Vol. 171, No. 10JOURNAL OF BACTERIOLOGY, OCt. 1989, p. 5551-55600021-9193/89/105551-10$02.00/0Copyright © 1989, American Society for Microbiology

Identification and Sequence Analysis of the Rhizobium meliloti dctAGene Encoding the C4-Dicarboxylate Carrier

THOMAS ENGELKE, DORIS JORDING, DIETER KAPP, AND ALFRED PUHLER*Lehrstuhl fur Genetik, Fakultat fur Biologie, Universitat Bielefeld, Postfach 8640,

D4800 Bielefeld 1, Federal Republic of GermanyReceived 20 March 1989/Accepted 5 July 1989

Transposon TnS-induced C4-dicarboxylate transport mutants of Rhizobium meliloti 2011 which could becomplemented by cosmid pRmSC121 were subdivided into two classes. Class I mutants (RMS37 and RMS938)were defective in symbiotic C4-dicarboxylate transport and in nitrogen fixation. They were mutated in thestructural gene dctA, which codes for the C4-dicarboxylate carrier. Class II mutants (RMS11, RMS16, RMS17,RMS24, and RMS31) expressed reduced activity in symbiotic C4-dicarboxylate transport and in nitrogenfixation. These mutants were mutated in regulatory det genes which do not play an essential role in thesymbiotic state. Thin sections of alfalfa nodules induced by the wild type and class I and class H mutants wereanalyzed by light microscopy. Class mutants induced typical Fix- nodules, showing a large senescent zone,whereas nodules induced by class II mutants only differed in an enhanced content of starch granules comparedwith wild-type nodules. Class I mutants could be complemented by a 2.1-kilobase SailI-HindI subfragment ofcosmid pRmSC121. DNA sequencing of this fragment resulted in the identification of an open reading frame,which was designated dctA because TnS insertion sites of the class I mutants mapped within this coding region.The dctA gene was preceded by a nifconsensus promotor and an upstream NifA-binding element. Upstream ofthe dctA promoter, the 5' end of the R. meliloti dctB gene could be localized. The amino acid sequence of theN-terminal part of the R. meliloti DctB protein shared 49% homology with the corresponding part of the R.leguminosarum DctB protein. The DctA protein consisted of 441 or 453 amino acids due to two possible ATGstart codons, with calculated molecular masses of 46.1 and 47.6 kilodaltons, respectively. The hydrophobicityplot suggests that DctA is a membrane protein with several membrane passages. The amino acid sequences ofthe R. melioti and the R. leguminosarum DctA proteins were highly conserved (82%).

In the Rhizobium-legume symbiosis, C4-dicarboxylatetransport plays an important role in the energy supply of themicrosymbiont. Both symbiotic partners, the plant and thebacteroid, seem to be involved in this transport process. AC4-dicarboxylate transport system was found in the peribac-teroid membrane of Glycine max (47). Other C4-dicarboxy-late transport systems are functional in several Rhizobiumspecies. For Bradyrhizobium jaconicum (40), Rhizobiumleguminosarum (3, 11, 12, 14), Rhizobium leguminosarumbiovar trifolii (37), and Rhizobium meliloti (5, 8, 49) it wasshown that succinate, fumarate, and malate are transportedby a common C4-dicarboxylate transport system. Addition-ally, it was reported that in R. meliloti, aspartate may betransported via the same system (49).The DNA region coding for C4-dicarboxylate transport

(dct) has been analyzed in detail in R. leguminosarum (35,36). The dct cluster contains the genes dctA, dctB, and dctDorganized in the two divergently transcribed operons dctAand dctBD. The dctB and dctD genes were suggested to codefor a two-component regulatory system activating togetherwith the RpoN protein the transcription of the structuraldctA gene in the free-living state (36, 38). In the symbioticstate, it is proposed that NifA can activate the dctA gene(36).The R. meliloti dct mutants analyzed in our laboratory

were all defective in free-living C4-dicarboxylate transport(Dctfl-) (8). Some of these mutants were unable to transportC4-dicarboxylates in the symbiotic state (Dcts-) and weredefective in nitrogen fixation (Fix-). Other mutants showedreduced symbiotic C4-dicarboxylate transport and fixed ni-

* Corresponding author.

trogen at reduced levels (Dctred Fixred). In this paper, weconcentrate on R. meliloti dct mutants which were comple-mented by cosmid pRmSC121. To localize the dct genes, thecosmid pRmSC121 was subcloned. The nucleotide sequenceof the dctA gene as well as the properties of the DctA proteinare reported.

(Parts of this work were presented at the 7th InternationalCongress on Nitrogen Fixation in Cologne, 1988 [7a].)

MATERIALS AND METHODSBacterial strains and plasmids. Strains and plasmids are

listed in Table 1.Media and growth conditions. Escherichia coli cells were

grown in LB or LBMM, and R. meliloti cells were grown inTY or in minimal medium as described before (8). Antibiot-ics were added at the following concentrations: streptomy-cin, 400 jig/ml; tetracycline, 5 ,ug/ml; chloramphenicol, 50jig/ml; kanamycin, 50 jig/ml; neomycin, 100 j,g/ml; andgentamicin, 50 j,g/ml for R. meliloti or 10 ,ug/ml for E. colicells.

Plant assays. Alfalfa plants were grown and inoculatedwith the appropriate R. meliloti strains as reported previ-ously (8). For light microscopy, 3-week-old nodules wereharvested, fixed, embedded, cut, and stained with toluidineblue as described previously (1). For quantitative analysis ofthe starch content of whole nodules, a photometrical methodwith the Lugol reagent was used as described before (25). Inparallel, nitrogenase activity of the nodulated plants wastested by the acetylene reduction assay with a HewlettPackard 5710A gas chromatograph (1).Recombinant DNA methods. Plasmid DNA from E. coli

was prepared on a small scale by a rapid isolation procedure

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5552 ENGELKE ET AL.

TABLE 1. Bacterial strains, phages, and plasmids

Strain, phage, or plasmid Relevant characteristics Reference

E. coliJM103 endA A(lac pro) thi strA sbcB hsdR subE F'(traD proB+ lacJqZlM15) 29K802 hsdR hsdM gal met supE 26S17.1 hsdR pro recA, containing an RP4-2-Tc::Mu integrated into the chromosome 43S605 thi thr leu supE lac met::Tn5 Kmr 23SM1o thi thr leu supE recA Kmr, containing RP4-2-Tc::Mu integrated into the chromosome 43

R. meliloti2011 Smr J. Denarie, FranceRMS11 2011, dctB::TnS 8RMS16 2011, dctB::Tn5 8RMS17 2011, dctB::Tn5 8RMS24 2011, dct::Tn5 8RMS31 2011, dctB::TnS This workRMS37 2011, dctA::TnS This workRMS938 2011, dctA::Tn5 8

PhagesM13mpl8 Cloning vector 50M13mpl9 Cloning vector 50

PlasmidspML123 Broad-host-range vector, Gmr Km", pKT210/pACYC184 derivative M. Labes, BielefeldpWKR56I Integration vector, Tcr Kmr, pACYC177 derivative 22pSUP205 Cmr Tcr, pBR325 derivative containing the X cos site and the RP4 mob site 43pSUP2021 Apr Cmr Nmr, pBR325 derivative 43pSUP5011 Nmr Tcr, pBR325 derivative containing TnS-Mob 43pRmSC121 Cosmid with replicon pSUP205, containing R. meliloti EcoRI fragments 8pRmSC121H6 Integration vector pWKR561, containing a 6.0-kb HindlIl fragment of cosmid This work

pRmSC121pRmSC121E6 Integration vector pSUP205, containing a 5.9-kb EcoRI fragment of cosmid This work

pRmSC121pRmSC121SH2 Broad-host-range vector pML123 containing a 2.1-kb SalI-HindIll fragment of cosmid This work

pRmSC121

(18, 20). Transformation of competent E. coli cells, freshlyprepared or stored at -70°C, and agarose gel electrophoresishave been described before (21). Restriction enzymes wereused according to the manufacturer's specifications (Boehr-inger Mannheim).Complementation experiments. R. meliloti C4-dicarboxy-

late transport mutants were complemented by cosmidpRmSC121 or its subclones. Complementing cosmids wereintroduced by conjugative transfer. Transconjugants wereselected on minimal medium containing 10 mM succinate asthe sole carbon source and in addition the antibiotics tetra-cycline and streptomycin. Bacterial matings were carried outas described previously (8).

Isolation of R. meliloti mutants by Tn5 mutagenesis andmapping of Tn5 insertion sites. TnS-induced R. melilotimutants were generated and isolated as described before (42,43). Mapping of TnS insertions of the mutants was carriedout as published previously (31). Hybridization experimentswere also described before (8).

Sequencing methods. The phage vectors M13mpl8 andM13mp19 were used for subcloning (50). Several deletionclones were generated by the following method. The frag-ment of interest was cloned in both orientations into thesequencing vector (M13) and cleaved with Sau3A in thepresence of ethidium bromide. The appropriate concentra-tion of ethidium bromide resulted in only one restriction cutper DNA molecule (32). Prior to ligation, the DNA wasprecipitated with ethanol and cleaved with an enzyme havingonly one recognition site between the annealing site of theuniversal primer and the insert DNA. This procedure re-sulted in a number of different deletion clones of the frag-ment to be sequenced. Additionally, defined fragments wereligated into the appropriate restriction sites of M13mpl8 or

M13mpl9. Single-stranded DNA from M13 phages wasisolated by published methods (28). Recombinant M13clones were sequenced by the dideoxy chain terminationmethod (41).

Analysis of the nucleotide sequence. The DNA sequencewas analyzed by the computer programs of Martinez (27)and Staden (44). The free energy (AG) of the secondarystructure formed by the terminator sequence was calculated(45).

RESULTS

Classification of R. meliloti dct mutants complemented bycosmid pRmSC121. A collection of TnS-induced R. melilotimutants defective in C4-dicarboxylate transport which couldbe complemented by cosmid pRmSC121 (8) are presented inTable 1. The collection contains dct mutants describedpreviously (8) as well as two new isolates (RMS31 andRMS37). According to their phenotypes, these mutantscould be subdivided into two classes.

Class I mutants (RMS938 and RMS37) could not take upC4-dicarboxylates in either the free-living (Dctfl-) or thesymbiotic state (Dcts-). They induced ineffective (Fix-)nodules in symbiosis with alfalfa plants. We compared themorphology of nodules induced by mutant RMS938 with thatof nodules induced by wild-type strain 2011. Thin sections oflongitudinal cuts through 3-week-old nodules were stainedwith toluidine blue and analyzed by light microscopy (Fig.1). In both nodule types, the meristematic, infection, andsymbiotic zones were present. The extension of the mer-istematic and the infection zone was similar in both noduletypes. In nodules induced by the wild-type strain 2011, allthe remaining space was occupied by the symbiotic zone

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R. MELILOTI dctA GENE 5553

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FIG. 1. Morphology of alfalfa nodules induced by R. meliloti wild type and dct mutants. (A, B, C) Median longitudinal thin sections of21-day-old alfalfa nodules induced by R. meliloti 2011 wild type (A), class I mutant RMS938 (B), and class II mutant RMS24 (C) were analyzedby light microscopy. The nodules exhibit meristematic (M), early symbiotic (ES), late symbiotic (LS), and senescent (5) zones. The adjacentroot (R) is also presented. Bar, 100 p.m. (D, E, F) Details of the late symbiotic zones and the adjacent nodule cortex (NC) are shown fornodules induced by wild-type 2011 (D), by class I mutant RMS938 (E), and by class II mutant RMS24 (F). Cells filled with bacteroids (b) andstarch granules (s) are indicated. In panel E, several host cells with degrading RMS938 bacteroids (db) from the senescent zone are alsovisible. Bar, 20 p.m.

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FIG. 3. Identification of the dctA coding region by sequencing a 2.3-kb SaIl-BglII fragment ot cosmid pRmSC121. The restriction map ofthis fragment including the Tn5 insertion sites and the sequencing strategy are presented. A detailed ORF analysis of the resulting nucleotidesequence (Fig. 4) with the coding probability program of Staden (44) is presented. Coding probabilities for the three reading frames in bothdirections are indicated by black curves. The dotted line indicates regions of high coding probability. Stop codons are indicated by verticallines, and ATG codons are indicated triangles. Thick black bars represent the dctA gene, containing two possible start codons, and the 5' endof the dctB gene upstream of the dctA gene. Restriction enzymes are abbreviated as follows: Bg, BgII; C, ClaI; E, EcoRI; H, Hindlll; P,PstI; S, SaII; Sm, SmaI; X, XhoI.

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5556 ENGELKE ET AL.

6T6CCO6VATTAACCGCGTGTACSCGYGCCCAST 66CAAAGTC6CATGCCTCCGACGGGRCCGA OTARpCGA LATOTGTSC

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6TCO6CGA"CCSTCC6CGAASCA76CCCAGAATGCTCGTCTC66GA6ACAAC66A6666CGCGCCTTCCCGAT6CTGAAGAGCAGGCTTCGr&3606O L SP AOP SL L AAGRI A AOOA L VL P L AA A EV VA ALL SA KL S

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CCSTOVAC"CTCG"$GCATC66TC6CCOCTCTGA66A6TT6ACCT6CAACAGGTGBDTM CCCGGACCCACACTCTSr.crGAAACCGACA 6010AAA A S AL A HPOS AE AP L K VMR VHH M

TCCA"ACOCAACGASACCCTCCAAAATSTATOGTTCATCGATAGCCTCCSCGCAACTGCACGATGTGCTACAGCTCACAAGCACCACGCTTCG 7222

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A PV I FL T V A TSIA 6 T 0L A K V AV A6k A MI Y FL A F S TLAKBCCGTGACTTCT"CTC6CACC6GATOCC6CATACC6TCTCCCA66TCCC6CTC6CGGCA66CAT6TCTATTTCC6CTTCTCACCTC66C10628

V V 6L V V A NV V OPG6A 6 HI OP A SL A KA V A TY A E K A HEE6TC6CGGCTC6C6TGCAAC6T6T6C6CC66C6666AT6CTAT6ATC66CTC6T66AGCCAGGCGTCCGACTATCCGAAAACGCASASA67CA 12020

T SF L NNI I PT TL V GA F A E BO1LQV LF I S VL FG I SLA I V 6ACCOWTCCTAT"AATCACCCGCOACCTCGCGGCCCTTGCCGAGGCACATCTSCGGTCTGTTATCTGGTGTCTTGGTACTCGTGGCATCSCUC 13222

KA E PV VOF L OA LTL PI F AL V A I L NKA APXI6A F GA NA FT IA"SC"SCCOTBOC"TTCCTGAGGCCTSCGCTCCGACTTCGGCTGTTGGATCTSASAASCCGCCCGACSGCCCTCGGCCCATGCCTCACCTC 14424

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L 5A A L SSE A V EAI P A V V P A E - aCTTTCGCACTCCGKSGA"OTCCTCSAGCCTCCCGC"COTCAGCCGCC"TAACTTCCTCCAGSCOAACCCACGTGGTCGCTGGCCBGCC 21626

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FIG. 4. Nucleotide sequence of the 2.3-kb Sail-BgiIl fragment of cosmid pRmSC121 containing the R. meliloti dctA gene and part of the

dctB gene. The sequence is shown in the 5'-to-3' direction. The ORF representing the dctA gene is indicated by the deduced amino acid

sequence written above the nucleotide sequence. The amino acid sequence of the dctB gene, which is transcribed in the opposite direction,

is written below the nucleotide sequence. Possible recognition sequences for NifA (wavy line) and RpoN (straight line) proteins are shown.

The potential ribosomal binding site and ATG start codons are boxed. A possible terminator structure is indicated by two vertical arrows.

(Fig. 1A). This zone was very small in nodules induced byRMS938 and followed by a large senescent zone (Fig. iB).

Bacteroids of RMS938 degraded before developing the typ-

ical morphology of mature bacteroids normally found in the

late symbiotic zone (Fig. iD and E). Additionally, starch

granules were packed in the symbiotic zone and in the

nodule cortex (Fig. 1E).Class mutants (RMS11, RMS16, RMS17, RMS24, and

RMS31) did not transport C4-dicarboxylates in the free-

living state (DctflV), but in the symbiotic state they showed

reduced transport activity (DctSred) The nitrogen fixation

rate was also reduced (FiXred) Examination of alfalfa nod-

ules induced by class II mutant RMS24 revealed a phenotype

similar to that of nodules induced by the wild-type strain

2011 (Fig. 1C), but starch granules also seemed to be

accumulated in these nodules (Fig. IF). By using the iodine-

starch assay, the starch concentration in nodules induced by

class I and class II mutants was found to be higher than in

nodules induced by the wild-type strain (data not shown).

Since class I mutants did not take UP C4-dicarboxylates,we assume that they are mutated in the structural gene

coding for the C4-dicarboxylate carrier, which was desig-

nated dctA. In contrast, class II mutants did take UP C4-

dicarboxylates in the symbiotic but not in the free-living

state. Therefore, we assume that they are mutated in the

regulatory gene dctB or dctD. This classification makes use

of data, recently published, concerning the genetics of

C4-dicarboxylate transport in R. leguminosarum (35, 36).

Mapping of TnS insertion sites and identification of clones

complementing R. meliloti dct mutants. For mapping the Tn.5

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R. MELILOTI dctA GENE 5557

A RmDctA MLPOWACHVEDIMIIEHSAEV---RGKTPLYRHLYVQVLAAIAAGILLGHFYPOIGTELKPLGoAFIRLVKMIIAPVIFL11 11 I 1111111 111111111111 11 i

RIOctA -----I-------IAAPLDAVAGSKGKKPFYSHLYVGVLVAIAAGILLGHFYPELGTOLKPLGOAFIKLVKMIIAPVIFL

SOS *S*RmOctA TVATGIAGMTDLAKVGRVAGKAMIYFLAFSTLALVVGLVVANVVOPGAGMHIDPASLOAKAVATYAEKAHEOSITGFLMN

111111II1 11 III11111 III HIM 11111111111 11i1i11 1111I II1111 IIIRlOctA TVATGIAGMSDLOKVGRVAGKAMLYFLTFSTLALIIGLIVANVVOPGAGMNIOPASLDPAAVATFAAKAHEQSIVGFLTN

-sRmOctA IIPTTLVGAFAEGOILOVLFISVLFGISLAIVGKKAEPVVOFLOALTLPIFRLVAILMKAAPIGAFGAMAFTIGKYGIAs

11111~~~~~I11I Itt I I I I I 11IIRlOctA IIPTTIVGAFAOGDILQVLFFSVLFGIALAMVGEKGEOVVNFLNSLTAPVFKLVAILMKAAPIGAFGAMAFTIGKYGVGS

RmOc tA IANLAMLIGTFYLTSFLFVFIVLGAVARYNGFSILSLIRYIKEELLLVLGTSSSEAALPGLMNKMEKAGCKRSVVGLVIP

RIOctA IANLAMLIGTFYITSLLFVFIVLGAVARYNGFSIVALLRYIKEELLLVLGTSSSEAALPGLMNKMEKAGCKRSVVGLVIP

RmDctA TGYSFNLOGTNIYMTLAALFIAOATOTPLSYGDOILLLLVAMLSSKGAAGITGAGFITLAATLSVVPSVPVAGMALILGI

RIOctA TGYSFNLDGTNIYMTLAALFIAGATGIHLSWGDOILLLLVAMLSSKGAAGITGAGFITLAATLSVVPSVPVAGMALILGI

r . .SRmOc tA DRFMSECRALTNFVGNAVATIVVAKWEGELOOAOLSAALGGEASVEAIPAVVOPAE

RDOctA DRFMSECRALTNLVGNAVATIVVARWENELDTVGLAAALGGOTGEOTSAAGLOPAE

B RmDc tB MHHVRM-VKLPAEASDPHALRSRARRSWLVFAAVALVLLAAGLLLARDYGRSOALAGLAGOSRIDASLKASLLRAVVERO., 11 1l1i1 1 1 1 111 1 11 1 11 1 11111 1RlDctB HHKSAMSVSOKLWPSLP--LOHRIRRMWWTYAALAFLAVVASLWTSGEIGGHRAEAALEEOARMDVTLNAALLRTVLEKY

* ~ O SOS

RmDctB RALPLVLADDAAIRGALLSPDRPSLORINRKLEALATSAEAAVIYLIDRSGVAVAASNWOEPTSFVGNOYAFRDYFRLAV1111 11 I 11 111 11 1111111 1111111 111ItII111 11 11 11

RIOctB RALPFVLSODTALAAALVGNDAGTFERLSOKLEILAAGTKAAVIYVIOKDGIAVSASNWREPTSFVGNOYRFREYFOGAV

RmOctB RDGMAEHFAMGTVSNRPGLYISRRVO ............I 11111 1111 111111

R1DctB ERGOAEHFALGTVSKKPGLYISORIS .........

FIG. 5. Comparison of the amino acid sequences of DctA and DctB of R. meliloti (Rm) with those of R. leguminosarum (Rl) DctA andDctB. (A) The amino acid sequences of DctA compared (C. W. Ronson, personal communication); (B) NH2-terminal part of the amino acidsequence of DctB is compared (34). Vertical bars indicate identical amino acids.

insertion sites of different dct mutants cosmid pRmSC121was used to pick up TnS insertions. This was achieved byintroducing the cosmid into R. meliloti det mutants and bytransferring the Tn5-loaded cosmid back into E. coli. In E.coli, the TnS insertion sites were determined by restrictionanalysis. For some mutants, mapping of TnS was alsocarried out by hybridizing TnS to total DNA isolated fromthe mutants. By this procedure, TnS insertion loci of the dctmutants could be mapped in a small region of cosmidpRmSC121 (Fig. 2). The Tn5 loci or class I and II mutantswere clustered in two groups, reflecting the division into twomutant classes. All the Tn5 insertions together spanned aregion of about 2 kilobases (kb).For complementation analysis, we generated three sub-

clones. These subclones carry fragments of the region con-taining the TnS insertions. Plasmid pRmSC121H6 is com-posed of a 6.0-kb HindIlI fragment and the integrationvector pWKR56I. Plasmid pRmSC121E6 contains a 5.9-kbEcoRI fragment and the integration vector pSUP205. Plas-mid pRmSC121SH2 consists of a 2.1-kb Sall-HindIIl frag-ment cloned into the broad-host-range vector pML123 (Ta-

ble 1, Fig. 2). By introducing these subclones into differentdct mutants, we obtained the following results. The 6.0-kbHindlll fragment complemented all class I and II mutants,while the 5.9-kb EcoRI and the 2.1-kb SalI-HindIII frag-ments only complemented the class I mutants RMS37 andRMS938 (Fig. 2). These results indicate that the structuraldctA gene is located on the 2.1-kb SalI-HindIII fragment,whereas the regulatory genes occupy a larger section of the6-kb HindIll fragment.

Sequence analysis of a DNA region encoding the detA gene.To prove the assumption that the dctA gene is located on thecentral Sall-HindIII fragment, a SalI-BgllI fragment wassequenced by the dideoxy sequencing method of Sanger etal. (41). In Fig. 3, the sequencing strategy and the openreading frame analysis are shown. The distribution of startand termination codons in the different reading framespermitted the identification of only one possible open read-ing frame (ORF). This ORF was assumed to represent thedctA gene because it covers the region in which the Tn5insertions of the class I mutants RMS938 and RMS37 weremapped. In the dctA sequence, two possible start codons

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0 so 100 150 200 250 300 350 400 450

FIG. 6. Analysis of the R. meliloti DctA protein as derived from the nucleotide sequence. The hydrophobicity plot (A) (24) is divided intohydrophobic (+) and hydrophilic (-) regions. The values were averaged by using a window of 11 amino acid residues. The distribution ofn-turns (B) and charged amino acids (C) is shown. The hatched areas represent clusters of 20 amino acids in very hydrophobic domains. TheDctA amino acid sequence is subdivided into parts I, II, and III.

(ATG) separated by 33 base pairs (bp) were detected (Fig. 4).Both were preceded by ribosome-binding sites. Each ATGcould be the putative start codon of the gene, resulting in alength of 1,359 and 1,323 bp, respectively. Upstream of thedctA coding region we found potential promotor elements. ATGT-N1O-GCA sequence 152 bp upstream of the first ATG isclosely related to the consensus sequence (TGT-N1O-ACA)proposed to interact with the NifA protein (2, 6). Thesequence CTGGCACG-N4-TTGCT, 60 bp upstream of thefirst ATG, shows strong homology to the nif consensussequence (CTGGCACPu-N4-TTGCA) (4, 7), probably inter-acting with the RpoN protein (17, 19). The dctA codingregion was terminated by an ocher stop codon. A rho-independent terminator was found 39 bp downstream of thestop codon (Fig. 4). It showed a stem-and-loop structurewith a free energy (AG) of -120 kJ/mol.

Examination of the nucleotide sequence upstream of theR. meliloti dctA coding region (Fig. 3) revealed an incom-plete ORF starting close to the promotor region of dctA. Thededuced amino acid sequence (Fig. 5B) of this incompleteORF showed 49% homology to the sequence of the R.leguminosarum DctB protein (36). Therefore, this ORF wasdesignated the R. meliloti dctB gene. The mapped TnSinsertions of the regulatory mutants are most probablylocated in or near the dctB coding region, suggesting thatthese mutants are dctB mutants.

Analysis of the DctA protein. The R. meliloti dctA geneencodes a protein of 441 or 453 amino acids (46.1 or 47.6kilodaltons [kDa], respectively) depending on the startcodon used. The amino acid sequence was analyzed for itshydrophobicity by the procedure proposed by Kyte andDoolittle (24). The protein was hydrophobic over its entirelength. Domains of high hydrophobicity, each coveringabout 20 amino acids, alternated with regions of lowerhydrophobicity. Charged amino acids and possible 3-turnswere concentrated in the less hydrophobic domains. Thesealternating domains were striking at the N-terminal end ofthe protein (part I), but they were less pronounced in themiddle (part II) and at the C-terminal end of the protein (partIII) (Fig. 6). The amino acid sequence of the R. meliloti DctAprotein was compared with that of the R. meliloti DctAprotein was compared with that of the R. leguminosarum

DctA protein (C. W. Ronson, personal communication).Strong homology (82%) was found between the R. melilotiDctA and R. leguminosarum DctA proteins (Fig. 5A).

DISCUSSION

In this work we describe two classes of R. meliloti mutantsdefective in free-living C4-dicarboxylate transport (Dctfl-).Class I mutants can neither transport C4-dicarboxylates norfix nitrogen in the symbiotic state (Dcts- Fix-). Similar R.meliloti mutants were also described by other groups (5, 49).We found that class I mutants are evidently defective in thestructural dctA gene coding for the C4-dicarboxylate carrierprotein. This conclusion was drawn from the followingobservations. (i) Class I mutants were complemented by a2.1-kb SalI-HindIII fragment of the cosmid pRmSC121 (Fig.2); (ii) the mutation sites of class I mutants are located withinan open reading frame of 1,323 or 1,359 bp present on the2.1-kb Sall-HindIll fragment (Fig. 3); (iii) the open readingframe probably encodes a membrane protein, since thederived amino acid sequence contains several hydrophobicregions which can be considered to act as membrane pas-sages (Fig. 6); and (iv) the derived amino acid sequence ofthe open reading frame is highly homologous (82%) to theamino acid sequence of the R. leguminosarum DctA protein(Fig. SA) (C. W. Ronson, personal communication).

In contrast, class II mutants transport C4-dicarboxylatesand fix nitrogen in symbiosis with alfalfa at a reduced level(Dctsred Fixred). Most of these mutants are mutated in theregulatory dctB gene, which was concluded from the follow-ing experiments: (i) class II mutants could not be comple-mented by the 2.1-kb Sall-HindIll fragment, but could by anextended 6.0-kb HindIII fragment (Fig. 2); (ii) the mutationsites of class II mutants are located in or close to anincomplete open reading frame adjacent to dctA and tran-scribed in the opposite direction (Fig. 3); and (iii) the aminoacid sequence encoded by this incomplete open readingframe shows 49% homology to the amino acid sequence ofthe R. leguminosarum DctB protein (Fig. SB).

Differences between class I and class II mutants were alsofound when the morphology of the alfalfa nodules inducedby these mutants was investigated. Class II nodules ap-

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R. MELILOTI dctA GENE 5559

peared like the wild type, whereas ineffective class I nodulesshowed the typical enlarged senescent zone reported forother Fix- nodules (1, 15, 16, 46, 48). Both nodule typescontained more starch granules than the wild-type nodule. Itis likely that the starch granules in the plant cells representthe energy reservoir, which is used for the nitrogen fixationprocess in the bacteroids. In the case of partial or deficientnitrogen fixation, the starch level in the mutant nodules washigher than in wild-type nodules. It should be mentioned thatWatson et al. (49) described an R. meliloti dct mutant whichinduced nodules similar to those induced by our detAmutants.The dctA gene in R. meliloti is preceded by a nifconsensus

promoter and an upstream element (NifA-binding site).These elements are found in front of many nitrogen fixationoperons (4). The sequence CTGGCACG-N4-TTGCT isclosely related to the nif consensus promoter sequenceCTGGCACPu-N4-TTGCA, which is thought to interact withthe RpoN protein (4). A nifA consensus promoter was alsofound upstream of the R. leguminosarum dctA gene (33, 34).The upstream sequence TGT-N1O-GCA is also related to itsconsensus sequence TGT-N1O-ACA, possibly interactingwith the NifA protein (2, 6, 30). The same imperfect NifA-binding site as in R. meliloti was found upstream of the R.leguminosarum dctA gene (33). The nif consensus sequenceand the upstream element indicate that transcription of thedctA gene may be regulated by both the RpoN and the NifAproteins.

In the noncoding region between dctA and detB in R.meliloti, several regulatory elements were found, and a totalDNA homology of 55% between R. meliloti and R. legumi-nosarum (36) was detected. This homology is about as highas that of the sequenced part of dctB, pointing to the generalimportance of this region or the existence of further commonregulatory sequences in this region.A model describing the transcriptional regulation of R.

leguminosarum dctA proposed by Ronson et al. (36) can beadapted to R. meliloti. The RpoN protein of R. meliloti isabsolutely necessary for dctA transcription, since R. melilotirpoN mutants cannot transport C4-dicarboxylates in thefree-living as well as in the symbiotic state (10, 38). Inaddition, we showed that the dctB gene is essential for dctAtransport in the free-living state. Up to now, the R. melilotidctD gene has not been identified. However, we assume thatthe dual regulatory system of R. leguminosarum, composedof DctD and DctB, is also working in R. meliloti. Accordingto this system, DctB is a C4-dicarboxylate sensor modifyingthe DctD protein, which may activate the transcription of thedctA gene. Since R. meliloti dctB mutants show reducedactivity in the symbiotic state (Dctsred Fixred), a furtherfactor must control the transcription of the detA gene. Theexistence of a NifA-binding site upstream of dctA indicatesthat NifA may control the transcription of the dctA gene incooperation with RpoN.The deduced amino acid sequence of the R. meliloti dctA

gene product contains several hydrophobic domains whichare connected by intervening hydrophilic or less hydropho-bic sequences. The intervening sequences include ,B-turnmotifs and carry a high percentage of charged amino acids.These properties suggest that the DctA protein is located inthe membrane, because the strong hydrophobicity divided inmore or less similar clusters is typical of membrane proteins(9, 13). The hydrophobic domains, containing about 20amino acids, may span the inner membrane of the R. meliloticell, resulting in several membrane passages which may bestabilized by the 13-turns and by charged amino acids in the

intervening sequences. The very high homology (82%) be-tween the R. meliloti and R. leguminosarum DctA proteinsindicates the specific relationship of these proteins in local-ization and transport function.

ACKNOWLEDGMENTS

We are very grateful to C. W. Ronson for providing the R.leguminosarum dctA sequence prior to publication. We thank M.Labes for kindly providing unpublished vector plasmids and W.Klipp, T. Dammann, and M. Keller for helpful discussions andcritical reading of the manuscript.

This work was financed by grant of the Federal Ministry forScience and Technology (No. 0318968A).

ADDENDUM IN PROOF

The nucleotide sequence of the dctA gene has beensubmitted to the EMBL Data Library and has been assignedthe code number M26399. The amino acid sequence of theDctA protein has been submitted to the Martinsried Institutefor Protein Sequence Data and has been assigned the codenumber S04815.

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