identiﬁcation of a plasmid-borne locus in rhizobium etli...

JOURNAL OF BACTERIOLOGY,0021-9193/99/$04.0010

Sept. 1999, p. 5606–5614 Vol. 181, No. 18

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Identification of a Plasmid-Borne Locus in Rhizobium etliKIM5s Involved in Lipopolysaccharide O-Chain

Biosynthesis and Nodulation ofPhaseolus vulgaris

PABLO VINUESA,1* BRADLEY L. REUHS,2 CHRISTELLE BRETON,3 AND DIETRICH WERNER1

FB Biologie, Fachgebiet fur Zellbiologie und Angewandte Botanik, Philipps-Universitat Marburg, D-35032 Marburg,Germany1; Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia2; and Centre de

Recherches sur les Macromolecules Vegetales, 38041 Grenoble cedex 9, France3

Received 19 February 1999/Accepted 21 June 1999

Screening of derivatives of Rhizobium etli KIM5s randomly mutagenized with mTn5SSgusA30 resulted in theidentification of strain KIM-G1. Its rough colony appearance, flocculation in liquid culture, and Ndv2 Fix2

phenotype were indicative of a lipopolysaccharide (LPS) defect. Electrophoretic analysis of cell-associatedpolysaccharides showed that KIM-G1 produces only rough LPS. Composition analysis of purified LPS oligo-saccharides from KIM-G1 indicated that it produces an intact LPS core trisaccharide (a-D-GalA-134[a-D-GalA-135]-Kdo) and tetrasaccharide (a-D-Gal-136[a-D-GalA-134]-a-D-Man-135Kdo), strongly suggestingthat the transposon insertion disrupted a locus involved in O-antigen biosynthesis. Five monosaccharides (Glc,Man, GalA, 3-O-Me-6-deoxytalose, and Kdo) were identified as the components of the repeating O unit of thesmooth parent strain, KIM5s. Strain KIM-G1 was complemented with a 7.2-kb DNA fragment from KIM5sthat, when provided in trans on a broad-host-range vector, restored the smooth LPS and the full capacity ofnodulation and fixation on its host Phaseolus vulgaris. The mTn5 insertion in KIM-G1 was located at the Nterminus of a putative a-glycosyltransferase, which most likely had a polar effect on a putative b-glycosyl-transferase located downstream. A third open reading frame with strong homology to sugar epimerases anddehydratases was located upstream of the insertion site. The two glycosyltransferases are strain specific, assuggested by Southern hybridization analysis, and are involved in the synthesis of the variable portion of theLPS, i.e., the O antigen. This newly identified LPS locus was mapped to a 680-kb plasmid and is linked to thelpsb2 gene recently reported for R. etli CFN42.

Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium,and Sinorhizobium strains, collectively known as rhizobia (59),are capable of inducing the formation of specialized symbioticorgans called nodules on the roots or stems of particular legu-minous host plants. Specific diffusible plant and bacterial me-tabolites (i.e., flavonoids and lipo-chitooligosaccharides, re-spectively) trigger the first steps of the nodulation process (20,21). The rhizobia colonize the central nodular tissues by meansof a complex infection process, leading to the formation of ahighly efficient N2-fixing association (7, 60). Surface polysac-charides, including lipopolysaccharides (LPSs), are involved inthe normal infection process of all rhizobial associations stud-ied so far (28, 35). Rhizobia nodulating hosts that form deter-minate nodules, such as beans (Phaseolus vulgaris) and soy-beans (Glycine max), require an intact LPS structure forcomplete nodule development and N2 fixation (29, 38). Alltransposon insertion mutants of Rhizobium etli, Bradyrhizo-bium japonicum, and B. elkanii isolated so far that are affectedin LPS synthesis fail to form normal infection threads. Theirgrowth is blocked either in the root hairs or in the underlyingcortical cells. These mutants have never been observed to bereleased into symbiosomes (37, 55), eliciting incompletely de-veloped nodules that do not fix N2 (Ndv2 Fix2 phenotypes).

LPS is a major and distinctive molecular component of theouter membranes of gram-negative bacteria, and lipid A, thehydrophobic moiety of LPS, forms much of the outer leaflet ofthe outer membrane. It is composed of an acylated disaccha-ride that is differently substituted in rhizobia and enteric bac-teria. Attached to this disaccharide is a nonrepeating core oligo-saccharide (core OS), the lipid A-proximal portion of which isconserved in structure among related bacterial species (52). TheLPS core region is also highly conserved between R. etli and R.leguminosarum strains, and both contain two core OS compo-nents that are released by mild acid hydrolysis. These aregenerally referred to as the core trisaccharide (a-D-GalA-134[a-D-GalA-135]-Kdo) and core tetrasaccharide (and a-D-Gal-136[a-D-GalA-134]-a-D-Man-135Kdo) (4, 23), and theyare easily separated by high-performance anion-exchangechromatography (HPAEC) (14). The lipid A moiety linked tothe core OS constitutes the rough LPS (R-LPS), because mu-tant strains producing only R-LPS display a characteristicallyrough (less glossy) colony appearance. The R-LPS is oftencapped by a hydrophilic O-antigen polysaccharide (O-PS),forming a mature LPS molecule termed smooth LPS (S-LPS).The O-PS is usually the most variable component of the LPS,being strain specific in Rhizobium and Bradyrhizobium strains,although not in Sinorhizobium spp. (22, 40). The variabilityresults primarily from differences in the sugar composition ofthe repeating units, as is the case for the members of the familyEnterobacteriaceae (65). Other variable factors include the re-peat unit size, the linkages between the monosaccharide com-ponents, and the presence of noncarbohydrate substituents.

* Corresponding author. Mailing address: FB Biologie, Fachgebietfur Zellbiologie und Angewandte Botanik, Philipps-Universitat Mar-burg, Karl von Frisch Str., D-35032 Marburg, Germany. Phone (49)6421 283476. Fax: (49) 6421 288997. E-mail: [email protected].

5606

The genetics of LPS biosynthesis in rhizobia has been onlypartially investigated, in contrast to the abundant informationon pathogenic Enterobacteriaceae. In these and many otherbacteria, the genes involved in O-chain and core biosynthesisare clustered on the chromosome, forming the wb* (formerlyrfb) and wa* (formerly rfa) regions, respectively (40, 42). Thisis not the case for R. etli and R. leguminosarum strains; at leastthree major genetic regions encoding enzymes involved in LPSbiosynthesis have been identified in R. etli CFN42T (5CE3)(38). Two of these lps regions, termed a and g regions, arelocated at distinct loci on the chromosome (15, 16, 38), and athird region, called b-lps, is plasmid borne (8, 16, 38). The largea-lps region is involved both in O-PS and core OS biosynthesis(12, 15), whereas the b-lps region encodes enzymes thought tobe involved in core OS synthesis (15, 24). Poole et al. (39) andAllaway et al. (1) reported on the presence of a distinct locusin Rhizobium leguminosarum bv. viciae involved in LPS coreOS synthesis that is not complemented by cosmid DNA fromany of the a, b, g regions of R. etli CFN42; it was, however,complemented with cosmid gene libraries derived from R. le-guminosarum bv. viciae and R. leguminosarum bv. phaseoli.This R. leguminosarum-specific LPS locus was named d-lps.Currently no sequence or enzymatic data are available forRhizobium spp. functions involved in O-PS synthesis. Here wereport on a novel plasmid-borne and strain-specific LPS locusof R. etli KIM5s involved in the synthesis of the variable O-PSof the LPS.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions. The strains used in thiswork are listed in Table 1. Rhizobial strains were grown in PY (36) at 28°C or onthe minimal medium (MM) described by Kingsley and Bohlool (30). Escherichiacoli strains were grown in antibiotic medium no. 3 (PA; Oxoid) at 37°C. Antibi-otics were added at the following concentrations: ampicillin (Ap), 75 mg ml21;kanamycin (Km), 50 and 75 mg ml21 for E. coli and Rhizobium strains, respec-tively; spectinomycin (Sp) and streptomycin (Sm), 50 and 75 mg ml21, respec-tively, for rhizobial strains and 50 mg ml21 for E. coli strains. Plasmids used in thiswork are listed in Table 2.

Transposon mutagenesis and conjugal transfer of plasmids. Derivatives of R.etli KIM5s carrying mTn5SSgusA30 insertions were obtained after biparental

matings using E. coli S17-1 l-pir (19) transformed with pCAM130 (66) as thedonor strain. Matings were carried out on nitrocellulose filters for 16 h on PYplates at 30°C. Selection of mTn5 insertion mutants was achieved by platingappropriate dilutions on MM containing Sp and Sm. The conjugal transfer ofpRePV7.2CMCS-21 into strain KIM-G1 was performed as described above,using E. coli S17-1 (53) as the donor strain, selecting the transconjugants on MMcontaining Km and Sm.

Cloning of the mTn5 insertion of strain KIM-G1. Genomic DNA of KIM-G1was extracted from broth cultures in late exponential phase by using a standardcetyltrimethylammonium bromide protocol (3). Purified DNA was digested withClaI and XhoI and then vacuum transferred onto nylon membranes by themethod of Southern (54). The Southern blots were probed with a gusA-specific,digoxigenin (DIG)-labelled PCR probe derived from pPVgusAESK, using theT3/T7 primers and a DIG-PCR labelling kit (Boehringer GmbH, Mannheim,Germany) as recommended by the manufacturer. Hybridization and posthybrid-ization washings were performed under stringent conditions (68°C and 0.13 SSC[13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate]) and signal detection wasperformed by using anti-DIG Fab fragments coupled to alkaline phosphatasewith colorimetric detection as instructed by the manufacturer (Boehringer). Toclone the hybridizing fragments, about 20 mg of genomic DNA from strainKIM-G1 was restricted with ClaI and XhoI and fractionated on a low-melting-point agarose gel (Biozym, Hess. Oldendorf, Germany), purifying the DNA fromappropriate gel fractions by enzymatic digestion of the agarose with an agarase(GELase; Epicentre Technologies). The purified DNA was ligated to pBluescriptSK1 with T4 ligase (USB-Amersham) and used to transform CaCl2-competentE. coli DH5a cells (49), selecting transformants resistant to Ap, Sp, and Sm.

Generation and screening of a partial genomic library of R. etli KIM5s size-fractionated DNA fragments. Plasmids pRePV1 and pRePV2 (see Fig. 3B) werechosen to generate DIG-labelled PCR probes (KIM-P#1 and KIM-P#2, respec-tively), using the T3/T7 primers, as described above. Probe KIM-P#1 was usedto screen Southern blots of genomic DNA from wild-type KIM5s restricted withClaI. To clone the hybridizing fragment, about 20 mg of genomic DNA of KIM5swas restricted with ClaI and fractionated on a low-melting-point agarose gel, andthe DNA of the size fraction corresponding to the hybridization signal waspurified as described above. This pool of DNA fragments was ligated intodephosphorylated pBluescript SK1 and used to transform E. coli DH5a. Alltransformants were streaked out in an ordered manner for colony lifting, usingnylon membranes as recommended by the manufacturer (Boehringer). Themembranes were screened with the internal 850-bp HindIII fragment from KIM-P#1, which had been gel purified. This purification step was required to removethe flanking vector sequences. Stringent hybridization and detection were per-formed as described above.

Filter blot hybridization of rhizobial plasmid profiles. Plasmid profiles ofselected rhizobial strains were visualized by the method of Hynes and MacGre-gor (26), using 0.6% horizontal agarose gels. Plasmids were blotted onto nylonmembranes by capillary transfer (49). Hybridization with probe KIM-P#1 wasperformed as described above, using a chemiluminescence detection system withCDP-Star (Boehringer).

TABLE 1. Bacterial strains used in this study

Strain Genotype or host/origin Source or referencea

Escherichia coliDH5a recA1 DlacU169 f80dlacZDM15 StratageneS17-1 thi pro hsdR hsdM1 recA, RP4 integrated in the chromosome,

2-Tc::Mu-Km::Tn7(Tpr Smr)53

S17-1 l-pir l-pir lysogen of S17-1 19GM2163 F2 ara-14 leuB6 thi-1 fhuA31 lacY1 tsx-78 galK2 galT22 supE44

hisG4 rpsL136 (Strr) xyl-5 mtl-1 dam13::Tn9(Camr) dcm-6 mcrB1hsdR2(rK

2mK1) mcrA

New England Biolabs

Rhizobium etli KIM5s derivativesKIM-G1 mTn5SSgusA30 insertion mutant, LPS2 Ndv2 Fix2 Smr Spr This studyKIM-G1/pRePV7.2CMCS-21 KIM-G1 complemented with pRePV7.2CMCS-21, Kmr Smr Spr This study

Wild-type rhizobiaa

R. etli KIM5s (Spr) Phaseolus vulgaris/Mexico J. Handelsman, University ofWisconsin, Madison

R. etli CFN42T P. vulgaris/Mexico E. Martınez, CFNR. etli CIAT7014 P. vulgaris/Colombia CIATR. tropici CIAT899T P. vulgaris/Colombia CIATSinorhizobium meliloti GRT3 Medicago sativa/Spain J. Sanjuan, CSIC

a CIAT, Centro International de Agricultura Tropical, Cali, Colombia; CIFN, Centro de Investigacion sobre Fijacion de Nitrogeno, Cuernavaca, morelos, Mexico;CSIC, Consejo Superior the Investigaciones Cientificas, Granada, Spain.

b Classification at the species level of strains KIM5s and CIAT7014 was performed by PCR-restriction fragment length polymorphism analysis of rrs and rrl genesas described elsewhere (61a).

VOL. 181, 1999 LPS BIOSYNTHESIS LOCUS IN R. ETLI 5607

DNA sequencing. Subclones of pRePV7.2CSK were made in pBluescript SK,and both strands of the resulting plasmids were subjected to cycle sequencing ina Gene Amp 2400 thermocycler (Perkin-Elmer), using IRD-labelled T3/T7 prim-ers (MWG-Biotech, Ebersberg, Germany) and a Thermosequenase fluorescentlabelled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Interna-tional, Braunschweig, Germany), based on the dideoxy-chain terminationmethod of Sanger et al. (50), as instructed by of the manufacturer. The cyclesequencing products were resolved, and the sequences were automatically readby using a model 4000 Li-COR automatic DNA sequencer and the correspond-ing sequence reading and editing software (MWG-Biotech).

Analysis of nucleotide and amino acid sequences. Contig assembly, sequenceediting, identification of open reading frames (ORFs), and determination ofpotential coding sequences and deduced amino acid sequences were carried outwith GeneCompar version 2.0 (Applied Maths, Kortrijk, Belgium). A remotesearch for potential promoter regions was performed at the Neural Network forPromoter Prediction (NNPP) server (41). Remote searches for sequence simi-larities were performed at the National Center for Biotechnology Informationserver, using the different BLAST programs (2). Multiple alignments of aminoacid sequences were performed with GeneCompar. Hydrophobic cluster analysis(HCA) plots of protein sequences were obtained from the HCA server, usingDRAWHCA (10).

DOC-PAGE analysis of crude LPS extracts. Crude LPS extracts were pre-pared from late-log-phase bacterial cultures by extraction of cell pellets with hotphenol-water as described elsewhere (11). The extracts were fractionated byelectrophoresis on 18% polyacrylamide gels (PAGE) assembled in the Bio-Rad(Richmond, Calif.) minigel casting system, using deoxycholic acid (DOC) as thedetergent for dissociating hydrophobic LPS aggregates, as described by Krauss etal. (32). Gels were silver stained by the procedure of Tsai and Frasch (58). Tovisualize capsular polysaccharides (KPS), an alcian blue prestain (18) is requiredprior to silver staining (43).

HPAEC-PAD of the LPS core OS. The extracted polysaccharides were sub-jected to mild acid hydrolysis in 1% acetic acid for 1 h at 105°C (12). The lipidA precipitate was removed by centrifugation, and the supernatant containing thewater-soluble polysaccharides and oligosaccharides, including the core fragmentsand O-PS, was analyzed by HPAEC on a Carbo Pac PA-1 column (Dionex)equipped with pulsed amperometric detection (PAD), as described elsewhere(12).

Purification and analysis of LPS oligosaccharides. The extraction and purifi-cation protocols used are presented in previous reports (43, 44). The cell pelletsfrom 2-liter cultures were extracted with hot phenol-water. The aqueous phasewas fractionated by size exclusion chromatography over Sephadex G-150 super-fine (Pharmacia, Uppsala, Sweden), and the column was eluted with 0.2 MNaCl–1 mM EDTA–10 mM Tris base–0.25% deoxycholic acid (pH 9.25). Thefractions (2 ml) were assayed colorimetrically for Kdo by the thiobarbituric acid(TBA) assay (63) and for uronic acid by the hydroxybiphenyl assay (5); LPS-containing fractions were positively identified by PAGE analysis. The LPS-containing fractions were then pooled, dialyzed, and freeze-dried (43), and theLPS pools were subjected to mild acid hydrolysis (1% acetic acid, 100°C, 90 min)followed by centrifugation to remove the insoluble lipid A. The oligosaccharideswere separated by gel filtration on Bio-Gel P-2 (Bio-Rad), using 50 mM ammo-nium formate (pH 5) as an eluent. The fractions were assayed for Kdo asdescribed above, and the pools were freeze-dried several times to remove theammonium formate.

Glycosyl composition and linkage analyses. Glycosyl residue compositionswere determined by gas chromatography-mass spectrometry (GC-MS) analysisof the trimethylsilyl methyl glycoside derivatives (68), using a 30-m DB-1 fusedsilica column (J&W Scientific, Folsum, Calif.) on a 5890A GC-MSD apparatus(Hewlett-Packard, Palo Alto, Calif.). The location of endogenous O-Me groupswas determined by GC-MS analysis of the alditol acetate derivatives, using a30-m SP-2330 column (Supelco). Inositol was used as an internal standard, andretention times were compared to authentic monosaccharide standards.

Plant nodulation assays. P. vulgaris cv. Saxa seeds (Kornhaus Colbe, Ger-many) were surface sterilized and germinated as described elsewhere (61a).Inoculum was derived from exponentially growing batch cultures that were di-luted in N-free LN leguminous nutrient solution (64) to reach about 5 3 106

CFU ml21. Seedlings were dipped into this suspension and transplanted togrowth pouches filled with N-free LN nutrient solution. Cultivation took place ina controlled-environment chamber (15 h of light and 9 h of darkness at 25 and18°C, respectively, and 75% relative humidity) for 21 days.

Nucleotide sequence accession number. The 2,709-bp sequence described inthis work has been deposited at GenBank nucleotide sequence database underaccession no. AF127522.

RESULTS

R. etli KIM-G1 is an R-LPS mutant. Strain KIM-G1 is aprototrophic mTn5 insertion derivative of wild-type strainKIM5s. It was identified as a putative LPS-defective mutant onPY or 20E plates (36, 64) due to its rough colony appearanceand tendency to flocculate (autoagglutinate) in liquid culture.It elicited few small, round, white nodules on P. vulgaris cv.Saxa that did not reduce acetylene (data not shown). In addi-tion, no bacteroids could be recovered from these structures,suggesting that strain KIM-G1 did not infect them. Thesephenotypic traits of R. etli mutants are commonly associatedwith defective LPS production (38). This was confirmed bycomparative DOC-PAGE analysis of hot phenol-water extractsof cell-associated polysaccharides from R. etli KIM5s and itsderivative KIM-G1. Figure 1A shows an alcian blue/silver-stained gel loaded with these extracts. The wild-type strainyielded a disperse band that represents R-LPS and severalforms of S-LPS containing sequential degrees of polymeriza-tion (DPs) of the O antigen. KIM5s seems to display a regu-lated (modal) distribution of O-antigen chain lengths since theLPS molecules with two attached O units are consistently pro-duced in lower quantity than molecules containing one, three,or four repeating units, as deduced from the relative bandintensities revealed by DOC-PAGE (Fig. 1). In contrast, strainKIM-G1 displayed only the R-LPS band, which had the same

TABLE 2. Constructs used in this study

Construct Relevant features Source or reference

pBluescript II SK1 Standard cloning and sequencing vector; Apr, lacZ1, fl(1) ori, ColE1 ori; high copy number;2,964 bp

Stratagene

pBBR1MCS-2 Mobilizable, replicative broad-host-range vector; Kmr; 5,144 bp 31pAM130 Plasmid used for mTn5gusA transposon mutagenesis. Sm/Sp, Ap; mTn5SSgusA30 (R. etli

nifH-gusA-trpA ter translational fusion in pUT/mini-Tn5 Sm/Sp66

pCAM140 Promoterless mTn5gusA transposon for promoter probing mutagenesis; Ap; Sm/Sp; loadedon pUT

66

pPVgusAESK Promoterless gusA gene from pCAM140 cloned as an EcoRI fragment into pSK This studypRePV-KG1 mTn5 insertion of strain KIM-G1 cloned as a 8.5-kb ClaI fragment in pSK This studypRePV1 pRePV-KG1 EcoRI subclone in pSK containing the I border of the mTn5 and 3.8 kb of

flanking genomic DNAThis study

pRePV2 pRePV1 XbaI subclone containing 1.3 kb of flanking genomic DNA linked to the I borderof the mTn5

This study

pRePV7.2CSK 7.2-kb KIM5s wild-type DNA fragment cloned in pSK, spanning the insertion site of themTn5

This study

pRePV7.2CMCS-21 7.2-kb insert of pRePV7.2CSK subcloned in broad-host-range vector pBBRMCS-2;complements strain KIM-G1, ORF2, -3, and -44 being in opposite transcriptionalorientation to the lacZ promoter of the vector

This study

5608 VINUESA ET AL. J. BACTERIOL.

electrophoretic mobility as the parental strain. Interestingly,strain KIM-G1, but not KIM5s, appears to produce high-mo-lecular-weight KPS which is visible only with alcian blueprestaining (43), migrating slower than the S-LPS bands of thewild type and displaying a characteristic ladder pattern withmultiple bands (Fig. 1A). This product was not examined fur-ther.

HPAEC and composition analysis of purified LPS fromKIM-G1 indicate that it has an intact core but lacks the Oantigen. To determine if the defect associated with strainKIM-G1 was in the biosynthesis of the LPS core or the Oantigen, a polysaccharide preparation was obtained from 5liters of cultured cells. The oligosaccharides from the LPS of R.etli KIM5s and KIM-G1 were released from the lipid A moietyby mild acid hydrolysis and analyzed by HPAEC-PAD. Thecore fingerprints of both strains were similar to each other andto those of other R. etli and R. leguminosarum strains (data notshown), yielding both the core a-D-GalA-134[a-D-GalA-135]-Kdo trisaccharide and the a-D-Gal-136[a-D-GalA-134]-a-D-Man-13Kdo tetrasaccharide that are highly con-served in these bacteria (12, 13, 27, 29). In addition,composition analysis of the Sephadex G-150-purified R-LPSindicated that the mutant produced all of the glycosyl residuesassociated with the core oligosaccharides. This demonstratesthat KIM-G1 is competent to synthesize an intact LPS core.

Purification and analysis of the O antigen from strainKIM5s. As the mutant did not produce any detectable O an-tigen (by PAGE), subsequent studies focused on the O antigenof the wild-type strain. The S-LPS of strain KIM5s was isolatedby size exclusion chromatography on Sephadex G-150 (datanot shown) and subjected to mild acid hydrolysis. The insolublelipid A was removed by centrifugation, and the released oligo-saccharides, both core and O antigen, were then separated onBio-Gel P2 (Fig. 2). The TBA assay (for Kdo) yielded three

major peaks; peaks P2-2 and P2-3 were shown by HPAEC andcomposition analysis to contain the tetrasaccharide and trisac-charide from the LPS core and were not studied further. P2-1(fractions 17 to 60) contained the higher-molecular-weight oli-gosaccharides, and the broad elution suggested a significantdegree of heterogeneity in size; PAGE analysis also showedthat the O antigen is heterogeneous in size (Fig. 1). An aliquotof pool P2-1 (fractions 17 to 60) was examined by HPAEC,which confirmed that there was no detectable core oligosac-charides in that preparation (data not shown). Therefore, poolP2-1 contained only O-antigen oligosaccharides.

Composition analysis of pool P2-1 showed a complex arrayof glycosyl components; however, five sugars accounted for81% of the detected carbohydrate. This fraction included Glc,Man, GalA, 3-O-Me-6-deoxytalose, and Kdo in approximatelya 2:1:1:1:1 molar ratio, and this most likely represents therepeating unit that yields the ladder pattern on the polyacryl-amide gel (Fig. 1). There were lesser amounts of five othersugars: GlcNAc, Fuc, Xyl, Gal, and quinovosamine constitutedthe remaining 19% of the detected carbohydrates. This indi-cates that these glycosyl residues are not part of the repeatingunit but are present in an inner O-antigen oligosaccharide oran oligosaccharide cap at the nonreducing terminus of the Oantigen, as has been found in the LPSs of other Rhizobiumspp. strains (10a, 23, 28).

Cloning of the mTn5SSgusA30 insertion of strain KIM-G1and of a wild-type fragment spanning the insertion site. En-zymatic digestion of the genomic DNA of KIM-G1 with ClaIand XhoI followed by Southern hybridization analysis with agusA-specific probe indicated that a single transposon insertionhad taken place, yielding single hybridization signals of about8.5 and 11.5 kb, respectively (data not shown). The ClaI frag-ment was cloned in pBluescript SK, yielding pRePV-KG1,which confers Ap, Sm, and Sp resistance (Fig. 3A). Restrictionmapping of this plasmid together with Southern hybridizationanalysis indicated that about 3.8 kb of genomic DNA fromKIM-G1 adjacent to the I border (19) of the mTn5 insertionwere cloned in pRePV-KG1 (data not shown). The 3.8-kbfragment was subcloned as an EcoRI fragment in pBluescriptSK to yield pRePV1. This plasmid was the source for pRePV2,which has the 1.3-kb XbaI fragment adjacent to the I bordercloned as an EcoRI-XbaI fragment (Fig. 3B). The cloned frag-ments were DIG labelled, and the resulting probes werenamed KIM-P#1 and KIM-P#2, respectively (Fig. 3B). A par-tial genomic DNA library of ClaI size-fractionated DNA frag-

FIG. 1. DOC-PAGE analysis of crude hot phenol-water extracts of cell-associated polysaccharides from wild-type R. etli KIM5s and its mTn5 insertionderivative KIM-G1. (A) Alcian blue/silver-stained gel loaded with the indicatedvolumes of extract, revealing the S-LPS phenotype of KIM5s and several DPs ofthe O unit (S-LPS). KIM-G1 exhibits only R-LPS, lacking the O-PS. KIM-G1,but not its parental strain, produces a high-molecular-weight polysaccharide thatstains specifically with alcian blue, migrating as a ladder pattern. This productmost probably corresponds to acidic Kdo-rich KPS. (B) Silver-stained LPSsproduced by strains KIM5s, KIM-G1, and the complemented strain KIM-G1/pRePV7.2CMCS-21. Synthesis of an intact O antigen is restored in the comple-mented strain.

FIG. 2. Elution profile of purified and hydrolyzed LPS core and O-antigenregions from strain KIM5s separated on a Bio-Gel P2 column. Peaks P2-2 andP2-3 were shown by HPAEC and composition analysis to contain the tetrasac-charide and trisaccharide from the LPS core. The P2-1 pool (fractions 17 to 60)contained the high-molecular-weight oligosaccharides corresponding to the Oantigen. Its broad elution suggests that the O polysaccharide is heterogeneous insize.


ments from R. etli KIM5s around 7 kb in size was screened withprobe KIM-P#2, yielding clone pRePV7.2CSK (Fig. 3C).

Complementation of KIM-G1 with pRePV7.2CMCS-21. Therecombinant plasmid pRePV7.2CSK carries a 7.2-kb DNAfragment from KIM5s spanning the insertion site of KIM-G1,as determined from sequence analysis (Fig. 3C). This wild-typeDNA fragment was subcloned into the broad-host-range clon-ing vector pBBR1MCS-2 (31), yielding pRePV7.2CMCS-21and pRePV7.2CMCS-22, respectively, depending on the insertorientation. E. coli S17-1 was transformed with pRePV7.2CMCS-21, which has the gene disrupted by the mTn5 insertion ori-ented in the opposite direction to the lacZ promoter of thevector and was conjugally transferred into KIM-G1. StrainKIM-G1/pRePV7.2CMCS-21 recovered the glossy colonymorphology of the parental strain and did not flocculate when

grown in liquid culture (data not shown), suggesting that thewild-type LPS had been restored in the complemented strain.This inference was confirmed by DOC-PAGE analysis of LPSextracts of KIM-G1/pRePV7.2CMCS-21, as shown in Fig. 1B.Finally, strain KIM-G1/pRePV7.2CMCS-21 was capable of fulland effective nodulation on P. vulgaris cv. Saxa (data notshown). Sections of the nodules formed by the complementedstrain presented the reddish color characteristic of leghemo-globin-containing N2-fixing nodules. These nodules had spe-cific nitrogenase activities comparable to that of nodules oc-cupied by the wild-type strain, as measured by the acetylenereduction assay (data not shown).

The mTn5 inserted in a glycosyltransferase (GT) locus. The2,709-nucleotide (nt) sequence spanning the site of insertion instrain KIM-G1 (Fig. 3C) encodes three ORFs, ORF2, ORF3,

FIG. 3. (A) Physical map of pRePV-KG1 carrying the cloned insertion of strain KIM-G1. (B) Physical maps of the subclones pRePV1 and pRePV2, carrying theI border of the mTn5 insertion along with the flanking genomic DNA fragments. These subclones were used for the generation of the DIG-labelled DNA probesKIM-P#1 and KIM-P#2. (C) Genetic and physical maps of the insert of pRePV7.2CSK, which spans over the mTn5 insertion site of KIM-G1, shown as a filled triangle.Transcriptional orientations of the ORFs are indicated by arrowheads. The approximate location of two potential promoter sequences detected by the NNPP isindicated by a flag. Only 1,682 nt of the 39 end of the lpsb2 gene are present on pRePV7.2CSK. (D) Nucleotide sequences of the two putative promoters predictedby the NNPP to be located in the region between ORF1 and ORF2, showing the presumed 210 and 235 sequences (underlined). The indicated residue positionscorrespond to those in GenBank accession no. AF127522.


and ORF4, which most likely form a single transcriptional unit.The mTn5 insertion was found in ORF3.

The start codon ATG of ORF2 is located 279 nt downstreamof the stop codon of ORF1 (not discussed in this report).NNPP (41) located two putative promoter sequences (Fig. 3D)with high correlation coefficients (r 5 0.9) within this inter-genic region. ORF2 is 816 nt long, encoding a 29.7-kDa proteinof 272 amino acids (aa). A potential 59-AAG-39 ribosomalbinding site (RBS) was located 7 nt upstream of the putativeATG start codon. The highest similarities of ORF2 were dis-played to (Mycobacterium tuberculosis EpiB [Z8034-3]) (83%identity) dTDP-glucose 4,6-dehydratase B. japonicum and Azo-rhizobium brasiliense and UDP-4-glucose-epimerases (AF039306and Z25478). Sequence similarity was particularly high at theN termini of these proteins, where the signature motifGxxGxxG (34) and other conserved residues characteristic ofbacterial NAD(P)-binding domains were identified in allaligned proteins, located between positions 8 and 14 of theKIM5s ORF2 product. Notable is that the product encoded byORF2 is some 50 residues shorter in its C terminus than therelated proteins.

The ATG start codon of ORF3 is located 14 bp downstreamof the TAG stop codon of ORF2 and is preceded by a potential59-AGGA-39 RBS located 12 bp upstream of the presumptiveATG start codon. ORF3 is 705 bp long, potentially encoding a235-aa protein of 26.6 kDa. It showed weak but significantsimilarities to sugar transferases, with the highest matches cor-responding to several a-GTs, such as WbpY, an a-1,3-D-rham-nosyltransferase involved in the synthesis of the A-band D-rhamnan O chain from Pseudomonas aeruginosa (48), and thea-mannosyltransferases MtfB and WbaW from E. coli andSalmonella choleraesuis, respectively, which are also involved inO-chain biosynthesis and are part of the rfb operons of theseenteric bacteria (8, 57). It is noteworthy that the product en-coded by ORF3 is shorter than these proteins, having a trun-cated N terminus. The KIM5s ORF3 product and related pro-teins display the signature sequence Ex7E (positions 144 to 152in ORF3) previously found in some a-GTs (6, 25). Interest-ingly, even though E. coli WbdP (62) was the protein with thehighest homology score in the BLASTP search (32% identityover 150 residues), it does not have the conserved Ex7E motifand therefore cannot be properly aligned in this region (datanot shown).

The site of insertion of the mTn5 in strain KIM-G1 wasprecisely mapped by sequencing over the I border cloned inpRePV2 (Fig. 3B). The insertion disrupted codon 16 (P) ofORF3 and therefore is located at the 59 end of the codingsequence.

The putative ATG start codon of ORF4 is located 54 ntdownstream of the TAA stop codon of ORF3 and is precededby a potential 59-AAG-39 RBS located 5 nt upstream of theATG codon. ORF4 is 828 nt long, encoding a putative 276-aaprotein of 31.1 kDa. Since no apparent termination or pro-moter sequences could be located between ORF3 and ORF4,the mTn5 insertion in ORF3 most likely has also a polar effecton ORF4. The product encoded by ORF4 displays significantsequence homology in its N-terminal part with many bacterialproteins, most of which are predicted to be GTs. The KIM5sORF4 product is similar over almost the entire length with afew proteins, particularly a hypothetical protein from Helico-bacter pylori HP0102 (AE000532) (;45% overall sequenceidentity). Among the proteins of known function, we foundenzymes belonging to different classes of b-GTs, i.e., b3-GlcT(WaaV [AF019746]) from E. coli, b3-GlcNAcT (Cps14I[X85787]) and b4-GalT (Cps14J [X85787]) from Streptococcuspneumoniae, and b6-GlcT (ExoO [L20758]) from Sinorhizo-

bium meliloti. All of this information strongly suggests thatORF4 encodes a b-GT. A comparison of peptide sequencesrevealed two conserved motifs, Ex4(D/N)x2SxD and Gx5NxGx5Gx9D (positions 35 to 45 and 68 to 92, respectively, ofORF4), which constitute a signature for this group of GTs.These peptide motifs comprise three invariant acidic residueswhich could play an important role in catalysis, since theseresidues possess appropriate side chain reactivity for glycosyltransfer (51). The three proteins HP0102, WbdO, and WcaEall share with ORF4 a high degree of conservation within thesecond motif (ExDxGYDYMNKGx5G), which could be char-acteristic of a subfamily of b-GTs.

The presence of many stop codons located at short intervalsin all three reading frames in the downstream sequence (datanot shown) strongly suggests that ORF4 is the last one in thepresumed transcriptional unit.

The mutated DNA region of strain KIM-G1 is located on aplasmid. Plasmid profiles of several Rhizobium spp. and Sino-rhizobium spp. strains were displayed by using a modified Eck-hardt procedure (Fig. 4A). The plasmids were blotted ontonylon membranes for probing with the DNA probe KIM-P#1(Fig. 3B). The Southern hybridization results presented in Fig.4B indicate that the hybridizing DNA region is plasmid en-coded. The plasmid profiles of R. etli KIM5s and CIAT7014had not been previously reported. The Eckhardt plasmid pro-filing experiments suggest that the two plasmid DNA bands ofKIM5s might actually correspond each to two plasmids ofsimilar size, as judged from the band intensities and visualinspection at high magnification of the profiles of replicatesamples (data not shown). Probe KIM-P#1 hybridized with theupper band (Fig. 4B), which was estimated to have a size ofabout 680 kb, based on the known sizes of the plasmids presentin CFN42 (34a). Importantly, strain CIAT7014 displays a plas-mid profile similar to that of KIM5s, although it has an addi-tional small plasmid of about 150 kb not present in KIM5s.Comparison of the plasmid profiles of 19 R. etli and R. tropicistrains indicated that KIM5s and CIAT7014 displayed the

FIG. 4. Eckhardt gel displaying plasmid profiles (A) and the correspondingSouthern hybridization signals (B) obtained with probe KIM-P#1 (Fig. 3B). Thetwo plasmid DNA bands displayed by strain KIM5s could each comprise twoplasmids of similar size, as might be also the case for CIAT7014, which has anadditional smaller plasmid of about 150 kb. The probe hybridizes only withplasmids from R. etli strains, those from KIM5s and CIAT7014 being about 680kb in size. The signal yielded by strain CFN42 is due to the lpsb2 region locatedon pCFN42b (of ca. 170 kb).


most similar plasmid profiles, suggesting that these strains haveclosely related plasmid backgrounds (61). The lowest plasmidband displayed by strain CFN42 actually comprises plasmidspCFN42a and pCFN42b (8). The latter carries the lpsb2 region(24), which is recognized by probe KIM-P#1 (Fig. 3). The lpsbregion was reported to be conserved among R. etli and R.leguminosarum bv. phaseoli strains (24).

The DNA region encoding GTs has a strain-specific distri-bution. pRePV2 was chosen as template to synthesize DNAprobe KIM-P#2, which is specific for the locus mutated by themTn5 insertion of KIM-G1, spanning the sequences of ORF3and ORF4 (Fig. 3B).

Southern blots of EcoRI-digested genomic DNAs of 18 beanmicrosymbionts were probed with KIM-P#2. The hybridiza-tion and the posthybridization washings were performed athigh stringency (0.53 SSC and 68°C). Under these conditions,only strains KIM5s and CIAT7014 yielded strong hybridizationsignals (data not shown). When the ionic strength of the wash-ing solution was reduced to 0.13 SSC, only these two strainshybridized with the probe (data not shown). These resultssuggest that the DNA region mutated in KIM-G1 has a strain-specific distribution, further supporting the notion that theputative GTs encoded by ORF3 and ORF4 are involved in thesynthesis of the variable O antigen and demonstrate that thehybridization signal yielded by the plasmid profile of strainCFN42 with probe KIM-P#1 (Fig. 4) is due to the lpsb2 genelocated on pCFN42b.

DISCUSSION

In this paper, we report on the mutagenesis, cloning,complementation, and sequence analysis of a novel DNA re-gion from R. etli KIM5s involved in LPS O-antigen biosynthe-sis. The mTn5 insertion derivative KIM-G1 exhibited a roughcolony appearance, tended to flocculate in liquid culture, andwhen inoculated on P. vulgaris cv. Saxa elicited pseudonodulesdevoid of leghemoglobin or bacteria. These phenotypic traitshad been previously shown to be characteristic for R. etli strainsdefective in LPS production (29, 36–38).

Comparative DOC-PAGE analysis of the LPS from wild-type strain KIM5s and KIM-G1 confirmed that the mutationaffected LPS production, as KIM-G1 appeared to produce onlyR-LPS, which had the same relative mobility as the R-LPS ofthe parental strain (Fig. 1). Subsequent HPAEC and compo-sition analyses showed that the mutant strain produced anintact core, with the core tri- and tetrasaccharides that arecommon in R. etli and R. leguminosarum (4, 12, 13, 29). NoO-chain residues were detected in LPS preparations fromstrain KIM-G1. From these results, we concluded that themutation in KIM-G1 specifically affects O-PS synthesis, leavingthe core OSs unaffected. Thus, the mutated locus is involved inLPS O-chain biosynthesis.

Although the structures of LPS molecules are not as wellestablished for rhizobia as they are for enteric bacteria, forclarity we have designated the tetrasaccharide and trisaccha-ride components of the R. etli (and R. leguminosarum) LPS asthe core, as has been done in other recent studies (23, 27). Thisdesignation is based on the fact that purified R-LPS of R. etlicontains only these moieties and an additional Kdo residue(10a, 47). The other carbohydrate residues, such as the Glc-NAc, Fuc, Xyl, Gal, and quinovosamine residues (i.e., theminor components), identified in this study are found only inassociation with S-LPS and therefore may be defined as O-antigen components. In addition, the tetrasaccharide and tri-saccharide components have been found in every strain of R.etli and R. leguminosarum studied to date (29), showing that

they are conserved in these species. In contrast, the othercomponents are strain specific, and this variable OS portiondefines the O antigens of many species, including R. etli and R.leguminosarum (29).

The O chains of R. etli and R. leguminosarum strains thathave been characterized, such as that from strain CFN42, con-tain complex repeating units and nonrepeating oligosaccha-rides (10a, 23, 29). Strain KIM5s also produces a complex Oantigen that appears to contain sequential DPS of a repeatingunit, as well as nonrepeating oligosaccharides. The apparentlymodal (regulated) distribution of O-chain lengths displayed byKIM5s (Fig. 1) is reminiscent of the situation found in theheteropolymeric O antigens produced, for example, by Salmo-nella enterica strains of serogroups B and E and could indicatethe action of a control system for O-chain polymerization suchas the Salmonella Rol protein (52, 65). However, there is noexperimental evidence indicating the existence of such a sys-tem in the rhizobia, and control of the O-chain length in thesebacteria may involve completely different mechanisms, such asO-methylation of the capping glycosidic residue(s) (29, 65).

The monosaccharide composition of the O-PS from KIM5sis clearly different from that of CFN42 (23), demonstratingthat it is a strain-specific antigen. The structural specificity ofthe O antigen indicates that each strain must carry uniquegenes involved in LPS biosynthesis. This notion is supported bygenetic evidence presented in this report based on Southernhybridization data for genomic DNAs from 18 bean microsym-bionts and probe KIM-P#2, which hybridized only with thehomologous strain KIM5s and one other strain, CIAT7014.This result suggests that the mutated region, which encodestwo GTs, appears to have a strain-specific distribution amongR. etli strains.

In this regard, Rhizobium spp. are quite different from Si-norhizobium spp., in which the LPS is a highly conserved an-tigen throughout the genus (22, 46). The K antigens are thestrain-specific antigens in the sinorhizobia (22, 43, 45, 46). Theproduction of K antigens by cultured cells of wild-type strainsof Rhizobium spp. has not been observed, which was also trueof R. etli KIM5s. In contrast, its mutant derivative KIM-G1 wasfound to produce a non-LPS, acidic polysaccharide that mi-grated as a low-mobility ladder pattern on polyacrylamide gels(Fig. 1A). This polysaccharide required an alcian blue prestainto appear on the gel, similar to the K antigens of Sinorhizobiumspp. (43). This phenomenon has been observed in other LPSmutants of rhizobia (47) and in a pb2 derivative from R. etliCFN42 (61). One possible explanation for the production of aspecific component by the mutant but not by the wild-typestrain is that by this means the cell can compensate in part forthe loss of the O antigen. It is not clear, however, why labora-tory-cultured cells of wild-type R. etli do not produce this classof polysaccharide. Conceivably the unique polysaccharide pro-duced by R. etli KIM-G1 is, in fact, a K antigen, as geneticanalysis of R. etli has shown that it carries the K-antigen-specific rkpABCDEF operon (47).

Three ORFs were identified in this report as the likely com-ponents of the putative transcriptional unit disrupted by themTn5 insertion in strain KIM-G1. ORF2, located upstream ofthe insertion site, probably encodes a sugar nucleotide epim-erase or dehydratase, containing the strictly conserved motifGxxGxxG at its N terminus. This motif is characteristic ofprokaryotic NAD(P)-binding domains (34, 67). The highesthomology was found to EpiB, a putative dTDP-glucose-4,6dehydratase from M. tuberculosis (17). Such an enzyme may beinvolved in the synthesis of 6-deoxy and dideoxy sugars (33),which are present in the O antigen of KIM5s.

ORF3 was disrupted at its 59 end by the mTn5 insertion in


strain KIM-G1, and complementation of KIM-G1 withpRePV7.2CMCS-2 unequivocally demonstrated that the inser-tion is the sole cause for the mutant phenotype. When pro-vided in trans, this plasmid restores the S-LPS, full nodulationand N2 fixation capabilities lost by the mutant. ORF3 couldencode for an a-GT, as it exhibits the ExE motif which is highlyconserved in the family of prokaryotic a-ManTs (25). Thismotif has also been found in other prokaryotic GTs, includinga3-GalTs, a6-GalTs, and a2-GlcNAcTs (6). Geremia et al.(25) have proposed that all prokaryotic a-mannosyltrans-ferases share the signature peptide ExFGxxxxE, a more spe-cific version of the ExE motif. This signature peptide is alsopresent in the R. leguminosarum core OS a-mannosyltrans-ferase LpcC (27). The putative a-1,3-D-rhamnosyltransferaseWbpY from P. aeruginosa (48) also shares this more specificmotif, indicating that it is not absolutely specific for a-manno-syltransferases. Even though ORF3 displays significant homol-ogy to several a-mannosyltransferases, and mannose was iden-tified as one of the components of the O-PS from KIM5s, itdoes not contain the FG residues of the diagnostic ExFGxxxxEsignature peptide. For these reasons, and as supported byHCA (data not shown), ORF3 is tentatively considered toencode an a-GT of unknown substrate specificity.

The mTn5 insertion in ORF3 probably has a polar effect onthe downstream ORF4 (Fig. 3C). ORF4 most likely encodes ab-GT, since it displays significant homology in its N terminuswith a number of known b-GTs. Two peptide motifs (Ex4(D/N)x2SxD and Gx5NxGx5Gx9D) similar to those found insome b-GTs (6, 51) were found to be conserved among nu-merous proteins. However, the position of the conserved acidicresidues is slightly different in the KIM5s ORF4 product andrelated proteins.

Upon HCA analysis, the KIM5s ORF4 product displayssimilarities over the entire sequence (data not shown) to pu-tative GTs WcaE and WbdO (56, 62) from E. coli. It alsocontains a more specific version (ExDxGIYDAMNKGx5Gx9D) of the second peptide motif described above, whichcould be a signature for a subfamily of b-GTs.

The lpsb2 gene, recently described by Garcıa de los Santosand Brom for R. etli CFN42 and located on plasmid pCFN42b(24), is found in KIM5s downstream of ORF4, in an oppositetranscriptional orientation (Fig. 3C). The complete lpsb2 geneand two other ORFs located further upstream were cloned inan overlapping 8.0-kb EcoRI fragment, which was found byusing the same strategy as for pRePV7.2CSK (61). The com-plete KIM5s Lpsb2 gene product shows 96% identity with thatof CNF42, has the same length, and complements strainCE168 (lpsb2::Tn5 derivative of CFN42) (15); consequently,these genes are considered to be allelic forms (61), and thusprobe KIM-P#1 hybridizes with pCFN42 (Fig. 4). Interest-ingly, the KIM5s lpsb2 allele is present as a monocistronictranscript, lacking the lpsb1 gene found in CFN42 (24), indi-cating a difference in gene organization around the lpsb2 locusin these two Mexican R. etli strains. Furthermore, since lpsb1and lpsb2 appear to be the only LPS biosynthesis genes onpCFN42b (24), and since probe KIM-P#2 hybridizes withgenomic DNAs from KIM5s and CIAT7014, but not withgenomic DNA from CFN42, the locus that we identified instrain KIM-G1 actually represents a novel plasmid-encoded lpsregion, mapped to a 680-kb plasmid present in strains KIM5sand CIAT7014, but not in other R. etli strains tested. Thenucleotide sequence of this region is the first one available forRhizobium spp. encoding for gene products involved in LPSO-chain synthesis.

ACKNOWLEDGMENTS

We thank J. A. Herrera and J. Sanjuan for help with the plasmidprofiling experiments, and we thank H. Thierfelder and E.-M. Kurz forexcellent technical assistance.

B. L. Reuhs was supported by National Science Foundation grantMCB-9728564 and by the U.S. Department of Energy-funded Centerfor Plant and Microbial Complex Carbohydrate Research under grantDE-FG02-93ER-20097. P. Vinuesa was the recipient of a TMR-Net-work grant from the EU. This work was supported by the DeutscheForschungsgemeinshaft through the SFB 395.

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