LuxR-type regulatory proteins. The visN and visR genes form an operon located centrally in the S. meliloti flagellar regulon between fliF and two operons (starting with flhB and motA, respectively) with opposite transcription polarity relative to all other genes (33). The derived polypeptide sequences of the structurally related 27-kDa VisN and VisR proteins revealed distinct similarities to the transcription activator LuxR (Fig. 1). LuxR-like factors typically consist of a receptor module with ligand-binding and oligomerization domains (located in the N-terminal half) and an activator module with the DNA-binding site featuring a helix-turn-helix motif and the transcription activation domain (located in the C-terminal half) (5). The entire LuxR with its autoinducer binding, oligomerization, and DNA-binding domains exhibits some 40% similarity (9) to the VisN and VisR polypeptides. The similarity to LuxR is more prominent among the DNA-binding domains with 39% identical and 60% similar residues for VisN and 42% identical and 60% similar residues for VisR, respectively. Both VisN and VisR contain the characteristic helix-turn-helix motifs, which have 36% identity and 60% similarity to each other. With respect to LuxR, the lowest sequence conservation prevails in the ligand-binding domains of VisN and VisR, suggesting a new specificity for a yet-unknown ligand.

View larger version (47K): [in this window] [in a new window]   FIG. 1.   Alignment of the LuxR (6) and VisN and VisR (33) polypeptide sequences. Comparisons of VisN and VisR each to LuxR with regard to identical (one-letter amino acid symbols) or similar residues (+; assigned according to the system of Henikoff and Henikoff [9]) are depicted above (VisN and LuxR) and below (VisR and LuxR) the sequences, respectively. LuxR-type functional domains are numbered as follows: 1, autoregulation; 2, ligand binding; 3, oligomerization; 4, transcription activation. The three components of the conserved helix-turn-helix DNA-binding motif (1, 9) are marked by black bars.

View larger version (76K): [in this window] [in a new window]   FIG. 2.   Effects on swarming of in-frame deletions of visR (A) and visN (B) and complementation by the wild-type alleles. (A) Swarm 1, RU11/001 (wild type); swarm 2, RU11/317 (visR); swarm 3, RU11/397 (visR/visR). (B) Swarm 1 RU11/001 (wild type); swarm 2, RU11/318 (visN); swarm 3 RU11/391 (visN/visN). Strains to be tested were transferred by micropipette (3 µl) onto Bromfield swarm plates and incubated at 30°C for 2 days. The diameter of a swarm ring reflects the motile proficiency of a given strain.

VisN and VisR are master controls of the flagellar regulon. Features of the visN and visR deletion mutants, notably the total absence of flagella, led us to ask whether VisN and VisR are global regulators of the entire cluster of chemotaxis, flagellar, and motility genes (flagellar regulon). We therefore tested the expression of representative basal-body, motor, flagellin, and chemotaxis genes by Western analysis (Fig. 3) and by lacZ fusions (Table 2) in wild-type S. meliloti and in defined tester mutant background. Polyclonal antibodies against recombinant FliM, MotC, CheY1, and purified flagellar filaments (Fla) were used to probe gene expression of the four "indicator" genes, i.e., fliM, encoding a constituent of the basal flagellar body; motC, encoding a flagellar motor protein; flaA to flaC, encoding the flagellin subunits; and cheY1, encoding a chemotaxis response regulator (23, 24, 31, 32, 33). The results, shown in Fig. 3 (lanes 2 and 3), confirm the presumed role of VisN and VisR as global regulators, since none of the three genes representing major operons and the fla genes were expressed, neither in the visN (RU11/318) nor in the visR (RU11/317) mutant background. In a control experiment with wild-type S. meliloti, each indicator gene produced a strong signal (lane 1). Since visN and visR mutants each failed to express the fli, mot, fla, and che genes, we postulate that VisN and VisR may form a functional heterodimer, VisNR, that acts as transcription activator on target promoters. This is an intriguing new function for a LuxR-like regulatory protein.

View larger version (38K): [in this window] [in a new window]   FIG. 3.   Western blot analysis of gene expression in wild-type and mutant S. meliloti by using polyclonal anti-FliM (-FliM), anti-MotC (-MotC), anti-flagellin (-Fla), and anti-CheY1 (-CheY1) antibodies. Equal amounts of total cell protein from strains 1 (RU11/001 [wild type]), 2 (RU11/317 [visR]), 3 (RU11/318 [visN]), 4 (RU11/801 [orf38]), 5 (RU11/800 [fliM]), 6 (RU11/802 [motA]), 7 (RU11/513 [motBC]), and 8 (RU11/011 [flaA-flaC]) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted on nitrocellulose, and detected with specific antibody as described in Materials and Methods. Note the lack of a signal where the indicator gene was deleted.

Genetic control of the vis operon. The combination of a lacZ fusion (pRU1770) and in-frame deletions of visN and visR used to probe transcription control of the vis genes (Table 2) has been extended to testing their expression at two extremes of bacterial growth. Exponentially growing motile cells and starved nonmotile cells (Table 3) with and without VisN or VisR were compared for expression of the vis operon. High -galactosidase activities observed under these two conditions indicate constitutive expression (again, with some 20% repression by VisN alone [Table 2]) of visN and visR throughout all stages of growth with a 25% up-regulation in response to starvation (low energy). On the other hand, immunoblots and observations of swimming cells (data not shown) tell us that flagellar and motility genes are preferentially expressed in a "window" between early and mid-exponential growth but not in stationary or low-energy phases. Therefore, an additional regulatory element that controls the onset and close of VisNR activity (controlling motility) during growth must exist. We propose that it is the binding of a hitherto unknown effector to VisN, VisR, or both that triggers the transcription of class II genes.

	DISCUSSION

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The stepwise assembly of enterobacterial flagella is reflected by a regulatory cascade of three classes of genes securing the sequential biosynthesis of flagellar and motor components as needed (20). Similarly, the present study of gene expression within the S. meliloti flagellar regulon yielded three major classes of operons and genes that are expressed in hierarchical order. However, the molecular nature of two LuxR-type global activators and the different class assignment of mot genes are distinguishing new features of the S. meliloti system. The interdependence of gene expression deduced from Western blots and reporter gene assays (Fig. 3 and Table 2) is diagrammed in Fig. 4. Class I, represented by the vis operon, encodes two LuxR-type master regulatory proteins, VisN and VisR, both required for the expression of all flagellar, motility, and chemotaxis genes tested. The vis operon itself is constitutively expressed, with possible modulation by VisN and low metabolic energy; it does not require other gene products for transcription. However, quasiconstitutive transcription of vis (Table 3), on one hand, and the fact that motility is limited mostly to exponential growth, on the other hand, are contradictory unless one assumes posttranslational activation of VisNR by the binding of an effector (see below). Class II includes genes encoding basal-body components and motor proteins. Their expression requires the function of visN and visR but of no other genes of the flagellar regulon. Class III comprises cheY1, representing the chemotaxis operon (33), and flaA, a principal flagellin gene. Their expression requires class I and class IIA genes. The mot genes are also contained in class II operons, but they do not control chemotaxis and flagellation and have thus been assigned to class IIB.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Regulation scheme of the S. meliloti flagellar, motility, and chemotaxis gene system falling into a hierarchy of three expression classes. The transcription polarities of operons and flaA are indicated by horizontal arrows drawn below the gene symbols (33). Translation to gene products (ellipsoids) is indicated by open white arrows, and positive regulatory controls are indicated by solid arrows. The postulated heterodimeric structure of VisNR, posttranslational activation by an unknown effector, and subclassification of basal-body (IIA) and motor (IIB) genes are included.

Although a similar hierarchy of gene expression exists within the enterobacterial flagellar regulon (20), there are new features in the S. meliloti regulatory cascade. The global regulators, VisN and VisR, are of the LuxR type without structural resemblance to the enterobacterial master controls, FlhC and FlhD (18). Since both VisN and VisR are needed for gene activation, it is plausible to assume that they function as a heterodimer, VisNR. Multimerization is similarly reported of LuxR, the transcription activator of Vibrio fischeri luminescence (2). Like LuxR (6), VisNR supposedly requires the binding of a yet unknown effector for function. Unlike in quorum sensing (6), such ligand molecules are not present among those excreted into the medium by motile or densely grown bacteria (34), since S. meliloti cells showed no response when concentrated culture medium from motile or from densely grown cells was added (data not shown). We are currently testing endogenous metabolites and chemotactic attractants as candidate effectors of VisNR. Another complication is introduced by dissimilar polypeptide structures of the ligand-binding sites of VisN and VisR (Fig. 1) that may reflect different ligand-binding specificities. Given the heterodimeric molecular structure of functional VisNR, two different receptor domains conceivably require two different ligands for activation. Obviously, the correct mixture of two ligand molecules may not be readily available in the cell. Alternatively, the heterodimeric receptor domains may cooperate in binding a single effector molecule at their common interface.

The Mot proteins (notably MotA and MotB) of S. meliloti are encoded by different class II operons (33), unlike in E. coli and S. enterica serovar Typhimurium, where they map in one operon assigned to class III (20). The S. meliloti motA gene (class IIB) is part of an operon that also contains the C-ring component genes, fliMNG (class IIA). The motBCD genes (class IIB), on the other hand, belong to a separate operon together with orf38 (class IIA), which may encode a structural component of the basal body. Does this genetic (and regulatory) separation of the motA and motBCD genes and their linkage to basal-body genes reflect known differences in the S. meliloti mode of flagellar rotation requiring a more complex motor structure (23)? It needs to be elucidated whether the basal-body (rotor) and the four motor (stator) genes require coassembly as a way of securing their correct positioning relative to each other inside and outside the cytoplasmic membrane.

While VisN and VisR directly control class II genes, their control over class III genes (flaA and the che operon) is most likely to be an indirect one. The dependence of class III gene expression on the completion of basal-body structure and flagellar export (class IIA genes) implies a similar mode of control to that which operates in enterobacteria (20). In E. coli and S. enterica serovar Typhimurium, ²⁸-mediated transcription of class III genes is inhibited by an anti-sigma factor, FlgM, which is expelled into the medium upon completion of the flagellar export apparatus, thus releasing ²⁸ for class III gene transcription. We expect that ongoing efforts toward completing the genetic map of the S. meliloti flagellar regulon (33) will, among other genes, also reveal class II genes that control class III transcription.