(Received for publication, May 25, 1995; and in revised form, July 24, 1995)
From the
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 factor production, but with the exception of sulfated factors (which are partially N-methylated and mono-carbamoylated), they are identical to those of the wild-type strain. Thus, Nod factor levels, their degree of oligomerization, and N-methylation are linked to the activity encoded by nodS.
Symbiotic soil bacteria of the genera Azorhizobium,
Bradyrhizobium, and Rhizobium (collectively termed
rhizobia) interact with the roots of legumes to form nodules in which
atmospheric nitrogen is reduced to ammonia. Signal exchange between the
symbionts regulates the expression of both bacterial 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 deformation 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
Nod mutants to enter the legume roots and to form
nitrogen-fixing nodules(4, 7) . All Nod factors so far
identified are
-1,4-linked tri- or tetra- or pentamers of N-acetyl-D-glucosamine, N-acylated 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 with D-arabinose, 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-acetylglucosaminyltransferase 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 NH 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 permit 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 Bradyrhizobium 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. japonicum(24) , and Azorhizobium caulinodans(25) , but no correlation
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 nodS mutant 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
similarities 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 introducing nodSU, nodS, and nodU into R. fredii USDA257
and by analyzing the Nod factors produced by the transconjugants.
Analysis of the Nod factors produced by nodS and nodU
mutants 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 expression
of the nodU gene.
Figure 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 ()
interposon in pA26 is also shown. B = BamHI, C = ClaI, E = EcoRI, H = HindIII, Hp = HpaI, K = KpnI, Ps = PstI, P = PvuI, Sa = SalI, S = SmaI. MCS =
multiple cloning site.
Figure 2:
Structure of the major Nod factors
produced by Rhizobium. sp. NGR234, R. fredii USDA
257, and various derivatives thereof. n is the number of N-acetyl-D-glucosamine residues. R1 represents acyl chains, the carbon length and double bonds of
which are indicated in the table (Carb = carbamate
group). The fatty acid compositions of these products were confirmed by
GC analysis using authentic standards and by H NMR (data
not shown). N-Acylated C
molecules were also
observed in Nod factors produced by the wild-type strain NGR234, but
not the over-producing strain NGR(pA28) (S. Jabbouri, unpublished
results).
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 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 nodSU mutant (=
NGR
25) (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 nodU
mutant produced similar amounts of Nod
factors to the wild-type bacterium. Together, the low amounts and
complex mixtures of the biologically active products produced by the nodSU
strain rendered their analysis
difficult. Nevertheless, the FAB-MS spectra in the negative-ion mode
gave [M-H]
ions predominately at m/z 1494, 1537, 1466, and 1509 which correspond to
non-N-methylated but fucosylated, sulfated,
C
-acylated, mono- or non-carbamoylated pentamers, and
non-methylated, fucosylated, sulfated, C
-acylated, mono-
or non-carbamoylated pentamers, respectively. Exhaustive butanol
extraction of the membrane fraction derived from NGR
25 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-carbamoylation of NodNGR factors.
Using the
tomato cell suspension assay, we found that R. fredii (pA16)
transconjugants produce 20 times more Nod factors 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 [M+Na] at m/z 1424 and an associated peak (5%) yielded
[M+Na]
at m/z 1410. The former
corresponds to Nod factors containing five N-acetyl-D-glucosamine residues, a C
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.
[M+Na]
ions 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
acyl chain. Sodium attached molecular ions from peak C [M+Na]
arise at m/z1454, 2 Da higher than those of peak B which
correspond to an acyl chain of C
rather than
C
. To locate the additional methyl group (14 Da), all
products were studied in the presence of an acidified matrix to enhance
the formation of MH
ions 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 MH
ions (B/E
scans) (Fig. 3B). The ion fragment at m/z 440
which arises from decomposition of the m/z 1430 (peak B)
corresponds to the non-reducing D-glucosamine end substituted
by a C
acyl chain and an additional methyl group (14
Da).
Figure 3:
Constant B/E scans of mass spectra of
[M+H] ions of the major products
produced by R. fredii transconjugants. A, scans of
the [M+H]
ion 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
[M+H]
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-methylfucosyl on
the reducing terminus. C, scans of the
[M+H]
ion at m/z 1473 Da which
is the major product of USDA257(nodSU) transconjugants. 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.
Localization of the methyl group was performed by (a) H NMR analysis, (b)
C NMR analysis,
and (c) using gas chromatography of the hydrolysis products
from peak B. The
C NMR spectra gave a signal at
= 27.3 ppm which corresponds to N-CH
.
In
H NMR (Fig. 4B), the singlet at
= 2.7 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) . This
H 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.
Figure 4:
A, C NMR spectrum (in deuterated Me
SO at 400 MHz)
of the major Nod factors produced by USDA257(nodSU)
transconjugants. The characteristic carbon shift of N-CH
is
at
= 27.3 ppm and of 6-O-CONH
is at
156.5 ppm. B,
H NMR spectrum of the major Nod
factors produced by USDA257(nodS) transconjugants. The
characteristic proton shift of N-CH
is at
=
2.7 ppm.
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 () 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 N-acetyl-D-glucosamine residues were found with
a predominance of the pentamer as in wild-type USDA257. Mass
spectrometry showed that the methylfucose was still present. Similarly,
fraction D gave B1 ions at m/z 441 and 398. The latter
corresponds to glucosamine bearing a C acyl 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 carbamoyl group.
Similarly, fractions E and Fcontain mixtures of
molecules that are either mono- or non-carbamoylated and possess either
C
or C
acyl chains and three, four, or
five N-acetyl-D-glucosamine residues (Fig. 2).
B/E spectra performed on the different [M+H]
ions (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
[M+H] ions 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
or C
or C
, 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
[M+H]
ion at m/z 1473
confirmed that these groups are attached to the non-reducing end (Fig. 3C). A
H NMR signal at
=
2.7 ppm corroborated the presence of an N-methyl group which
also gave a signal at
= 27.2 ppm in
C NMR (Fig. 4A). The same spectrum confirmed the presence of
a 6-O-carbamoyl group at
= 156.5 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-methylated (data not shown).
Figure 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 C acyl 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 complete
eliminations 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 NGR
26 (= nodU
). The main daughter ion at m/z 422 (-61 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.
elimination
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).
elimination shows the
loss of water.
Constant B/E scan spectra
of the ion fragment at m/z 483 of Nod factors
(mono-carbamoylated species) produced by NGR26 (=
NodU
) shows an ion fragment at m/z 422 which
originated from
elimination of carbamic acid (Fig. 5C). This indicates that the carbamoyl group
produced by NodU
strains is at least partially
located on C-3 or C-4. Periodate oxidation followed by reduction with
NaBD
(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 NaBD
(data not shown). In other
words, mutational analysis suggests that nodU codes for an
enzyme with 6-O-carbamoyltransferase-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 at
= 156.1 and
= 155.7 ppm in the
C spectra of nodU
mutants of
NGR234 (data not shown).
Additional proof that nodU directs
C6-O-carbamoylation was obtained by examination of the
metastable ion spectra performed 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.
elimination yielded ion fragments at m/z 451 and 465,
respectively, which originate from the loss of 18 Da (=
H
O), 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.
C NMR spectra of the major product of R.
fredii(pA18) transconjugants gave a singlet at
=
156.5 (Fig. 4A) which suggests a single carbamoyl
group. The resonance of carbon C-6, identified by DEPT (distortionless
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 -CH
OH group was not
observed in supernatants obtained from NGR
26 (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-CH
proton group (
= 0.03 ppm).
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, GenBank, 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 carbamoylated -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 product is involved in N-linked methyltransferase
activity, while 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 functions. Mutations in nodS drastically reduce Nod factor production, 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 carbamoyl 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 pentameric species
occurs. NodU causes partial 6-O-mono-carbamoylation 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. Surprisingly, the activity of either
gene is sufficient to confer nodulation of Leucaena species.
Indeed, there is no apparent combination of N-linked methyl
and carbamoyl groups with the number of saccharide repeating units
which permits nodulation 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 nodSU mutants of other (brady)rhizobia, including NGR234(24) .