Rem, a New Transcriptional Activator of Motility and Chemotaxis in Sinorhizobium meliloti

Bacteria and plasmids.Derivates of E. coli K-12 and S. meliloti MV II-1 (25) and the plasmids used are listed in Table 1.

Media and growth conditions. E. coli strains were grown in Luria broth (29) at 37°C. S. meliloti strains were grown in TYC (0.5% tryptone, 0.3% yeast extract, 0.13% CaCl₂ · 6H₂O [pH 7.0]) (41) at 30°C for 2 days. Motile cells for β-galactosidase assays were grown for 2 days in TYC, diluted in 15 ml of rhizobium phosphate buffer (RB) to an optical density at 600 nm (OD₆₀₀) of 0.05, layered on Bromfield agar plates (53), and incubated on a slowly rotating platform at 30°C for 16 h to an OD₆₀₀ of 0.15 to 0.25. Motile cells for growth phase-dependent β-galactosidase assays and Western blot analysis were grown in Sinorhizobium motility medium (RB, 0.2% mannitol, and 2% tryptone-yeast extract [3, 20]). Cells from a stationary-phase culture grown in Sinorhizobium motility medium were diluted in 10 ml of fresh medium to an OD₆₀₀ of 0.05 and incubated at 30°C for 2 h to 64 h to a final OD₆₀₀ of 0.06 to 1.2. Flasks for growing motile cells were pretreated with chrome sulfuric acid for 24 h and rinsed three times with distilled H₂O to obtain highly motile cells. 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 3 days. Percentages of wild-type swarm diameters were determined after deduction of the 6-mm inoculum. The following antibiotics were used at the indicated final concentrations: for E. coli, kanamycin (50 mg/liter), tetracycline (10 mg/liter), and gentamicin (10 mg/liter); and for S. meliloti, streptomycin (600 mg/liter), neomycin (120 mg/liter), tetracycline (10 mg/liter), and gentamicin (40 mg/liter). For o-nitrophenyl-β-d-galactopyranoside (ONPG) tests, tetracycline was used at 3 mg/liter and gentamicin at 13 mg/liter.

DNA methods. S. meliloti DNA was isolated and purified as described previously (53). Plasmid DNA was purified with a NucleoSpin (Macherey Nagel, Düren, Germany). DNA fragments or PCR products were purified from agarose gels by use of a QiaEX DNA purification kit (QIAGEN, Hilden, Germany). PCR amplification of chromosomal DNA and Southern blotting were carried out according to published protocols (41, 55). DNA was sequenced with an ABI 310 automatic sequencer (Applied Biosystems, Weiterstadt, Germany). Sequences were aligned and compared by using GCG sequence analysis software (11).

Gene replacement and complementation.Deletions and amino acid substitutions were generated in vitro by overlap extension PCR as described by Higuchi (21). PCR products containing the deletions or amino acid substitutions were cloned into the mobilizable suicide vector pK18mobsacB and used to transform E. coli S17-1. Clones were sequenced to ascertain the accuracy of PCR-generated portions. Filter matings of E. coli S17-1 and S. meliloti were performed according to Simon et al. (51), followed by sequential selections on neomycin and 10% sucrose, as previously described (53). Confirmation of allelic replacement and elimination of the vector was obtained by gene-specific primer PCR, Southern blotting, and DNA sequencing. Complementation with pBBR1MCS-based recombinant rem and visNR followed established protocols (26, 27).

Recombinant expression and purification of Rem.Recombinant Rem protein was overproduced from plasmid pRU2365 in E. coli ER2566 (Table 1). Cells were grown at 37°C in Luria broth containing 100 mg of ampicillin per liter to an OD₆₀₀ of 0.7, and gene expression was induced by 0.3 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Cultivation was continued for 16 h at 16°C until harvest. Cells from 2-liter cultures were resuspended in 20 ml column buffer (20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 1 mM EDTA) containing 10 μg/ml DNase I and lysed by two passages through a French press at 20,000 lb/in². Cell membranes and debris were removed by centrifugation at 48,000 × g and 4°C for 30 min. The supernatant was loaded on a chitin agarose (NEBiolabs, Beverly, MA) column (2.6 by 5.0 cm) equilibrated with column buffer and washed thoroughly with 5 bed volumes of column buffer at 4°C. Intein-mediated cleavage at the intein cleavage site was elicited by equilibration of the column with 2 bed volumes of cleavage buffer (50 mM dithiothreitol in column buffer) and further incubation at 4°C for 16 h. Rem was eluted with column buffer, and pooled fractions were dialyzed against 10 mM Tris-HCl (pH 8.0), 250 mM NaCl, 1 mM dithiothreitol (DTT), 50% glycerol and concentrated by ultrafiltration on a regenerated cellulose membrane (10-kDa cutoff) to a protein concentration of 5 mg/ml. In vitro phosphorylation experiments of purified Rem with [³²P]acetyl phosphate and [³²P]phosphoamidate were performed according to McCleary and Stock (34) and Buckler and Stock (5), respectively.

Immunoblots.Polyclonal antibodies raised against S. meliloti flagellar filaments; recombinant MotC, MotE, FliM, and Rem; and the N-terminal receiver domain of Rem were isolated from whole serum by affinity purification using 100 μg protein by following previously described protocols (48). Whole-cell extracts were separated in 10% or 15% acrylamide gels, transferred to nitrocellulose, and probed using purified anti-S. meliloti MotC, MotE, FliM, and Rem polyclonal antibody at a 1:100 dilution and anti-S. meliloti Fla polyclonal antibody at a 1:500 dilution. CheY1 was detected by using whole serum at a 1:1,000 dilution. Antibodies were purchased from Pineda (Berlin, Germany), except for an antiserum raised against Rem by Eurogentec (Belgium) that was used for the immunoblot shown in Fig. 2B.

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FIG. 2.

Kinetics of rem gene expression in wild-type and Δrem mutant S. meliloti during growth. (A) Averaged growth curves (OD₆₀₀) (•) and rem promoter (Prem) activity (in Miller units) monitored by the Prem-lacZ reporter gene construct pRU2361 in strains RU11/001 (wild type; ▪) and RU11/555 (Δrem; □). Points represent the mean of two experiments (6% average deviation). The horizontal bar at the top marks the period of motility; the arrows at the bottom define the period of sampling for Western blot analysis shown below. (B) Samples of 4.7 × 10⁸ cells of wild-type strain RU11/001 were taken at the intervals indicated at the bottom (in hours). Equal amounts of total cell protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted on nitrocellulose, and detected with a specific anti-Rem antibody (1:100) described in Materials and Methods.

Construction strains carrying promoter-lacZ fusions and β-galactosidase assay.For analyzing the expression of genes in the flagellar regulon, the broad-host-range vector pPHU234 and two derivates, pPHU235 and pPHU236 (Table 1), served as vehicles for translational fusions to a promoterless lacZ (23). Nine lacZ fusion plasmids (Table 1) were used to transform E. coli S17-1 and then conjugally transferred to the S. meliloti wild type and Δrem mutant by a streptomycin-tetracycline double selection, as described by Labes et al. (27). β-Galactosidase was assayed by the hydrolysis of ONPG (Sigma) as described by Miller (36).

DNase I footprint experiments.Footprinting experiments were performed essentially as described by Dickneite et al. (12). The pRU2916 (PflgB), pRU2917 (PfliF), pRU2918 (Porf38), and pRU2919 (Prem) plasmids (Table 1) containing the named promoter regions were first digested with EcoRI and then subjected to dephosphorylation by calf intestine phosphatase (Roche). Approximately 4 pmol of each plasmid was then 5′ end labeled using 5 pmol of [γ-³²P]ATP (6,000 Ci/mmol; Amersham) with T4 polynucleotide kinase (New England Biolabs) and digested with HindIII. The promoter fragment was labeled at one terminus, extracted from a 4% polyacrylamide gel, and eluted in 4 ml of elution buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 300 mM sodium acetate [pH 5.2], 0.2% sodium dodecyl sulfate [SDS]) at 30°C overnight. The eluted probe was extracted once with an equal volume of phenol-chloroform (1:1) and then ethanol precipitated. Approximately 150,000 cpm of the labeled probe was used in each reaction mixture for the footprinting experiment. Protein-DNA complexes were formed in 50 μl of binding buffer (10 mM Tris-HCl [pH 8.0], 100 mM NaCl, 2 mM MgCl₂, 0.1 mM CaCl₂, 10 mM KCl, 1 mM DTT) supplemented with glycerol to a final concentration of 20% (vol/vol) for 20 min at 25°C. DNase I digestion was carried out by the addition of 1 μl of DNase I (10 U/μl) in 1× binding buffer for 1 min at 25°C. The reaction was stopped by the addition of 140 μl of stop buffer (192 mM sodium acetate [pH 5.2], 32 mM EDTA, 0.14% SDS, 9 μg of yeast tRNA). Samples were then extracted once with an equal volume of phenol-chloroform (1:1), ethanol precipitated, and resuspended in 5 μl of sequencing loading buffer (97.5% deionized formamide, 10 mM EDTA [pH 8.0], 0.3% xylene cyanol FF, 0.3% bromophenol blue). After denaturation at 80°C for 3 min, samples were subjected to electrophoresis on 10% polyacrylamide-7 M urea gels at 1,100 V and autoradiographed. A G+A sequence reaction was performed in parallel with the labeled DNA probe and electrophoresed on the same gel (33).

Preparation of RNA.Total RNA was essentially prepared according to Delany et al. (8). Motile cells from a 400-ml culture with an OD₆₀₀ of 0.19 were lysed by boiling for 5 min in 20 ml of 1% SDS, 2 mM EDTA, and 100 mM Tris-HCl (pH 7.5) and immediately chilled on ice for 5 min in the presence of 80 mM KCl. After precipitated DNA was removed by centrifugation at 12,000 × g for 10 min, 1.3 g of CsCl per milliliter of supernatant was added and dissolved. RNA was sedimented by centrifugation in swing-out buckets in an SW40 rotor at 50,000 × g and 22°C for 20 h. The pellet was resuspended in 400 μl Tris-EDTA, extracted with 1 volume of phenol-chloroform, and precipitated after the addition of 1/10 volume of 3 M sodium acetate (pH 5.2) and 2 to 2.5 volumes of ethanol. RNA was sedimented by centrifugation at 16,000 × g and 4°C for 20 min, washed with 600 μl of 70% ethanol, and centrifuged at 16,000 × g and 4°C for 10 min. The RNA pellet was resuspended in 50 to 100 μl Tris-EDTA, kept for 15 min on ice, aliquoted, and stored at −80°C.

5′-RACE.Rapid amplification of 5′ cDNA ends (5′-RACE) was essentially carried out using a GeneRacer kit (Invitrogen) according to the manufacturer's instructions with minor modifications. Briefly, 5′-triphosphates were converted to monophosphates by treatment of 5 μg total RNA with 0.5 U tobacco acid pyrophosphatase at 37°C for 60 min in a total reaction volume of 10 μl containing 50 mM sodium acetate (pH 6.0), 1 mM EDTA, 0.1% β-mercaptoethanol, and 0.01% Triton X-100. The reaction was stopped by phenol-chloroform extraction followed by ethanol-sodium acetate precipitation. Precipitated RNA was redissolved in water, mixed with 250 ng of GeneRacer RNA oligonucleotide (5′-CGA CUG GAG CAC GAG GAC ACU GAC AUG GAC UGA AGG AGU AGA AA-3′), heat denatured at 65°C for 5 min, and quick-chilled on ice. The GeneRacer RNA oligonucleotide was ligated at 37°C for 1 h with 5 U of T4 RNA ligase in a reaction mixture containing 33 mM Tris-acetate (pH 7.8), 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DTT, 1 mM ATP, and 40 U of RNaseOut. After phenol-chloroform extraction and ethanol precipitation, the RNA was reverse transcribed at 45°C for 60 min with 2 pmol of reverse transcriptase (RT) primer specific for flgB (5′-GGT CTT CAT CAA TTC CTC TTC-3′) or rem (5′-CAG CGT CGA TAT CGG AAT C-3′) and 200 U of SuperScript III reverse transcriptase according to the manufacturer's instructions. After inactivation at 70°C for 15 min, the RT reaction was treated with 2 U of RNase H at 37°C for 20 min. One microliter of the RT reaction product was used for PCR amplification with 10 pmol of PCR primer specific for flgB (5′-GCT TTC GAA GGG CTG AAC GTC TTT C-3′) or rem (5′-GGA ATC GGC GGC GGA GCT TAC-3′) and 20 pmol of the GeneRacer 5′ primer (5′-CGA CTG GAG CAC GAG GAC ACT GA-3′). The 50-μl PCR contained 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 2.5 mM MgCl₂, 0.1% Triton X-100, 0.2 mM concentrations of each of the four deoxynucleoside triphosphates, and 2 U of Taq polymerase (Promega). Cycling conditions were as follows: 95°C for 3 min; 28 cycles of 95°C for 40 s, 68°C for 40 s, and 72°C for 1 min; 72°C for 8 min. PCR products were separated on a 2% agarose gel, and the band of interest was excised, purified with a GenElute agarose spin column (Sigma), and cloned into pCR4-TOPO vector (Invitrogen) with a TOPO TA cloning kit for sequencing (Invitrogen) according to the manufacturer's instructions. Recombinant plasmids were purified using a QIAprep Spin Miniprep kit (QIAGEN) and sequenced using a Thermo Sequenase Primer cycle sequencing kit (Amersham Pharmacia Biotech) and ALFexpress II automated DNA sequencer (Amersham Pharmacia Biotech).

Rem is an OmpR-like transcriptional activator.The monocistronic rem gene maps within the flagellar regulon between the mot operon (15, 41) and nine flagellar genes (19) assigned by sequence similarities to known enterobacterial orthologs (31), as diagrammed in Fig. 1A. The derived 223-residue Rem polypeptide sequence (25 kDa) is a member of the OmpR family of transcriptional regulators, as revealed by sequence alignments with HP1043 (25% identity) from Helicobacter pylori (8, 46) and the prototype OmpR (22% identity) from E. coli (32) as shown in Fig. 1B. OmpR-like response regulators are characterized by an N-terminal receiver or regulatory domain with a conserved Asp residue (Asp55 in OmpR) serving as a phosphate acceptor and a C-terminal DNA-binding domain containing the three-helix pattern of a “winged helix-turn-helix” motif (32). The good correlation between secondary structure elements in the C-terminal domain of Rem (analyzed by PSIPRED [35]) and the corresponding elements derived from the crystal structure of OmpR (32), as diagrammed in Fig. 1B, underlines the close relationship of these DNA-binding proteins. The recognition helix α3 interacts with the major groove of DNA, α2 acts as a positioning helix, and the recognition wing W1 (and probably W2) interacts with the minor groove (32). Therefore, Rem belongs to the winged helix-turn-helix proteins. Unlike OmpR and CheY-like response regulators (4) that are activated by the phosphorylation of one particular aspartate, the regulatory domains of Rem and HP1043 have substitutions at some positions in the conserved signature aspartyl residues (Fig. 1B, arrowheads), suggesting that they may exert their function in the absence of regulatory domain phosphorylation, an assumption that has been confirmed (see below).

Rem and motility have similar growth phase-dependent expression profiles.A rem knockout mutant (RU11/555) generated by allelic exchange (45) provided an initial insight into the function of the gene product Rem. The mutant cells were nonmotile on swarm plates and under phase-contrast microscopy, while electron microscopy revealed that RU11/555 cells were nonflagellated. These data suggested that Rem is required for the production of flagella.

In liquid culture, the motility of wild-type S. meliloti cells is confined to a “window” during exponential growth. The global regulator VisNR (class IA) by itself cannot account for this restriction, as it is expressed throughout exponential- and stationary-phase growth (52). Attempts to uncover a VisNR-activating transient effector have failed so far. As an alternative, we studied the expression kinetics of rem as a possible key to the growth-dependent control of motility. To this end, rem promoter (Prem) activity was monitored in growing cultures using a plasmid-borne lacZ reporter construct (pRU2361) introduced into the wild-type (RU11/001) and the Δrem mutant (RU11/555) genetic backgrounds. Figure 2A (filled squares) reveals a clear-cut dependence of wild-type Prem activity on exponential growth. Promoter activity increases rapidly at the onset of exponential growth (at 4 h) and enters a plateau at mid-exponential growth (at 9 h) that is maintained until the onset of stationary growth (at 20 h), when it suddenly drops by 75% followed by a tapering to below 10% final activity. These transcription kinetics of rem are matched by Rem concentrations present in the cell. The Western blot shown in Fig. 2B records cellular levels of Rem protein sampled throughout exponential growth of wild-type S. meliloti. The Rem protein band was first detectable shortly after the start of rem transcription (6 h), and the band intensities peaked between 9 h and 20 h and then declined rapidly parallel to transcription (Fig. 2A). This rapid decay of protein is noteworthy and suggests that Rem may be the target of a specific proteolytic degradation.

Parallel to the wild type, we determined the kinetics of Prem activity in the Rem-deficient background of strain RU11/555 (Δrem) (Fig. 2A, open squares). Compared to the wild-type kinetics, this curve shows an even steeper increase of Prem activity during an initial incline (4 to 7 h) and an early decline of activity in the progressing exponential phase (at 8 to 17 h) without an extended plateau period. These data suggest that the transcription of rem is subject to autoregulation by its wild-type product, Rem, to maintain a high level of transcription throughout exponential growth. The two transcription profiles seen in the presence and absence of Rem (Fig. 2A) are believed to result from two different promoters acting on rem gene expression, as is described further below.

Phosphorylation of signature aspartyl residues is not required for Rem activity.Rem is an orphan OmpR-like response regulator (Fig. 1B) for which no cognate histidine kinase has been identified. On the other hand, the Rem regulatory domain contains signature residues (Asp6, Asp7, Asp43, Asp45, and Asp47 [Fig. 1B]) suggestive of a CheY-like acidic pocket that accommodates Mg²⁺ and a phosphate group bound to a conserved aspartate for activation (4, 28, 59). Therefore, we tried to phosphorylate recombinant Rem protein in vitro with ³²P-labeled acetyl phosphate and phosphor- amidate, substances known to act as small phosphoacyl donors to many response regulator proteins (5, 34). However, neither was capable of phosphorylating the Rem protein, although a CheY2 control exhibited good phosphorylation under the given conditions (not shown).

In another approach, we exchanged putative phosphorylation sites, namely, Asp43, Asp45, and Asp47, singly and in combination, for Asn. If Rem activity depends on the phosphorylation of any of these residues, an Asp-to-Asn exchange would eliminate the regulatory function of Rem and hence the swimming and swarming proficiency of the mutant cells. When assessing motile proficiency on swarm plates, mutant D43N exhibited a minute reduction of swarm diameter (95% of wild type [wt]), whereas mutants D47N (75% of wt) and D45N (50% of wt) exhibited moderate losses of their swarming potentials, but in no case was a complete loss of swarming (as seen in the Δrem control) observed (Fig. 3). Only when two (D45N/D47N) or three (D43N/D45N/D47N) substitutions were combined did swarm diameters drop to 29% or 0%, respectively, compared to the wild type (Fig. 3). The gradual loss of function caused by several charge-neutralizing substitutions introduced in the regulatory domain of Rem does not result from impaired protein stability (data not shown) but rather reflects conformational changes that affect the function of the regulatory domain. However, there is no evidence for phosphorylation of a single aspartyl residue as a requirement for function. In this, Rem differs from most other two-component regulatory proteins (40), but it is consistent with the OmpR-like response regulators HP1043 and HP1021 of H. pylori (Fig. 1B) (8, 46). Taken together, our data show that transcription control is exerted by the unphosphorylated Rem protein.

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FIG. 3.

Histogram of relative swarm sizes of six rem mutants compared to wild-type cells. wt, wild-type S. meliloti (RU11/001); Δrem, in-frame deletion of rem (RU11/555); D43N (RU13/006), D47N (RU13/557), and D45N (RU11/007), single amino acid substitutions; and D45/47N (RU13/009) and D43/45/47N (RU13/224), double and triple substitutions. Percentages of the wild-type swarm diameter on 0.3% Bromfield agar are the means of results from five replicates.

Rem is a transcriptional activator of class II genes.Features of the Rem-deficient (Δrem) mutant, notably the total absence of flagella, led us to ask whether regulation by Rem is limited to flagellar synthesis or if it acts on the entire cluster of flagellar, motility, and chemotaxis genes. We thus tested the expression of representative basal body, motor, flagellin, and chemotaxis genes by Western blot analysis (Fig. 4) and by lacZ fusions (Table 2) in wild-type S. meliloti and in the regulatory mutant background. Polyclonal antibodies raised against recombinant FliM (C-ring protein), MotC (periplasmic motor protein), MotE (periplasmic chaperone), purified flagellar filaments (Fla), and CheY1 (response regulator) were used to probe the expression of genes representing relevant transcription units of the flagellar regulon (55). The results shown in Fig. 4 confirm the presumed role of Rem as a master regulator, since FliM, MotC, and MotE, representatives of major class II operons, were expressed at below 5% of wild type in the Δrem mutant background and flagellin and CheY1, representatives of class III operons, were expressed at below 10% of wild type in the Δrem mutant background.

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FIG. 4.

Western blot analysis of gene expression in wild-type S. meliloti (RU11/001) and a rem deletion mutant (Δrem; RU11/555) by using polyclonal anti-FliM (α-FliM), anti-MotE (α-MotE), anti-MotC (α-MotC), anti-flagellin (α-Fla), and anti-CheY1 (α-CheY1) antibodies. Cell extracts of the wt and Δrem strains were electrophoretically separated, blotted, and analyzed with polyclonal antibodies as described in Materials and Methods.

This scheme has been confirmed and extended by probing transcription from the promoters that correspond to seven indicator genes and to genes encoding the regulators VisNR and Rem. Table 2 lists the β-galactosidase activities of the different lacZ fusions measured in Δrem and ΔvisNR tester strains expressed in proportion to the activities determined in wild-type S. meliloti RU11/001. Ideally, the ratio of mutant to wild type is 1 if the mutant allele exerts no control over the promoter tested, and it is close to 0 if the wild-type allele is needed for gene expression. Accordingly, both visNR and rem are required for the transcription of basal body (fli and flg), motor (mot), flagellin (fla), and chemotaxis (che) genes. Notably, VisNR exerts control over rem gene transcription, whereas Rem exerts no control over visNR. Moreover, when probed in the absence of Rem (strain RU11/555) during early exponential growth (as in Table 2), rem transcription exceeds that of the wild type (Rem present) by 24%, indicative of Rem autoregulation. We conclude that Rem is a transcriptional activator of class II and, indirectly, of class III genes, while its own transcription is controlled by VisNR and modulated by autoregulation.

Rem is the actual regulator of motility.The data so far indicate that two transcriptional activators, VisNR and Rem, control the motility of S. meliloti cells (Table 2). Do these regulators act in parallel, i.e., synergistically, or in a sequential order? A decision between these alternatives was reached by the following complementation experiment. The rem gene placed under the control of a constitutive lac promoter on the broad-host-range plasmid pBBR1MCS-2 (to yield plasmid pRU2818 [Table 1]) was conjugally transferred to strains RU11/555 (Δrem) and RU11/814 (ΔvisNR). Complementation by constitutive expression of the native rem allele from plasmid pRU2818 restored the swimming and swarming proficiencies of the two strains, as recorded in Table 3. Conversely, even an overexpressed visNR operon (from plasmid pRU2803) in a Δrem genetic background was incapable of restoring motility of that host, while the same plasmid could restore the motility of strain RU11/814 (ΔvisNR) (Table 3). These data provide genetic evidence that the rem gene product acts in trans and, most importantly, that it overrides the control by VisNR on motility but not vice versa. Therefore, the two master controls clearly act in a hierarchy, with VisNR at the top activating the transcription of rem, whose gene product, Rem, in turn activates the transcription of class II genes in the flagellar regulon. In the regulatory cascade determining the order of gene transcription in the flagellar regulon, we have assigned visNR to class IA (formerly class I) and rem to class IB.

It may be noticed that the motility restored by complementation with a Rem-overproducing plasmid reached only 50% or 60% of the wild-type reference (Table 3). By implication, this may be a consequence of surplus Rem regulatory protein that leads to a dosage imbalance. In support of this reasoning, an immunoblot comparing the levels of Rem in the wild type and three pRU2818-bearing strains revealed threefold-elevated levels of Rem protein in the latter strains as well as a faint leading band indicating fast degradation of Rem (Fig. 5).

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FIG. 5.

Western blot showing the levels of Rem protein in the S. meliloti wild type and different mutant strains. Strains RU11/001 (Wt), RU11/555 (Δrem), and RU11/814 (ΔvisNR) with and without plasmid pRU2818 (constitutively expressing the rem gene) were tested. Equal amounts of total cell protein were electrophoretically separated, blotted, and detected with a polyclonal anti-Rem antibody specified in Materials and Methods. Overexpressed Rem (arrow) comigrates with a faint degradation band. The slow-moving, unspecific cross-reaction band served as a loading control.

Mapping the transcription initiation sites of flgB and rem.Transcription initiation is the primary stage at which gene expression is regulated. Therefore, to better understand the molecular mechanism by which Rem activates class II genes and its own transcription, we (i) mapped the transcription initiation sites of two representative genes, flgB and rem, (ii) determined the DNA-binding sites of Rem in the promoter regions of four typical genes, and (iii) deduced possible promoter motifs from the consensus of the aligned Rem binding sites and by their relative distances to the transcription start. The 5′ ends of flgB and rem mRNAs (defining the respective transcription initiation sites) were mapped by 5′-RACE, a method based on reverse transcription of oligonucleotide-tagged mRNA and PCR amplification of the mRNA-derived cDNA (see Materials and Methods). Amplified cDNAs representing the 5′ sequences of the flgB and rem transcripts, each framed by specific 5′ and 3′ oligonucleotides, were analyzed by gel electrophoresis (Fig. 6A), cloned, and propagated in E. coli DH10B. The sequence of four individual clones each of the flgB-derived (199 bp) and rem-derived (202 bp) cDNAs yielded consistent results that are shown in Fig. 6B and C, respectively. Typically, both transcripts start with an A (+1) located 17 bp (flgB) and 25 bp (rem) upstream of the ATG start codons. Before a detailed appreciation of these findings, we will first describe footprinting experiments that revealed the DNA-binding sites of Rem.

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FIG. 6.

5′-RACE analysis of the flgB and rem transcription start sites. (A) PCR-amplified cDNA reverse transcribed from the 5′ ends of flgB and rem mRNAs; the sizes of the standard marker bands (S) are indicated. (B) Nucleotide sequence of the flgB mRNA-derived cDNA (capital letters) including 5′ and 3′ primers (lowercase letters). The transcription start site (+1) is marked by an arrow, and the ATG start codon is shown in boldface. (C) Nucleotide sequence of the rem mRNA-derived cDNA including primer sequences. Symbols are the same as in panel B.

Rem protein binds to class II and rem gene promoter DNA.Promoter probes of three representative class II genes, flgB, fliF, and orf38, were used in DNase I footprinting experiments with purified Rem protein in the presence of excess nonspecific competitor DNA. The results shown in Fig. 7 indicate that the addition of increasing amounts of Rem resulted in the protection of nucleotides spanning 33 bp of flgB and fliF promoter DNA and 26 bp of the orf38 promoter DNA. An alignment of the three sequences protected by Rem revealed 17 of 26 conserved nucleotides forming an imperfect tandem repeat as the possible recognition motif for Rem (Fig. 8, large arrows). Two other class II genes, motA and orf20, whose upstream sequences contain the same binding motif were included in the alignment (Fig. 8). This sequence is absent from the upstream regions of all inspected class III genes, suggesting that these genes are not under the direct transcriptional control of Rem but may be indirectly linked to Rem through class II-encoded flagellar export (see Discussion).

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FIG. 7.

DNase I footprinting of four promoter DNAs using Rem. Shown is the protection of three class II (A to C) and rem (D) promoter DNAs from DNase I by the binding of increasing amounts of Rem protein. Numbers refer to the number of micrograms of Rem protein in each sample. Bars indicate the position of Rem binding in each sequence. G+A is the sequencing ladder used to index the sites of protection. The arrow marks a DNase I-hypersensitive site in the rem regulatory sequence (D).

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FIG. 8.

Promoter sequences aligned relative to Rem binding. The promoter regions of flgB, fliF, and orf38 examined by footprinting (Fig. 7) were aligned with those of two other class II genes (marked by asterisks) and with the rem promoter. Nucleotides protected from DNase I digestion by Rem are underlined, and the hypersensitive site in the rem sequence is marked by an arrowhead. Conserved positions are highlighted (black, 100% identity; gray, 67% identity), and the direct repeats are delineated by two horizontal arrows. The derived P1 promoter consensus (P1 con) is shown at the top of the alignment, with −35 and −10 boxes marked accordingly. The entire 5′ untranscribed region of rem is shown at the bottom to illustrate the Rem binding site, promoters P1 and P2 (highlighted in black), and the P1 transcription start (+1). The transcription start site of flgB (+1) as determined by 5′-RACE (Fig. 6) is marked by a bent arrow, putative start sites at corresponding positions below are shaded, and ATG start codons are written in boldface.

Footprinting also served to confirm the predicted autoregulatory function of Rem (Fig. 2). When using a Prem DNA probe and increasing amounts of Rem protein (Fig. 7D), a large, 47-bp sequence was protected from DNase I digestion, with one DNase I-hypersensitive site (Fig. 7D, arrow) demonstrating that Rem binds to its own promoter. Compared to the five class II promoter sequences, the aligned Prem sequence contained only 12 of the 17 conserved positions and a less perfect tandem repeat (Fig. 8). These differences correspond to the autoregulatory function of Rem, which rather than an all-or-nothing control implies fine-tuning and a partial control of rem gene transcription.

OmpR-like activator proteins usually bind immediately upstream or inside of the promoter sequences and are believed to function by directly contacting RNA polymerase (6). We therefore examined the region inside and immediately downstream of the second repeat for conserved nucleotides that might constitute the −35 and −10 sequence elements of class II and rem gene promoters. This scrutiny suggested a putative promoter consensus for class II genes and rem, as shown in Fig. 8 (top). Accordingly, the −35 box partially overlaps with the proximal repeat sequence, and it is separated by a 7- to 11-bp spacer from the −10 box. This dense arrangement of the activator and the proposed RNA polymerase binding sites is the basis for an efficient interaction of the bound proteins in transcription initiation.

The assignment of a new promoter consensus in S. meliloti was substantiated by mapping the flgB transcription initiation site at an A (Fig. 6) 5 nucleotides downstream of the −10 promoter box (Fig. 8). Purine nucleotides at corresponding positions of all aligned 5′ untranslated regions listed in Fig. 8 suggest that these A's and G's (shaded) mark the transcription starts of fliF, orf38, motA, and orf20. While we believe that the same holds for the rem gene, 5′-RACE positioned the rem transcription start 102 bp further downstream (Fig. 6). However, this unexpected result was rationalized when an almost perfect σ⁷⁰-like rhizobial promoter, CCCGAT-N₁₇-CTATAT (30), was detected 9 nucleotides upstream of the mapped transcription start (Fig. 8, bottom). Therefore, we propose that the rem gene is under dual control of a weaker upstream promoter (P1) autoregulated by Rem and a stronger, σ⁷⁰-like downstream promoter (P2) that produced the signal seen by 5′-RACE. Despite various attempts to map the P1 transcription start of rem, our RT primer seems to have exclusively selected the more abundant P2 mRNA species. The activities of both P1 (on top of the control by Rem) and P2 are controlled by the global regulator VisNR (Table 2). The obvious next step is to identify VisNR binding sites upstream of P1 and P2, using standard techniques.