Vol. 171, No. 10

Conservation between Coding and Regulatory Elements ofRhizobium meliloti and Rhizobium leguminosarum dct Genes

JIN JIANG,1t BAOHUA GU,' LISA M. ALBRIGHT,2 AND B. TRACY NIXON`*Department of Molecular and Cell Biology, The Pennsylvania State University, University Park, Pennsylvania 16802,1

and Department of Genetics, Harvard Medical School, and Department of Molecullar Biology,Massachusetts General Hospital, Boston, Massachlusetts 021142

Received 6 April 1989/Accepted 27 June 1989

Complementation of Rhizobium leguminosarum dct mutants with a cosmid bank yielded Rhizobium melilotihomologs of the dctA, dctB, and dctD genes. The genes dctB and dctD are thought to form a two-componentsystem which responds to the presence of C4-dicarboxylates to regulate expression of a transport proteinencoded by dctA. DNA sequence analysis showed that det coding and intergenic regions, including putativebinding sites for the dctD protein and r54-RNA polymerase, were highly conserved between these twoRhizobium species. Mutation of R. meliloti dctD showed that it was not essential for symbiotic nitrogen fixationbut was needed for growth on succinate and the expression of a dctA-lacZ fusion gene in free-living cells.

Hybridization of R. meliloti genomic DNA with probes representing the central portion of dctD potentiallyidentified more than 20 similar regulatory genes, all of which are likely to depend upon the alternative sigmafactor encoded by rpoN and stimulate transcription in a manner very similar to ntrC activation of glnA inenteric bacteria.

Rhizobia are soil bacteria that can differentiate into nitro-gen-fixing endosymbionts of a number of leguminous plants.The differentiated bacteria, called bacteroids, utilize nutri-ents provided by the host to create and fuel a cell metabolismdevoted to the reduction of N2 gas. Resulting ammonia isthen exported to the plant. In recent years, C4-dicarboxylicacids such as succinate, fumarate, and malate have becomelikely candidates for the major energy source provided to thebacteroids (2, 8, 21, 24, 59). Isolated bacteroids are able totake up these compounds and increase their output ofreduced nitrogen. Moreover, mutant bacteroids that cannottransport C4-dicarboxylates are unable to fix nitrogen.

In Rhizobium leguminosarum, three genes have beendetermined by genetic and sequence analysis to play a role indicarboxylate transport, dctA, dctB, and dctD (56-58). Inresponse to the presence and/or transport of C4-dicarboxyl-ates, the products of dctB (DCTB) and dctD (DCTD) are

believed to regulate expression of a transport protein en-

coded by dctA (DCTA). By homology to the products ofenteric ntrB and ntrC genes (58, 61), DCTB has beenproposed to contain a C-terminal kinase domain that resultsin phosphorylation of an N-terminal domain of DCTD. Acentral domain of phosphorylated DCTD is then believed tointeract with RNA polymerase to promote transcription ofdctA, probably by transforming a closed polymerase com-

plex to an open one (48, 52). Because insertion mutations ofdctA result in constitutive, high-level transcription at thedctA promoter (56, 71), the transport gene product is likelyto also have a role in suppressing its own synthesis in theabsence of C4-dicarboxylates. This may involve an interac-tion between DCTA and DCTB, which, from its hydropathicprofile, has been suggested to be a transmembrane protein(58).Homology between the dct and ntr regulatory gene prod-

ucts was also suggested to indicate that transcription of the

* Corresponding author.t Present address: Department of Biological Science, Columbia

University, New York, NY 10027.

dctA promoter depends on an analog of the enteric alterna-tive sigma factor r54 encoded by rpoN (also called ntrA andginF) (60). Indeed, a DNA sequence very similar to the ur5consensus promoter (28) was observed in the region betweenthe R. leguminosarum dctA and dctB genes (56). Screening a

transposon TnS insertion library for cells that were unable toexpress a dctA-IacZ fusion gene in the presence of succinateallowed the identification and subsequent characterization ofrhizobial rpoN (60). However, this screen entailed a com-

plex heterologous system in which R. leguminosarum dctgenes were present in R. meliloti cells. The nature andorganization of R. meliloti dct genes remain unknown.We have undertaken the isolation and characterization of

R. meliloti dct genes, hoping that conservation between twoclosely related species would allow us to focus on keyregulatory elements. Moreover, in R. meliloti the rhizobialrpoN gene and other two-component regulation systems(ntrB/ntrC [66] and fixLlfixJ [14]) have been characterized;thus, this work provides the basis for future comparativestudies of the predicted signal-transducing domains andpotential cross-talk interactions between the systems. Wefound that R. meliloti contains identically arranged ho-mologs of the R. leguminosarum genes dctA, dctB, anddctD. Blocks of conserved DNA sequence that are separatedfrom each other by nonconserved residues in the dctA-dctBintergenic region suggest elements that are probably in-volved in the regulation of transcription of these genes,among which are two DCTD-binding sites that precede a C54consensus promoter for dctA (H. Ledebur, B. Gu, and B. T.Nixon, manuscript in preparation). We also found that in R.meliloti, dctD is required for basal and induced expression ofdctA and that R. meliloti may contain dozens of genes thatfall into the subclass of regulators that need rpoN to stimu-late transcription.

MATERIALS AND METHODS

Bacterial strains, plasmids, and media. The bacterialstrains and plasmids used in this study are listed in Table 1.

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TABLE 1. Strains and plasmids

Source orStrain or plasmid Relevant characteristics reference

E. coliHB101 ara xyl mtl pro leu thi lacY supE rpsL endA recA hsdR hsdM H. BoyerJM101 A(lac-pro) supE thi (F' traD36 proAB+ lacIq lacZAM15) J. MessingMC1061 ara leu lac gal hsdR rpsL M. CasadabanMM294 pro thr endA hsdR supE M. Meselson

R. leguminosarum534 dctA::TnS Strr Nmr C. W. Ronson535 dctB::TnS Strr Nmr 58538 dctD::TnS Strr Nmr 58

R. meliloti1021 Wild type, Strr 396000 'dctD-(dctA-lacZ)-dctD' Tcr Strr This study

PlasmidspRK290 IncP Tcr 17pLAFR1 pRK290 with lambda cos 26pRK2013 rep (ColEl) Mob' Tra+ Kmr 17pDCT1, -2, -5, -16 pLAFR1 cosmids with R. meliloti dct inserts This studyM13mpl8 70M13mpl9 70pUC13 70pUC19Cm Derivative of pUC19, Cmr L. M. AlbrightpSUP202 rep (ColEl) Mob' Tcr 65pSDZ-13 pSUP202 with the EcoRV-StuI portion of R. meliloti dctD and R. leguminosarum B. Gu

dctA-lacZ fusion (codons 1-12:8-end)pWB5A pRK290 with polylinker EcoRI, ClaI, Hindlll, XbaI, BglII, and BamHI sites, Tcr W. J. BuikemapHP45Ql Apr spr; source of fQ fragment 25, 53pBG-Q pWB5A with tet replaced by fl, Tcs Spr B. GupLA114 pUC19Cm with BamHI-PstI fragment from R. leguminosarum dct locus; contains L. M. Albright

part of dctA and all of dctB and dctDpBG-1 pUC13 with BamHI-HindIII fragment of pLA114; DctB+ DctD+ in R. meliloti This studypBG-lfl Biphasic plasmid combining pBG-fl and pBG-1 at Hindlll sites This study

An existing gene bank of R. meliloti 1021 DNA cloned in thecosmid vector pLAFR1 (26) was used to complement R.leguminosarum strains carrying TnS insertions in dct genes

(59). Complex media were LB for Escherichia coli, LB or

TY for R. meliloti, and TY for R. leguminosarum. Definedmedium contained RDM salts and vitamins, 10 mM sodiumsuccinate, 5 mM ammonium chloride, and 50 mM MES(morpholineethanesulfonic acid, pH 6.1) as described before(59). Streptomycin (250 ,ug/ml), tetracycline (3 ,ug/ml), neo-

mycin (50 plg/ml), and spectinomycin (100 ,ug/ml) were

included as needed to select for Rhizobium strains.Bacterial crosses. Triparental spot matings were performed

between recipient cells, donor strains, and the helper strainMM294(pRK2013) (17). Cells which had plasmids that com-

plemented mutations in dctA, dctB, and dctD were found bystreaking the exconjugants on medium that had succinate as

the sole carbon source and the drugs streptomycin andtetracycline. A second mating was used to move the plas-mids back into E. coli.DNA manipulations. Large- and small-scale plasmid prep-

arations were made by the alkaline lysis method (7). South-ern blots of DNA were cross-linked to GeneScreen (DuPont) filters as described before (12). DNA fragments were

labeled by the random primer method (22). Hybridization to

106 cmp of restriction fragment probes per ml was performedat 60°C in plaque screen buffer (0.2% polyvinylpyrrolidone,0.2% Ficoll-400, 0.2% bovine serum albumin, 50 mM Trishydrochloride [pH 7.5], 5.8% NaCl, 0.1% sodium pyrophos-phate, 1% sodium dodecyl sulfate [SDS]) that contained 10%dextran sulfate. The filters were washed at 60°C for 5 and 30min with plaque screen buffer and then three times for 30 min

each with 0.5x SSC-1% SDS (lx SSC is 0.15 M NaCl plus0.015 M sodium citrate).

Nuclease Bal31 was used to generate two sets of nesteddeletions (51) to yield sequence data for both strands of theDNA. The fragments were cloned into M13mpl8 andM13mpl9 (70) so that the DNA could be sequenced by thedideoxy method as described before (4), except that[35S]dATP was used in lieu of [32P]dATP. In all of thereactions, deaza-dGTP was used instead of dGTP to mini-mize compressions (44). The inclusion of 30% formamide inthe gels was sometimes needed to obtain interpretable data(38), and six specific oligonucleotides were used as primersto provide accurate data for regions not close to Bal31deletion endpoints. A small PstI fragment containing theEcoRI site common to the two sets of deletions was inde-pendently cloned and sequenced to permit merging of thedata.DNA sequences were aligned by eye to derive consensus

data that were analyzed with programs from the Universityof Wisconsin (16). The program FASTP (37) was used torank the NBRF data base by similarity to the dct geneproducts. Pairwise comparisons of the proteins were madewith the ALIGN program, using the mutation data matrixand a gap penalty of 16 (15). The scores from this program,given as SD units, are the number of standard deviations thatdivide a given match from the average that arises from 100random permutations of the sequences. For comparisonpurposes, the alignments are reported together with themaximum possible SD score obtained by aligning one of thesequences to itself. A 3-residue average of the mutation datamatrix values assigned to each position in illustrated align-

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5246 JIANG ET AL.

-3 -2 -1 0 1 2 3 4 5 kb

- dctA dctB dctD.4-~~~~~~~~0

EcoRI Hindill SstiIj EcoR Stul EcoRIEcoRI

Pstl PstI EcoRV EcoRV

regions sequenced

FIG. 1. Organization of R. meliloti dct genes. The region ofoverlap between cosmid plasmids pDCT1, pDCT2, pDCT5, andpDCT16 is illustrated together with various restriction enyzme sitesthat were used to provide material for DNA sequence analysis(HindIII-EcoRI, PstI-PstI, EcoRI-EcoRI), hybridization (EcoRV-EcoRV), and the construction of a DctD- strain (reading left toright, EcoRV-Stul).

ments is compared with a corresponding moving average ofself-alignment to simplify visualization of the extent ofconservation. Hydropathy profiles were calculated over awindow of 19 residues with the values of Kyte and Doolittle(36).

P-Galactosidase assays. Cultures of growing cells werewashed with minimal medium containing glucose or succi-nate as a carbon source. Cells were suspended in the same

medium and diluted 15 x for growth with one of the twocarbon sources. After incubation at 32°C for 24 or 48 h, cellswere washed and suspended in ice-cold Z buffer (40) andsonicated for 10 s with the microtip of an Artek SonicDismembrator (model 150). Supernatants were cleared bycentrifugation for 10 min at 10,000 x g. The 0-galactosidaseactivity, given as nanomoles of o-nitrophenyl-p3-D-galacto-pyranoside (OPNG) hydrolyzed per minute per milligram ofprotein, was measured for samples of supernatant with aBeckman DU 50 spectrophotometer. The protein content oflysates was measured by the Bio-Rad assay (Bio-Rad Lab-oratories).

Plant assays. Assay of effective nodulation of alfalfa seed-lings was done as described previously (66).

RESULTS

Cloning of the dct locus. We isolated 18 plasmids from a

pLAFR1-based cosmid library of the R. meliloti genome (26)that complement Tn5 insertion mutations of the R. legumi-nosarum genes dctA, dctB, and dctD (mutant strains 534,535, and 538 [58]). The restriction fragment patterns result-ing from digestion of these plasmids with EcoRI (data notshown) suggested that there were four distinct plasmids,typified by those we named pDCT1, pDCT2, pDCT5, andpDCT16. Each plasmid had genomic EcoRI fragments of6,000, 3,000, and 500 base pairs. The dct genes were locatedwithin these pieces of DNA by ligating the mixture of EcoRIfragments from pDCT5 into M13mpl8 and determining thesequence of their endmost 200 to 300 bases. Comparison ofthe data with those for R. leguminosarum (58) showed that a

dctB-like gene spanned the 6- and 3-kilobase (kb) fragments.We inferred and later showed that a dctD-like gene was alsopresent on the 3-kb piece and a dctA-like gene existed withinthe 6-kb fragment (summarized in Fig. 1).

Nucleotide sequences of dctA, dctB, and dctD. To charac-terize further the R. meliloti dct locus, we sequenced bothstrands of DNA from a portion of the 6-kb fragment and allof the 3-kb fragment. Nuclease Ba3l3 was used to createnested deletions that were cloned in M13. The data (Fig. 2)

revealed three putative dct genes (Fig. 2 and 3). The first1,323 base pairs sharing 80% identity with R. leguminosarumdctA (C. Ronson, personal communication) would encode aprotein of 441 amino acids that is 83.7% homologous withDCTA from R. leguminosarum (Fig. 3, top). If conservativeamino acid changes are considered, the homology of DCTAproteins was almost total (106.5 of 140.7 possible SD units byALIGN analysis; note 3-residue average in Fig. 3). A poten-tial site for ribosome binding (GGAGG) preceded a likelystart codon for the dctA open reading frame. Like the R.leguminosarum dctA gene product (C. Ronson, personalcommunication), R. meliloti DCTA had many groups of veryhydrophobic amino acids flanked by more hydrophilic ones(Fig. 3). However, the FASTP and ALIGN programs failedto detect significant primary sequence similarity betweenDCTA and known transport proteins or any others listed inthe NBRF data base.A second open reading frame spanned 1,861 base pairs

that were 65% identical to those of R. leguminosarum dctB.This gene is predicted to encode a DCTB protein that is59.7% homologous to that of R. leguminosarum (100.9 of160.2 possible SD units; Fig. 3, middle). R. meliloti dctB maybegin at either of two possible start codons separated by 12bases, as also observed in R. leguminosarum. However,there was little homology between the N-terminal-mostportions deduced for either open reading frame. Also, inboth species, ribosome-binding sites could not be easilyidentified ahead of either initiation codon. Only 33% of theresidues of region 1A and 60% of those in 1B of DCTB wereidentical between R. leguminosarum and R. meliloti, but allfour regions encoded hydrophobic residues that might forma helix capable of spanning a lipid bilayer. The C-terminalthird of DCTB was strongly conserved between the twospecies (Fig. 3, box 2). This region of the DCTB proteins issimilar to that of other two component monitor proteins suchas NTRB and CHEA (58), and hence is likely to be ahistidine kinase domain (30, 31, 35, 46, 68).A third open reading frame began 4 base pairs beyond

dctB. It covered 1,380 base pairs, 73% of which were thesame as those of R. leguminosarum dctD. This open readingframe would encode 460 amino acids which are 76.6%homologous with R. leguminosarum DCTD (117.5 of 142.8possible SD units by ALIGN analysis; Fig. 3, bottom). Asseen with the R. leguminosarum dct genes, a likely ribo-some-binding site (AGAAGG) that overlapped the last twocodons of R. meliloti dctB lay 8 bases before the dctDtranslation start. The portions of DCTD that are hypothe-sized (58) to act as a regulatory domain (box 3), interact withthe transcription apparatus (box 4), bind nucleotide (boxes5A and 5B), and bind to DNA (box 6) were also very similarbetween the two species. The hydropathy profile of R.meliloti DCTD (Fig. 3, bottom) was also very similar to thatof the R. leguminosarum protein (data not shown).

Potential regulatory elements are conserved between Rhizo-bium dctA and dctB genes. A comparison of the intergenicDNA sequences between dctA and dctB revealed severalblocks of conservation separated by highly divergent ones

(Fig. 4). Upstream of dctA, adjacent to dctB, was a partialdyad symmetry that is repeated in both species. About 80base pairs closer to dctA was a conserved region that ishomologous with other r54-dependent promoters (28). Threeadditional areas of homology were present in the dctB-dctAintergenic region, one near the U54 consensus, one about 30base pairs ahead of the beginning of the dctA coding region,and one immediately preceding dctA.

Transcription of R. meliloti d4tA depends on dctD. It seemed

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CONSERVATION OF RHIZOBIUM dct GENES 5247

O TTATTCGGSCG6=TGACGAEYCGYCX>900 T6GTCCACGcACGCCTOCGCCCGAGAOC±TCGGAAAGC*OGCOCTGAT&OAOCTCOCC6TCCCACTTc6cCAcCAcrATCOTCGC441 E A P Q V V A P I A E V S A E G G L A A S L Q A Q D L E G E W K A V V I T A

AACCGCGTTOCGACGAAATTOGTCAGGGCGOCGTTCCGACATGAAOGTGTGOOGA S ATCAOCGAGOGCGV A N G V F N T L A R C E S M F R D I GL IL AMGAV PV S PV V S L T A A LCGT TTGCGOCAGCATTGCGACGAGGAGCAOCAGAATCTGOTCOccTATGAOAOcGOGTATCOOTCCTOGCOATOAACAOT I F G A G T I G A A G K 8 8 L M A V L L L L I Q D G YSLPTDT Q IF LOOGcOCMGZOTCATOTAOATATTOOTOG~OTCCAGOTTOAAC 4ATMccOZGT$COMTACOAAAccOAcG_C TCoAoTGTOCAOCcoO~CTTCTcCATCTTGTTCATGAOCGGA A L T MY IN T GD L N F S Y G T PI VL GVV S RK C OAK E M K N M L G PAAGAGCCGCC G C iTCTCCTTOATOTAGCSATGAC0AGG AGMOcCOTTATO COG M GA CL A A E S S S T G L VLLL E E K I F VGAGAGAAACGACGTCAGATAGAAGGTGCCGATCAGCATGGCGAGATTGGCGATCGAGGCGATACCTACTTGGATGTGAAGGCCATGGCGCCGAAGGCGCCGATCGCGCCTTF L F S T L Y F T G I L M A L N A I S A I G Y K G I T F A M A G F A G I P A A K

CATCA*OATcOC4_$GCIOGAMcOOSCATGOCATCAGCGCTCAGGAAATCGACCACGGGCTCGGCTTTCTTGCCGACGATCGCCAG CGAGATACCGAAGACCGAGATGMCAGM L I A V L R F I PLT L A Q L F D V V P E A K K G V I A L S I G F L V S I F LGACTGCAATGaCTTCCC A TCC TTTCATACTCATTTTCTCGGCATAGGTCGCGACCGCCTTGGCV Q L I D G E A F A G V L T T P IIN M L F G T I S Q E B A K E A Y T A V A K AGTC6AcTccGCOGGAOCGATAcCcTAOUCOCTAGCTGACAGTGAGOAAGAAGGTACATCGCCTTGCCGGCD L S A P D I H M G A G P Q V V N A V V L T SF AL F Y I M A K G AGACRGGVCCGAcCTGGCGAGATCGGTCATOCCGGCAATCCCGTCGCGOATCAOOGAGATCACCOOCOCGATGATCATCTTCACGAOCCTGATGAAGGCGTCCAAGCGGTTTGTGV ROGV KA L D T MKGA I10 T A V T L F I V P AIlI M KV L R I F ADOG L P K LCTCCGTcCGATATCAGGATAGAAATGCCCGAGGATAOAATGMACATAGAGATOC TAAAGGGGTGTCTTGCCGGACCTCCGCGGAATGTTCE T G I D P Y F H G L L I G A A I A A L V Q V Y L H R Y L P T K G R V E A S H EGATGATCATGATATCCTCCACGTGOCAGOCCCAGTCCGACATOaOGGCCTCCCGGGCCGCTTTCCGAAGATCOACAGCAGTGOCTOcTTGTGGAGCTTGTCAGCMCATGCGTG

CCAGTiTTGCGCGGAGCWCTATGCGATTGAATCCATGACTTTTTZGTGTCGCTTGCCGTTCGTGGATATGTGCGGATTTCCCAGAGTGCTGGCGTCCTGGGCGAACATGCACCATGTTCGCATGGTCAA CTTCCTGAAGGGGTCCGCATG=TCGCAGTM=AGTCCTGGCTCGTTTTCGCG GCGTCGCGCTGGTCCTTCTCGGCAGCC

M V K L P A E A S D P H A L R S R A R R S W L V F A A V A L V L L A AGGCCTCTTCTTTGCGCGAACTACGOCCOGTCOCAGCGCTCOCCGOCTTGCCGGTCAGAGCwOATCGACGCCAGCCTGAAAGCCTCOCTTCTTCGAGCAGTCGTGGAACGGCAGCGCG L L L A R D Y G R S Q A L A G L A G Q S R I D A S L K A S L L R A V V E R Q RGCCCTOCCGCTTCOTCTCCCGACGACGCAOCCATT CGTGGCOCATTGCTTTcOCCGACAOOC CTOCTCGAMcCATCAACOTMGCTCGAGGCTGGCGACAAGCGCCGAMGCCA L P L V L A D D A A I R G A L L S P D R P S L D R I N R K L E A L A T S A E AGCGOOTCATTTATCT'GATCGACCGGAGCGcTCGCCGTcCOOGCAGCACTGGCAGGACC CG AOCTTTGTGTOCAACGACTATOCCTTCCGCGAATTATTTCCGGCTCGCCGTCCGCA V I Y L ID R S GOVA V AA S N W Q E P T SFP VOGN D Y A F R D Y F R L AV R

GACGGCATGG =OAACATTTCOCCATGGOCACOGTCAGCAATCOOCCCG = TTTAATTTTCCGCG T CCG T TGATCGTCG GTTCD G K A E H F A K G T V S N R P G L Y I S R R V D G P G G P L G V I V A K L E F

GACGOOOTCGAGGCGGATTGOCAGOCCTCGGGCAAGCCGCTATGTCACC GCACcCOCGTCGTCCTCATCACCAOCCTGTCTCTOOCGCTTCATGACGACG AGCCGATCGCCD G V E A D W Q A S G K P A Y V T D R R G I V L I T S L P S W R F K T T K P I AGAGCCTCCC TTCGC GCCTGC GCTCTGTTCCGAAGATCGAAGCCOCCCGATGOCTCCTCCACOCTCGACGCCCTGCTGE D R L A P I R E S L Q F G D A P L L P L P F R K I E A R P D G S S T L D A L LCCOGCGCACTCCACCGCAGCCTTCCTGCGCGTGGAAACCATOGTGCCGTCGTTGCCTOCG CT ACC CAGP G D S T A A F L R V E T M V P S T N W R L E Q L S P L K A P LA A G A R E A QCTCCTCACCCTTGCCGCGCTCTCCGCCGCTTGCTCTTGGTTGAGGCGL L T L A A L V P L L A L A A L L L R R R Q V V A M R S A E RL R N A L E A

AGCGTC ACG CGCGACCTTCGTCCAS V E E R T R D L R M A R D R L E T E I AD R Q T T E K L Q A V Q Q DLV Q AAACC6TGCCATGCTCGCATGCCCGGGTTG CATGAGATCACWTCCCCACMTATGCGGATAATGCCGCACCTTTCTCC ACCGCGGCCAGACCN R L A I L G Q V A A G V A H E I N Q P V A T I R A Y A D N A R T F L H R Q T

* _~~~~~~~~~~~~~CTCAC__CGACACTGCGCCGCT M:GCAGGCCATTTC CGCGATGV T A A E N K E S I A E L T E R V G A I T D E L R R F A R K G H F AA G P T A MMAGAGGTCGTCGAGGTOCOCTCATGCTOCTTCGCTCATGCTOCAGTiC GK E V V E G A L M L L R S R F AGRK D A R L D L P P D G L Q A L G N R I R LGAGCAGGTCTTGATCMCCTTCTGCAGMTGCACTGAAGCGATCOGCGACAGCGAGGACOGCCGATCCAGOTGCOCTGCGAGGAOGMcOGGCATCOCTGACCGTCGCCGACE Q V L I N L L Q N A L E A I G D S E D G A I Q V R C E E A A G G I A L T V A DMTOOCCCGGOOATTGGTCCOTATGTCCTTTCACOGTTCAACACTTCGAAGGAAGACGGGCTGOcCTCGGTCTCOCOATCTCCAAGGAOATCGTCTCCACTATGGCN G P G I A A D V R E E L F T P F N T S K E D G L G L G L A I S K E I V S D Y GGGCACGATCGAOOTCGAGAGAOOCCCCTCCSGAACGACATTTOGTCAATCTCAAGACCTGAGCGATGAGCOCCATCCGTGTTCCTGATCOATGACGACCGCOACTTGCGCG T I E V E S G P S G T T F A V N L K K A HM A A P 8 V F L I D D D R D L RAAGCCACCAGACCTTGACTATTTGCACCGCCTCGTCCTATTCATCAATTTTCCCCGTACKATQQ T L EL A OFGTV 8S A SATE A LA G S AD F AOI V IS DIR

K P OKM DOG LA L F R K I L A L D P D L PKM I L V TO HOGD I P K A V Q AlI Q D

G A Y D F I A K PFAADRLVQ S ARRAE EKRRL VKE NR S L RRAAE

A A * i G L P L I O TT P V LK R L R f T Lir LII A D T D V D V L V A G E T G SGGCMCGOAGOTCGTWCOACr0CTGCTGCACCAAT .IACGAT GCOO CTGCAG K zGV V A^( T L L BQwSRRR V A L NIIC G A LP T G HGAGCCGCSCTF TTCCTCGACGAGACGAt CATCp p AATCQAGGTGAAGATG

E P O AF T GOA V KKR I EOliA SOOT L F L D ElI AKH P P A T Q VKKCW W _T SA CG_TCGGUCTCA T G C TL R V L E A R E I T PLT N L T R P V D I R V V A A V DL G D P A A R G DTTCCGCGAGGATCTCTATTACCOOCTGAACGTCGTGA TCTCTGA CCCTTTCTTCGGAACC

TTWCCAGGCCCACC OOOACTCOCCGAAGOTGOCOCTGOGGTGGAGF GOR EV P A I S A AKR A YLAT S W POGN V RE L S H F A E R V AL GOVEOOMk=TGr,GAGGAGCCA RP^ACGCCAA CAWCTCAXOOCGCATTGGACGTCOGN L 0 V PA A A PASS OATL PERLE RYE A DI L K Q AL TA H COD VMAGGCCOAGCTOCTCCOAGCCTOACACCOG CAGA0TTATGT,CGAACQO(CAGGCcCOGCRTCCCAATGCCATA

116404236364356324476284596244716204836164956124

107684

119644

13164

14351

15551675

351795

751915115

2035155

2155195

2275235

2395275

2515315

2635355

2755395

2875435

2995475

3115515

3235555

3355595

3475616, 17

359557

371597

38351373955177

4075217419525743152974435337455537746754174795A alX I L Q A L G I P R T F Y K L Q RX v IS N RK A D Y V E R A ID F 1r r n A 1 45

TCGOATTOA 4807S K T * 460

FIG. 2. DNA and derived amino acid sequences of R. meliloti dctA (bases 1325 to 0), dctB (bases 1571 to 3421), and dctD (bases 3425 to4807) genes. DNA bases and amino acid residues are numbered at the right margin. These data have been submitted to GenBank underaccession number M26531.

likely that the det genes function the same way in R. meliloti internal piece of the dctD gene (EcoRV-StuI; see Fig. 1), onas their homologs do in R. leguminosarum. For other work pSUP202. This plasmid can be mobilized into R. meliloti butwe had fused the 12th codon of R. leguminosarum dctA to will not replicate there (65). This plasmid also contains athe 8th of lacZ (Gu and Nixon, unpublished). We made gene that confers resistance to tetracycline. By selecting forpSDZ-13 by cloning this reporter gene, together with an tetracycline-resistant transconjugants, we obtained strain

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OL In a E. %7 " r A. L w 4 a Jr v L's so EL As SL %J & " 86 LA J6 St Af A LO V AO V &O V JM %F &A & %- W

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RMDCTA 1-112RLDCTA 1-115

RMDCTA 113-227RLDCTA 116-230

RMDCTA 228-342RLDCTA 231-345

RMDCTA 343-441RLDCTA 346-444

M -A Ik Al A3 RESIDUE AVERAGE

A AL -A . A..._. I

HYDROPATHY OF RH DCTA19 RESIDUE AVERAGE

RMDCTB 1-111RLDCTB 1-115

RMDCTB 112-226RLDCTB 116-230

M4VKLPAEASDPAV""' SDRPSIRMH6SA_SVS_KL__ S 6TF__ER L#

A_

RMDCTB 227-340RLDCTB 230-344

KIEARP*STLDAL TA AMVRNLGELWDVVEI HGI Il

RMDCTB 341-455 SAERLDCTB 346-460 ISS

RMDCTB 456-569RLDCTB 461-575

RMDCTB 570-616RLDCTB 576-622

3 RESIDUE AVERAGE

HYDROPATHY OF RM DCTB19 RESIDUE AVERAGE

V F yrv

LA A.AL &.LAL a.Ok A r-nA L.. -L &iLdL ]Ar-_b-lA- -IB-

RMDCTD 1-115 ISAAPSRLDCTD 1-115 pTLMP

RMDCTD 116-230RLDCTD 116-230

RMDCTD 231-345

RLDCTD 231-345

RMDCTD 346-460 I

RLDCTD 346-448 FOY

I-I_l

a 1 . I A.A la_ . lb k .A

3 RESIDUE AVERAGE

HYDROPATHY OF RN DCTD19 RESIDUE AVERAGE

3-

- A . . im odm No

_S-SA 5B

FIG. 3. Homology between R. meliloti (RM) and R. leguminosarum (RL) dctA, dctB, and dctD genes (3-residue average is for the mutationdata matrix homology values assigned to each position in the alignments [15]). Unshaded, R. meliloti gene products aligned to self; shaded,interspecies alignments. Also shown are hydropathic profiles (19-residue moving average; Kyte and Doolittle [36]; hydrophobic [+]hydrophilic [-]). lA/1B, Putative membrane-spanning domains of DCTB; 2, conserved C-terminal domain of DCTB; 3, conserved N-terminaldomain of DCTD; 4, conserved central domain of DCTD; 5A/5B, putative nucleotide-binding domain of DCTD; 6, putative DNA-bindingdomain of DCTD as identified previously (58).

ll. Ab --J= -&.Aa-m& AMML-A - - -

I ..I.

-- -.--& 7=6GRPNAISKT

.- -- - - - -- - ,MA, A&ML-

I&& &

NIMLA 0, 0 A A . 0--

- 'IIPW ""r lqmm - MIT '- -1

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-4

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CONSERVATION OF RHIZOBIUM dct GENES 5249

> -- - - --- -->

RL CATGGC66AM6TGCATTTTTCT6CACGAAAC6CAAAT66AMGTGCGGAMCCGCATTGCTTAGTliii IllIllIll III 1111111 111HIM 111111

RN CATGCGAACATGGTGCAT6TTTTCGCCAG6ACGCCA. GCACTTCTTGCG6B TCCGCACATATCCAC-DCTB -

->

-

RL TAGTTGTTAGCAGTCTCGTAAAT.TTTTCATTAATAAATTCAATCG.GTTGGTTGGCGACTTAAAACTGGIII I I I 1I IIIIIIIII I 1I 1111111

RN GMCGGCAAGCGABCACCrCTCCCAAMTGTCATGGATTCAATCGCATAGGCCTTCCGCGGCAMCTGG-CONSENSUS1

RL CACGGCGATTGCGAAGGBTGG6CAACAACGGCTGAGCTGTTGGACTTGMAGCGACGCTCG6GAGBCCliii liii II I I 11 I II I 11 1111111

RM CACGCATGTT6CTGACAAGCTCCACAA6GCA6CCACTGCTGTCGATCTTC66AAAGCGGCCCGGGAG6CC

RL GGAGTTCGTTCCGGA..... CGAGCCACTAG6AGGACATCATGII 111111 11111 HIM11 HIM,

RN CGVCATGTTGCCGGACTGGGCCTGCCAGTGGAGGATATCATBL-DCTA-.

FIG. 4. Comparison ofR. leguminosarum (56, 58) and R. melilotidctAldctB promoter regions. Bases read 5' to 3', left to right;vertical bars denote homology between species; arrows indicaterepeated bases (solid) and partial dyad symmetries (broken). Theputative o54 consensus promoter (28) is also labeled. The R.leguminosarum (RL) dctB start codon, previously suggested to bewithin the first half of the first partial dyad symmetry, is shown alongwith that of R. meliloti (RM) as beginning at the leftmost 5'-CAT-3'sequence.

Rm6000, which, upon hybridization was found to have thedctA-lacZ-containing plasmid inserted into dctD. The inser-tion event also created two truncated forms of dctD.When used to inoculate seedlings of alfalfa, this strain

formed bacteroids that were able to reduce acetylene andthus probably fix nitrogen (data not shown). However,free-living cultures of this strain could not use succinate as asole carbon source, and they had only background levels of,-galactosidase activity, which are detected even in wild-type cells that contain no additional lacZ DNA (Table 2).The ability to utilize succinate for free-living growth wasrestored when the mutant strain harbored pBG-lQ, apRK290-based plasmid that carries both dctB and dctD fromR. leguminosarum (see Fig. 5 for the structure of pBG-lfl).When these cells were grown on glucose, expression ofdctA-lacZ was 20- to 40-fold higher than background levels(Table 2). The P-galactosidase activity increased another17-fold when succinate replaced glucose as the sole carbon

TABLE 2. ,B-Galactosidase activity due to dctD-mediatedexpression of dctA-1acZ

P-Galactosidase activitya (nmol of ONPGhydrolyzed/min per mg of soluble cell protein)

Strain(description) Minimal Minimal

glucose succinate LBmedium medium

RmlO21(pBG-lQ) (wild 24 ± 8 23 ± 2 26 ± 4type plus R. legumi-nosarum dctB-dctD)

Rm6000(pBG-4) 19 ± 3 53 ± 3['dctD-(dctA-1acZ)-dctD' insertion mu-tant plus carrier plas-mid]

Rm6000(pBG-lQ) 1,007 ± 37 17,830 ± 1,231 8,600 ± 360['dctD-(dctA-1acZ)-dctD' insertion mu-tant plus R. legumi-nosarum dctB-dctD]a Values represent averages for three cultures of a typical experiment

repeated three times.

dcaBam__

dctBamp pBG-1

\ .6kbJColE1ori z daD

PsdnHindm

a (spc)

FIG. 5. Structure of pBG-lQl. pBG-1 was made by ligatingpUC13 and an R. leguminosarum BamHI-PstI fragment that con-tains part of dctA and all of dctB and dctD (58). pBG-Ql was made byligating pWB5A that had been digested with SmaI, removing aninternal portion of the tet gene, to the HindIll fl fragment (25, 53)whose ends were rendered blunt by treatment with Klenow frag-ment. The fQ fragment confers resistance to spectinomycin (spec).The biphasic plasmid pBG-lfl was constructed by ligating pBG-1and pBG-fl after digestion with HindIll. The relative orientation ofthe two plasmids is undetermined.

source (Table 2). Growth of these cells in LB mediumresulted in about 50% of the succinate-induced expression ofthe reporter gene.DCTD may belong to a large family of rpoN-dependent

regulatory genes. Based on deductions about the homologousportions of ntrC, nifA, and dctD gene products, it has beenproposed that the central domains of these proteins interactwith a"5 RNA polymerase to activate transcription (re-viewed in reference 28; see also reference 52). From varioushybridization experiments, it has been suggested that theremay be 6 to 10 genes in R. meliloti that interact with therpoN gene product to affect transcriptional control (58, 60;T. Adams, Ph.D. thesis, Michigan State University, EastLansing, 1986). To identify such genes, we hybridized ge-nomic DNA from R. meliloti with probes of R. meliloti dctD,ntrC (66), and nifA (10) (Fig. 6). A single PstI digestion of R.meliloti DNA, which would generate a unique hybridizingfragment for each probe, was split and used to preparemultiple blots. One-hour exposures to film showed the singlefragments expected for each probe. Exposing film for 16 h tothe blot hybridized with the nifA probe showed that diges-tion was complete. This fragment of nifA, beginning in theregion encoding the last 11 residues of its central domain(residue 366 of DCTD, SHFAER; Fig. 3, bottom) andterminating 28 base pairs beyond the gene, encompasses itsputative C-terminal DNA-binding domain (19). Only oneadditional fragment hybridized weakly to this nifA probe.However, the same exposure to blots reacted with probescontaining other portions of dctD (corresponding to residues55 to 405, DIRMPG-/-RYEADI, Fig. 3) and ntrC revealedmore than 30 bands of varied intensity, many of whichappeared to comigrate. Because hybridization between ntrCand dctD was not very strong, even bands of low intensitymay reflect the presence of homologous genes. Although thentrC probe includes DNA encoding the very C-terminal partof NTRB and the N-terminus of NTRC, the region commonto both it and the dctD probe was that which encodesresidues 55 to 262 of DCTD (DIRMPG-/-MLRVLE). This

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~TC

FIG. 6. Hybridization of R. meliloti genomic DNA with probes

of R. meliloti nifA (base pairs 1248 to 1653 [10, 19]), dctD (base pairs

3847 to 4639, Fig. 2), and ntrC (base pairs 1 to 1020 [66]) and

Bradyrhizobium sp. [Parasponiae] ntrC (ntrC*; base pairs 1625 to

1996 [49]). Putative functional domains encoded by each probe are

as illustrated (refer to text and Fig. 3 for more detail). Blots were

exposed to film for 1 h (A) or 16 h (B).

portion of dctD encodes only part of the N-terminal domain

(box 3, Fig. 3, bottom) and about half of the central domain

proposed to interact with &54 RNA polymerase. Also within

this region was the putative nucleotide-binding site (boxes

5A and SB, Fig. 3, bottom) and an additional run of 17

residues that were nearly 100% conserved among the NIFA,

NTRC, and DCTD proteins (58). A probe made of DNA

from only this central region of a Bradyrhizobium sp. [Para-

sponiae] ntrC gene (49), encoding amino acids corresponding

to DCTD residues 139 to 264 (ASEGLP-/-RVLEAR), also

revealed more than 30 DNA fragments. This Bradyrhizo-

bium DNA fragment was 83, 62, and 53% homologous to the

R. meliloti ntrC, dctD, and nifA counterparts, respectively.

DISCUSSION

We have identified R. meliloti dctA, dctB, and dctD genes

needed for the utilization of the C4-dicarboxylic acid succi-

nate. These genes are able to replace those of R. legumi-

nosarum in complementation assays, suggesting that expres-

sion and function of dct genes are similar in both bacteria.

Restriction analysis of the R. meliloti dct locus that we

characterized suggests that it is the same as those recently

identified genetically by others (5, 21, 24, 67, 71). Finan et al.

(24) have shown by transduction analysis that this dct locus

is adjacent to thi genes on the megaplasmid pRmeSU47b.Sequence data show that another putative two-componentregulatory system lies in opposition to R. meliloti dctA instrain 1021 (J. Sojda and B. T. Nixon, unpublished). It is notknown whether this system is functional or, if so, whatgene(s) it regulates. In fact, because one of the open readingframes begins beyond the EcoRI site downstream of dctA,the intact system may not even be present in the related R.meliloti strain JJ1c1O, whose DNA from this region does nothybridize with that of strain 1021 (67).

In this paper we present DNA sequence data showing thatdct genes are highly conserved between R. leguminosarumand R. meliloti; in both species, dctB and dctD are tran-scribed divergently from the putative transport gene dctA.Recently, homology has been detected between deducedamino acid sequences of procaryotic transport proteins thatmediate uptake of phosphorylated hexoses (uhpT [691),phosphoglycerates (pgtP [27]), and glycerol-3-phosphate(glpT [20]) on the one hand, and xylose (xylE) and arabinose(araE) on the other (reviewed in reference 3; the latter groupof proteins are also homologous to passive glucose transportproteins of mammals). At least uphT and pgtP are regulatedby gene products that have homology with portions of theC-terminus of DCTB and the N-terminus of DCTD (69).Although we did not find significant homology betweenDCTA and any of these or other transport proteins whosesequences might be in the NBRF data base, DCTA doespossess numerous potential membrane-spanning domainstypical of transport proteins.By monitoring the expression of a dctA-lacZ fusion gene,

we found that free-living R. meliloti cells rely on dctD forbasal and induced transcription of dctA. This result confirmsthe recent data of Yarosh et al. (71), who also demonstratedthat in R. meliloti the expression of dctA depends on dctBand rpoN. These workers also note that, as previously seenfor R. leguminosarum (56), mutation of dctA results in veryhigh expression of dctA even in cells that are not exposed toC4-dicarboxylates; this uncontrolled expression of dctA wasfound to depend upon a functional dctD gene. These resultsare consistent with the current model for dctA regulation(58), in which DCTB modifies the functional state of thetranscriptional activator DCTD in response to the presenceor absence of C4-dicarboxylates. As noted (60, 71), the dataalso suggest that DCTA plays a role in regulating expressionof dctA by affecting the functional state of DCTD, perhapsvia interactions with DCTB.Another feature of the current model for detA regulation is

the expectation that, like the proteins NTRC (1, 29, 32, 41,48, 54) and NIFA (9, 45), DCTD binds upstream of a (54consensus promoter in order to activate RNA polymerase. Acomparison of the intergenic regions between dctB and dctAin R. meliloti and R. leguminosarum revealed potentialDCTD-binding sites in the form of a conserved partial dyadsymmetry that is repeated about 80 base pairs upstream of a

(r54 consensus sequence. In other work we have found thatthe repeats do indeed bind DCTD protein and that when theyare placed upstream of the R. meliloti nifH &54 consensuspromoter, they cause succinate-sensitive expression of nifH(H. Ledebur, B. Gu, and B. T. Nixon, manuscript inpreparation).

It is the C-terminal portions of &4-dependent regulatoryproteins like NTRC, NIFA, and DCTD that are believed toform a-helix-r-turn---helix DNA-binding domains (19, 58).The results of mutational analysis of ntrC are consistent withthis notion (11, 52). Another block of strong conservationprecedes this portion of the DCTD proteins (GALT

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CONSERVATION OF RHIZOBIUM dct GENES 5251

Fig. 3). The sequence of this region predicts a p-turn andadditional a-helix that may also contribute to the DNA-binding domain. When the last 50 or so residues of R.meliloti NIFA are fused randomly to C-terminal residues ofDCTD, some of the resulting chimeric proteins respondnormally to succinate but regulate transcription of nifHrather than dctA (B. Gu and B. T. Nixon, manuscript inpreparation). The portion of NIFA that causes this change ofspecificity contains sequence that is also predicted to beginwith a ,-turn followed by an additional a-helix.One or another of the additional regions conserved be-

tween Rhizobium dctA and dctB genes may represent a dctBpromoter. Further study is needed to verify the currentassumption that dctB and dctD are cotranscribed, but it hasbeen shown (56, 71) and we confirm that dctD is not essentialfor expression of dctA in bacteroids. It is not known whetheranother symbiotic promoter is available to dctA or anothera54-dependent regulatory protein such as the nifA gene

product substitutes for DCTD in bacteroids. Several DNAfragments have been found to hybridize with probes of nifAand ntrC genes in R. meliloti, E. coli, Bradyrhizobium sp.

(Adams, Ph.D. thesis; B. T. Nixon, unpublished) and Ses-bania rostrata (50). We found that more than 20 R. melilotiDNA fragments hybridized to the DNA encoding 125 resi-dues common to the central core of the DCTD, NTRC, andNIFA proteins. A nucleotide-binding domain has been pro-

posed to reside within these amino acids (58); thus, thesefragments may simply represent genes whose products pos-

sess a similar domain. However, additional sequence homol-ogies not known to be associated with a nucleotide-bindingsite exist within this probed region. This central portion isbelieved to encode a domain that interacts with a"4 RNApolymerase (10, 19, 33) to promote transition from a closedto an open complex (48, 52); we found that a truncated R.meliloti DCTD protein lacking the N-terminal domain droveexpression of R. leguminosarum dctA::lacZ in E. coli (B. Guand B. T. Nixon, unpublished). Many of these potentialgenes may therefore also encode members of a family ofrpoN-dependent proteins.Most of these potential regulatory genes have not yet been

characterized. One that is known, the tyrR gene of E. coli(13), is unusual in that it appears to represss rather thanenhance the expression of several genes involved in thebiosynthesis and transport of aromatic amino acids. Two ofseveral genes involved in the anaerobic expression of for-mate hydrogenlyase, fdhF (6) and hydB (62), need &54, andthefdhF promoter has a a54 consensus region. The transcrip-tion of these genes is very likely controlled by a dctD-likegene, perhaps JfhlA or hydF, which flank hydB and are now

known to be needed for expression offdhF and an associatedhydrogenase activity (63, 64). Recently, rpoN-like genes

have been identified in Alcaligenes eutrophus and Pseudo-monas facilis (55). In these organisms, the rpoN gene isimplicated as being essential for hydrogen oxidation, nitrateand urea assimilation, denitrification, and various substratetransport systems. Among the other genes that may dependon rpoN are toluene and xylene degradation genes (xyl [18,34]) and carboxypeptidase G2 (42) of Pseudomonas spp. andflagellin genes expressed during development of Caulobactercrescentus swarmer cells (41, 48).

ACKNOWLEDGMENTSWe thank Clive Ronson, Fred Ausubel, Sydney Kustu, and

Turlough Finan for sharing materials, methods, data, and ideas priorto publication and Donald Bryant for generously making oligonucle-otides and critically reading the manuscript.

This work was supported by a Project Initiation Grant from thePennsylvania State University and Public Health Service grantGM40404-01 from the National Institutes of Health to B.T.N.L.M.A. was supported by Hoechst A.G.

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