Rhixobium leguminosarum exoB Mutants Are Deficient in the Synthesis of UDP-glucose 4’-Epimerase*

Rhizobium leguminosarum bv. viciae Exo- mutant strains RBL5523,exo7::Tn5,RBL5523,exo8::Tn5 and RBL5523,exo52::Tn5 are affected in nodulation and in the syntheses of lipopolysaccharide, capsular polysaccharide, and exocellular polysaccharide. These mutants were complemented for nodulation and for the syntheses of these polysaccharides by plasmid pMP2603. The gene in which these mutants are defective is functionally homologous to the exoB gene of Rhizobium meliloti. The repeating unit of the residual amounts of EPS still made by the exoB mutants of R. leguminosarum bv. viciae lacks galactose and the substituents attached to it. The R. leguminosarum bv. viciae and R. meliloti exoB mutants fail to synthesize active UDP-glucose 4'-epimerase.

The gene in which these mutants are defective is functionally homologous to the exoB gene of Rhizobium meliloti. The repeating unit of the residual amounts of EPS still made by the exoB mutants of R. leguminosarum bv. viciae lacks galactose and the substituents attached to it. The R. leguminosarum bv.
In recent years the role of polysaccharides in nodulation by Rhizobium bacteria has once again gained interest. Several groups described the isolation of mutants of various Rhizobium strains defective in the production of particular polysaccharides. Some of these mutants are pleiotropic, e.g. Diebold and Noel (1989) recently described the isolation of two mutants of Rhizobium leguminosarum bv. phuseoli defective in the syntheses of both exopolysaccharide (EPS)' and lipopolysaccharide (LPS). The best characterized pleiotropic Exemutants are the exoB mutants of Rhizobium meliloti (Long et al., 1988), which were shown to be defective in the syntheses of both LPS and EPS (Leigh and Lee, 1988). Like the other Exe-mutants of R. meliloti (Leigh et al., 1985), the exoB mutants form ineffective nodules. Genes functionally homologous to the R. meliloti exoB gene were shown to be present in Agrobacterium (Cangelosi et al., 1987) and Azospirillum (Michiels et al., 1988).
R. leguminosarum bv. uiciae strains synthesize EPS with a different chemical structure (Robertsen et al., 1981) in comparison with the succinoglycan synthesized by R. meliloti (Jansson et al., 1977). Furthermore, R. legumirwsarum bv. uiciae strains synthesize a neutral gelling capsular polysaccharide (CPS; Zevenhuizen and Van Neerven, 1983b (Canter Cremers et al., 1988a). In order to identify mutants not only defective in the synthesis of EPS, the LPS, CPS, and cyclic @(l-2) glucan synthesized by these strains were analyzed.
Most Exe-mutants produce a LPS profile identical to that of parental R. leguminosarum bv. vi&e strain RBL5523 (Fig.  1, lane a). The LPS profile of this strain consists of two functions, LPSI and LPSII. LPSII presumably consists of lipid A and core LPS, whereas LPSI contains in addition O-antigen (De Maagd et al., 1988). Mutant strains RBL5523,exo?:Tn5, RBL5523,exo&:Tn5 and RBL5523, exo52::Tn5 produce an LPS (Fig. 1, lane b), which consists mainly of LPSII and a few intermediate bands, which may consist of LPSII to which a low number of O-antigen units are attached. Zevenhuizen and Van Neerven (1983b) described that R. leguminosarum bv. viciae strains produce a CPS. The amount of CPS produced by strain RBL5523 and most of the Exomutants was about 0.15 g of CPS/g of cellular protein. However, the Exe-mutants RBL5523,exoE:Tn5, RBL5523, exo&:Tn5, and RBL5523,exo52::Tn5 produce no CPS. These latter mutants are thus affected in the syntheses of EPS, LPS, and CPS. Zevenhuizen and Van Neerven (1983a) described that R. leguminosarum strains synthesize and secrete cyclic /3( l-2) glucan. We investigated the presence of p(l-2) glucan in strain RBL5523 and in its pleiotropic mutants. Therefore culture supernatants of these strains were fractionated using an Amicon Hollow Fiber apparatus as described previously (Djordjevic et al., 1986). When the fraction containing molecules with an apparent size between 500 and 3000 Da isolated from the culture supernatant of strain RBL5523 or any of the three pleiotropic mutants was eluted from a Sephadex DEAE A-25 column, only one evident neutral hexose-containing peak was obtained. After further purification the oligosaccharides isolated from these peaks were analyzed by 13C NMR spectroscopy.
The spectra obtained were identical to that published previously for the neutral cyclic /3(1-2) glucan isolated from Rhizobium strain NGR234 (Batley et al., 1987). Similarly, also the fractions containing molecules with an apparent molecular size between 3000 and 100,000 Da isolated from the culture supernatant of these four strains were assayed for the presence of cyclic p(l-2) glucans. In all these fractions cyclic /3(1-2) glucan was present as a neutral polysaccharide only. We therefore concluded that both R. leguminosarum bv. viciae strain RBL5523 and the three pleiotropic mutants synthesize only neutral cyclic p( l-2) glucan.
Symbiotic Characterization of the Pleiotropic Mutants of R. leguminosarum bv. viciae-When inoculated on V. sativa, V. hirsuta or Pisum sativum plants, the three pleiotropic mutant strains RBL5523,exo?:Tn5, RBL5523,exo&:Tn5, and RBL5523,exoZ?:Tn5 failed to induce marked root hair curling, nodules, or nodule-like structures. The mutants induce some root hair deformation and rare abortive infection threads. Hence the three pleiotropic mutants are impaired in an early step of infection.

Complementation of the Pleiotropic Exe-Mutants-To
isolate the complementing DNA fragment(s), a DNA library of a Sym plasmid-cured derivative of R. leguminosarum bv. viciae strain RBL5523, namely strain RBL5515, was constructed in IncP plasmid pTJS133, which carries the Tc' gene. The DNA library was introduced in Exe-mutant strain RBL5523, eao&:Tn5, and the resulting strains were inoculated on forty V. sativa plants. On two plants nodules appeared after 2 weeks. About 98% of the bacteria reisolated from these nodules were tetracycline-resistant. Unlike the original RBL5523,exo&:Tn5 mutant strain, these tetracycline resistant colonies were mucoid on YMB plates. From these colonies, plasmid pTJS133, harboring five Hind111 additional DNA fragments with a total size of 14.8 kb, could be isolated. When this plasmid, pMP2602, was introduced into either of the Exe-mutant strains RBL5523,exo?:Tn5, RBL5523,exo8:: Tn5, or RBL5523,exo52::Tn5, they acquired a mucoid colony morphology and induced nitrogen-fixing nodules on V. sativa and V. hirsutu as fast as parental strain RBL5523. The three mutant strains were also complemented for the synthesis of LPS (Fig. 1, lane d) and CPS. So, plasmid pMP2602 functionally complements all three pleiotropic mutant strains. Analysis of Plasmid pMP2602-To analyze which part(s) of the DNA fragment present in pMP2602 is involved in complementation, the five Hind111 fragments were subcloned into IncP plasmid pMP92. Of these, only plasmid pMP2603, which harbors an additional DNA fragment of 7.1 kb, complements all three Exe-mutants for mucoidy, the synthesis of CPS and LPS (Fig. 1, lane f), as well as for nodulation on V. sativa and V. hirsuta plants.
In order to define more precisely which area of the DNA insert of pMP2603 is involved in complementation, pMP2603 was mutagenized with transposon Tn5. A total of 14 derivatives of pMP2603 with Tn5 inserted at different sites in the complementing DNA fragment were isolated (Fig. 2) and separately introduced into the pleiotropic mutant strains by conjugation.
Of these strains, only plasmid pMP2603,n3::Tn5 failed to complement the three mutant strains (Fig. 2). In addition we determined the location of Tn5 in the DNA of these three Exe-mutants. Total DNA was isolated from these strains, followed by digestion with restriction enzymes EcoRI, HindIII, or both of these enzymes. After gel electro-phoresis and transfer of the DNA to nitrocellulose filters, these filters were hybridized with radioactively labeled DNA either from Tn5 or from the 7.1-kb HindIII insert present in plasmid pMP2603.
Based on the results, we concluded that the Tn5s in these Exe-mutant strains are inserted: 1) in a Hind111 DNA fragment similar in size to the 7.1-kb DNA insert present in plasmid pMP2603 and 2) in the region with a size of about 1 kb, defined by the locations of the Tnb present in plasmid pMP2603;n2::Tn5 and pMP2603$4::Tn5. Complementation by R. meliloti exe Genes-Several cosmid clones, on which exo genes of R. meliloti are present, have been described Long et al., 1988). We used three of these clones, pD56, pEX154 and pEX312, which together harbor nearly all described exo genes of R. meliloti, for complementation studies of the three pleiotropic mutants. Only plasmid pD56, which carries exo genes exoB, exoJ, exoG, exoF, and exoQ, was able to complement the three R. leguminosarum bv. viciae Exe-mutants for colony morphology, synthesis of CPS and LPS (Fig. 1, lane e), as well as nodulation of V. sativa or V. hirsuta plants.
To determine which of the R. meliloti exo genes is involved in the complementation, we tested whether plasmid pMP2602 was able to functionally complement R. meliloti Exe-mutants. Plasmids pMP2602 and its smaller derivative pMP2603 were able to complement only R. meliloti Exe-mutant strain Rm7094 (exoB) ( Table 2). In order to locate the complementing locus more precisely, derivatives of pMP2603 in which Tn5 was inserted at various sites (Fig. 2) were introduced into R. meliloti mutant strain Rm7094. The resulting strains were tested for fluorescence on plates containing calcofluor and for nodulation on Medicago sativa. The only strain that was not complemented for both these characteristics was strain Rm7094 harboring pMP2603,03::Tn5 (Table 2). A gene functionally homologous to the exoB gene of R. meliloti is thus present on plasmid pMP2603 in a locus defined by pMP2603,93::Tn5 (Fig. 2). We propose to designate this gene as the exoB gene of R. leguminosarum bv. viciae strain RBL5523.
Chemical Structure of the EPS of exoB Mutants-Colonies of R. leguminosarum bv. viciae exoB mutants on YMB plates are not completely rough. We therefore checked whether the mutants still produce some EPS.
After growth for 6 days in B-minimal medium, parental strain RBL5523 produces about 0.8 g of EPS/g protein present in the culture. Under these circumstances, the exoB mutants of R. leguminosarum bv. viciae produce approximately 0.07 g of EPS/g of protein. The sugar composition of this residual EPS produced by the exoB mutants was analyzed on TLC plates. In the hydrolyzed EPS of parental strain RBL5523, spots indicating the presence of glucuronic acid, glucose, and galactose were observed. The EPS of all three exoB mutants appeared to lack galactose. Therefore the structure of the EPS produced by one of these exoB mutants was further analyzed by NMR spectroscopy.
To facilitate the spectroscopy, the EPS was depolymerized by phage RL38 and purified by column chromatography.
In comparison to the depolymerized EPS of strain RBL5523 (Fig.  3A), that of strain RBL5523,exo&:Tn5 eluted earlier from the Sephadex DEAE A25 column (Fig. 3B ) (Fig. 4A) was identical to that of the repeating unit of R. leguminosarum bv. trifolii strains ANU843 (Hollingsworth et al., 1988) and LPR5 (McNeil et al., 1986). The chemical structure of the repeating unit of RBL5523 is therefore identical to that of these strains (Fig. 5). When the 13C NMR spectrum of the repeating unit of the EPS of strain RBL5523, exo&:Tn5 (Fig. 4B) is compared with that of RBL5523 (Fig. 4A), it is apparent that several peaks are absent. Carboxyethylidene signals at 26.1 and 101.8 ppm and galactose peaks for C2 to C6 at 71.3, 66.7, 71.9, 72.5, and 65.8 ppm, respectively, are missing. Furthermore, C3 of the glucose carboxyethylidene residue is no longer glycosylated, and the peak at 80.3 ppm assigned to it has moved several parts/million upfield. The remaining peaks can all be assigned as in the 13C spectrum of the repeat unit from strain RBL5523. It may therefore be concluded that R. leguminosarum bv. uiciae Exe-mutant strain RBL5523,exo&:Tn5 produces small amounts of EPS, similar in structure to that of strain RBL5523, but lacking the terminal galactose carboxyethylidene from the side chain (Fig. 5). Methylation analysis confirmed these findings.
The 3-hydroxybutanoate that is usually esterified to a proportion of the galactose rings was also absent in the mutant EPS. The small peak at 23.0 ppm and others in the 60-70 ppm region were absent from the spectrum of the Exe-mutant (Fig. 4B). In the 'H spectrum, the 3-hydroxybutanoate peak at 1.51 ppm was also absent.
Small peaks in the 13C spectrum at 90.4 and 95.2 ppm showed the presence of some acetate on the sugar at the reducing terminus of material from strain RBL5523, as was also reported for R. leguminosarum strains 128C53 (Kuo and Mort, 1986) and LPR5035, a derivative of LPR5 (Philip-Hollingsworth et al., 1989). Mutant strain RBL5523,exo&:Tn5 had the same acetylation pattern. The 'H spectra also showed the additional complexity in the acetate methyl signal at 2.2 ppm that was reported by Hollingsworth et al. (1988) as being an indication of acetylation of glucose at the branch point, but the carbon spectra have the advantage that the assignment is unequivocal and do not require confirmation by methylation analysis.
Finally, the structure of the repeat unit of EPS produced by RBL5523,exo&:Tn5 harboring either pD56 or pMP2603 was determined. Hydrolyzed EPS from both strains gave spots Gk, glucose; GlcA, glucuronic acid; HB, 3-hydroxybutanoate; Gal, galactose; p, pyranose. The repeating unit of the EPS formed by mutant strain RBL5523,ezo&:Tn5 lacks the galactose residue h and consequently the substituents attached to this residue. *, intermediates in the Leloir pathway; A, intermediates in the De Ley-Douderoff pathway; 0, intermediates in the Entner-Douderoff pathway. Abbreviations used: GGPDH, glucose-gphosphate dehydrogenase; KDG, 2-keto-3-deoxygalactonate; KDPG, 2-keto-3-deoxy-6-phosphogalactonate;AcCoA, acetyl coenzyme A. All other abbreviations are explained in the legend to Table 3. on TLC plates indicating the presence of galactose as well as glucose and glucuronic acid. When the depolymerized EPS of these strains were purified over a Sephadex DEAE A25 column, peaks eluted at the same time as the repeating unit from strain RBL5523 (Fig. 3). The 13C NMR spectrum from the oligosaccharides obtained from these peaks were identical to that of strain RBL5523. Summarizing, 1) the three Exomutants synthesize residual amounts of EPS that lacks the terminal galactose residue h (Fig. 5) and the substituents attached to it and 2) the synthesis and structure of the EPS is restored to wild type after complementation of these strains by plasmids pMP2602, pMP2603, or pD56.

Analysis of the Activity of Enzymes Involved in Galactose
Metabolism-Generally bacteria use UDP-galactose for the incorporation of galactose into polysaccharides (Stoddart, 1984). Therefore we analyzed the exoB mutants of R. leguminosarum bv. viciae and R. meliloti for the presence of enzymes involved in the synthesis of UDP-galactose (Fig. 6).
In the extracts of the three exoB mutants of R. leguminosarum bv. viciae, as well as in the R. meliloti exoB mutant strain Rm7094, UDP-glucose 4'-epimerase activity is absent (Table 3), whereas the other tested enzymes activities were present (Table 3). In the cell-free extracts prepared from R. legumirwsarum bv. viciue and R. meliloti exoB mutant strains harboring either one of the complementing plasmids pMP2602, pMP2603, or pD56, UDP-glucose 4'-epimerase activity was present (Table 3). DISCUSSION In the present paper we describe three indistinguishable pleiotropic mutants of R. leguminosarum bv. viciae with the following characteristics. 1) They are non-mucoid as a result of a strongly reduced amount of EPS. The residual EPS lacks the terminal galactose residue (Fig. 5, residue h) and the substituents attached to it. In addition, these mutants fail to synthesize CPS, whereas their LPS profile is also affected. Furthermore, these mutants synthesize unsubstituted neutral cyclic p(1-2) glucans indistinguishable from that synthesized by the parental strain.
2) The mutants are defective in an early step of the nodulation process, since they only induce root hair deformation but not marked root hair curling and only rarely infection threads which are abortive. 3) The three mutants are affected in a DNA region only about 1 kb in size, whereas the characteristics of the mutants are indistinguishable. It is therefore likely that these mutants are affected in the same gene. 4) The same region of plasmid pMP2603 is involved in the complementation of the three R. leguminosarum bv. viciae Exe-mutants as well as a R. meliloti exoB mutant. A clone harboring the exoB gene of R. meliloti is able to complement the three pleiotropic mutant strains of R. legumirwsarum bv. viciae, for nodulation and the syntheses of EPS, LPS, and CPS. We therefore designated the mutant strains as R. leguminosarum bv. viciae exoB mutants.
Function of the exoB Gene-The EPS of R. leguminosarum bv. viciae contains galactose as a structural element (Fig. 5).
In R. leguminosarum, galactose is also a structural element of CPS (Zevenhuizen and Van Neerven, 1983b) and of LPS (Carlson et al., 1987;Zevenhuizen et al., 1980). The R. leguminosarum bv. vi&c exoB mutants are affected in the syntheses of all three polysaccharides.
In addition, both types of EPS isolated from R. meliloti, EPSI and EPSII, contain galactose, and it was reported that exoB mutants of R. meliloti fail to synthesize either one of these EPS species (Glazebrook and Walker, 1989). If the exoB gene is involved in some step in the synthesis or polymerization of UPD-galactose, the defects seen in the EPS, LPS, and CPS isolated from exoB mutants of R. meliloti and R. leguminosarum can be understood.
The exoB mutants of both R. leguminosarum bv. viciae and R. meliloti grow on galactose as sole carbon source.3 This indicates that in these mutants all the enzymes required for the uptake and conversion of galactose are functioning.
Initially we were misled by this observation and the knowledge that in Escherichia coli galactose is lethal for galE mutants (Fukasawa and Nikaido, 1961). However the situation in Rhizobium differs from that in E. coli as it was reported previously that R. leguminosarum bv. trifolii (Ronson and Primrose, 1979) and R. meliloti (Arias and Cervenansky, 1986) make use of the De Ley-Douderoff pathway for the conversion of galactose (Fig. 6). In the strain used in the present study, namely R. leguminosarum bv. vi&c strain RBL5523 and its derivatives, a crucial enzyme of the De Ley-Douderoff pathway, galactose dehydrogenase, is present (Table 3), indicating that strain RBL5523 also makes use of this metabolic pathway. Furthermore, one of the enzymes of the LeLoir pathway (Fig. 6), galactokinase, is absent (Table 3). In strain RBL5523, UDP-galactose can therefore only be formed by the epimerization of UDP-glucose (Fig. 6), a reaction which is catalyzed by UDP-glucose 4'-epimerase (Maxwell et al., 1961). The fact that our R. leguminosarum bv. viciae exoB mutants still produce polysaccharides that contain glucose indicates that these mutants are still able to synthesize UDP-glucose. The exoB mutants of R. leguminosarum bv. vi&e and R. meliloti, however, lack UDP-glucose 4'-epimerase activity (Table 3) and are thus unable to synthesize UDP-galactose (Fig. 6). The most likely explanation for our results is that the exoB genes of R. leguminosarum bv. viciae and R. meliloti are structural genes for UDP-glucose 4'-epimerase. We plan to sequence the R. leguminosarum bv. vi&e exoB gene to see ' H. C. J. Canter Cremers, unpublished data.
whether it shares homology with the E. coli galE gene. The Role of exoB in Nodulution-Our results point out that there is an effect of the mutation in the exoB gene on the early steps of the nodulation process, even though the attachment of the mutants to root hairs of V. sativa plants is comparable with that of the parental strain RBL5523.3 Our data indicate that the exoB mutants are not able to synthesize any molecule which requires UDP-galactose for its synthesis. A possible explanation is that the R. leguminosarum bv. vicinc signal molecule that induces root hair curling contains galactose. However, this explanation implies that this compound is different from its R. meliloti counterpart which lacks galactose (Lerouge et al., 1990).