Molecular Microbiology (1991) 5(6), 151&-1530 ADONIS 0950382X91001676

The Rhizobium meiiioti exoZlexoB fragment ofmegaplasmid 2: ExoB functions as a UDP-glucose4-epimerase and ExoZ shows homology to NodX ofRhizobium ieguminosarum biovar viciae strain TOM

A. M. Buendia,^ B. Enenkel, R. Kdplin, K. Niehaus, W.Arnold and A. Piihler'*'Lehrstuhi fur Genetik, Universitat Bielefeld, Postfach8640. D-4800 Bielefeld, Germany.

Summary

A 2.6kb C/at-BamHI DNA fragment of megaplasmid 2of Rhizobium meiiioti 2011 was found to carry genesinvolved In exopolysaccharide synthesis and infectionof alfalfa nodules. The analysis of the nucleotidesequence of this ONA fragment revealed the existenceof two open reading frames (ORFs) running in oppositedirections. Plasmid integration mutagenesis showedthat these ORFs are organized as two monocistronictranscription units. One ofthe ORFs represents a newexo gene designated exoZ, which is involved in, but notessential for, the production of acidic exopotysaccha-ride. However, exoZ is not necessary for noduleformation with alfalfa. The ExoZ protein was found toshow homology (23.3%) to the NodX protein of the RIeguminosarum biovar viciae strain TOM, known to beessential for nodulating the primitive Afghanistan pea.The second identified ORF corresponds to the exoBlocus. The deduced amino acid sequence of the ExoBprotein is homologous (39.6%) to that of the Escher-ichia coli GalE protein. In R meiiioti, exoB codes for aUDP-glucose 4-epimerase. A deficiency In the activityof this enzyme fully accounts for all the multiplecarbohydrate defects that have been observed in exoBmutants.

Introduction

A compiex symbiotic interaction between rhizobia andspecific leguminous plants results in the formation ofnitrogen-fixing root nodules. In the course of this interac-tion, the bacteria associate with the host's epidermal root

Received 28 November, 1990; revised 15 February. 1991. tPresentaddress: Departamento de Microbiologia, Facultad de Biologia, Universi-dad de Sevilla, 41080 Sevilla, Spain. 'For correspondence. Tel. (521) 1065607; Fax (521) 106 5626.

hairs, and penetrate by formation of a specialized invasionstructure called the infection thread. Mitoses and cellgrowth in the plant root cortex lead to the formation of aroot nodule, in which bacteria infect host cells anddifferentiate into bacteroids that fix nitrogen {Bauer, 1981;Halverson and Stacey, 1986; Long, 1989). The develop-ment of the symbiosis is directed by signals from theenvironment and signal exchange between the symbioticpartners. The Rhizobium cell-surface components areinvolved in many of these stages but their exact role hasnot been elucidated. The results from a number ofprevious studies have indicated that rhizobial exopolysac-charide (EPS) production is necessary for successfulnodulation on legumes that develop an indeterminate typeof nodule, e.g. alfalfa, peas, clovers, and Leucaenaieucocephala (Borthakur e^ ai, 1986; Chakravorty et ai,1982; Chen et ai. 1985; Leigh et ai, 1985; Muller et ai,1988). Acidic EPS does not seem to be essential for bean-and soybean nodulation (Diebold and Noel, 1989; Ko andGayda, 1990). Lipopolysaccharides are essential for theinfection of determinate nodules such as Phaseotus beansand soybeans. Mutants of Rhizobium meiiioti that fail tosynthesize its acidic Calcofluor-binding exopolysaccha-ride, often referred to as succinoglycan or EPS I, formineffective nodules on alfalfa (Finan et ai, 1985; Leigh efa/., 1985; Muller ef a/., 1988). These mutants are unable toelicit infect ion-thread formation (Inf phenotype) and thenodules induced by these mutants do not containintracellular bacteroids. They are therefore called 'emptynodules'. Although numerous mutants affected in terms ofEPS production have been isolated, little is known aboutthe biochemical functions of individual gene products.

Mutations affecting production of EPS have been clus-tered in a 22 kb DNA region on megaplasmid 2 in R. metilotistrain SU47 and fall into 12 complementation groups(Long etal., 1988). Muller etai (1988) obtained EPS Inf"mutants of R. meiiioti 2011 following Tn5 mutagenesis.Several complementing cosmids were isolated and physi-cally mapped (Muller et ai. 1988). Tn5 insertions werelocalized on a 7.8 kb EcoRI DNA fragment of megaplasmid2, where at least three regions involved in EPS synthesisand nodule infection could be identified (Keller et ai,1988). In this paper we describe a further inf/exo DNA

1520 A M. Buendia et al.

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Rg. 1. Genetic anafysis o( a DNA region of megaplasmid 2 of R. meliloti 2011 carrying genes for EPS synthesis and nodule infection.A. Above the restriction map of cosmid pRmPM551 the approximate locations of the exo complementation groups (Long et ai, 1988) are shown. The7.8kb EcoRI fragment complementing two Inf EPS mutants (Muller ef a/.. 1988) is marked along with the sequenced 2.6kb C/al-SamHI fragment.B. The locations of three deletions as well as the phenotype ot the corresponding mutants are indicated. The deleted regions were replaced by aninterposon confemng gentamicin resistance.C. In the restriction map of the sequenced 2.6kb DNA fragment only relevant sites are shown. The location of the coding regions o( exoZ and exoB areindicated by bars. DNA fragments deleted in mutants RmH90 [exoZ) and RmH89 {exoB) are indicated by black bars. These DNA fragments werereplaced by an interposon conferring gentamicin resistance.Abbreviations of restriction enzymes are; B, BamHi. Bg. BglW: C, Clal; E, fcoRI; S, Sa/I; X. Xhot. Other abbreviations are: Cfw. Calcofluor-whitephenotype; Inf. infection phenotype.

region overlapping the right-hand border of the 7.8kbEcoRI DNA fragment on megaplasmid 2 of R. metitoti2011. We will present the nucleotide sequence of this DNAfragment and demonstrate that two genes. exoZ andexoS, are located on this fragment. By means of anenzymatic assay, it will be shown that exoB codes for aUDP-glucose 4-epimerase.

Results

A 2.6kb Cla\-BamH\ DNA region adjacent to the 7.8kb

Eco RI fragment of R. metiloti megaplasmid 2 is essentialfor EPS production and nodule infection.

Several Tn5-induced EPS" Inf" mutants of R melilotiwere complemented by three different cosmids(pRmPM551, pRmPM552, and pRmPM5511) which spana 43kb DNA region of megaplasmid 2 (Muller ef a/., 1988).The complementing region of the three cosmids could bereduced to a 7.8kb EcoRI fragment (Fig. IA). To analysewhether other DNA regions of cosmid pRmPM551 (Fig.IA) are also involved in EPS production and noduleinfection, several cosmid derivatives carrying deletions in

exoB and exoZ genes of Rhizobium meliloti 1521

this region were constructed. Three different cosmidderivatives (pBEl, pBE35. and pBE36) were obtained.with a gentamicin-resistance fragment replacing deletedDNA regions (Fig, IB). The deletions of the cosmidderivatives were established in the R. meiiioti genome,resulting in the three deletion mutants RmHl. RmH35, andRmH36. These deletion mutants were tested for theirability to produce acidic EPS and to form effective noduleson alfalfa. Mutants RmH35 and RmH36 failed to synthe-size acidic EPS {EPS ) and therefore did not fiuoresce onplates containing Calcofluor {Cfw"). When alfalfa wasinoculated with these two mutants, empty ineffectivenodules resulted. Using the phenol-clearing method (Nie-haus and Puhier, 1988) it was shown that these noduleslacked infection threads {Inf^). As a consequence, thesenodules showed no plant cells invaded by bacteria andwere therefore Fix .

In contrast, deletion mutant RmHl was Cfw* andproduced the same amount of EPS as the wild type. Thismutant was still able to induce nitrogen-fixing nodules onalfalfa which were undistinguishable from the nodulesinduced by the wild type. The fact that deletion mutantRmHl is not affected in terms of EPS synthesis andsymbiosis with alfalfa indicates that no genes involved inEPS synthesis or Infection are located to the right of theBamHl site in megaplasmid 2 {Fig. IB). Deletion mutantRmH36 indicates that a further inf/exo DNA region islocatedtotheleft of the SamHI site and outside the 7.8kbEcoRI DNA fragment on megaplasmid 2 {Fig. IB). R.melitoti mutant RmH36 was complemented for EPS pro-duction and nodule infection by a plasmid (pBE62) carry-ing a 2.6kb C/al-SamHI DNA fragment that overlaps the7.6kb EcoRI fragment mentioned above {Fig. IA). Com-parison of the location of exo genes of R. melitoti SU47{Long et ai, 1988) with our restriction map of cosmidpRmPM551 has indicated that the deletion of mutantRmH36 corresponds to the exoS complementation group.

Sequence analysis of the 2.6kb C\al-BamHt DNA regionof megaplasmid 2 revealed two ORFs designated exoZand exoB

In order to obtain detailed genetic information about thegene region adjacent to the R. metitoti 7.8kb EcoRIfragment, the nucleotide sequence of a 2.6kb C/al-SamHI DNA fragment was determined. The location of thesequenced DNA fragment and its restriction map areshown in Fig. 1C. The DNA sequence of this regionspanning 2604 bp is shown in Fig. 2. By using thecomputer programs of Staden (1986) and R. melitotispecific codon usage (see the Experimentai procedures),two possible ORFs with high coding probability wereidentified. TTie location of the identified ORFs and theextension and direction of their coding regions are shown

in Fig. IC. ORFI is located between nucleotide positions228 and 1181 and contains a putative GTG initiation codonpreceded by a potential ribosomal binding site — 5'-GGCAA-3' (Fig. 2). This 0RF1 was called exoZand coversthe region overlapping the 7.8kb EcoRI fragment extend-ing to the right. The predicted translational product ofexoZ is a protein of 317 amino acids with a calculatedmolecular weight of 33764. Upstream of this codingregion we could not identify any typical Escherichia coliconsensus promoter sequence.

By comparing our restriction map with the map alreadypublished by Long etai (1988) for fl. me///o(/strain SU47.we found that the second identified ORF running from rightto left corresponds to the exoB complementation group(see Fig. IA). This second ORF {exoB) extends fromnucleotide 2255 to 1269 and runs in the opposite directionrelative to exoZ. It also contains a GTG start codon and ispreceded by a potential ribosomal binding site — 5'-AGAGG-3' {Fig. 2). The exoB coding region encodes aprotein of 328 amino acids with a molecular weight of36081. According to Fig. 1, the deletion in mutant RmH36comprises the first 150bp of exoB and 300bp of theupstream region. The two identified ORFs are separatedby a short intergenic region of 88 nucleotides.

Exopolysaccharide production and nodule formation ofdefined exoZ and exoB mutants

In order to obtain more information about the function ofthe exoZ gene we constructed a defined interposondeletion mutant. A 1.1kb Cla\-Xho\ DNA fragment con-taining most of exoZwas cloned (pBE70). and the internalEcoRI fragment encompassing 360bp of the exoZcodingregion was replaced by a gentamicin-resistance (Gm'^interposon. The resulting deletion carrying the Gm" inter-poson was introduced into the R. metitoti genome byhomologous recombination, resulting in mutant RmH90{Fig. 1C). The same strategy was used to construct theinterposon deietion mutant, RmH89 (exoB). A 600bpSa/l-EcoR( DNA fragment was replaced by a gentamicincassette and this deletion was introduced into R. meliloti2011 by homoiogous recombination {Fig. IC).

Colonies of the exoZ mutant RmH90 exhibited dimfluorescence on LB-Calcofluor plates when comparedwith those of wild-type strain Rm2011. However, thesecolonies became fully fluorescent after several days.Mutation in exoZ caused a delay in the appearance of theCatcofluor-bright phenotype. Isolation of EPS indicatedthat the exoZmutant RmH90 produced less EPS after fourdays in comparison with wild-type Rm2011. This indicatedthat the exoZ gene product is not essential for acidicexopolysaccharide synthesis but has some influence inthe amount of EPS produced. On the other hand, exoBmutant RmH89 was no longer able to tluoresce on agar

1522 A. M. Buendia et al.

ATCBATATTCTCACCCCCtACCACIlTCSeGTCCTTCCTCTTBTACTTCACGGCAGGCAAACTGACBATCCCGCGCCGCCGGCGGGCASCBACGCTGCTCTGBCCGGCAfiGACTCGATCCGIP ao 30 '0 90 60 70 BO 90 100 110 120

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T I G A A G v a V P F V I S B F I H M V I S O *

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TC»TCGCC8ATC8TGCCCGCC0CATCSTGCCCBTCT*TTGGC7CGCG»CCGGC0TBAteCTCSCGGGGGCACTGGC5SfiTCTGTTCCCC*ATCTGGTGCrGACACTCG*AC»TGTTCTGG370 380 3 9 0 400 4 tO 4Z0 *30 440 490 460 ' 7 0 'SO

C0TCGCTGTTC7TCGTGCCCGCGCGCTC5CCGAGCA0TGGA0AGATTTGGCCTGTGCTCBTSCAGBCCIGGACGCTCAATTTCGABATGCTCITCTACGCGGTCTTCGCCGOCTCGCTGT49Q 300 3 1 0 920 330 940 530 960 370 3flO 9 9 0 6 0 0

H P n N M H L P V V S B L F I . A 1 . . V I A B H V V A F 0 0 A V M 1 . T T T R P V I LTCATGCC5CGCAACTSGC5GCTCCCCGTCGTCTCGGGSCTGTTCCT*GCGTTGGTGATCGCCBGCCGCST6BTCGCCTTCGACGATGCBBTGAtGCTSACCTATAC5CG6CCGGTCArTC

SIO eaO 630 e40 GSO 6 6 O E 7 O BBO 6 9 O 7 0 0 7 1 0 7 2 0

C F V A G H I J G E P M L K G n V P P L A V S S A L r t C S L S G F A L I G V LTGGA*TTCGTCGCCGGCATG*TCATCGGCGAGTTCTGGTTGAAGGGCAGBGTACCGCCACTCGCTGTCOGTTCCGCGCTGTTCGCCTGCTCGCTCGGCGGCTTCGCTCTCATCGGTBTGC

730 740 730 7B0 770 700 790 BOO 810 630 B30 B40

TCGGTCTCCCATTCGACGAACTGACGACCGSGCCGCTBBCGGTQCTGCTTGTCATCGGCGTGCTCTCTCTGGAGGCGAACGGCTGCGTGCGGGCGCTGTCGCTGCCGGGGCTACTCGGCGeSO BBO B 7 0 SeO B90 900 910 920 930 940 930 960

A S Y S I Y L W H T F A I S V V A K A B L A I G L G A P A T M F A A V L S G T LAC0CCTCCTATTCGATTTATCTCTGGCACACTTTTGCBAT7TCGBTCGTGBCBAACGCAGGACTTGCGATCGGTCTCBBCGCBCCCGCAACGATGTTCGCABCGGTTCTTTCGGGAACGC

S70 9 8 0 990 1000 1010 1020 1030 1040 lOSO lOSO 1070 1060

T C A T A G G C A T C G C C G C T T A C A T G A T G C T C G A G C G C G C G C T G C T G C G G C G C G G C C G C G C C C B G C B A G T C A C G G C A G G C C T G G C A G G C C G A G C G G C C B A A T A G A G C B C C T T G A G O A A A A G G C

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TSABCGGTTTGSAAAGAAGACCGGBCAACCCATCGTTCCGGCASCCACGGTCGCTBGTCGCGAAAeTGAGCCGCCCTGATTTCTGCGCGAGTGCCAGTTCCATGCCGATTCBGTAATCGC1210 1220 1230 1240 1290 12S0 1270 1280 1290 1300 1310 1320

A L O Y O P E K O L V O n A K O N N A V L T T S D B E f l R E A Y G I N F P P K ATBCGAGATCBTArTBCGGCTCCCABCCTASAACCTSACGCGCCTTBTCATTBTTGBCGACAABCBTGGTCBABTCBCCTTCBCGCCGCTCBGCGTAGCCGATGTTGAACGGTCBCTTGGC

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V K e i A O L L E K V T T G T G T B L M L A V S E G B C L L Y Q V A B V H A O ACACTTTCTCGATCBCGTCCAGCAACTCCTYCACCGTBGTACCBGTGCCBGTCCCGABATTSAGCGCAACGCTCTCGCCBCCCTCGAGBABATAGTCGACCGCBCBCACATGBGCATCCGC

1490 1460 1 4 7 0 1 4 8 0 1 4 9 0 1 3 0 0 1 9 1 0 1 3 2 0 1 3 3 0 1 3 4 0 1 3 3 0 1 3 6 0

L D L V H I Y O n V C T G D R T Q Y O T ' S F V K F B E R n B L A A O I A L P I ACABATCGAGGACBTGGATATAGTCCCGCACBCACBTTCCGTCGCGCGTGTCGTAGTCTGTCCCGAAGACCnGAAGCCTTCBCBCCGCCCAAGTBCCBCATCGATCGCGAGCGGGATCGC

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I B A A L A A S L L T L T B I V M U D Y F A A B O K V S E B W E I M A A F H U IQATGCCCGCGGCAAGCGCCSCAGACAAGAGGGTCAGCGTGCCBATGACGTTGTTGTCATAGAAGGCGGCCGGBTCCTTBACCGACTCGCCCACCTCBATCATCGCCGCGAAGTBCAGBAR

1930 1940 1990 1960 1970 1960 1990 2000 2010 20Z0 2030 2O40

CGCCCGCBGCTTGTGCCGCGCAA0AACCTCGTCAABACGCTGCCTBTCACGGATATCBCCCTTTTCGAGAACGCCCCACTTBACBAATTCCTCGTGGCCGT7GGAGA6GTTBTCGTAAAC

2 0 9 0 2 0 6 0 2070 2060 2090 2100 2110 2120 2130 2140 Zi90 ZIBO

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2 2 9 0 2300 2310 2320 2330 2340 2390 23B0 Z370 23S0 2390 2400

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ABBACCTCAACTATAAGCCBTCCTTCGGCAATCAGATCAGGGAGGTTACBATGAABBCATTTTCTTCTCCTACGBBGCBGATCC2330 2340 2330 2360 2370 2360 2390 2600

exoZ

exoB

Fig. 2. Nucleotide sequence of a 2.6kb Cla\-BamH\ tragment of megaplasmid 2 containing the R meliloti sxoZand exoB genes. The nucleotidesequence of one strand of the DMA Iragment ts presented in the 5-to-3' direction. The two identified ORFs are indicated by the deduced ammo acidsequence written above th>e nucleotide sequence. The arrows indicate the direction of transcnption. The putative GTG start codons are boxed and thepotential ribosomal binding sites are underlined. These sequence data will appear in the EMBL/GenBank/DDBJ Nucleotide Sequence Data Librariesunder the accession number X58126 [Rhizobium exoZ a.n6 exoB).

exoB and exoZ genes of Rhizobium meliloti 1523

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Fig. 3. Operon analysis by ptasmid integration inlo the R. meliloti exoZ-exoB region.A. The coding regions of exoZand exoB are indicated by arrows, and the assumed putative promoters are marked by soiid circles. For abbreviations ofrestriction sites, refer to the legend of Fig. 1. The DNA fragments which were cloned into mobiliz^le vector plasmids (see the Bxparimerital proceduresand Table 2) and used for integration mutagenesis are shown.B. The relevant genomic structures resultir>g from the integration of each plasmid into the R. meliloti genome by a single crossing over are given. Thevector part is marked by heavy lines and the coding regions are indicated by arrows and boxes. Abbreviations: Z. exoZ, B, exoB. The designations ofthose genes which are inactivated by plasmid integration are given in brackets. The filled arrows indicate'genes that allow undisturbed transcription ofexoZ and exoB from the promoters located in tront of the genes. The Calcofluor-white (Cfw) phenotypB of R. meliloti strains canving the correspondingplasmid integrated in the genome is listed on the right-hand side. d. delayed fluorescent phenotiflie.

plates containing Calcofluor and no EPS production couldbe detected by precipitation using cetylpyridinlumchloride (CPC). Leigh and Lee (1988) previously reportedthat exoB mutants had multiple carbohydrate defects andwere also affected in terms of lipopolysaccharide (LPS)synthesis. Sodium dodecyl sulphate gel electrophoresisof LPS revealed that exoB mutants RmH89 and RmH36had a different pattern from that observed in wild-typeRm2011. The banding pattem of the exoZmutant RmH90was similar to that of Rm2011 (data not shown).

The exoB mutants RmH89 and RmH36 induced emptyineffective nodules on alfalfa. In contrast, exoZ mutantRmH90 formed normal, infected. N2-fixing nodules onalfalfa, indicating that exoZdid not affect the bacterium'ssymbiotic phenotype on alfalfa.

Operon analysis by plasmid intergation mutagenesisrevealed that exoZ and exoB are organized into twomonocistronic transcription units

Sequence analysis indicated the presence of two ORFsrunning in opposite directions. To determine if exoZ andexoB carry a promoter in front of their coding region wecarried out plasmid integration mutagenesis. To perfoim

this mutagenesis procedure various subfragments of the2.6kb Cla\-BamH\ DNA region were cloned into mobiliza-ble piasmid vectors which cannot replicate in Rhizobium(Fig. 3). Selection for the vector-encoded antibiotic resist-ance results in integration of the plasmid into the R. metitotigenome by single crossing over. Depending on the clonedfragment, this plasmid integration caused the inactivationof certain genes and the disruption of the operon struc-ture. Transconjugants carrying the integrated plasmidswere tested for their Calcofluor phenotype. R. melitoti2011 with plasmid pAB62 integrated in exoZ exhibited adelay in the production of fluorescence on agar platescontaining Calcofluor because no intact exoZ gene waspresent (Fig. 3. strain RmAB62). Plasmids pAB60 andpAB61 carrying the 400bp C/al-£coRI fragment with theputative exoZ promoter in both orientations were inte-grated in the Rm2011 genome. The resulting mutants,RmAB60 and RmAB61, showed In both cases a Cfw* Inf"phenotype. Since it is unlikely that in both strains avector-specific promoter is responsible for the transcrip-tion of exoZ, we concluded that a promoter is locatedwithin the cloned fragment (Fig. 3).

Similar results were obtained for exoS. Integration ofplasmid pAB64 resulted in an EPS" phenotype (RmAB64)

1524 A. M. Buendia et al.

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Fig . 4 . Al ignment of the ammo acid sequence deduced from e x o Z w i t h

that of R. Ieguminosarum bv, viciae strain T O M nodX gene product . The

amino ac id sequences derived f rom the R. meliloti exoZ gene and from

the R. Ieguminosarum bv. viciae strain TOM nodX gene (Davis et ai.

1988) were compared with the FASTA computer program (Lipman and

Pearson, 1965). Identical residues are marked by vertical iines. Similar

amino acKls are indicated by dots.

The hydropathic plot (Kyte and Doolittle. 1982) of theprotein encoded by exoZindicated that this protein is alsohighly hydrophobic, with several putative transmembraneregions (data not shown).

The ExoB protein of R. meliloti is structurally andfunctionatty retated to the UDP-glucose 4-epimerase(GaiE) of E. coi\

A comparison of the amino acid sequence derived fromthe exoB nucleotide sequence with the NBRF Protein DataBank revealed signficant homology (39.6%) to the E ooliGalE protein (Fig. 5). gaiE encodes a UDP-glucose 4-epimerase interconverting UDP-glucose and UDP-galactose.

In order to determine if the exoS gene actually codes fora UDP-glucose 4-epimerase, the enzymatic assay for thisenzyme was carried out as described by Fukasawa etal.(1980). Figure 6 shows that the UDP-glucose 4-epimer-ase activity in R. metitoti20^ 1 varied very little when grownin minimal medium containing glucose or galactose assole carbon source, indicating constitutive expression ofexoB. The exoB mutants RmH36 and RmHU9 showed nodetectable enzymatic activity relative to R. metitoti 2011(Fig. 6). indicating that the exoB gene prciduct is funcrtionally equivalent to the UDP-glucose 4-epimerase.

RmexoS

because no intact copy of exoB was present. In contrast,integration of plasmids pAB67 and pAB68 had no influ-ence on EPS synthesis (RmAB67 and RmAB68; Fig. 3),showing that a promoter is located within the EcoRI-SamHI DNA fragment upstream of exoB. These resultsdemonstrate that both exoZ and exoB are organized asmonocistronic transcription units.

The ExoZ protein of R. meliloti 2011 shows homology tothe Rhizobium Ieguminosarum b/ovar viciae strain TOtvinodX gene product

The R. metiloti predicted ExoZ protein consists of 317amino acids with a calculated moiecuiar weight of 33764.The deduced amino acid sequence of ExoZ was com-pared with sequences held in the NBRF Protein SequenceDatabase. We found homology between ExoZ and theRhizobium Ieguminosarum biovar viciae NodX gene pro-duct. Beth aminc acid sequences showed an overallhomology of 23.3% (Fig. 4). In R. Ieguminosarum bv. viciaestrain TOM, nodX encodes a hydrcphcbic protein whichmay be associated with the bacterial membrane, but thespecific function of NodX is not known (Davis et ai., 1988).

10 20 ]a (0 soVQNNNILVVGGACYICSKTCLOLAAICGYOPVVYDNLSNGKEEFV KWGVLEKG

- - I -EcgalE HRVLVIGGSGYlGSHTCVaLLQNGHOVIILDHLCHSKRSVU'VIERLGGKHPTFVEG

10 2Q 30 40 SD

GO 70 80 90 100 n oRmeKoB OIRDRQRLOEVLARHKPRAILHFAAHIEVGESVKDPAAFYDHHV GTLTLL5AALAAGI0

I I I - ' " M I - - . M l - . - I M I 1 - - I - . M i l l I I I M I 1 1 - . .EcgalE OlBNEALHTElLHOHAIOTVIHFAGLKAVSESVQKPLEYYDNNVItGTLRLISAHRAANVK

60 ; o eo 90 loo no

IZO 130 140 150 160 170RmexoB AFVFSSTCAnGLPOSVp.HDESHltqAPlMPYGRIKWICEQALKE'YGLYKGLRSV-lLRY, , - I - I M . . . I I . -.1 1 . 1 1 1 . - 1 . II 1-1 . .|. . I l lEcg»U KFIFSSSATVYGOOPKiPYVESFPTGTPOSPYGKSKLMVEOlLTUOKACPOWSIALLRY

120 130 140 ISO 160 170

ISO 190 200 Z]0 220RwexoB FNAAGADFEGRIGE-WHEPETHAIPLAIDAALGRREGFKVFGTDVDTRDGTCVRDYIHVL

EcgalE FNPVGAHPSGDMGEDPQGlPWLKPYlAOVSVGRRDSLAiFGNOtPTEDGTGVROYIHVH180 190 200 210 220 230

240 250 260 270 280RmexoB DLADAHVRAVDYLLEGGESVALNLGTGTGTTVKELLDAIEKVAKSPFNICYAERREGDST

EcgalE DLADGHVVAMEKLANKPGVHIYNLGAGVGNSVLDVVliAFSKACGiiPVNYHFAPRREGDLP240 Z50 260 270 2S0 290

300 310 320RmexoS THfAHNOKAR0VLGW£PI)YDLAA!TESAWKWHSHRN0GG

EcgalE AYWADASKADaELNWRVTRTLOEMAODTWHWQSfiHPOGYPD300 310 320 330

Fig. 5. Alignment of the amirro acid sequence deduced from exoB withthat of the E. coli UDP-glucose 4-epimerase igalE). The emino acidsequences derived from the R. metiloti exoB gene and frtim the E. coliga'C gene (Lemaire and Muller-Hill. 1986) ware comparec with theFASTA computer program (Lipman and Pearson, 1985). I denticalresidues are marked by vertical lines. Similar residuas are marked bydots.

exoB and exoZ genes o^ Rhizobium meliloti 1525

Rm 2011 RmH89 Rm H36

Rhizobium strains

Fig. 6. UDP-glucose 4-epimerase activity of R. meliloti wild-type strain2011 and the exoS deletion mutants RmH89 and RmH36. The UDP-glucose 4-epimerase activity was determined in R. meliloti wild-typestrain Rm2011 and the two exoB deletion strains RmH89 and RmH36.after growth on medium containing galactose or glucose as the carbon

Discussion

In this paper we report on the structural and functionalanalysis of a 2.6kb DNA fragment of the R melitotimegaplasmid 2 adjacent to a 7.8kb EcoRI DNA fragment,both of which are involved EPS biosynthesis and in noduleinfection (Muller etat., 1988; Keller ef a/.. 1988). Two ORFsrunning in opposite directions were identified on the 2.6 kbC/al-SamHi fragment. ORF1, running from left to right,was designated exoZ. An exoZ deletion mutant exhibiteda delay in the appearance of fluorescence on Calcofluorplates and a decrease in EPS production, but was notimpaired in terms of formation of effective nodules onalfalfa. Therefore we concluded that exoZ is involved in,but not essential for, EPS biosynthesis. However, theamino acid sequence of the exoZ gene product revealedhomology to the gene product of the nodulation genenodXoi R. teguminosarum bv. viciae strain TOM, which isnecessary for nodulation of Afghanistan pea, a cultivar notnormally nodulated by conventional R. ieguminosarum bv.wc/aestrains (Davis etai. 1988). Since R. melilotia\so hasthe potential to nodulate many species of Trigoneila,Melilotus, and Medicago genera, one may speculate thatexoZ coutd also be involved in the host-specific nodu-lation process,

The second identified ORF. which runs from right to left,was designated exoB since it corresponds to the exoBcomplementation group reported by Long e( ai (1988).

The direction of transcription and the phenotype of thedeletion mutants constructed support this idea. Thepredicted translational start codon of exoB is GTG, whichis in accordance with findings for other rhizobial genes(Scott, 1986; Borthakur and Johnston, 1987; Borthakur efai, 1988). Results of the plasmid integration mutagenesisdemonstrated that there is a promoter within a 300 bpregion upstream of the exoB coding region. Conse-quently, the exoS complementation group forms a mono-cistronic transcription unit. The deduced amino acidsequence of exoB shows a significant degree of homology(39.6%) to the galEgene product of E. coti, suggesting thatexoB codes for a UDP-glucose 4-epimerase. Thisenzyme interconverts UDP-glucose and UDP-galactose.The inability to synthesize UDP-galactose because of alack of UDP-glucose 4-epimerase activity fully accountsfor the multiple surface carbohydrate defects of exoBmutants. In R. melitoti, galactose has been found to bepresent in the repeating unit of the exopotysaccharidesEPS I (Aman et at., 1981) and EPS II (Glazebrook andWalker, 1989; Zhan et ai, 1989), and in the saccharideportion of LPS (K. Niehaus, unpublished results). The latterobservation can explain the altered phage resistancepattern of exoB mutants (Leigh and Lee, 1988). The factthat UDP-glucose 4-epimerase activity could not bedetected in exoB deletion mutants demonstrated that theexoB gene codes for this enzyme. A gene functionallyhomologous to the exoB gene of R. metiioti has beenidentified recently in R. ieguminosarum bv. viciae strainRBL5523 (Canter Ceemers ef ai, 1990). These authorshave determined that the EPS produced by R. iegumino-sarum bv. viciae exoB mutants lacks the terminal galac-tose residues. They have also shown that theUDP-glucose 4-epimerase activity was absent in exoBmutants of R. teguminosarum bv. viciae and R. metitoti

In E coli, galactose as unique carbon source is lethal forgalE mutants (Adhya, 1987), while in R. metitoti, exoBmutants are able to grow on galactose as sole carbonsource. This is in accordance with a report from Arias andCervenansky (1986), who showed that the De Ley-Doudo-roff pathway serves an essential role in the metabolism ofgalactose in R. metitoti L5-30. In E coti, Salmonelta, andKlebsielta, the ga/Egene belongs to the galactose operon,which is inducible by galactose (Adhya and Shapiro,1969). In contrast, we have shown for R. metitoti 2011 thatthe exoB gene is organized as a single transcription unitthat is constitutively expressed. This has also beenreported for Erw/n/a Stewart//(Dolph etai. 1988), in whichgalE is not linked to galTK and the epimerase activity isconstitutive. These differences might reflect the import-ance of the enzyme UDP-glucose 4-epimerase in the EPSand LPS biosynthetic pathway. This is also obvious whenwe consider that DNA cloned from Azospiritum brasitense(Michiels et ai, 1988) or from Agrobacterium tumefaaens

1526 A. fi/1. Buendia et al

Table 1. Strains used or constructed in thra work.Strain

E. coli

JM83

MM29'1

S17.1

R. metiloti

Rm2011RmHl

RmH35

RmH36

RmH89

RmH90

RmAB60

RmAB61

RmAB62

RmAB64

RmAB67

RmAB68

Relevant characteristics Source/reference

F'. ara. thi-1, (lao-pro). strA: host for pUC andpSVB plasmids

F , supE-44, endA-1, thi-1. pro. recA. hsdSi7\used for transformation

E. CO//MM294, RP4-2-Tc::MLi-Km::Tn7chromosomalty integrated

Wild type, Nod , Fix •. Cfw •. Sm"Mutant of Rm2011 with a deletion of 29.5 kb in

megaplasmid 2 (see Fig. 1B). Cfw", EPS . Fix'Gm"

Mutant of Rm2011 with a deletion of 24 kb inmegaplasmid 2 (see Fig. IB). Cfw .EPS , FixGm"

Mutant of Rm2011 with a delBtion of 0.4 kb inmegaplasmid 2 (see Fig. IB), Cfw .EPS .FixGm"

Mutant of Rm2011 with a deletion in exoB (seeFig. IC), Cfw ,EPS , Fix . Gm"

Mutant of Rm2011 with a deletion in exaZ(seeFig. IC),Cfw-' .EPS-. Fix'. Gm"

Rm2011 with plasmid pAB60 integrated into thegenome

Rm2011 with plasmid pAB61 integrated into thegenome

Rm2011 with plasmid pAB62 integrated into thegenome

Rm2011 with plasmid p A ^ 4 integrated into thegenome

Rm2011 with plasmid pAB67 integrated into thegenome

Rm2011 with plasmid pAB68 integrated into thegenome

Vieira and Messing(1982)

F. Schoffei (Bielefeld)

Simon era/. (1963)

Cass6 etai (1979)This work

This work

This work

This work

This work

This work

This work

This work

This work

This work

This work

Sm". streptomycin-resistant: Gm". gentamicin-resistant.

(Cangelosi et ai, 1987) can suppress the EPS" Inf"

phenotype of exoB mutants of R. melitoti, indicating that

this locus has been well conserved during evolution.

Experimental procedures

Bacteria and plasmids

Strains and plasmids used in this work are listed in Tables 1 and 2.

Media and growth conditions

E. CO//strains were grown in Penassay broth (Difco Laboratories)or LB medium (Maniatis ef al.. 1982) at ST^C. R melitoti strainswere grown on TY (Beringer. 1974), Vincent medium (Vincent,1970) or LB (Maniatis etai, 1982) at 30"C. For some experiments,Rhizobium minimal medium (RMM) and M98 medium were used(Keller ef a/., 1988).

Nodulation and nitrogen-fixing assays forR. melilotistrains

Alfalfa seeds (Medicago sativa cv. "Du Puits") were surface-ster-

ilized and germinated as described by Muller ef al. (1988). Theseedlings were inoculated with late log-phase bacteria. Theplantlets were maintained on nitrogen-free medium as describedby Rolfe ef al. (1980) and inspected for nodulation after 3-4weeks. The occurrence of infection threads was checked usingthe phenol-clearing method of Niehaus and Puhier (1988).Nftrogenase activity was measured using the acetylene-reductionassay, as previously reported by Aguilar ef at. (1985).

Staining procedures ofR. meliloti colonies

Acidic EPS production by R. metitoti strains was tested by platingsingle colonies on M98 agar or LB containing Calcofluor white, aspreviously described by Hynes ef al. (1986).

Ouantitative anaiysis of EPS production

Quantitative analysis of EPS production was as described byMullerefa/. (1988). R. me///o(/strains were grown for 10d in 10mlof Vincent minimal medium. EPS was isolated by precipitationfrom the culture supernatant with cetylpyridinium chloride(Sigma) and ethanol. The amount of acidic EPS was determinedby using the sulphuric acid-anthrone method (Nowotny. 1969).

exoB and exoZ genes of Rhizobium meliloti 1527

Table 2. Plasmids used and constructedin this work. Plasmid Relevant characteristics Source/Reference

PSUP102PSUP202PSUP205PWKR56IpWKR136pWKR189ll

pJO47pSVB23

pSVB28

pSVB30

PSVB31

pRmPM551

pBElpBE35pBE36

pBE52

pBE62

pBE70pAB60

pAB61

pAB62

pAB64

pAB67

pAB68

pACYCI84 derivative. Mob. Tc", Cm"pBR325 derivative. Mob. Ap", Tc", Cm"pBR325 based cosmid. Mob. Cm", Tc"PACYC177 derivative. Mob. Km", Tc"PACYC177 derivative. Mob, Ap". Km", Gm"pUC8 derivative carrying a SamHI Gm'^/Spec"

fragmentpKi8 denvative. ^4ob. Km"Ap"

Ap". Lac*

Ap". Lac'

Ap«

Cosmid based on pSUP205, earring a 43kbcontiguous region ot EcoRi fragments of B.meliloti 2011 megaplasmid 2

Deletion derivative of pRmPM551. Gm"Deletion derivative of pRmPM551. Gm"S.2kb Cla\ tragment sublconed in pWRK56l wrth

a Gm" cassette replacing a 400bp EcoRt-SamHI fragment

3,7kb Cla\-Pst\ fragment containing the 2.6kbC/af-Sam HI-DNA region cloned in pSVB23

2.6kb Cla\-BamH\ fragment subk:oned inPWKR56I

1.1 kb Cla\-Xho\ fragment cloned in pWKR56lEcoRI fragment from pBE52 carrying the 350bp

Ctel-EcoRI fragment cloned in pSUP202Same plasmid as pAB60 with the EcoRI fragment

in opposite orientation360bp EcoRI fragment from pBE52 cloned in• pJQ47

600bp Xho\-EcoB\ fragment from pBE52 ckinedinpSUP102

470bp EcoH\-Bamh\ fragment from pBE52cloned In pS\/B31 and recloned as a Hin6\\\-Sal\ fragment in pWKR56l

470bp EcoRI-eamHI fragment from pBE52cloned in pSVB31 and recloned as a Hindlll-SamHI fragment in pWKR56l

Simon etai (1983)Simon e( a/, (1983)Simon ef a/, (1983)Klipp era/. (1988)W. Klipp (Bielefeld)W. Klipp (Bieiefeld)

J. Quandt (Bielefeld)Arnold and Puhier

(1988)Arnold and Puhtef

(1988)Arnold and POhler

(1988)Amold and Puhier

(1988)Muller crt a/. (1988)

This workThis workThis work

This work

This work

This workThis work

This work

This work

This work

This work

This work

Tc", tefracydine-resistant; Cm", chlorarnphenicol-resistant; Ap", ampicillin-resistant; Km", kanamy-cin-resistarrt.

DAM biochemistry

Total DNA of R. meliloti was isolated according to the method

published by Meade et al. (1982). Plasmid DNA was isolated as

described by Priefer (1984). Restriction enzyme analysis, agarose

electrophoresis. and cloning procedures were performed using

established protocols (Maniatis ef at.. 1982). All enzymatic

reactions were carried out as recommended by the manufac-

turers. Transformation of E. coli cells with plasmid DNA was

performed as described by Morrison (1977). Visualization of

plasmid DNA was carried out using a modified Eckhardt tech-

nique, as described by Hynes et al. (1985),

DNA sequencing

Subclcnes for DNA sequencing were obtained by cloning of

defined restriction fragments tnto pSVB sequencing vectors

(Amold and Puhier, 1988). TTie sequence data were obtained forboth strands by thechain termination method (Sanger eta/.. 1977)using double-stranded DNA (Amold ef a/,. 1988). For some of thesubclones, the sequence of the complementary strand wasobtained by the chemical degradation method described byMaxam and Gilbert (1980) (with some modifications; Arnold etal.,1986).

Sequence analysis

The DNA sequence was analysed using the sequence analysis

programs of Staden (1986). The coding probability was calcu-

lated by means of the codon-usage method (Staden and

McLachlan, 1982), For calculation of the codon-usage table,

several sequenced R. meliloti genes were used: dctA (Engetke et

al., 1989). dcrSand dcfD(Jiang etai, 1989), flaAmd flaS(Pleier

1528 A. M. Buendia et ai.

arrd Schmitt. 1989). nodC(Jacobs etal., 1985), nodPand nodQ(Schwedock and Long, 1969). and trpEG (Bae ef al., 1989). Theamino acid sequences deduced from the nucleotide sequenceswere compared with the actual version in the NBRF Protein DataBank using the FASTA programs (Lipman and Pearson, 1985).

Transfer of plasmids between E. coli and R. meliloti

E. coli strain S17-1 (Simon et al., 1983) was used as a broadhost-range mobilizing strain to transfer hybrid plasmids to R.meliloti. The transfer was carried cut by means of filter matings(Simon, 1984).

Construction of R. meliloti deletion mutants usingreverse mutagenesis

Subcloned DNA fragments were obtained which carried differentdeletions and a gentamicin resistance gene from plasmidpWKR189ll replacing the deleted megaplasmid 2 DNA region.The resulting plasmids were mobilized from B. coli SI 7-1 to R.meWotf 2011. Since the constructed plasmids cannot replicate inR meliloti, selection on gentamicin and subsequent testing forloss of the vector-encoded resistance yielded transconjugants inwhich integration of the plasmid had occurred by homologousrecombination. The correct recombination of the deletions wasverified by Southem hybridization of total DNA isolated fromthese R metitoti mutants.

Plasmid Integration mutagenesis

This mutagenesis procedure was carried out as described byKlipp ef al. (1989). Various DNA fragments were cioned intomobiiizable vector plasmids that cannot replicate in Rhizobium.Integration of these plasmids into the R meliloti genome by singlecrossover was selected by vector-encoded antibiotic resistance.Transconjugants were tested for EPS phenotype on agar platescontaining Calcofluor.

Enzyme assay for UDP-glucose 4-epimerase

Bacteria were grown on rhi2obial minimal medium containinggtucose or galactose as sole carbon source. Cells were disruptedby sonication. Debris was removed by centrifugation. After gelfiltration (PD10 column, Pharmacia) the protein extracts wereused to measure the UDP-glucose 4-epimerase activity accord-ing to Fukasawa et al. (1980), The reaction mixture, in a totalvolume of 1ml, contained lOOmM Glycine/NaOH pH 8.9 and100 1 of cell-free extract. The reaction was started by the additionof 20^1 of 50mM UDP-galactose (Boehringer). Aliquots of 200[j-iwere taken after 0, 15, 30, and 45 min and the reaction wasstopped by immersing the tubes in boiling water for 90s. Theamount of UDP-glucose produced was determined by directNAD-coupled oxidation catalysed by UDP-glucose dehydroge-nase (Boehringer). The reaction mixture, in a total volume of 1 ml,contained lOOmM Glycine/NaOH pH 8.9,2 mM NAD and 100 \i.\ ofsample. The reaction was started by the addition of 0.1 UUDP-glucose dehydrogenase. The irreversible oxidation of UDP-glucose yields a proportional amount of NADH which is measuredby the Increase in optical density at 340nm. One unit of

UDP-glucose 4-epimerase causes the conversion of 1 jimol ofUDP-galactose to UDP-glucose and thus the formation of 2 jimolof NADH per min. Specific activity of UDP-glucose 4-epimeraseis given as mU mg protein '. Protein concentration of crudeextracts was determined using a Micro BCA Protein AssayReagent (Pierce),

Acknowledgements

We are very grateful to Dr W. Klipp and J. Ouandt for helpfulsuggestions and for kindly providing some of the vectors andptasmids used in this work. We thank A, Kleickmann for excellenttechnical assistance. We would also like to thank Dr J. Kalinowskifor help with the sequence analysis, and M. Keller for helpfuldiscussions. A. Buendia was supported by a training contract inthe Research Action Programme — Biotechnology by the Com-mission of the European Communities. This work was financiallysupported by Deutsche Forschungsgemeinschaft Pu 28/13-2.

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