In Sinorhizobium meliloti, choline is the direct precursor of phosphatidylcholine, a major lipid membrane component in the Rhizobiaceae family, and glycine betaine, an important osmoprotectant. Moreover, choline is an efficient energy source which supports growth . Using a PCR strategy, we identified three chromosomalgenes [choXWV] which encode components of an ABC transporter:ChoX [binding protein], ChoW [permease], and ChoV [ATPase].Whereas the best homology scores were obtained with componentsof betaine ProU-like systems, Cho is not involved in betainetransport . Site-directed mutagenesis of choX strongly reduced[60 to 75%] the choline uptake activity, and purification ofChoX, together with analysis of the ligand-binding specificity,showed that ChoX binds choline with a high affinity [K_D, 2.7µM] and acetylcholine with a low affinity [K_D, 145 µM]but binds none of the betaines . Uptake competition experimentsalso revealed that ectoine, various betaines, and choline derivativeswere not effective competitors for Cho-mediated choline transport.Thus, Cho is a highly specific high-affinity choline transporter.Choline transport activity and ChoX expression were inducedby choline but not by salt stress . Western blotting experimentswith antibodies raised against ChoX demonstrated the presenceof ChoX in bacteroids isolated from nitrogen-fixing nodulesobtained from Medicago sativa roots . The choX mutation did nothave an effect on growth under standard conditions, and neitherNod nor Fix phenotypes were impaired in the mutant, suggestingthat the remaining choline uptake system[s] still present inthe mutant strain can compensate for the lack of Cho transporter.

 
Choline is a common constituent of eukaryotic membranes in theform of phosphatidylcholine [PC] and therefore should be widespreadin different environments, including the soil and the rhizosphere. Indeed, significant amounts of choline are readily liberatedinto the environment from plant and animal residues [15] . Sinorhizobiummeliloti, a plant root-associated bacterium, possesses distincttransport activities for choline uptake [27] and has the abilityto oxidize choline to glycine betaine via the bet operon [34,24] . In contrast to Escherichia coli and Bacillus subtilis [25, 2], S . meliloti can use choline for growth . This depends ona functional bet locus [34, 24] associated with catabolism ofglycine betaine which is absent in E . coli and B . subtilis.This catabolism is reduced under hyperosmotic conditions, andunder these conditions glycine betaine accumulation is favored[34] . Moreover, due to the presence of a PC synthase in S . meliloti, which directly condenses choline to CDP-diacylglyceride, cholineis a direct precursor of PC, as recently demonstrated for otherbacteria, including Agrobacterium, Brucella, and Pseudomonas [6, 19] . In addition to this PC synthase pathway, S . melilotipossesses a methylation pathway for PC biosynthesis which functionsby threefold methylation of phosphatidylethanolamine with S-adenosylmethionineas a methyl donor [5] . Whereas the presence of PC is rather unusual in bacterial membranes, PC is a major lipid membrane component in the Rhizobiaceae family, and PC biosynthesis is required for normal growth of S . meliloti [5] . To fulfill therequirement for choline, S . meliloti requires effective transportsystems to take up this trimethylammonium compound from exogenoussources.

In E . coli, import of choline is mediated at a low concentration by the high-affinity BetT transporter and at a high substrate concentration by the low-affinity multicomponent ABC uptakesystem ProU [36, 16] . In B . subtilis, choline uptake occursby two evolutionarily highly conserved ABC transporters, OpuBand OpuC, that probably evolved through the duplication of aprimordial gene cluster . Despite the close sequence relatednessof the two systems, these high-affinity transporters exhibitvery different substrate specificities [13] . In S . meliloti,three kinetically distinct transport activities for cholineuptake have been identified; one constitutive activity has lowaffinity, and two activities have high affinity, and they areeither inducible by choline or constitutively expressed [27]. While choline has multiple functions in this symbiotic bacterium, nothing is currently known at the molecular level about the route of choline transport . The present study was initiatedto gain some understanding of the mechanisms of choline uptakein S . meliloti, and our results provide the first identificationand detailed analysis of a high-affinity choline-binding protein-dependent transport system [Cho] in this species . We also demonstrated the high level of specificity of the binding protein and its expression in bacteroids from nodules of Medicago sativa, the host plant of S . meliloti.

 
Chemicals. Choline and glycine betaine were purchased from Sigma-Aldrich[Saint Quentin Fallavier, France], and proline betaine was purchasedfrom Extrasynthčse [Genay, France] . Radioactive [methyl-¹⁴C]choline [2.04 GBq/mmol] and [methyl-¹⁴C]acetylcholine [0.68 GBq/mmol]were purchased from Amersham Corp . [Little Chalfont, United Kingdom] and Sigma-Aldrich, respectively . [methyl-¹⁴C]glycine betaine was prepared from [methyl-¹⁴C]choline as previouslydescribed [24], and [U-¹⁴C]proline betaine [4.6 GBq/mmol] wasobtained from the Commissariat ŕ l'Energie Atomique [Gif-sur-Yvette,France] . Ni²⁺-nitrolotriacetic acid resin was obtained fromQIAGEN [Courtaboeuf, France] . Rabbit anti-ChoX antibody wasprepared by Eurogentec [Angers, France], and rabbit anti-immunoglobulinG alkaline phosphatase conjugate was obtained from Sigma-Aldrich.

Bacterial strains, plasmids, and growth conditions. The bacterial strains and plasmids used in this study are listedin Table 1 . The E . coli DH5 and MT616 strains were used forsubcloning of the pF1 insert and as a helper strain for triparentalmating, respectively . The E . coli BL21[DE3][pLysS] strain wasused for overexpression of the choX gene from the T7 promoterin plasmids pETNE and pETNX . S . meliloti strains were routinelygrown at 30°C in Luria-Bertani [LB] medium containing 5g of NaCl per liter, 2.5 mM MgSO₄,and 2.5 mM CaCl₂ . For uptakeexperiments and periplasmic protein extraction, cells were grownin MCAA medium containing 0.1% sodium malate, 0.1% CasaminoAcids [technical], and minerals as described previously [34].For physiological analysis of the role of Cho and for the choXexpression study [Western blotting], cells were grown in M9minimal medium [20] supplemented with 0.2% mannitol or 0.2%choline as a carbon source . The osmolarities of the variousmedia were increased by addition of 0.3 M NaCl . When necessary,glycine betaine was added at a concentration of 1 mM, and cholinewas used at a concentration of 7 mM, which allowed maximal stimulationof choline oxidase [34] . The antibiotics ampicillin, tetracycline,chloramphenicol, and spectinomycin were used in E . coli culturesat final concentrations of 100, 20, 20, and 100 µg/ml,respectively . Rifampin and spectinomycin were used in S . meliloticultures at final concentrations of 20 and 100 µg/ml,respectively.

 
DNA manipulation and cloning of the cho locus. Restriction analysis, ligation, transformation, plasmid DNA extraction, and Southern hybridization were carried out by standard techniques [21, 31] . DNA probes were labeled by using the Prime-a-generandom priming system [Promega, Charbonničres, France]and [-³²P]dCTP [Amersham Corp.] . A PCR strategy was used toamplify an internal fragment of the choV gene . The PCR mixturescontaining each degenerate primer and Rm5000 genomic DNA werecycled automatically by using a Biometra thermocycler [T gradientmodel; Biometra GmbH, Göttingen, Germany] through temperatureand time cycles as follows: denaturation at 95°C for 1 min,annealing at 40°C for 1 min, and extension at 70°C for1 min . The sequences of the two degenerate primers used were5'-GAR ATI TTY GTI ATI ATG GG-3' [bup1] and 5'-CAT DAT IGC DATICK RTC ICC-3' [pro4] . The resulting fragment, which was theexpected size [564 bp], was cloned into pGEMT to obtain pGQ5 and was sequenced . The latter plasmid was used as a probe to screen, by colony hybridization, a genomic DNA library of S. meliloti obtained by partial Sau3A digestion of S . meliloti 2011 DNA cloned into pLAFR3 and kindly provided by D . Kahn [Laboratoire de Biologie moléculaire des Relations Plantes-Microorganismes, CNRS-INRA, Castanet-Tolosan, France] . One clone containing a recombinant cosmid, designated pF1, strongly hybridized withthe probe . The 16-kb insert of pF1 was subcloned, and the regionof interest was sequenced by MWG Biotech [Ebersberg, Germany]by using the fluorescent ABI dye-labeled deoxy terminator method.DNA and protein sequences were analyzed by using BLAST protocols[1].

Mutagenesis of S . meliloti. The choX gene was mutated by insertion of a BamHI-digested interposon [Sp^r/Sm^r] into the BglII restriction site of pSUP6.5H,which corresponded to a 6.5-kb HindIII fragment from pF1 clonedat the HindIII site of the suicide vector pSUP202 [Table 1].Triparental spot mating was used to introduce recombinant plasmidsfrom E . coli into S . meliloti as previously described [7, 8].The insertion was finally recombined into the S . meliloti Rm5000 genome, and correct recombination of the interposon in the genomic choX gene was verified by Southern hybridization.

Overproduction and purification of ChoX. The choX gene under the control of the T7 promoter from pET20-b[+]was overexpressed in E . coli BL21[DE3][pLysS] [Table 1] . Twoconstructs were made . The first construct [pETNE], which allowed overproduction of a native ChoX protein without any extra amino acid residues, resulted from a PCR fragment digested by NdeIand EcoRI and cloned into the pET20b vector restricted withthe same two restriction enzymes . The PCR fragment was obtainedby using S . meliloti RCR2011 DNA as the template, Pfu polymerase,and primers PxNde [5'-AGG GGA ACG ACG CAT ATG ATA AGG A-3';yielding an NdeI site] and PxEco [5'-AGT CAG GAA TTC CAC GAAACA GGG T-3'; overlapping an EcoRI site] . The second construct[pETNX], which allowed overproduction of a ChoX protein witha C-terminal His₆ tag, resulted from a PCR fragment obtainedby amplification of RCR2011 DNA with primers PxNde and PxXho[5'-TGC CGC CGA CTC GAG GCC GAG G-3'], which created a XhoIsite . This PCR fragment, digested with NdeI and XhoI, was clonedinto pET20b . The purification steps used for ChoX overexpressedfrom pETNX in the E . coli BL21[DE3][pLysS] strain were thosedescribed by Novagen [Merck KGaA, Darmstadt, Germany] . Briefly,E . coli recombinant cells were grown at 37°C in LB medium[200 ml] with ampicillin [50 µg/ml] and chloramphenicol[30 µg/ml] until the A₆₀₀ was 0.8, and this was followedby a 2-h expression period initiated by addition of 0.4 mM isopropyl-ß-D-thiogalactopyranoside [IPTG] . The cells were centrifuged, resuspended in 4 ml of lysis buffer [50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole [pH 8.0],1 mg of lysozyme per ml], and incubated at 4°C for 30 min before sonication on ice [eight times for 30 s each time] . After centrifugation, the protein content of the soluble fraction [supernatant] was determined by the Bradford method [3] . Purificationby Ni²⁺-nitrilotriacetic acid affinity chromatography was performedas described by the supplier [QIAGEN] . ChoX was eluted fromthe column with 200 mM imidazole in buffer A [50 mM NaH₂PO₄,300 mM NaCl; pH 8.0].

Transport assays. Cells were harvested at an A₆₀₀ of 1.5, washed twice in thefresh medium used for the culture, and diluted to obtain a finalA₆₀₀ of 0.5 . All assays were carried out at 30°C with 1ml of cell suspension for 1 min, and radioactive substrates[100,000 dpm] were used at the following concentrations: 10µM for choline, 1 and 40 µM for glycine betaine, and 10 µM for proline betaine and proline . Uptake was determined by rapid filtration through GF/F glass microfiberfilters [Whatman, Maidstone, United Kingdom], which were rinsedwith 3 ml of the corresponding medium . The radioactivity remainingon the filters was determined with a liquid scintillation spectrometer[model LS6000SC; Beckman Instruments, Villepinte, France] . Thetransport rate was linear during the 1-min assay, and therewas no inhibition by the intracellular choline accumulated bythe cells, in agreement with previous results [27] . For competition experiments, cold competitors were added at a final concentrationof 100 µM or 1 mM to a 10 µM [¹⁴C]choline solution[100,000 dpm] . Competition uptake experiments were performedwith a 1-min incubation period before filtration.

Periplasmic protein extraction and binding assays. E . coli BL21[DE3][pLysS] was grown to an A₆₀₀ of 0.6 in LB mediumcontaining 0.4 mM IPTG, and S . meliloti Rm5000 and M1A weregrown to an A₆₀₀ of 1.5 in MCAA medium supplemented with 7 mMcholine . Cells were collected by centrifugation [10,000 x g, 10 min, 20°C] and resuspended in 10 mM Tris-HCl [pH 7.5]. Periplasmic proteins were released by cold osmotic shock asdescribed by Neu and Heppel [22] and were concentrated by ultrafiltrationby using the standard procedure [17] . To determine binding activities,100 µg of periplasmic proteins was incubated overnightwith 5 nmol of [¹⁴C]choline [500,000 dpm] in 10 mM Tris-HClbuffer [pH 7.5] at 4°C, separated by nondenaturing polyacrylamidegel electrophoresis [PAGE], and autoradiographed, as describedpreviously [17] . For determination of the substrate-bindingaffinities for choline and acetylcholine, binding assays withthe purified ChoX were performed by using ammonium sulfate precipitation[29] . Samples containing 8 µg of protein [final concentration,5 µM] were incubated at 25°C for 15 min with variousconcentrations of ¹⁴C-labeled substrates [1 to 40 µM cholineand 1 to 120 µM acetylcholine] in 50-µl reactionmixtures containing 10 mM Tris-HCl buffer [pH 7.4] . The proteinswere precipitated by adding 950 µl of an ice-cold saturatedammonium sulfate solution, and after incubation for 15 min onice, the precipitated ChoX protein was collected by rapid filtrationonto GF/F glass microfiber filters [Whatman] . Each filter wasthen washed twice with 3 ml of an ice-cold ammonium sulfate solution, and the radioactivity retained by ChoX was determined by scintillation counting . For each substrate concentration, measurements were obtained in triplicate in order to determinethe binding constant . Alternatively, an analysis of the specificityof the binding activity of ChoX was performed by gel filtration.A 100-fold excess of unlabeled competitors was added into thebinding assay mixture, and the radioactivity retained by ChoXwas separated from the unbound [¹⁴C]choline on a gel filtrationcolumn [Sephadex G-25; Amersham Biosciences Europe GmbH, Orsay,France] that was eluted with 100 mM Tris-HCl buffer [pH 7.5].

Immunological analysis. Total cell proteins, periplasmic proteins, and purified ChoXprotein were separated by sodium dodecyl sulfate [SDS]-PAGEand transferred to nitrocellulose membranes [Hybond protein;pore size, 0.2 µm] by electroblotting . Immunoblottingwas performed by using a 1/20,000 dilution of a polyclonal serumraised against the purified ChoX protein of S . meliloti . Theimmunoblots were developed with rabbit anti-immunoglobulin Galkaline phosphatase conjugate [Sigma-Aldrich], as instructedby the manufacturer.

Nodulation, nitrogen fixation assays, and bacteroid preparation. The symbiotic proficiency of S . meliloti strains was assayed by using alfalfa [M . sativa L . cv . Europe] seedlings grown in a sterilized mixture of vermiculite and sand and inoculatedwith the appropriate strains 1 week after sowing, as describedpreviously [18] . The number and the mass of nodules were determined3, 5, 6, and 7 weeks after inoculation . Nitrogen fixation capacitywas determined by C₂H₂ reduction by using a gas chromatograph[38] . Freshly harvested nodules [4 weeks old] were used to isolate bacteroids as described by Trinchant et al . [38], and proteinswere extracted for SDS-PAGE analysis.

Nucleotide sequence accession number. The nucleotide sequence of the cho locus has been depositedin the GenBank database under accession number AF360731.

 
Cloning strategy and genetic organization of the cho locus. The presence of periplasmic glycine betaine and choline-binding proteins in S . meliloti [4, 17] suggests that ABC-type transportersmight contribute to trimethylammonium uptake in this species.In order to isolate such transporters, a PCR strategy basedon domains specifically conserved in ATPases of well-characterizedbacterial ABC ProU-like transporters and ProV-like orthologuespresent in databases was used . Two stretches of amino acids,EIFVIMG [positions 65 to 71] located upstream of the WalkerA motif, and GDRIVLM [positions 243 to 249] located downstreamof the Walker B motif in the OpuAA system from B . subtilis,were selected to design degenerate primers, which were designatedbup1 and pro4 [see Materials and Methods] . A PCR fragment ofthe expected size was obtained by using Rm5000 genomic DNA asthe template and was cloned into the pGEMT vector before a genomicDNA library of S . meliloti 2011 was screened . A recombinantcosmid [pF1] bearing a 16-kb EcoRI insert was isolated by colonyhybridization . By using restriction analysis and Southern hybridization,the region homologous to the PCR-amplified fragment was subclonedin pBS-SK [Fig . 1A] and sequenced . Analysis of the sequencedDNA [4,311 bp] revealed the presence of five open reading frames[accession number AF360731] . The genetic organization of thisregion [Fig. 1A] indicated that there were two divergently transcribed sets of genes, with the tdk gene encoding a putative thymidine kinase located 288 bp from the choXWV genes and open reading frame Y04457 having an unknown function . Analysis of the S. meliloti genome showed that these genes are present on the chromosome [SMc02737 to SMc02739] and are located upstream of the nolR gene [http://sequence.toulouse.inra.fr/meliloti.html] . Withinthe cho locus, the first gene, choX, is separated from choWby a large 160-bp noncoding region, while choW and choV overlapby 4 bp in the ATGA sequence, suggesting that there is translationalcoupling between these two genes . The choXWV genes had the characteristicsof genes encoding an ABC transporter, and the genetic organizationof the cho locus followed the binding protein first rule, whichis mainly encountered in a binding protein transport operon.The choX gene encodes a hydrophilic protein containing 309 aminoacid residues [M_r, 32,904] which exhibited the characteristic signature of a signal peptide for secretion into the periplasm[26] . ChoX has a positively charged N-terminal sequence [MIR⁺TLSLK⁺], followed by a hydrophobic stretch of amino acids [MLAGAVCMATLTA] and the motif GSAFA, in agreement with the consensus GXAXA sequence of a signal peptidase I site [Fig . 1B] . Comparison of the ChoXprotein sequence with protein sequences in the data librariesrevealed that the highest amino acid identity [28%] was obtainedwith the glycine betaine-binding proteins BusAB, GbuC, and OpuACfrom Lactococcus lactis, Listeria monocytogenes, and B . subtilis,respectively [Fig . 1B] . As usually observed for the ligand-bindingproteins involved in ABC transporters [37], ChoX is the lessconserved protein encoded by the cho locus . ChoW [M_r, 30,323; 281 amino acids] is highly hydrophobic, with six putative transmembrane domains, and corresponds to the integral inner membrane permease component of the transporter . ChoW is homologous to the glycine betaine permease BusAB of L . lactis, OpuAB of B . subtilis, and ProW of E . coli [45 to 50% amino acid identity] . However, ChoW is considerably smaller than ProW [354 amino acids], mostlyas the consequence of the lack of the N-terminal region of ProWthought to be exposed in the periplasmic space [39] . It should also be mentioned that the choW start codon codes for a valine. The last gene, choV, encodes a 349-amino-acid hydrophilic protein with a predicted M_r of 37,615 . The ChoV protein is likely theATPase of the transporter and has motifs characteristic of suchproteins [i.e., Walker A and B boxes, a linker peptide, and a switch motif] . The highest levels of homology [about 50% amino acid identity] of ChoV with proteins in the databases were the levels of homology with ATPases of glycine betaine ProU-typesystems, including ProV of E . coli, BusAA of L . lactis, OpuAAof B . subtilis, and GbuA of L . monocytogenes.

 
cho encodes a choline transporter. The highest levels of homology for cho-encoded proteins wereobtained with the components of glycine betaine-proline betaineProU-like systems, which suggested that the S . meliloti transporteris involved in trimethylammonium uptake . In order to preciselydefine the substrate of Cho, transport activities for glycinebetaine, proline betaine, and choline, the major quaternaryammonium compounds, were measured in wild-type strain Rm5000and mutant strain M1A obtained by reciprocal recombination witha choX:: construction [Fig . 1A] . Surprisingly, there were no significant differences in betaine uptake activities betweenthe two strains [data not shown], whatever the growth mediumcomposition [with or without added 0.3 M NaCl, in the presenceor the absence of 1 mM glycine betaine, or a combination ofthe two conditions], the growth phase of the culture [exponentialor stationary phase], and the substrate concentration used foruptake measurement [1 or 40 µM] . In contrast, when 10µM [methyl-¹⁴C]choline was used as a substrate in transportexperiments, clear differences in uptake activities were observedbetween strains Rm5000 and M1A grown until the stationary phase[Fig . 2] . The mutation in choX reduced by 75 and 60% the cholineuptake activity in cells grown in control MCAA medium and inMCAA medium containing 7 mM choline, respectively . It is alsointeresting that in both strains, choline uptake was inducedabout sixfold when choline was added to the growth medium, whereasaddition of 0.3 M NaCl alone resulted in a very low level ofcholine accumulation . In choline-induced cells, addition ofsalt did not result in uptake inhibition, and a mutation inChoX reduced by 35% choline uptake activity at high osmolarity [0.3 M NaCl] . These results suggested that Cho is a high-affinity choline ABC transporter, which has an overall activity thatis stimulated by choline but not by salt stress . Interestingly,the presence of choline in the growth medium alleviated theinhibition observed in salt-stressed cells . In addition, Choactivity might be growth phase dependent since no differencein uptake activity was detected between the wild-type and mutantstrains when exponentially grown cells were used [data not shown].The remaining choline transport activity in mutant strain M1Aindicated that there is another system or other systems forcholine uptake in S . meliloti.

 
The specificity of choline transport in the wild-type strainwas assessed in competition experiments performed in the presenceof 10- and 100-fold molar excesses of potential competitors.Different compounds known to be compatible solutes in soil bacteria[ectoine, glycine betaine, proline betaine, -butyrobetaine, and carnitine] were tested together with choline derivatives [acetylcholine, phosphorylcholine, and choline-O-sulfate] . Dimethylthetine,an S-methyl homologue of glycine betaine, trigonelline, andspermidine were also tested . The polyamine was chosen sinceit is transported by E . coli via the PotA system, whose ATPase[14] shows significant homology with ChoV [43% identical aminoacid residues] . At a competitor/substrate ratio of 10/1, acetylcholinewas the only competitor of choline uptake; 53% inhibition and32% inhibition were observed in cells grown in the presenceof choline and maintained at low and high osmolarities, respectively.Increasing the competitor/substrate ratio to 100/1 showed thatacetylcholine was the only substrate that competed with cholineuptake in the wild-type strain . In order to evaluate the roleof Cho in acetylcholine uptake, [¹⁴C]acetylcholine transportactivity was measured in the wild-type and M1A mutant strainsgrown at low osmolarity in the presence of choline and collectedat the stationary phase . At substrate concentrations of 10 and100 µM, no significant difference was observed [data not shown], indicating that under the growth conditions tested,Cho might not be involved in acetylcholine transport . Thus,the Cho transporter seems highly specific for choline and isnot involved in glycine betaine or proline betaine uptake, despiteits high levels of homology with betaine transporters.

ChoX purification and binding activity. The results presented above clearly showed that S . melilotiCho behaved like a choline transporter . To subsequently investigatethe role of the periplasmic binding protein, ChoX was overproducedand purified . The choX gene was overexpressed in E . coli BL21[DE3][pLysS] under the T7 promoter of pET20-b[+] in a fusion with a His tag-specifying sequence . The recombinant plasmid pETNX [Table 1] allowed overproductionof ChoX, which was purified by Ni²⁺ chelate affinity chromatographyas described in Materials and Methods . SDS-PAGE analysis ofthe purified fraction revealed a single band corresponding toa protein with an apparent molecular mass of 33 kDa, in agreementwith the expected size of the His₆-tagged ChoX without its peptidesignal [M_r, 32,188] . Overall, when we started with an extractcontaining 63 mg of soluble proteins, 1.3 mg of the highly purifiedperiplasmic form of ChoX was obtained.

This purified protein was used for binding assays performedwith ¹⁴C-labeled substrates, and the complex formed between ChoX and the substrate was separated from the unbound substrateby gel filtration . Of the four compounds tested [choline, acetylcholine, glycine betaine, and proline betaine], only choline and acetylcholine were bound to ChoX . In the presence of [methyl-¹⁴C]choline as the substrate, addition of a 100-fold excess of unlabeled choline was followed by total disappearance of the label associated with ChoX, demonstrating the specificity of the binding phenomenon [data not shown] . Addition of a 100-fold excess of unlabeled acetylcholine significantly decreased the intensity of the labeling by about 70%, whereas, as expected, addition of unlabeled glycine betaine and proline betaine had no effect, confirming that the specificity of the binding phenomenon is very narrow . The maximal binding capacity for choline, as determined by ammonium sulfate precipitation, was 4.8 nmol/mg of protein with a free ligand concentration of 40 µM . The calculated K_D for choline was 2.7 µM, whereas it was much higher [145 µM]for acetylcholine . The binding activity was also detected bydirect PAGE of the ¹⁴C-labeled ligand-protein binding complexin nondenaturing conditions as described previously [17] . Briefly,the purified ChoX protein and periplasmic fractions from the E . coli BL21 and BL21[pETNE] strains were incubated with [methyl-¹⁴C]choline and subjected to PAGE, followed by autoradiography [Fig . 3A]. Since high-affinity choline uptake in E . coli depends only on the betaine choline carnitine transporter BetT [16], no choline-bindingactivity was detected in periplasmic proteins from E . coli strainBL21 . However, induction of choX gene expression in the E . coliBL21 strain carrying the choX gene on the recombinant plasmidpETNE [Table 1] directed the synthesis of a [¹⁴C]choline-bindingprotein . Thus, ChoX was expressed and translocated to the periplasm,meaning that its signal peptide was successfully recognizedby the secretion machinery of E . coli . In addition, it is interestingthat the presence of a C-terminal His tag on ChoX had no effecton the choline-binding activity since the ¹⁴C-labeled purified protein showed the same electrophoretic mobility as the ¹⁴C-labeled untagged native overproduced ChoX protein produced in E . coli complemented with the recombinant plasmid pETNE . These binding assays indicated that the narrow range of substrates transportedby Cho seems to be linked to the high binding specificity ofChoX . While ChoX can bind [¹⁴C]acetylcholine in vitro, results presented above suggested that acetylcholine was not transportedby Cho . This discrepancy might be explained by the very lowaffinity of ChoX for acetylcholine [K_D, 145 µM], whereasuptake experiments were performed with a substrate concentrationof 100 µM.

 
ChoX is a periplasmic choline-binding protein present in free-living and symbiotic forms of S . meliloti. The results presented above clearly show that heterologous expressionof the choX gene from S . meliloti results in a choline-bindingprotein . Using nondenaturing gel electrophoresis followed byautoradiography, we subsequently investigated the presence ofsuch a protein in periplasmic extracts of S . meliloti wild-typestrain Rm5000 grown at low osmolarity in the presence of choline.A strong radioactive band corresponding to the [¹⁴C]choline-choline-binding protein complex and having the same electrophoretic mobilityas the [¹⁴C]choline-purified ChoX protein complex was detected in periplasmic extracts from the Rm5000 strain . In contrast, analysis of crude periplasmic extracts from the M1A mutant strain grown in the same conditions did not show that any label wasbound [Fig . 3B] . Thus, in the growth conditions used here, ChoX was the only choline-binding protein present in the periplasm of S . meliloti.

To analyze the effects of growth conditions on the presenceof ChoX in S . meliloti and to identify ChoX in bacteroids, the differentiated symbiotic form of the bacterium present in alfalfa nitrogen-fixing nodules, a polyclonal antibody specificallyraised against purified ChoX was produced . By immunoblotting,this anti-ChoX antibody was able to detect a 32-kDa proteinin total extracts from wild-type strain Rm5000 grown in MCAAmedium supplemented with choline [Fig . 4A] . As expected, thisprotein was slightly smaller than the purified recombinant His₆-tagged ChoX . No signal was detected with extracts from the choX:: M1Amutant strain . While we could not totally eliminate the presence of a ChoX homologue in S . meliloti Rm5000 which could not react with the ChoX antibody, the data are in full agreement withthe results presented above and obtained after radiography ofthe nondenaturing gel [Fig . 3B] . Since choline is a ubiquitous molecule in plants and has been identified in alfalfa nodules[18], we wanted to determine whether this transporter was presentin bacteroids . Therefore, bacteroids from nodules produced onM . sativa roots by strains Rm5000 and M1A were purified, andthe ChoX protein level was estimated by immunoblotting . As shownin Fig. 4A, a protein of the expected size, which corresponded to ChoX, was present in wild-type bacteroids, whereas no signal was detected in the mutant choX bacteroids . This result demonstrated that the ChoX protein is synthesized by the symbiotic form of S . meliloti and suggested that the whole Cho system is probably functional in bacteroids.

 
Uptake experiments have indicated that the rather low constitutive Cho activity was significantly induced by the presence of the substrate in the growth medium, independent of the osmolarity[Fig. 2] . To get a better understanding of the regulation of this system, immunodetection of ChoX in total protein extracts of S . meliloti grown in M9 medium after addition of choline[7 mM] or salt [0.3 M NaCl] was performed [Fig . 4B] . In the absence of choline, a very low level of ChoX was observed, particularly in cells grown in the presence of 0.3 M NaCl, in which the choline-binding protein could not be detected . Addition of choline, independent of the presence of 0.3 M NaCl, led to an increase in the ChoXlevel . Thus, the availability of choline highly induced ChoXexpression, whereas salt stress alone had a negative effecton ChoX synthesis . The level of ChoX detected in this experimentis in good agreement with the choline uptake activity of Cho[Fig . 2] . Additional experiments showed that a choline concentrationof 1 mM was sufficient to induce ChoX biosynthesis [data notshown].

Phenotypes of S . meliloti choX mutant. In order to precisely define the phenotype of a choX-deficientS . meliloti mutant, the growth properties of the free-living heterotrophic bacterium and the efficiency of the endosymbioticform were evaluated . Since S . meliloti can use choline as an osmoprotectant and as an energy source after it is convertedto glycine betaine [34], the growth parameters were studied in high-osmolarity medium [0.3 to 0.7 M NaCl] supplemented or not supplemented with choline [10 µM, 100 µM, and7 mM] and in minimal medium containing choline [14 mM] as theonly carbon and/or nitrogen source . Significant differencesin growth rates and final cell yields between wild-type strainRm5000 and mutant strain M1A were never observed [data not shown].Obviously, other choline uptake system[s] can compensate forthe lack of the Cho transporter . Indeed, in the presence ofcholine in the growth medium, 65 and 40% of the choline uptakeactivity of the wild-type strain still remained in the mutantstrain grown at high and low osmolarities, respectively [Fig. 2] . Thus, it is not very surprising that the choX mutation didnot have an effect under standard laboratory growth conditions.

The effects of the choX mutation on nodulation of the alfalfa host plant and on nitrogen fixation activity were also tested. Seedlings were inoculated with the wild-type and mutant strains,and the nodulation efficiency was monitored for 7.5 weeks . Nodifference was observed in the kinetics of nodulation betweenthe Rm5000 and M1A strains, and the weights of 7.5-week-oldnodules obtained with the two strains were comparable [datanot shown] . In addition, the acetylene reduction activitiesmeasured at various times after bacterial infection [4, 6, and7.5 weeks] for M1A-nodulated plants and Rm5000-nodulated plantsindicated that the nitrogen fixation activity was not altered[data not shown] . Thus, the nodulation and nitrogen-fixing phenotypesof the M1A mutant strain were Nod⁺ and Fix⁺, and the maximumacetylene reduction activity was 25 nmol of ethylene per h permg [fresh weight] of nodules . As observed for the free-livingbacterium, the absence of a functional Cho system is not crucialfor the endosymbiotic form of S . meliloti.

 
Because of the competition among soil bacteria for carbon and nitrogen sources, the symbiotic bacterium S . meliloti is subjected to major nutrition challenges . Consequently, this organism is equipped with a very large number of transport systems, whichare probably efficiently regulated . As shown by the entire annotated sequence of S . meliloti [11], genes encoding transport systemsconstitute the largest class of genes [12%], and most of thesegenes are ABC transporters, which are still uncharacterized. The data presented here describe the first isolation and characterization of an S . meliloti locus which encodes a trimethylammonium, high-affinity,multicomponent, binding protein-dependent transport system,Cho . While Cho is closely related to glycine betaine ProU-liketransport systems, its substrate is not betaine but rather choline.It has been shown previously that choline is an efficient carbonand nitrogen source for this bacterium, after it is converted into glycine betaine, which is further catabolized [34] . However,the mechanisms of choline transport are not known, and no geneshave been assigned to these functions yet . In this study, we isolated and sequenced an S . meliloti DNA fragment composed of three genes, choXWV, which encode a typical ABC transporter with a soluble, ligand-binding, periplasmic protein [ChoX],an integral inner membrane component [ChoW], and an ATPase linkedto the membrane [ChoV] . Inactivation of the choX gene had noeffect on high-affinity glycine betaine or proline betaine uptakeactivity, whereas it caused a strong reduction in high-affinitycholine transport . Since choW- and choV-deficient mutants were not constructed, the corresponding genes were designated basedon in silico data and their physiological proximity to choX.The remaining choline uptake activity in the choX-deficientmutant indicated that there is at least one other transporterfor this quaternary ammonium compound, in agreement with previousresults showing distinct transport activities for choline uptakein S . meliloti [27] . Indeed, on the basis of the annotated sequenceand considering the homology with previously characterized cholinetransporters [BetT from E . coli and OpuB from B . subtilis],analysis of the entire genomic sequence revealed the presenceof other potential choline transporters, either an ABC systemor a symporter . Purified ChoX binds selectively and with high affinity [K_D, 2.7 µM] to choline, but none of the betainesappear to be recognized . Only acetylcholine can also be boundby ChoX, but it is bound with a very low affinity [K_D, 145 µM].Since the periplasmic protein is thought to ensure the substratespecificity and directionality of the overall transport reaction,Cho can be defined as a strict high-affinity choline transporter.Surprisingly, while ChoX binds choline, no significant homologywas found with either OpuBC or OpuCC, the choline-binding proteinsof B . subtilis . In contrast, the highest levels of homologyfor ChoX were found with glycine betaine-binding proteins of the L . lactis BusA, L . monocytogenes Gbu, and B . subtilis OpuAtransporters and to a lesser extent with ProX, the periplasmic protein of the E . coli glycine betaine ProU transporter [Fig. 1B] . The high-resolution crystal structure of the ProX protein,in complex with each of its ligands [i.e., ProXglycine betaineand ProXproline betaine], was just determined recently [32].The binding pocket is formed by the indole groups of three tryptophan residues, Trp⁶⁵, Trp¹⁴⁰, and Trp¹⁸⁸ . This crystallographic studyrevealed that cation- interactions between the positive chargesof the quaternary amines of the ligands and the indole groupsof the three tryptophan residues are the key determinants ofthe high-affinity binding of betaines by ProX . In addition,the entire motif C¹³⁶XPGWGC¹⁴² is strictly conserved among severalclose homologues of ProX from various bacteria . While the overallstructure of the S . meliloti choline-binding protein is stillunknown, it is more likely that choline interacts with ChoXby using the positive charge of the quaternary amine group.However, if two tryptophan residues from ProX, at positions65 and 188, are well conserved in ChoX, the Trp residue at position140 is replaced by an Asn residue, and the two ProX cysteineresidues, Cys¹³⁶ and Cys¹⁴², are absent in ChoX [Fig . 1B] . Thus,the arrangement of the binding site for choline, which possessesa hydroxylic group, is obviously different from the arrangementof the binding site for glycine betaine, which has a carboxylicgroup . A crystallographic study of ChoX would be very informativeand should allow us to precisely define the structure of thecholine-binding site.

Characterized ABC choline transporters are rather scarce . Toour knowledge, the only choline ABC transporters in bacteriathat have been fully characterized are the OpuB and the OpuCsystems from B . subtilis [13, 12] . These two systems are closelyrelated and evolved from a primordial gene cluster duplication.Regardless of the identity, but considering the functionality,Cho is physiologically more similar to OpuB, which is highlyspecific for choline, than to OpuC, which is involved in the entry of a large variety of compounds, including choline, choline-O-sulfate, glycine betaine, -butyrobetaine, crotonobetaine, ectoine, carnitine,and probably some other substrates . Western blotting experimentswith a polyclonal antiserum cross-reacting with the presumedsubstrate-binding proteins from both the OpuB and OpuC transportershave suggested that expression of the opuB and opuC operonsis regulated in response to increasing osmolarity of the growthmedium [13] . Our studies show that Cho activity is stronglystimulated by the presence of choline in the growth medium,whereas elevated osmolarity has no effect [Fig . 2] . Such resultsare in full agreement with immunodetection of ChoX, which indicatedthat there was clear induction in choline-grown cells and avery low level in NaCl-grown cells [Fig . 4B] . In contrast toB . subtilis, S . meliloti uses choline as a carbon and nitrogen source, and expression of the cho and opuB genes is obviouslyregulated differently . Whereas in the gram-positive bacteriumOpuB contributes to osmotic adjustment [13], it is more likelythat choline taken up by S . meliloti via Cho is catabolizedafter subsequent conversion into glycine betaine and/or is usedas a direct precursor of PC . This phospholipid is crucial forS . meliloti since it is required for normal growth [5] and alsofor a successful interaction with the host plant, alfalfa [35].In this context, it is interesting to highlight the presenceof ChoX in differentiated bacteroids [Fig . 4A] . Choline is indeedavailable in alfalfa, and significant amounts of choline haverecently been found in the cytosol of nodule cells, in the peribacteroidspace of the symbiosome, and also in bacteroids [18] . The choline concentration in bacteroids was estimated to be approximately1 mM, a concentration sufficient to induce ChoX synthesis . Inaddition, these results indicate that choline provided by thehost plant is transported into symbiosomes and bacteroids throughthe peribacteroid membrane . In fact, preliminary experimentswith purified symbiosomes confirmed that there is choline transportthrough this membrane [data not shown], and previous data haveindicated that there is choline transport activity in isolatedbacteroids [9].

At present, the physiological effect of the choX mutation on the phenotype of S . meliloti, either as a free-living cell or as an endosymbiotic form, is not clear . Significant residual levels of choline transport activity in the mutant suggest thatthere must be an alternative route[s] for choline uptake . InB . subtilis, for example, double mutations in the opuB and opuCloci are required to abolish osmoprotection by choline, sinceeach of the ABC transporters, OpuB and OpuC, is able to providethe cell with enough choline to sustain growth under unfavorablecircumstances [13] . Thus, identification of the other cholinetransporter system[s] should help workers evaluate the importanceof choline for S . meliloti, both as a heterotrophic soil bacteriumand during the establishment and maintenance of symbiosis.

 
Financial support for this study was provided by the CentreNational de la Recherche Scientifique.

We are grateful to the colleagues who generously provided strains and the S . meliloti genomic bank used in this study . We thank R . Krämer for the gift of cold ectoine.

 
* Corresponding author . Mailing address: Unité Interactions Plantes-Microorganismes et Santé Végétale, UMR6192 CNRS-INRA-Universitéde Nice Sophia Antipolis, Centre INRA Agrobiotech, 400 Route des Chappes, BP167, 06903 Sophia Antipolis Cédex, France . Phone: [33] 492 386 630 . Fax: [33] 492 386 587 . E-mail: leruduli@unice.fr.

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