Control of Inducer Accumulation Plays a Key Role in Succinate-Mediated Catabolite Repression in Sinorhizobiummeliloti

The symbiotic, nitrogen-fixing bacterium Sinorhizobium melilotifavors succinate and related dicarboxylic acids as carbon sources.As a preferred carbon source, succinate can exert cataboliterepression upon genes needed for the utilization of many secondarycarbon sources, including the ${alpha}$ -galactosides raffinose and stachyose.We isolated lacR mutants in a genetic screen designed to findS. meliloti mutants that had abnormal succinate-mediated cataboliterepression of the melA-agp genes, which are required for theutilization of raffinose and other ${alpha}$ -galactosides. The loss ofcatabolite repression in lacR mutants was seen in cells grownin minimal medium containing succinate and raffinose and grownin succinate and lactose. For succinate and lactose, the lossof catabolite repression could be attributed to the constitutiveexpression of ß-galactoside utilization genes in lacRmutants. However, the inactivation of lacR did not cause theconstitutive expression of ${alpha}$ -galactoside utilization genes butcaused the aberrant expression of these genes only when succinatewas present. To explain the loss of diauxie in succinate andraffinose, we propose a model in which lacR mutants overproduceß-galactoside transporters, thereby overwhelming theinducer exclusion mechanisms of succinate-mediated cataboliterepression. Thus, some raffinose could be transported by theoverproduced ß-galactoside transporters and causethe induction of ${alpha}$ -galactoside utilization genes in the presenceof both succinate and raffinose. This model is supported bythe restoration of diauxie in a lacF lacR double mutant (lacFencodes a ß-galactoside transport protein) grown inmedium containing succinate and raffinose. Biochemical supportfor the idea that succinate-mediated repression operates bypreventing inducer accumulation also comes from uptake assays,which showed that cells grown in raffinose and exposed to succinatehave a decreased rate of raffinose transport compared to controlcells not exposed to succinate.

INTRODUCTION

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Introduction

Bacteria belonging to the genera Sinorhizobium, Rhizobium, andBradyrhizobium are members of the ${alpha}$ -proteobacteria, a fascinatinggroup of organisms, many of which are intracellular symbiontsor pathogens. Sinorhizobium meliloti can grow in soil as free-livingorganisms but can also live as nitrogen-fixing symbionts insideroot nodules of alfalfa and a few other plants belonging tothe family Leguminosae (3, 9, 15, 19, 22, 34).

Free-living S. meliloti, like many heterotrophic bacteria, utilizesa wide variety of compounds as sources of carbon for growth.S. meliloti can utilize ${alpha}$ -galactosides in laboratory medium andalso when growing in the rhizospheres of host and nonhost plants(4). The utilization of ${alpha}$ -galactosides requires genes which arepart of an operon located on pSymB, a 1.7-Mb plasmid, in S.meliloti (Fig. 1) (7, 12). The agpA gene encodes a 77-kDa periplasmicprotein that is required for ${alpha}$ -galactoside transport and thatis similar to periplasmic binding protein components of theoligopeptide family of permeases (Fig. 1) (12). Immediatelydownstream of the agpA gene is agpB, which encodes a proteinsimilar to transmembrane proteins of the oligopeptide familyof permeases. Downstream of agpB are genes encoding a secondtransmembrane permease and an ATP binding protein. Thus, thisoperon appears to encode a complete ATP binding cassette-typetransport system for ${alpha}$ -galactosides. The agpA gene is precededby the melA gene, which encodes a 55-kDa ${alpha}$ -galactosidase (12).The melA and agp genes are cotranscribed and are referred toin this study as the melA-agp operon. Induction of the melA-agpoperon by ${alpha}$ -galactosides requires the action of an AraC-typetranscriptional activator, AgpT, which is encoded upstream ofmelA (5).

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FIG. 1. Map of the region of pSymB involved with the transport and utilization of ${alpha}$ -galactosides and ß-galactosides. ORFs are indicated along with the official ORF names (top row) and names indicating the functions of the proteins encoded by the ORFs (bottom row). Regulatory roles of AgpT and LacR are indicated above the map.

S. meliloti favors succinate and related dicarboxylic acidsas carbon and energy sources. Succinate, fumarate, and malateare brought into the cell via membrane-bound permease DctA (28,38). The expression of dctA is inducible by dicarboxylic acidsand structurally related compounds and requires the action ofa two-component signal transduction system composed of DctB,the sensor kinase, and DctD, the response regulator (25, 27,29, 38). As a favored carbon source, succinate often exertscatabolite repression upon genes needed for the utilizationof secondary carbon sources. Secondary carbon sources includecompounds such as glucose, fructose, galactose, lactose, myo-inositol,and several pentoses and polyols (16, 17, 24, 37). This preferencefor succinate can manifest itself as diauxie when S. melilotiis grown on succinate plus a secondary carbon source (16, 37).

Succinate also exerts catabolite repression on the consumptionof ${alpha}$ -galactosides in S. meliloti. The expression of the melA-agpoperon is repressed by succinate (12), and S. meliloti showsdiauxie when grown on a combination of succinate and the ${alpha}$ -galactosideraffinose or stachyose. While the phenomenon of succinate-mediatedcatabolite repression in S. meliloti is well documented, mostof the molecular details of its operation remain to be elucidated.This is also true for organisms of other genera, such as Pseudomonas,which utilize succinate and other tricarboxylic acid cycle intermediatesin preference to other carbon sources (8, 14, 23, 35).

It is known that succinate transport is important for the establishmentof catabolite repression in S. meliloti. S. meliloti dctA mutantsare unable to repress the expression of the lac operon in thepresence of succinate plus lactose, indicating that succinatetransport, or at least the interaction of succinate with theDctA permease, is required for the establishment of cataboliterepression (17).

In order to find genes needed to establish succinate-mediatedcatabolite repression, we screened for mutants unable to repressthe melA-agp operon when grown on a combination of succinateand raffinose. lacR mutants, which constitutively overexpressß-galactoside utilization genes, were isolated inthis screen. We show that the effects of lacR mutations on thesuccinate-mediated catabolite repression of the melA-agp operonare due to the overexpression of ß-galactoside transportgenes, which likely overwhelms inducer exclusion or inducerexpulsion mechanisms (26, 30, 36). The genetic and biochemicalexperiments presented in this study support this idea and supportthe conclusion that succinate-mediated catabolite repressionacts, in part, through the regulation of intracellular induceraccumulation.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains, plasmids, and growth media. Bacterial strains, plasmids, and their relevant characteristicsare listed in Table 1. Cells were grown in tryptone-yeast (TY),Luria-Bertani, or M9 minimal medium with various carbon sources(32). Antibiotics were used at the following concentrations:ampicillin, 50 µg/ml; chloramphenicol, 5 µg/ml forS. meliloti and 100 µg/ml for Escherichia coli; gentamicin,30 µg/ml; neomycin, 200 µg/ml; spectinomycin, 100µg/ml; and streptomycin, 500 µg/ml.

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TABLE 1. Strains and plasmids

Construction of S. meliloti strains. Strain RB21 (agpA::TnphoA lacR::Tn5lacZ) was isolated from thefollowing genetic screen. S. meliloti strain SG2001 (agpA::TnphoA)was mutagenized by triparental mating with E. coli strain S17-1 ${lambda}$ pir/pB22 (Tn5lac) and E. coli helper strain MT616. The threestrains were streaked together on a TY plate without antibioticsand incubated for 24 h at 30°C. Swaths of bacteria werepicked from the plate, suspended in 100 µl of M9 salts,and spread on M9 plates containing 0.2% succinate, 0.02% raffinose,80 µg of 5-bromo-4-chloro-3-indolyl phosphate (X-Phos)/ml,neomycin, and gentamicin. Deep blue colonies were retained forfurther study; one of these was strain RB21.

Strain RB33 (lacR::Tn5-233) was also isolated from a geneticscreen. S. meliloti wild-type strain Rm1021 was mutagenizedby mating with MM294a/pRK607; pRK607 is a self-transmissibleplasmid harboring Tn5-233. The strains were streaked togetheron a TY plate without antibiotics and incubated for 24 h at30°C. Swaths of bacteria were picked from the plate, suspendedin 100 µl of M9 salts, and spread on M9 plates containing0.2% succinate, 40 µg of 5-bromo-4-chloro-3-indolyl-ß-D-galactoside(X-Gal)/ml, streptomycin, and gentamicin. The lacR mutants wereexpected to be blue on this medium because they constitutivelyexpressed the endogenous S. meliloti lacZ gene. One of the resultantdeep blue colonies was strain RB33. Southern hybridization confirmedthat the Tn5-233 transposon was in the lacR gene of strain RB33(data not shown).

Strain RB45 is a lacF mutant constructed by inserting suicideplasmid pRB69 into the lacF gene of S. meliloti wild-type strainRm1021. Suicide plasmid pRB69 was constructed as follows. A576-bp internal piece of the lacF gene was generated by PCRwith primers 5'GCGTGGTCGCTCTGGATGT and 5'TCGTCGAAATGACTGTCGTGAA.The resulting amplification product was inserted into T/A cloningvector pGEM-T Easy. This piece was then removed by EcoRI digestion,and the 594-bp fragment was cloned into broad-host-range suicidevector pMB438, resulting in plasmid pRB69. pRB69 was mobilizedfrom strain XL1Blue/pRB69 into strain Rm1021 by triple matingwith E. coli helper strain MT616 and selected on Luria-Bertanimedium plus streptomycin and neomycin.

Strain RB46 (lacF lacR) was constructed by transducing the lacFmutation from strain RB45 into strain RB33 (lacR::Tn5-233) withphage N3 (20). Strain RB47 (lacF agpA) was constructed by transducingthe lacF mutation from strain RB45 into strain DG73 (agpA::TnphoA-Sp^r)with phage N3.

Diauxic growth curves. S. meliloti strains were grown overnight in M9 minimal mediumplus antibiotics and with succinate as the sole carbon source.A quantity (1 ml) of cell culture was pelleted, washed theetimes with M9 salts to remove residual succinate, and resuspendedin 100 µl of M9 salts. Ten microliters of this suspensionwas used to inoculate 10 ml of M9 minimal medium with 0.05%succinate or with 0.05% succinate plus 0.1% raffinose, maltose,or lactose. Cultures were incubated in 125-ml flasks at 30°Cwith shaking. Cell density was determined by measuring the absorbanceof 100 µl of culture at 415 nm with a Bio-Rad 550 platereader. Optical densities determined with the plate reader shouldbe multiplied by three to approximate the optical density determinedwith a spectrophotometer with a standard 1-cm path length.

Quantitation of pmelA::gfp expression. Bacterial strains were grown overnight in M9 minimal mediumwith succinate as the sole carbon source. Cells (25 ml) werepelleted, washed three times in M9 salts, and resuspended in1 ml of M9 salts. Twenty microliters of this cell suspensionwas used to inoculate 200 ml of M9 minimal medium with 0.1%succinate plus 0.1% raffinose. Growth was monitored by measuringthe absorbance of 100 µl of culture at 415 nm with a Bio-Rad550 plate reader. Periodically, a quantity of culture (1 to10 ml, depending on the culture density) was pelleted, resuspendedin 15% glycerol, and stored at -80°C until all samples hadbeen taken. After all samples were collected, the fluorescenceof the pellets was measured by using a CytoFluor 4000 fluorimeter(PerSeptive Biosystems, Foster City, Calif.) with excitationat 485 nm and emission at 508 nm. Relative fluorescence wasdetermined by dividing sample fluorescence by sample opticaldensity at 415 nm. Optical density at 415 nm was determinedby using a Bio-Rad 550 plate reader.

Catabolite repression of the pmelA::gfp fusion was measuredby growing test strains in tubes (18 by 150 mm) containing 2.5ml of M9 minimal medium with 0.4% succinate plus 0.4% raffinose.Under these conditions, some succinate remains when cells enterstationary phase and can exert catabolite repression on thepmelA::gfp fusion. After 72 h, 100 µl of culture was removed,and its relative fluorescence was determined as described above.

Raffinose uptake assays. Bacterial strains were grown overnight in M9 minimal mediumwith 0.4% succinate or 0.4% raffinose as the sole carbon source.Cells grown in succinate were used to inoculate 25 ml of M9minimal medium containing 0.4% succinate plus 0.4% raffinose,and cells grown overnight in raffinose were used to inoculate25 ml of M9 minimal medium containing 0.4% raffinose. Cultureswere grown to mid-exponential phase at 30°C with shaking.The raffinose culture was split, and succinate was added toone of the portions to a final concentration of 0.4%. All cultureswere incubated for an additional hour. One to 2 ml of culturecontaining raffinose plus succinate was washed once with M9salts plus 0.4% succinate, resuspended in 100 µl of M9salts plus 0.4% succinate, and incubated at room temperaturefor 5 min. Nine hundred microliters of M9 salts plus 0.4% succinateand 10 µl of 10 mM [³H]raffinose (0.1 mCi/ml) (AmericanRadiolabeled Chemicals, Inc.) were added to the cell suspension.Raffinose-grown cells were prepared in the same way, exceptthat they were washed and suspended in M9 salts without succinate.Samples (100 µl) were removed every 4 min and filteredthrough 45-µm-pore-size nitrocellulose filters. The filterswere immediately rinsed three times with 5 ml of M9 salts containing10 mM raffinose, and counts were determined with a scintillationcounter. Counts were normalized by dividing counts per minuteby the optical density of the cell suspension.

RESULTS

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Results

Genetic screen for mutants unable to establish succinate-mediated catabolite repression of the melA-agp operon. To conduct a screen for catabolite repression mutants, we madeuse of the fact that an agpA::TnphoA reporter is highly expressedwhen S. meliloti is grown on the ${alpha}$ -galactoside raffinose butis repressed when the organism is grown on a combination ofsuccinate and raffinose. S. meliloti strain SG2001 (agpA::TnphoA)was mutagenized with Tn5lac and plated on M9 plates containingselective antibiotics plus succinate, raffinose, and X-Phos.Most colonies were pale blue, indicating that succinate-mediatedrepression had down-regulated the agpA::TnphoA reporter in spiteof the presence of raffinose. However, 12 colonies out of the20,000 screened were deep blue and were candidates for mutantsthat had altered succinate-mediated catabolite repression. Transductionalmapping and Southern analysis of the Tn5lac mutations showedthat two were linked to agpA (data not shown). Sequencing showedthat the insertions were in open reading frame (ORF) Y21650,which encodes a lacI-type repressor just downstream of agpT(Fig. 1) (data not shown).

The orientation of Tn5lac in the two ORF Y21650 mutants wassuch that the lacZ reporter should not have been transcribed.However, we believed that it was still possible for the heterologouslacZ gene, carried on Tn5lac in these strains, to confound theinterpretation of experiments investigating galactoside utilization.For this reason, we reisolated an insertional mutation in ORFY21650 by using Tn5-233. The resulting strain, RB33, was usedin the following experiments.

ORF Y21650 encodes the lac repressor LacR. Jelesko and Leigh reported that LacZ activity in S. melilotiis controlled by a repressor protein, LacR, and that mutationsin lacR result in the constitutive expression of ß-galactosidaseactivity (17). The genome sequence of S. meliloti (1, 6, 11)indicates that ORF Y21650 encodes a LacI-type repressor andis about 1,000 bp upstream of the first gene in an operon containingfive genes similar to the genes involved in ß-galactosidetransport and catabolism (http://sequence.toulouse.inra.fr/meliloti.html)(Fig. 1). We believed that it was likely that the ORF Y21650gene was the same as the lacR gene characterized genetically,but not sequenced, by Jelesko and Leigh (17). The fact thatthe Tn5-233 mutation in ORF Y21650 caused the constitutive expressionof ß-galactosidase activity (data not shown) lentweight to this idea. To confirm that ORF Y21650 was the sameas lacR, we partially sequenced plasmids pJGJ54 and pJGJ86,which complemented lacR mutations (17), and found that theycarried the ORF Y21650 gene. Because the phenotype of an ORFY21650 mutant is the same as the phenotype of a lacR mutantand because ORF Y21650 is carried on plasmids which can complementlacR, it is likely that ORF Y21650 is the same as the lacR locusdescribed first by Jelesko and Leigh (17).

Mutations in lacR cause aberrations in succinate-mediated catabolite repression of ${alpha}$ -galactoside and ß-galactoside utilization. S. meliloti wild-type strain Rm1021 showed diauxie when grownin M9 minimal medium containing a combination of succinate andeither raffinose, lactose, or maltose (Fig. 2A). lacR mutantRB33 failed to show diauxie when grown on succinate plus raffinoseor on succinate plus lactose. Diauxie on succinate plus maltosewas normal, indicating that the abnormal diauxie seen in thisstrain was not a general phenomenon affecting the utilizationof all secondary carbon sources.

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FIG. 2. The lacR::Tn5-233 mutation abolishes succinate-mediated diauxic utilization of ${alpha}$ - and ß-galactosides. (A) Strains Rm1021 (wild type [w.t.]) and RB33 (lacR::Tn5-233) were grown in medium containing 0.05% succinate and 0.1% the indicated secondary carbon source. Growth was monitored by measuring the optical density at 415 nm (OD₄₁₅). (B). Strain RB33 (lacR::Tn5-233)/pRB27 (pmelA::gfp) does not exhibit diauxie or repression of the melA::gfp reporter when grown in medium containing succinate plus raffinose. Strains Rm1021/pRB27 (pmelA::gfp) and RB33 (lacR::Tn5-233)/pRB27 (pmelA::gfp) were grown in M9 minimal medium containing 0.1% succinate plus 0.1% raffinose. The OD₄₁₅ and relative green fluorescent protein (GFP) fluorescence were determined. The vertical broken line marks the time at which growth on succinate stopped in the wild-type strain. The inset shows the relative fluorescence of the strains during the first phase of diauxic growth. (C) The lacR mutation does not cause constitutive expression of the melA-agp promoter. Strains Rm1021/pRB27 (pmelA::gfp) and RB33 (lacR::Tn5-233)/pRB27 (pmelA::gfp) were grown in M9 minimal medium containing 0.4% succinate (Suc), 0.4% raffinose (Raf), or 0.4% succinate plus 0.4% raffinose (Both). After 72 h, cells were harvested, and the relative GFP fluorescence was determined. Numbers above the bars indicate the relative fluorescence value for each culture.

Plasmid pRB27 (pmelA::gfp) contains a transcriptional fusionof the melA promoter to gfp. This fusion was overexpressed inthe lacR mutant, relative to the wild type, when succinate andraffinose were present together (Fig. 2B and C). In the experimentsdepicted in Fig. 2B, the fusion was induced in wild-type cellsduring the diauxic lag only after succinate had been consumed;however, the fusion was expressed throughout the growth curvefor cells of strain RB33 (lacR::Tn5-233), including early times,when succinate was still present.

The lacR::Tn5-233 mutation caused the constitutive expressionof ß-galactoside utilization genes. If it also causedthe constitutive expression of ${alpha}$ -galactoside utilization genes,then that would explain its ability to alleviate succinate-mediatedcatabolite repression in medium containing succinate plus raffinose.The data presented in Fig. 2C show relative fluorescence fromthe pmelA::gfp fusion when strain Rm1021/pRB27 and strain RB33(lacR::Tn5-233)/pRB27 were grown for 72 h in M9 minimal mediumcontaining either 0.4% succinate, 0.4% raffinose, or 0.4% succinateplus 0.4% raffinose. In the wild-type strain, the pmelA::gfpfusion was expressed when cells were grown in raffinose butnot when cells were grown in succinate or in succinate plusraffinose. In strain RB33 (lacR), the fusion was not expressedwhen cells were grown in succinate, but it was expressed whencells were grown in raffinose or in succinate plus raffinose.In addition, the pmel::gfp fusion was not expressed in eitherstrain when the medium contained glycerol as the sole carbonsource (data not shown). Thus, the lacR::Tn5-233 mutation doesnot cause merely constitutive expression of the melA-agp genesbut causes aberrant expression of these genes when succinateand raffinose are present together.

Abnormal succinate-mediated catabolite repression of the melA-agp operon in a lacR mutant requires a functional ß-galactoside transport system. If succinate-mediated catabolite repression acts, at least inpart, by preventing the intracellular accumulation of raffinose,then the effect of lacR mutations on the catabolite repressionof the melA-agp operon could be explained as follows (Fig. 3).In lacR mutants, ß-galactoside transporters are overproduced(17). These may allow some ${alpha}$ -galactoside molecules into the cell,where they can interact with AgpT and cause the induction ofthe melA-agp operon. The overproduction of the ß-galactosidetransporters may overwhelm the ability of the succinate-mediatedcatabolite repression system to inhibit the accumulation of ${alpha}$ -galactoside inducers. If ${alpha}$ -galactoside transport through overproducedß-galactoside transporters is key for the eradicationof succinate-mediated control of the melA-agp operon in lacRmutants, then inactivating a ß-galactoside transportgene in the lacR::Tn5-233 mutant should alleviate the abnormalcatabolite repression of the melA-agp operon caused by the lacRmutation.

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FIG. 3. Model to explain the effects of lacR mutations on succinate-mediated repression of ${alpha}$ -galactoside utilization. The model depicts the ${alpha}$ -galactoside ( ${alpha}$ -gal) and ß-galactoside (ß-gal) transport systems when wild-type (top) and lacR mutant (bottom) cells are growing on succinate plus an ${alpha}$ -galactoside. The model postulates that succinate-mediated catabolite repression of the melA-agp operon is due, in part, to succinate causing inducer exclusion of ${alpha}$ -galactosides. In the lacR mutant, the overproduction of ß-galactoside transporters would allow some ${alpha}$ -galactosides into the cell, thus short-circuiting succinate-mediated repression. The details of the model are described in the text.

The lacF gene, which is postulated to encode a permease portionof a lactose ATP binding cassette transport system, was mutatedby insertion of a suicide plasmid. Strains carrying this mutationgrew on lactose with a doubling time of 30 h, compared to 4h for the wild-type parental strain (Fig. 4A). The lacF mutationwas combined with a lacR mutation to produce the double-mutantstrain RB46 (lacR lacF), which also grew slowly on lactose.Unlike strain RB33 (lacR), strain RB46 (lacR lacF) showed diauxiewhen grown in M9 minimal medium containing a combination ofsuccinate and raffinose (Fig. 4B). Thus, the double mutant,with defects in lacR and ß-galactoside transport,appears phenotypically normal with respect to succinate-mediatedrepression of ${alpha}$ -galactoside utilization genes. This finding indicatedthat the transport of some raffinose through ß-galactosidetransporters is required for the abnormal catabolite repressionphenotype seen when a lacR strain is grown on succinate plusraffinose. Even though enough raffinose appears to cross intothe cell via the Lac transport system to trigger the inductionof the melA-agp genes, the amount must be rather small, becauseagpA (12) or agpB mutants cannot grow on ${alpha}$ -galactosides (datanot shown). Even when the Lac transport system is fully induced,not much raffinose crosses into the cell via this system, becauselacR agpA double mutants are also unable to grow on ${alpha}$ -galactosides(data not shown).

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FIG. 4. The lacR lacF double mutant shows wild-type growth in succinate-plus-raffinose medium. (A) lacF mutants grow very slowly on lactose. The indicated strains were grown in M9 minimal medium containing lactose as the sole source of carbon. OD₄₁₅, optical density at 415 nm. (B) Succinate-mediated diauxie of ${alpha}$ -galactoside utilization is restored in a lacR lacF double mutant. The indicated strains were grown in M9 minimal medium containing 0.05% succinate plus 0.1% raffinose. The lacR mutant failed to undergo diauxic growth, whereas the lacR lacF double mutant exhibited diauxic growth which was indistinguishable from that of wild-type (wt) strain Rm1021.

${alpha}$ -Galactoside accumulation is inhibited by succinate. The experiments described above suggested that succinate repressionof the melA-agp genes operates by preventing inducer accumulation.We tested this idea directly by conducting raffinose uptakeassays with strain Rm1021 grown in M9 minimal medium containing0.4% raffinose, 0.4% raffinose with 0.4% succinate present continuously,or 0.4% raffinose with 0.4% succinate added 1 h before the uptakeassay was started. The results showed that cells grown in raffinosewith succinate present continuously did not transport raffinose(Fig. 5B). Cells grown in raffinose and exposed to succinatefor 1 h before the uptake assay were restricted in their abilityto transport raffinose compared to raffinose-grown cells notexposed to succinate. Such cultures showed a 45 to 80% reductionin the accumulation of raffinose compared to control culturesnot exposed to succinate (Fig. 5A).

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FIG. 5. Succinate inhibits the accumulation of raffinose. (A) Strain Rm1021 (wild type [w.t.]) was grown in M9 minimal medium plus 0.4% raffinose. A portion of the culture was exposed to 0.4% succinate for 1 h, and then the uptake of [³H]raffinose was measured in both the raffinose and the raffinose-plus-succinate cultures. Error bars indicate one standard error. (B) Strains Rm1021 and RB33 (lacR::Tn5-233) were grown in M9 minimal medium containing 0.4% raffinose plus 0.4% succinate. Succinate was present continuously as cells were grown in preparation for uptake measurements.

Unlike wild-type strain Rm1021 (Fig. 5A), lacR mutant strainRB33 was able to transport raffinose when grown in M9 minimalmedium containing raffinose with succinate present continuously(Fig. 5B).

The biochemical data presented above do not indicate whethersuccinate decreased raffinose uptake by preventing raffinosefrom entering the cell or by accelerating its efflux from thecell. That is, succinate may decrease raffinose uptake throughinducer exclusion, inducer expulsion, or a combination of thetwo.

DISCUSSION

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Discussion

Succinate is able to repress the utilization of many differentcarbon sources, including ${alpha}$ -galactosides and ß-galactosidesin S. meliloti. In order to better understand this phenomenon,we conducted a screen to identify genes involved in the succinate-mediatedrepression of ${alpha}$ -galactoside utilization. This screen revealedthat lacR mutants no longer repressed the melA-agp operon whensuccinate was present along with raffinose. The mutations alsoeradicated the diauxie normally seen when cells are grown inM9 salts containing succinate plus raffinose. The eliminationof succinate-mediated repression of the melA-agp operon in lacRmutant strains was not caused by a general derepression of themelA-agp operon, because these mutants did not exhibit constitutiveexpression of the melA-agp operon when grown on single carbonsources such as glycerol or succinate.

Experiments were done to determine whether the altered succinate-mediatedcatabolite repression seen in lacR mutants was general or restrictedto the melA-agp operon. These experiments showed that the lacRmutation also eradicated succinate-mediated catabolite repressionin a medium containing succinate plus lactose but did not doso in a medium containing succinate plus maltose. The lacR mutantstrains constitutively expressed LacZ activity in all mediatested. Thus, with succinate plus lactose, the altered succinate-mediatedrepression seen in the lacR mutants was likely due to the factthat the genes needed for lactose transport and utilizationare always overexpressed.

While it is easy to understand how the lacR mutations gave riseto a loss of succinate-mediated repression with respect to lactose,it is not so easy to explain the effects of these mutationson the succinate-mediated repression of ${alpha}$ -galactoside utilization.This is because the melA-agp operon is aberrantly expressedonly when an ${alpha}$ -galactoside is present along with succinate. Ourdata best support the idea that succinate normally preventsraffinose from accumulating in the cell when both are presenttogether. The overproduction of ß-galactoside transportersin the lacR mutants (17) may overwhelm the ability of the succinate-mediatedcatabolite repression system to inhibit some Lac transporter-dependentaccumulation of ${alpha}$ -galactoside inducers, even though the succinate-mediatedrepression system itself is fully operational. The fact thatlacR lacF double mutants are normal with respect to the succinate-mediatedrepression of the melA-agp genes indicates that the overexpressionof the Lac transport system is critical for the aberrant succinate-mediatedrepression of the melA-agp genes observed in the lacR mutants.

Raffinose uptake assays supported the hypothesis that succinateprevents raffinose from accumulating when cells are grown inthe presence of both succinate and raffinose. Wild-type cellsgrowing in raffinose and exposed to succinate for 1 h exhibiteda 45 to 85% decrease in their rate of raffinose accumulation.Cells grown in raffinose and succinate have a doubling timeof about 6 h; thus, the decrease in raffinose uptake seen followingthe addition of succinate for 1 h cannot be accounted for bya halt of ${alpha}$ -galactoside transporter synthesis followed by dilutionof the remaining transporters by cell growth. If synthesis ofthe transporters were shut off completely for 1 h, then onewould expect a one-sixth (16%) decline in the rate of raffinoseaccumulation, because the number of active transporters perculture mass would decline 16%.

Wild-type cells grown in raffinose with succinate continuallypresent did not accumulate raffinose to any significant extent(Fig. 5). The difference in ${alpha}$ -galactoside transport between cellsgrown in raffinose with succinate present continuously and thosegrown in raffinose and exposed to succinate for 1 h is similarto the effect of preinduction on catabolite repression of thelac system by glucose in E. coli (7a). The more complete shutdownof raffinose transport seen in the cells exposed to raffinoseand succinate continuously may have resulted from one or moreof the following: (i) active down-regulation of melA-agp expressionby transcriptional factors, (ii) down-regulation of melA-agpexpression caused by lower internal concentrations of inducerresulting from nearly complete inhibition of ${alpha}$ -galactoside transporteractivity, or (iii) succinate-dependent degradation of proteinsrequired for ${alpha}$ -galactoside transport.

In bacterial species which exhibit glucose-mediated cataboliterepression, glucose status is linked to gene repression and/orinducer exclusion by the Hpr or GlcIIA proteins. These two proteins,which are directly involved in glucose transport, also mediateinducer exclusion, cyclic AMP synthesis, and repression of catabolicgenes (31, 36). How the information concerning succinate statusis transmitted to the regulated transport systems in rhizobiais not yet known. Jelesko and Leigh showed that the dicarboxylicacid transporter DctA was required for the succinate-mediatedcatabolite repression of the Lac system (17). It is possiblethat DctA bound to succinate can meditate inducer exclusiondirectly or that DctA is needed only to bring succinate intothe cell, where the presence of succinate then can be signaledby a variety of means: intracellular concentration of tricarboxylicacid cycle intermediates, flux of reducing equivalents throughthe electron transport chain, or proteins that bind dicarboxylicacids and cause inducer exclusion or expulsion directly.

We have shown that succinate establishes catabolite repressionof the melA-agp genes, at least in part, by preventing intracellularinducer accumulation. Currently, we are working on identifyingother mutants that are altered in this process. We hope to findmutants that are altered in the systems that convey informationabout the succinate status of the cell and transmit that informationto secondary carbon source transporters. Such mutants shouldprovide information on how succinate-mediated catabolite repressionoperates in rhizobia and may eventually shed light on how thisprocess works in other species which use dicarboxylic acidsas preferred carbon sources.

ACKNOWLEDGMENTS

We thank John Leigh for providing plasmids pJGJ54 and pJGJ86,the S. meliloti sequencing consortium for making preliminarydata available, Charlie Giardina for use of a fluorescence microplatereader, and Robert Bender for pointing out the work of M. Cohnand K. Horibata.

This work was supported by Department of Energy contract DE-FG02-01ER15175,by a University of Connecticut Research Foundation grant toD.J.G., and by a Heinz Herrmann Graduate Fellowship in CellBiology from the University of Connecticut Graduate School toR.M.B.

FOOTNOTES

* Corresponding author. Mailing address: University of Connecticut, Department of Molecular and Cell Biology, 75 N. Eagleville Rd., U-44, Storrs, CT 06269. Phone: (860) 486-3092. Fax: (860) 486-1784. E-mail: gage{at}uconnvm.uconn.edu.

REFERENCES

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