Involvement of nodS in N-Methylation and nodU in 6-O-Carbamoylation of Rhizobium sp. NGR234 Nod Factors

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Article

Reference

Involvement of nodS in N -Methylation and nodU in 6- O -Carbamoylation of Rhizobium sp. NGR234 Nod Factors

JABBOURI, Said, et al.

Abstract

Although Rhizobium sp. NGR234 and Rhizobium fredii USDA257 share many traits, dysfunctional nodSU genes in the latter prohibit nodulation of Leucaena species. Accordingly, we used R. fredii transconjugants harboring the nodS and nodU genes of NGR234 to study their role in the structural modification of the lipo-oligosaccharide Nod factors. Differences between the Nod factors mainly concern the length of the oligomer (three to five glucosamine residues in USDA257 and five residues only in NGR234) and the presence of additional substituents in NGR234 (N-linked methyl, one or two carbamoyl groups on the non-reducing moiety, acetyl or sulfate groups on the fucose). R. fredii(nodS) transconjugants produce chitopentamer Nod factors with a N-linked methyl group on the glucosaminyl terminus.

Introduction of nodU into USDA257 results in the formation of 6-O-carbamoylated factors.

Co-transfer of nodSU directs N-methylation, mono-6-O-carbamoylation, and production of pentameric Nod factors. Mutation of nodU in NGR234 suppresses the formation of bis-carbamoylated species. Insertional mutagenesis of nodSU drastically decreases Nod [...]

JABBOURI, Said, et al . Involvement of nodS in N -Methylation and nodU in 6- O

-Carbamoylation of Rhizobium sp. NGR234 Nod Factors. Journal of Biological Chemistry , 1995, vol. 270, no. 39, p. 22968-22973

DOI : 10.1074/jbc.270.39.22968

Available at:

http://archive-ouverte.unige.ch/unige:113176

Disclaimer: layout of this document may differ from the published version.

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Involvement of nodS in N-Methylation and nodU in 6-O- Carbamoylation of Rhizobium sp. NGR234 Nod Factors*

(Received for publication, May 25, 1995, and in revised form, July 24, 1995)

Saı¨d Jabbouri‡, Re´my Fellay‡, Franck Talmont§, Philippe Kamalaprija^¶, Ulrich Burger^¶, Biserka Relic´‡, Jean-Claude Prome´§, and William J. Broughton‡i

From the ‡L. B. M. P. S., Universite´ de Gene`ve, 1 ch. de l’Impe´ratrice, 1292 Chambe´sy/Gene`ve, Switzerland, the

§Laboratoire de Pharmacologie et Toxicologie Fondamentales, CNRS, 205 Route de Narbonne, 31077 Toulouse Cedex, France, and the¶De´partement de Chimie Organique, Universite´ de Gene`ve, Sciences II, 30 quai Ernest-Ansermet, 1211 Gene`ve 4, Switzerland

Although Rhizobium sp. NGR234 and Rhizobium fredii USDA257 share many traits, dysfunctional nodSU genes in the latter prohibit nodulation of Leucaena species.

Accordingly, we used R. fredii transconjugants harbor- ing the nodS and nodU genes of NGR234 to study their role in the structural modification of the lipo-oligosac- charide Nod factors. Differences between the Nod factors mainly concern the length of the oligomer (three to five glucosamine residues in USDA257 and five resi- dues only in NGR234) and the presence of additional substituents in NGR234 (N-linked methyl, one or two carbamoyl groups on the non-reducing moiety, acetyl or sulfate groups on the fucose). R. fredii(nodS) transcon- jugants produce chitopentamer Nod factors with a N- linked methyl group on the glucosaminyl terminus. In- troduction of nodU into USDA257 results in the formation of 6-O-carbamoylated factors. Co-transfer of nodSU directs N-methylation, mono-6-O-carbamoyla- tion, and production of pentameric Nod factors. Muta- tion of nodU in NGR234 suppresses the formation of bis-carbamoylated species. Insertional mutagenesis of nodSU drastically decreases Nod factor production, but with the exception of sulfated factors (which are par- tially N-methylated and mono-carbamoylated), they are identical to those of the wild-type strain. Thus, Nod fac- tor levels, their degree of oligomerization, and N-meth- ylation are linked to the activity encoded by nodS.

Symbiotic soil bacteria of the genera Azorhizobium, Brady- rhizobium, and Rhizobium (collectively termed rhizobia) inter- act with the roots of legumes to form nodules in which atmo- spheric nitrogen is reduced to ammonia. Signal exchange between the symbionts regulates the expression of both bacte- rial and plant genes involved in nodule development (1, 2).

Flavonoids excreted by the legume roots activate both common and host-specific nod genes which direct the synthesis of lipo- oligosaccharide Nod factors. Secretion of these hormone-like substances into the plant rhizosphere induces root hair defor- mation and curling (3, 4), the formation of preinfection threads (5), and nodule-like structures (6). On a number of legumes,

Nod factors permit various natural or engineered Nod² mutants to enter the legume roots and to form nitrogen-fixing nodules (4, 7). All Nod factors so far identified areb-1,4-linked tri- or tetra- or pentamers of N-acetyl-D-glucosamine, N-acy- lated at the non-reducing end, and N-acetylated on the other residues (1, 8). Essential differences among the Nod factors of the various species concern the substituents linked to both ends of the chitinic backbone. Among the substitutions found on the terminal non-reducing N-acetyl-D-glucosamine are an N-methyl group, carbamoyl groups, acetyl groups, and various fatty acids. Similarly, the reducing N-acetyl-D-glucosamine residue may be substituted with a sulfate group or withD-arabi- nose, L-fucose, or 2-O-methylfucose. Furthermore, this additional saccharide may be acetylated or sulfated (1, 9).

Mutations in the nodABC genes, which are common to all rhizobia, abolish Nod factor production. nodC shares homology with chitin synthases (10), an observation which has been supported by in vitro studies (11). It thus seems likely that the first step in Nod factor synthesis involves the assembly of N-acetyl-D-glucosamine subunits by the N-acetylglucosaminyl- transferase coded by nodC (12). When the growing chain reaches three to five residues, NodB probably removes the N-acetyl moiety of the non-reducing end (11). NodA is an N- acyltransferase which links the acyl chain to the free NH₂ group on the oligomers synthesized by the NodC and NodB proteins (13, 14).

Host-specific nod genes are involved in the addition of extra groups to the core lipo-oligosaccharides. Modified factors per- mit nodulation of specific plants (16). For example, the nodH and nodPQ genes of R. meliloti are involved in the sulfation of the oligosaccharide signals (16). NodL shares homology with O-acetyltransferases; in vitro studies showed that NodL 6-O- acetylates the non-reducing glucosaminyl residue (17). nodE and nodF are involved in synthesis of the lipid chain (18, 19), while nodZ and nolO play a role in fucosylation of Bradyrhizo- bium japonicum nodulation signals (20, 21). The nodSU genes of Rhizobium. sp. NGR234 form an operon, the activity of which is required for nodulation of Leucaena leucocephala (22).

nodSU are also present in R. fredii USDA257 (23), B. japoni- cum (24), and Azorhizobium caulinodans (25), but no correla- tion exists between the ability to nodulate Leucaena and the presence of nodSU in these organisms (23). Moreover the nodSU genes from B. japonicum are unable to complement a nodS²mutant of NGR234 (24). On the other hand the transfer of nodSU from NGR234 to R. fredii USDA257 confers on the latter the ability to nodulate Leucaena (23). NodS shares sim- ilarities with S-adenosylmethyl transferases (25, 26). Support for this latter function has been obtained by in vitro labeling studies. The role of NodU remains to be elucidated.

Functions of nodS and nodU of NGR234 were determined by

* This work was supported by the Erna och Victor Hasselblads Stif- telse, Fonds National de la Recherche Scientifique Projects 31–

30950.91 and 31–36454.92, the Universite´ de Gene`ve, the Re´gion de Midi Pyrene´es, and the EEC Science Programme. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”

in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

iTo whom correspondence should be addressed: LBMPS, Univer- site´ de Gene`ve, 1 chemin de l’Impe´ratrice, 1292 Chambe´sy/Gene`ve, Switzerland. Tel.: 0041-22-7320420; Fax: 0041-22-7320734.

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at Université de Genève on January 22, 2019http://www.jbc.org/Downloaded from

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introducing nodSU, nodS, and nodU into R. fredii USDA257 and by analyzing the Nod factors produced by the transconju- gants. Analysis of the Nod factors produced by nodS² and nodU²mutants of NGR234 confirmed that the product of the nodS gene is involved in N-methyltransferase activity and that the 6-O-carbamoyltransferase activity is dependent upon ex- pression of the nodU gene.

EXPERIMENTAL PROCEDURES

Bacteriological and Molecular Methods—Rhizobia, along with their relevant characteristics, are listed in Table I. Microbiological tech- niques were performed as described in Lewin et al. (22). Nodulation tests were conducted in Magenta^TM-type Leonard jars (22). DNA sequences were determined using the dideoxy chain termination proce- dure using double-stranded templates and the Sequenase II^TMkit (U. S.

Biochemical Corp.).

Placement of nodU Under Control of the nodSU nod Box—The cohe- sive ends of the 250-base pair PvuI-EcoRI fragment containing the nod box of the nodSU operon were filled in and ligated into the SmaI site of pBS(1). Orientation of the insert in the resulting plasmid pNBSU was checked by sequencing. Concomitantly, the nodS gene (as well as the accompanying upstream sequences) was deleted from pA18 by double digestion with BamHI and EcoRI and replaced by the nod box of pNBSU (Fig. 1). The resulting plasmid pRAF25 was introduced into R.

fredii USDA257 using the helper plasmid pRK2013.

Purification of Nod Factors—Nod factors were isolated from apige- nin-induced (10²⁶M) culture media. Solid-phase extraction onto large scale C18reverse-phase columns, followed by washing with ethyl ace- tate and preparative HPLC¹chromatography was performed essen- tially as described previously (27). Fractions reacting positively with anthrone were repurified on a C18reverse-phase analytical column. A 20 –90% methanol gradient was used for elution. Further purification was achieved on a propylamine-linked analytical column using a linear gradient of 100% acetonitrile to water/acetonitrile (80:20 (v/v)).

Tomato Cell Suspension Assay—Fifty ml of the appropriate cultures in RMM3 (27) were centrifuged (4,000 3 g, 4 °C), applied to 3-ml Sep-Pak C18 cartridges (Waters Associates, Milford, MA), and the Nod

factors eluted with methanol. Dried samples were dissolved in Me2SO/

ethanol, and appropriate dilutions added to suspension cultures of tomato cells (28).

Analytical Methods—Gas chromatography was performed on an OV1-bound capillary column (0.32 mm330 m, Spiral, France) using a temperature gradient of 100 –280 °C (2 °C/min). Mass spectra were recorded on an Autospec instrument (Fisons, VG-Analytical, Manches- ter, United Kingdom). The acceleration voltage was 8 kV. A glycerol matrix (1ml), either acidified with 0.13Mtrifluoroacetic acid (1ml) or spiked with 10% sodium iodide (1ml), was used in the positive-ion mode.

Glycerol-thioglycerol (1:1) was the matrix used for ionization in the negative-ion mode. B/E-linked scans were performed to study metastable ion decay in the first field-free region without the use of a collision gas. NMR spectra were recorded on a Bruker 400 MHz spectrometer (9.4 Tesla) using deuterated dimethyl sulfoxide as the solvent and as the secondary reference (¹³C,d539.5 ppm).

RESULTS

Nod Factors Produced by nodU² and nodSU² Mutants—

Reverse-phase HPLC analysis of the supernatants from NGRV26 (5nodU²) (see Fig. 2) cultures revealed the same two Nod factor peaks as in the overproducing strain. The first peak corresponds to a mixture of sulfated compounds, the second to non-sulfated Nod factors. FAB-MS spectra of NodNGR factors of the mutant strains were compared with those previously published (27). In the positive-ion mode, the spectra of compounds from peak A gave [M2H1Na]¹ions by sodium attach- ment of m/z 1597, 1569, 1554, and 1526 which are character- istic of sulfated Nod factors. Fragmentation of the non-reducing sugar yielded similar molecular ions which correspond to the glucosaminyl residue at the non-reducing end. They differ from analogous fragments of Nod factors originating from the over- producing strain NGR(pA28) by the absence of bis-carbamoy- lated ion products (lacking m/z 526 and 498 which correspond to species with two carbamates and C_18:1or C_16:1, respectively).

Peak B contains Nod factors with an acetylated or non- substituted 2-O-methylfucose. As with the sulfated molecules, they differ from those of the NGR(pA28) by the presence of only one carbamoyl group (Fig. 2). Again, this is clearly seen on the

1The abbreviations used are: HPLC, high performance liquid chromatography; ppm, parts/million; FAB-MS, fast atom bombardment- mass spectroscopy.

TABLE I

Strains and plasmids used in this study

The abbreviations used are as follows:^Rsignifies resistance against ampicillin (Ap), kanamycin (Km), rifampicin (Rif), spectinomycin (Sp), and tetracycline (Tc).

Strain/plasmid Characteristics Source/refs.

E. coli

pBS(1) pMB1, phage f1, lacaZ¹, Ap^R Stratagene, La Jolla,

CA

pRK2013 Tra1helper plasmid for mobilization (Km^R) 33

pRK7813 Broad host range, IncP1 costramid (Tc^R) 34

Rhizobium sp.

NGR234 Rif^Rderivative of Rhizobium sp. NGR234 22

NGRV25 NGR234 carrying anVinsertion in nodS

(Rif^RKm^R)

22

NGRV26 NGR234 carrying anVinsertion in nodU

(Rif^RSp^R)

22

pA28 2.2 kilobase EcoRI-PstI fragment containing

nodD1 of NGR234 in pRK7813 (Tc^R)

27

pA18 EcoRI-HindIII fragment containing nodSU

of NGR234 cloned in pRK7813 (Tc^R)

22

pA16 EcoRI-PstI fragment containing nodS of

NGR234 cloned in pRK7813 (Tc^R)

22

pA25 pA18 containing anVinsertion in the

BamHI site of nodS (Km^RTc^R)

22

pA26 pA18 containing anVinsertion in the HpaI

site of nodU (Tc^RSp^R)

22

pRAF25 nod box::nodU of NGR234 cloned in

pRK7813 (Tc^R)

This work

R. fredii

USDA257 R. fredii USDA257S1. Km^Rderivative of

wild-type strain USDA257. Contains a silent Tn5 insertion in the pSym.

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Role of Rhizobium nodSU Genes in Nod Factor Biosynthesis 22969

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B1 oxonium ion fragment (data not shown). Thus, a mutation in nodU seems to suppress the production of bis-carbamoylated products (Fig. 2).

Analyses of the supernatants of the nodSU² mutant (5 NGRV25) (22) revealed a dramatic reduction in Nod factor production in comparison with the wild-type or overproducing strains (data not shown). This was shown both by thin layer chromatography and by direct measurement of the amounts of chitin oligomers in the supernatants using the tomato cell suspension assay (28, 29). In contrast, the nodU²mutant produced similar amounts of Nod factors to the wild-type bacte- rium. Together, the low amounts and complex mixtures of the biologically active products produced by the nodSU² strain rendered their analysis difficult. Nevertheless, the FAB-MS spectra in the negative-ion mode gave [M2H]²ions predomi- nately at m/z 1494, 1537, 1466, and 1509 which correspond to non-N-methylated but fucosylated, sulfated, C_18:1-acylated, mono- or non-carbamoylated pentamers, and non-methylated, fucosylated, sulfated, C_16:1-acylated, mono- or non-carbamoylated pentamers, respectively. Exhaustive butanol extraction of the membrane fraction derived from NGRV25 cells after lysis with a Frensh press did not reveal significant amounts of Nod factors. Together, these data show that expression of nodS and nodU modulates the synthesis, methylation, and bi-car- bamoylation of NodNGR factors.

Nod Factors Produced by R. fredii Transconjugants Harbor- ing nodSU, nodS, and nodU—To circumvent problems linked to mutagenesis (e.g. operon structure, duplication of genes, etc.), we studied Nod factors produced by R. fredii transconju- gants harboring nodSU (pA18) or nodS (pA26) and a truncated nodU (pA16) (Fig. 1). All three transconjugants are able to nodulate Leucaena (22). As the published structures of the USDA257 factors (30) and those ascertained in this laboratory differed only by an additional molecule containing a C_16:1acyl chain, this approach is valid (Fig. 2).

Using the tomato cell suspension assay, we found that R.

fredii (pA16) transconjugants produce 20 times more Nod fac- tors than wild-type USDA257. HPLC analysis of the supernatants revealed three peaks (A-C) in the ratios 0.35:1:0.6, respectively. FAB spectra measured from the peaks showed a fragmentation sequence separated by 203 mass units which is characteristic of N-acetyl-^D-glucosamine oligomers. Peak A gave [M1Na]¹ at m/z 1424 and an associated peak (5%) yielded [M1Na]¹at m/z 1410. The former corresponds to Nod factors containing five N-acetyl-D-glucosamine residues, a C_16:1 acyl chain, 2-O-methylfucose, and an additional methyl group.

The smaller peak (5%) lacks the 2-O-methyl substitution on the fucose seen in wild-type factors. [M1Na]¹ions of peak B arose at m/z 1452. Again, this mass is 14 Da more than that of chitopentameric Nod factors of wild-type USDA257 possessing a C_18:1acyl chain. Sodium attached molecular ions from peak C [M1Na]¹arise at m/z 1454, 2 Da higher than those of peak B which correspond to an acyl chain of C_18:0rather than C_18:1. To locate the additional methyl group (14 Da), all products were studied in the presence of an acidified matrix to enhance the formation of MH¹ions and their fragmentation. It is clear that the methyl group is borne on the ion fragment corresponding to the non-reducing sugar (oxonium B1 ion). This result was confirmed by the metastable decay of MH¹ions (B/E scans) (Fig.

3B). The ion fragment at m/z 440 which arises from decompo- sition of the m/z 1430 (peak B) corresponds to the non-reducing

D-glucosamine end substituted by a C_18:1 acyl chain and an additional methyl group (14 Da).

Localization of the methyl group was performed by (a) ¹H NMR analysis, (b)¹³C NMR analysis, and (c) using gas chro- matography of the hydrolysis products from peak B. The¹³C NMR spectra gave a signal atd527.3 ppm which corresponds to N-CH₃. In¹H NMR (Fig. 4B), the singlet atd 52.7 ppm corresponds to the chemical shift of a methyl group bound to an amide nitrogen as in NodNGR and other N-methylated Nod factors (26, 27). This¹H NMR singlet is absent from the Nod factors of R. fredii USDA257 (30). Moreover, comparison of the gas chromatograph retention times of peracetyl derivatives of authentic N-methyl-^D-glucosamine and of the acid hydrolysates of peak B, confirmed the presence of an N-methyl group on the glucosaminyl end.

Another observation of relevance to the role of nodS concerns the fact that wild-type USDA257 produces a majority of pentamers with relatively small amounts of tetramers and trimers (30). Introduction of nodS into R. fredii results not only in N-methylation of the Nod factors, but also in the complete disappearance of products containing three or four glucosamine residues (Fig. 2). Even though pA16 lacks only the carboxyl terminus of nodU, and the Omega (V) insertion in pA26 is very close to the N terminus, USDA257 transconjugants harboring either pA16 or pA26 have the same phenotype (22). Given these data, it is not surprising that the Nod factors produced by R.

fredii (pA16) and R. fredii (pA26) are identical.

To discriminate between the roles played by nodS and nodU, the nod box of the nodSU operon was fused to the nodU gene.

This way, the nodU gene could be expressed independently of nodS but under the control of the same promoter (Fig. 1). The resulting plasmid pRAF25 confers the ability on USDA257 to nodulate Leucaena leucocephala (data not shown). In contrast to USDA257(nodS) transconjugants, R. fredii containing nodU (pRAF25) produces comparable amounts of Nod factors to those made by the wild-type USDA257 (tomato cell suspension culture assay). The HPLC profile of USDA257(pRAF25) transconjugants shows three peaks, D-F, which are present in the proportions 1:0.4:0.2, respectively. Using similar techniques to those described above, components with three, four, and five FIG. 1. Schematic representation of the way in which the nodU

gene was placed under control of the flavonoid-inducible nod box promoter of nodSU. nodU was cloned from pA18 which contains the complete nodSU operon, which is truncated in pA16 (22). The site of insertion of the spectinomycin resistant Omega (V) interposon in pA26 is also shown. B5BamHI, C5ClaI, E5EcoRI, H5HindIII, Hp5 HpaI, K5KpnI, Ps5PstI, P5PvuI, Sa5SalI, S5SmaI. MCS5 multiple cloning site.

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N-acetyl-D-glucosamine residues were found with a predomi- nance of the pentamer as in wild-type USDA257. Mass spec- trometry showed that the methylfucose was still present. Sim- ilarly, fraction D gave B1 ions at m/z 441 and 398. The latter corresponds to glucosamine bearing a C_16:1acyl chain as in Nod factors of wild-type USDA257. In the former, the shift up of 43 mass units is indicative of the presence an additional carbam-

oyl group. Similarly, fractions E and F contain mixtures of molecules that are either mono- or non-carbamoylated and possess either C_18:1or C_18:0acyl chains and three, four, or five N-acetyl-D-glucosamine residues (Fig. 2). B/E spectra performed on the different [M1H]¹ions (Fig. 3A) confirmed the presence of an additional 43 mass units on the non-reducing sugar. Thus, introduction of nodU into R. fredii induces a partial mono-carbamoylation at the non-reducing end.

Conjugation of both nodS and nodU (pA18) into R. fredii produced Nod factors than can be separated into three HPLC peaks which gave [M1H]¹ions at m/z 1445, 1473, and 1475, indicating a chitopentameric backbone (data not shown). B1 ions of these peaks at m/z 455, 483, and 485 correspond to N-acetyl-D-glucosamine possessing acyl chains of C_16:1or C_18:1 or C_18:0, respectively, that are shifted up by 57 mass units in comparison with ions from USDA257 Nod factors. This 57 Da difference corresponds to the sum of the mass of two groups: 43 Da for carbamate and 14 Da for the N-methyl group. B/E spectra performed on the [M1H]¹ion at m/z 1473 confirmed that these groups are attached to the non-reducing end (Fig.

3C). A¹H NMR signal atd52.7 ppm corroborated the presence of an N-methyl group which also gave a signal atd527.2 ppm in¹³C NMR (Fig. 4A). The same spectrum confirmed the pres- ence of a 6-O-carbamoyl group atd5156.5 ppm (Fig. 4A). The minor products (5% of the total) detected by FAB-MS also correspond to products containing five N-acetyl-^D-glucosamine residues which are partially carbamoylated but not 2-O-meth- ylated (data not shown).

Location of the Carbamoyl Groups—¹³C NMR analysis in FIG. 2. Structure of the major Nod

factors produced by Rhizobium. sp.

NGR234, R. fredii USDA 257, and var- ious derivatives thereof. n is the num- ber of N-acetyl-D-glucosamine residues.

R1 represents acyl chains, the carbon length and double bonds of which are in- dicated in the table (Carb5 carbamate group). The fatty acid compositions of these products were confirmed by GC analysis using authentic standards and by¹H NMR (data not shown). N-Acylated C18:Omolecules were also observed in Nod factors produced by the wild-type strain NGR234, but not the over-producing strain NGR(pA28) (S. Jabbouri, unpub- lished results).

FIG. 3. Constant B/E scans of mass spectra of [M1H]¹ions of the major products produced by R. fredii transconjugants. A, scans of the [M1H]¹ion at m/z 1256 which is the major product of USDA258(nodU) transconjugants. This chitotetrameric component bears one carbamoyl group, a vaccenyl residue on the non-reducing end, and a 2-O-methylfucose on the reducing sugar. B, scans of the [M1H]¹ ion at m/z 1430, which is the major product of USDA257(nodS) transconjugants. This chitopentameric component bears an N-methyl group, a vaccenyl residue on the non-reducing sugar, and a 2-O-meth- ylfucosyl on the reducing terminus. C, scans of the [M1H]¹ion at m/z 1473 Da which is the major product of USDA257(nodSU) transconju- gants. This component bears a single carbamoyl group, an N-methyl, and a vaccenyl residue on the non-reducing end together with a 2-O- methylfucosyl group on the reducing sugar.

FIG. 4. A,¹³C NMR spectrum (in deuterated Me2SO at 400 MHz) of the major Nod factors produced by USDA257(nodSU) transconjugants.

The characteristic carbon shift of N-CH3is at d527.3 ppm and of 6-O-CONH2is at 156.5 ppm. B,¹H NMR spectrum of the major Nod factors produced by USDA257(nodS) transconjugants. The characteris- tic proton shift of N-CH3is atd52.7 ppm.

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deuterated-Me₂SO of Nod factors produced by the over-producing strain NGR234(pA28) gave three signals at 156.5, 156.1, and 155.7 ppm, indicative of carbamate groups possibly at C-3, -4, or -6 (data not shown). In addition, FAB-MS spectra of the non-sulfated products showed B1 ion fragments at m/z 526, 483, and 440, which correspond to bis-, mono-, and non-car- bamoylated species, respectively, that are N-methylated and possess a C_18:1acyl chain. Since it was not possible to separate these compounds by HPLC, all attempts to identify the exact positions of the carbamoyl groups failed. Accordingly, another approach based onb-elimination of the C-3 (or C-4) substituents on the B1 oxonium ion, and using factors produced by different strains, was chosen. Indeed, the H-C(2) or H-C(5) proton of B1 ions generated by fragmentation of [M1H]¹ions is acidic and leads to facileb-elimination of the C-3 (or C-4) substituent. Thus, the metastable ion spectra (B/E scans) of the ion fragment at m/z 483 corresponding to the mono-carbamoy- lated species (of NodNGR(pA28) factors) gave fragments at m/z 465 and 422 (Fig. 5B). This loss of 61 Da (carbamic acid) or 18 Da (H₂0) suggests that a carbamoyl group is partially present at C-3 or at C-4. Data obtained bybelimination do not exclude, however, that carbamoyl groups in mono-carbamoy- lated NodNGR factors could be at C-6. Nod factors of Azorhi- zobium caulinodans (which are carbamoylated at C-6) were used to verify this technique (see Fig. 5A).

Constant B/E scan spectra of the ion fragment at m/z 483 of Nod factors (mono-carbamoylated species) produced by NGRV26 (5NodU²) shows an ion fragment at m/z 422 which originated frombelimination of carbamic acid (Fig. 5C). This indicates that the carbamoyl group produced by NodU²strains is at least partially located on C-3 or C-4. Periodate oxidation followed by reduction with NaBD₄ (26) did not give cleavage products containing a carbamoyl group, ruling out carbamoylation at position C-6. In contrast, periodate cleaves the non- carbamoylated Nod factors which are also present, as revealed by a 4 mass units shift up of the B1 ion following reduction by NaBD₄(data not shown). In other words, mutational analysis suggests that nodU codes for an enzyme with 6-O-carbamoyl- transferase-like activity. It is evident, however, that 3-O- (and/or 4-O-) carbamoyl transferases must also exist in NGR234. This is confirmed by the two singlets atd5156.1 and d5155.7 ppm in the¹³C spectra of nodU²mutants of NGR234 (data not shown).

Additional proof that nodU directs C6-O-carbamoylation was obtained by examination of the metastable ion spectra per- formed on m/z 469 and 483 of mono-carbamoylated B1 ions formed in the FAB spectrum of Nod factors produced by USDA257 containing nodU (USDA257(pRAF25)) as well as USDA257 and nodSU (USDA257(pA18)) transconjugants. The latter ion species bear an additional N-linked methyl group.b elimination yielded ion fragments at m/z 451 and 465, respec- tively, which originate from the loss of 18 Da (5H₂O), showing that the carbamate cannot be at C-3 or C-4 (Fig. 5, D and E).

Moreover, these sugar rings can be oxidized by periodate which is indicative of free OH groups at C-3 and C-4.¹³C NMR spectra of the major product of R. fredii(pA18) transconjugants gave a singlet atd5156.5 (Fig. 4A) which suggests a single carbamoyl group. The resonance of carbon C-6, identified by DEPT (dis- tortionless enhancement by polymerization transfer) analysis, showed a down-field shift of 3 ppm. This confirms that the carbamoyl group is positioned on C-6. This shift, relative to a free -CH₂OH group was not observed in supernatants obtained from NGRV26 (which bear a carbamoyl group at C-3 or C-4).

Moreover, carbamoylation at position C-3 affects the proton resonance of the neighboring N-CH₃ proton group (d 50.03 ppm).

DISCUSSION

We previously noted the striking nucleotide and amino acid sequence homologies between nodS of NGR234 and USDA257 (22, 23). Similarly, the nodU gene of NGR234 (EMBL, Gen- Bank, and DDBJ accession number X89965) shows significant similarities to the amino acid sequeces of NodU of R. fredii USDA257 (23), B. japonicum USDA110 (24), A. caulinaudans (25), and ORF10 of Nocardia lactamdurans (31) (data not shown). ORF10 of Nocardia is implicated in the biosynthesis of the cephamycin family of carbamoylatedb-lactam antibiotics.

Nevertheless, the fact that such well conserved and apparently functional genes behave differently in Rhizobium sp. NGR234 and B. japonicum USDA110 suggest that the NodU protein also may act on other substrates such as lipopolysaccharides as has recently been reported (32). We are currently investigating this possibility.

Our physical/chemical data suggest that the nodS gene prod- uct is involved in N-linked methyltransferase activity, while FIG. 5. Constant B/E scans of B1 oxonium ions originating from fragmentation of the non-reducing end of various Nod factors. A, scans of the B1 ion at m/z 483 of A. caulinodans Nod factors. These factors are N-methylated, possess a C18:1acyl chain, and are C6-O-carbamoylated. They were used as a reference compound. The main daughter ion of m/z 483 is due to loss of water at m/z 465. Losses of 61 mass units were not detected. B, scans of the B1 ion m/z 483 of the overproducing strain, Rhizobium sp. NGR(pA28) Nod factors. Two com- pletebeliminations are seen representing either loss of water (m/z 465) or of carbamic acid (61 Da) at m/z 422. C, scans of the B1 ion at m/z 483 of Nod factors produced by Rhizobium sp NGRV26 (5nodU²).

The main daughter ion at m/z 422 (261 Da) shows that the carbamate group is at C-3 (or C-4). D, scans of the B1 ion at m/z 483 of Nod factors produced by USDA257(nodSU) transconjugants. The main ion shows the loss of water at m/z 465. E, scans of the B1 ion at m/z 469 of Nod factors produced by USDA257(nodbox::nodU) transconjugants. These factors differ from those of Azorhizobium by the absence of the N- methyl group.belimination generated an ion (m/z 451) which resulted from the loss of water. F, scans of the B1 ion at m/z 440 of Nod factors produced by USDA257(nodS).belimination shows the loss of water.

(7)

that of the nodU gene is probably a 6-O-carbamoyltransferase.

An impediment to elucidating the role of nodSU in NGR234 is the probable existence of other genes encoding similar func- tions. Mutations in nodS drastically reduce Nod factor produc- tion, but suppression of nodU has no effect on Nod factor levels (28). Thus, definitive analysis of nodS could only be obtained by introducing it into USDA257. Although both nodSU exist in USDA257, a deletion in the promoter region drastically reduces transcription of the operon (23). In spite of the extraordinary similarities between the two genomes, USDA257 produces Nod factors that differ from those of NGR234 by the absence of acetyl and sulfated groups on the 2-O-methylfucose moiety and which lack both the N-linked methyl group as well as carbam- oyl groups on the non-reducing sugar. In R. fredii harboring nodS of NGR234, tri- and tetrameric Nod factors are no longer produced, while N-methylation of the acyl chain of the pentam- eric species occurs. NodU causes partial 6-O-mono-carbamo- ylation of the non-reducing sugar but does not control the length of the oligomer. Together, nodSU yield pentamers that are N-methylated and mono-carbamoylated on C-6. Surpris- ingly, the activity of either gene is sufficient to confer nodula- tion of Leucaena species. Indeed, there is no apparent combi- nation of N-linked methyl and carbamoyl groups with the number of saccharide repeating units which permits nodula- tion of Leucaena. This contrasts starkly with the requirement of sulfated Nod factors of R. meliloti for nodulation of Medicago sativa (16). Nevertheless, mutations in nodS prevent NGR234 from nodulating Leucaena sp. (22, 23). It should be noted, however, that nodSU of both B. japonicum and R. fredii are unable to complement nodSU²mutants of other (brady)rhizobia, including NGR234 (24).

Acknowledgments—S. Relic´ and D. Gerber are thanked for their assistance with many aspects of this work.

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Involvement of <i>nodS</i> in <i>N</i>-Methylation and <i>nodU</i> in 6-<i>O</i>-Carbamoylation of <i>Rhizobium</i> sp. NGR234 Nod Factors