(Received for publication, July 6, 1995; and in revised form, September 26, 1995)
From the
By in vitro and in vivo studies with Escherichia coli expressing different combinations of the nodABCS genes of Azorhizobium caulinodans, Nod factor intermediates were identified and their structures determined using mass spectrometry. Substrate-product relationships were studied by time course experiments, and the Nod factor biosynthetic pathway was partially resolved. E. coli strains, harboring nodA and/or nodB, did not produce Nod metabolites, whereas the strain expressing nodC produced chitooligosaccharides. Thus, the first committed step was the production of the carbohydrate backbone. Bacitracin and tunicamycin did not affect this step, suggesting that undecaprenyl pyrophosphate-linked intermediates were not involved. The second step was the deacetylation of chitooligosaccharides by NodB since the E. coli strain expressing nodBC produced chitooligosaccharides, deacetylated at the non-reducing end and since the NodC products were precursors of the NodBC products. A strain expressing nodBCS produced N-methylated oligosaccharides, whereas a strain expressing nodCS produced unmethylated oligosaccharides. Time course experiments showed that methylation occurred after deacetylation. Thus, NodS acted after NodB. The NodBCS metabolites were partially converted to lipo-chitooligosaccharides when the nodABCS genes were expressed, showing that NodA was involved in the acylation and acted after NodS.
The unique ability of rhizobia to nodulate leguminous plants
largely depends on signal molecules that trigger the
nodule-developmental program. These regulatory molecules, called Nod
factors, consist of a lipo-chitooligosaccharide (LCO) ()core, modified with several substitutions(1) . The
core contains a tri- to pentamer of
-1,4-linked GlcNAc residues
and a fatty acyl moiety that replaces the N-acetyl group of
the nonreducing end. This structure is common to all Nod factors,
independent of their origin. However, the type of modifications, the
length of the sugar chain, and the nature of the fatty acid differ from
strain to strain. For example, the Nod factors produced by Azorhizobium caulinodans strain ORS571 are pentamers carrying
a vaccenoyl or stearoyl chain. On the nonreducing end, the
oligosaccharide is modified with a N-methyl and O-carbamoyl group, and the reducing end is branched with a D-arabinosyl sugar (2) .
For the production of Nod factors, rhizobia are dependent on their nodulation (nod) genes. The nodABC genes are absolutely required for the synthesis of Nod factors and, hence, for nodulation. NodC produces chitooligosaccharides(3, 4) , NodB removes the N-acetyl group of their nonreducing end(5) , and NodA adds an acyl chain(6, 7) .
Modifications of the Nod
factor core are dependent on nod genes that are strain
specific. A. caulinodans has an operon containing the genes nodABCSUIJZorf9(8, 9) . ()We have
shown that the nodS gene is involved in Nod factor methylation
and that the NodS protein is an AdoMet-dependent methyl transferase
that uses deacetylated chitooligosaccharides as
substrate(9, 10) . nodU is involved in
carbamoylation of Nod factors and nodZ and/or other
downstream-located genes in arabinosylation.
The
biochemical activities were ascribed to the NodABCS proteins based on in vitro experiments using synthetic substrates. The
occurrence of the substrates and/or products had not been demonstrated in vivo. Here, we describe the isolation and chemical
characterization of Nod factor intermediates produced in Escherichia coli expressing nodABCS in different
combinations. Our results allow us to propose a biosynthetic pathway
for Nod factors in A. caulinodans.
Figure 1:
Map of the different
constructs used in this study. The nod genes located on the
different plasmids are indicated in the name. The constructs were
introduced into the E. coli strain DH5. The numbers indicate the position of the primers used for cloning. Restriction
enzymes used are Ba, BamHI; M, MscI; Sa, SalI; P, PstI; Pp, PpuMI; and Sp, SphI. bp, base pairs.
The
water phase was loaded on a Sephadex A-25 anion exchange column
(Pharmacia Biotech, Uppsala, Sweden) (gel volume, 500 µl)
equilibrated with water. The column was washed with 2 ml of water, and
the unbound fraction was vacuum dried. Samples were analyzed by silica
gel TLC(3) . The C-labeled standards were obtained
by reacetylation of hydrolyzed chitin with
[1-
C]acetic anhydride (30 mCi/mmol, Amersham).
The n-butyl alcohol phase was washed three times with 100 µl of water, vacuum dried, and dissolved in 500 µl of water. This solution was washed three times with 500 µl of ethyl acetate and vacuum dried. The samples were analyzed by octadecyl silica TLC (2) .
For labeling experiments in the presence of
antibiotics, tunicamycin (10 µg/ml, Sigma) or bacitracin (150
µg/ml, Sigma) were added at the time of induction. For the
experiment, described in Fig. 6, a DH5(pUCNABCS) culture
was grown at 25 °C in the presence of
[
C]GlcNAc (5 µCi/ml). The incubations were
stopped at different time points. Quantification was done with a
PhosphorImaging system from Molecular Dynamics, using the Image
Quant
software.
Figure 6:
Production of NodBCS and NodABCS
metabolites by DH5(pUCNABCS) in function of time. Nod metabolites
were labeled in vivo using [
C]GlcNAc,
extracted from cells at the indicated times, analyzed by TLC, and
quantified with a PhosphorImager.
Figure 5:
Conversion of NodBC metabolites to NodBCS
metabolites. A, TLC analysis of Nod metabolites produced in vivo from [C]GlcNAc (lanes
2-4) or [
H]methionine (lanes
5-7). Lane 1, chitooligosaccharide standards; Nod
metabolites were extracted from the following: DH5
(pUCNCS) (lanes 2, 5), DH5
(pUCNBC) (lanes 3, 6), and DH5
(pUCNBCS) (lanes 4, 7). B, TLC analysis of an in vitro time course experiment
showing conversion of NodC metabolites to NodBC metabolites to NodBCS
metabolites in function of time. Protein extracts of ORS571(pRG70) were
incubated with [
C]GlcNAc
at 30
°C. Reactions were stopped at different time points indicated in minutes under the lanes. In the first lane,
chitooligosaccharide standards were loaded. The positions of 1
(GlcNAc
), 2 (GlcNGlcNAc
), and 3
(GlcNCH
GlcNAc
) are indicated with arrows. The arrowheads indicate the origin of the
TLCs.
The NodABCS metabolites were further purified as was done for Nod factors of ORS571(pRG70)(2) . The elution times of the NodABCS metabolites in the final HPLC purification step are given in Table 1.
The
aqueous solutions containing the NodC, NodBC, or NodBCS metabolites
were vacuum concentrated to 10 ml, and trichloroacetic acid was added
to a final concentration of 10%. The precipitate was centrifuged, and
the supernatant was concentrated to 1 ml. This was loaded on a
Bio-Gel P-2 column (Bio-Rad; 90
1.5 cm, 20 ml/h, eluent 0.1 M pyridine-acetate in water, pH 6.0), and fractions of 2 ml
were collected. Nod metabolites were finally purified using an Econosil
NH
5-µl, 250
4.6-mm HPLC column (Alltech,
Deerfield, IL) with a gradient from 80 to 50% CH
CN in 30
min. Elution times (in minutes) of the different Nod metabolites are
11.8, 15.0, 18.3, and 20.1 for the NodC metabolites GlcNAc
,
GlcNAc
, GlcNAc
, and GlcNAc
,
respectively, 24.4 for the NodBC metabolite GlcNGlcNAc
, and
27.5 for the NodBCS metabolite GlcNCH
GlcNAc
.
Fatty
acids were analyzed as methyl esters with gas chromatography using a
5890A instrument (Hewlett-Packard, Wilmington, DE) equipped with an
Ultra 2 silica capillary column coated with methyl phenyl silicone (25
m 0.2 mm
0.33 µm; Hewlett-Packard).
Figure 2:
In vivo and in vitro synthesis of radioactively labeled Nod metabolites. A,
direct phase TLC analysis of water-soluble products, produced in
vivo from [C]acetate. Nod metabolites were
produced by the following: lane 1, DH5
(pUCNA); lane
2, DH5
(pUCNB); lane 3, DH5
(pUCNAB); lane
4, DH5
(pUCNC); lane 5, DH5
(pUCNBC); lane
6, DH5
(pUCNABC); lane 7, DH5
(pUCNBCS); lane
8, DH5
(pUCNABCS); lane 9, chitooligosaccharide
standards. B, reverse-phase TLC analysis of butyl
alcohol-soluble products, produced in vivo from
[
C]acetate (lanes 1-8, 13 and 14), [
C]GlcNAc (lanes
9-10), or [
H]methionine (lanes
11-12). Nod metabolites were produced by the following: lane 1, DH5
(pUCNA); lane 2, DH5
(pUCNB); lane 3, DH5
(pUCNAB); lane 4, DH5
(pUCNC); lane 5, DH5
(pUCNBC); lane 6, DH5
(pUCNBCS); lane 7, DH5
(pUCNABC); lane 8,
DH5
(pUCNABCS); lane 9, DH5
(pUCNABC); lane
10, DH5
(pUCNABCS); lane 11, DH5
(pUCNABC); lane 12, DH5
(pUCNABCS); lane 13, ORS571 (induced
with 10 µM naringenin); lane 14, ORS571
(uninduced). LCO indicates the position of lipo-chitooligosaccharides. C, direct-phase TLC analysis of in vitro synthesized
Nod metabolites from UDP-[
C]GlcNAc. Lane
1, chitooligosaccharide standards; Nod metabolites were produced
by protein extracts from the following: lane 2,
DH5
(pUCNC); lane 3, DH5
(pUCNBC); lane 4,
DH5
(pUCNBCS); lane 5, DH5
(pUCNBCS) in the presence
of AdoMet; lane 6, ORS571(pRG70); lane 7,
ORS571(pRG70) in the presence of AdoMet; lane 8,
ORS571-1.59S(pRG70-1.59S) in the presence of AdoMet; lane 9, ORS571-1.31U(pRG70-1.31U) in the presence
of AdoMet. Arrow 1 indicates the position of
GlcNAc
, arrow 2 of GlcNGlcNAc
, and arrow 3 of GlcNCH
GlcNAc
. Arrowheads indicate the origin of the
TLCs.
Figure 3:
CID
mass spectra of Nod metabolites. A, NodC metabolites with
[M+H] at m/z 1034. B,
NodBC metabolites with [M+H]
at m/z 992. C, NodBCS metabolites with
[M+H]
at m/z 1006. D,
NodABCS metabolites with [M+H]
at m/z 1244.
Bacterial polysaccharides such as peptidoglycan and exopolysaccharides are synthesized on a undecaprenyl phosphate lipid carrier(12) . We investigated whether the NodC metabolites are also synthesized on such a carrier. Several attempts to isolate lipid-linked intermediates failed. Moreover, the antibiotics tunicamycin, which inhibits the transfer of GlcNAc-1-phosphate from UDP-GlcNAc to undecaprenyl phosphate, and bacitracin, which inhibits the dephosphorylation of undecaprenyl pyrophosphate, had no effect on the synthesis of the NodC metabolites neither in vitro nor in vivo (data not shown).
Figure 4:
Conversion of NodC metabolites to NodBC
metabolites. A, TLC analysis of an in vitro time
course experiment showing conversion of NodC metabolites to NodBC
metabolites in function of time. Protein extracts of ORS571(pRG70) were
incubated with UDP-[C]GlcNAc at 30 °C.
Reactions were stopped at different time points indicated in minutes under the lanes. B, TLC analysis of
an in vitro incubation with ORS571(pRG70) protein extract at
30 °C during 90 min. The reaction mixture was divided in two parts.
One part served as control(-); the second part was chemically N-acetylated (+). Positions of chitooligosaccharide
standards are indicated with a bar. The arrowheads indicate the origin of the TLCs.
The NodB protein of Rhizobium meliloti deacetylates the nonreducing end residue of
chitooligosaccharides(5) . We investigated whether the novel
metabolites were the deacetylated forms of the NodC metabolites and
whether they were made by NodB. The in vitro and in vivo synthesis of the NodC metabolites and/or the novel metabolites was
analyzed in the strains DH5(pUCNBC) and DH5
(pUCNB) (Fig. 1). These experiments (Fig. 2, lanes A2, A5, and C3) showed that indeed the NodB protein was
involved in the synthesis of the novel metabolites and that their
synthesis also depended on the NodC protein. Therefore, they were
called the NodBC metabolites.
Chemical N-acetylation of the
NodBC metabolites resulted in the formation of products that migrated
on TLC (Fig. 4B) or HPLC (data not shown) at the
position of GlcNAc standards and NodC metabolites,
suggesting that the NodBC metabolites are indeed deacetylated
chitooligosaccharides. To prove this and to localize the GlcN residue,
the NodBC metabolites were synthesized in vivo by the
DH5
(pUCNBC) strain. The putative pentasaccharide was purified and
analyzed by mass spectrometry. The
[M+H]
pseudo-molecular ion was
determined at m/z 992 in agreement with the mass of a
pentasaccharide of four GlcNAc residues and one GlcN residue. The
CID-MS spectrum (Fig. 3B) showed fragment ions at m/z 771, 568, 365, and 162. This fragmentation corresponded to
GlcNAc
, which is deacetylated at the nonreducing end.
The chemical structure of a pentameric NodBCS metabolite was
determined by FAB-MS and CID-MS (Fig. 3C). The
[M+H] pseudo-molecular ion was at m/z 1006, and fragment ions were at m/z 785, 582,
379, and 176. These ions were 14 mass units higher than the ions of the
NodBC metabolites, in agreement with the presence of a methyl group at
the nonreducing end of a deacetylated chitopentasaccharide.
The NodS
protein methylates in vitro deacetylated chitooligosaccharides
but not chitooligosaccharides(10) . An in vivo labeling experiment with the strains DH5(pUCNBCS),
DH5
(pUCNCS) (Fig. 1), and DH5
(pUCNBC) and using the
precursors [
C]GlcNAc or
[
H]methionine (methionine is a precursor for
AdoMet) was performed (Fig. 5A). The three strains
incorporated the [
C]GlcNAc label in
chitooligosaccharides. In contrast, the
[
H]methionine label was only incorporated in the
chitooligosaccharides produced by DH5
(pUCNBCS). This observation
showed that methylation by NodS needs the prior action of NodB. An in vitro time course experiment further confirmed this. As
substrate, chemically synthesized
[
C]GlcNAc
was used. TLC analysis
showed that the substrate was first converted to the deacetylated form
and then to the methylated, deacetylated form (Fig. 5B).
Therefore, the involvement of the azorhizobial NodA protein in
acylation was studied by in vivo labeling experiments using
DH5(pUCNABC) and DH5
(pUCNABCS) (Fig. 1). Radioactive
precursors used were [
C]acetate,
[
C]GlcNAc, or
[
H]methionine, and the production of
lipo-chitooligosaccharides was analyzed by reverse-phase TLC. Both
strains incorporated [
C]acetate or
[
C]GlcNAc into molecules that comigrated on the
TLC system with the Nod factors produced by ORS571(pRG70) (Fig. 2B). With [
H]methionine,
however, these metabolites could only be labeled in the strain
DH5
(pUCNABCS). The DH5
strains containing nodA, nodAB, nodB, nodC, nodBC, or nodBCS did not produce these Nod metabolites (Fig. 2B). These results demonstrated that NodABC was
absolutely required and sufficient for the production of the Nod factor
core and that the NodA protein was involved in the acyl transfer.
The NodABCS metabolites were purified from DH5(pUCNABCS) cells.
The HPLC chromatogram of the final purification step displayed several
peaks eluting in the region where azorhizobial Nod factors elute. These
peaks were analyzed with FAB-MS and CID-MS, and in eight of them, LCOs
were identified. As an example, the CID-MS spectrum of one of the major
products is shown in Fig. 3D. The
[M+H]
pseudo-molecular ion was at m/z 1244, and the fragment ions were at m/z 1023,
820, 617, and 414. This pattern corresponded to a pentasaccharide of
GlcNAc with, on the nonreducing end, a C
and a methyl
substitution. The CID-MS spectra of the other products were exactly the
same (loss of 221, followed by three losses of 203 and with the ion
derived from two GlcNAc losses as the most intense) except that the
masses differed. This means that all the molecules consisted of five
GlcNAc but that the substitutions at the nonreducing end differed. To
obtain additional structural information, the fatty acids were
determined by gas chromatography. The results, together with the data
from the mass spectrometry, are summarized in Table 1. The HPLC
peaks corresponding to the major NodABCS products (with a
C
, C
, or C
fatty acid) and
some of the minor products were accompanied with a slightly slower
migrating peak with approximately the same intensity. Products in these
peaks showed the same MS spectrum as the products in the preceding peak
except that they had a mass that is 42 units higher. This might
indicate that these products were NodABCS metabolites, O-acetylated at the nonreducing end, a substitution that might
be the result of an endogenous O-acetyl transferase.
From
the analysis of the water-soluble products of the in vivo labeling experiments with the strains DH5(pUCNABC) and
DH5
(pUCNABCS), it is clear that the NodBC and NodBCS metabolites,
respectively, were still present, even after overnight incubation (Fig. 2A, lanes 6 and 8). The in
vivo production of the NodBCS and NodABCS products was followed in
function of time. DH5
(pUCNABCS) cells were labeled with
[
C]GlcNAc, and, at regular time intervals,
samples were analyzed. Both the NodBCS and NodABCS metabolites
increased equally before reaching a maximum level (Fig. 6) at
which they remained constant, even after 24 h of incubation (data not
shown). The accumulation of NodBCS metabolites was not due to a stop in
Nod metabolite synthesis. A control experiment, in which cells were
incubated with [
C]acetate or
[
C]GlcNAc at the time point where the NodBCS
metabolites reached their constant level, showed that Nod metabolite
synthesis was still going on (data not shown).
nod genes of rhizobia code for proteins that are
involved in the biosynthesis of lipo-chitooligosaccharide Nod factors.
Azorhizobial nodABCS genes were introduced in various
combinations into E. coli strain DH5, and we found that nodABC genes are necessary and sufficient to produce the Nod
factor core. In the case of the nodABCS construct, the LCOs
are pentamers acylated at the nonreducing end with fatty acids varying
in length from C
to C
. The MS data,
together with the [
H]methionine in vivo labeling studies, further show that the NodABCS metabolites are
methylated at the nonreducing end, except for two minor compounds (see Table 1). This confirms that NodS is a methyl transferase
involved in Nod factor methylation(9, 10) .
Data obtained from analysis of the Nod factor intermediates produced by E. coli strains expressing nodC, nodBC, nodBCS, nodABC, and nodABCS are in agreement with the described in vitro enzymatic activities of the proteins encoded by these genes and show that these enzymes produce in vivo the same products as in vitro: NodC is a N-acetylglucosaminyl transferase synthesizing chitooligosaccharides with polymerization degree from 2 to 5, NodB deacetylates these products at the nonreducing end, NodS methylates the deacetylated products, and NodA transfers an acyl chain to the free amine at the nonreducing end. The variety of acyl chains found in the NodABCS metabolites produced in an E. coli background shows that the NodA protein of A. caulinodans has low specificity for the chain length of the fatty acid.
Bacterial polysaccharides are often synthesized on the lipid carrier undecaprenyl phosphate(12) , and it was suggested that such lipids could be involved in Nod factor synthesis(1, 4) . However, we found no evidence for this hypothesis. First, we never detected lipid-linked intermediates in vitro nor in vivo. Second, the antibiotics tunicamycin and bacitracin had no effect on the synthesis of Nod factors or NodC metabolites in vivo and in vitro in A. caulinodans and E. coli. Tunicamycin specifically inhibits the transfer of GlcNAc-1-phosphate from UDP-GlcNAc to polyprenyl monophosphate acceptors. For example, in E. coli, the in vitro synthesis of GlcNAc-pyrophosphoryl-undecaprenol, an intermediate in the synthesis of enterobacterial common antigen, is totally inhibited by tunicamycin (13) as is the in vivo synthesis of enterobacterial common antigen(14) . Bacitracin is a polypeptide antibiotic, inhibiting the dephosphorylation of polyprenol-pyrophosphate, thereby preventing the use of the lipid carrier(15) .
NodC is homologous to yeast chitin synthases (16) and hyaluronan synthase of group A streptococci(17) . Also for these enzymes, no lipid-linked intermediates could be identified, and tunicamycin had no effect on the activity(18, 19, 20, 21) .
The in vitro time course ( Fig. 4and Fig. 5B) and the in vivo labeling of Nod
metabolites in the strains DH5(pUCNCS) and DH5
(pUCNBCS) (Fig. 5A) show the substrate-product relationship for
the NodC-NodBC and NodBC-NodBCS products and that NodC, NodB, and NodS
act in this order. The strain DH5
(pUCNABCS) produces both NodABCS
and NodBCS metabolites, suggesting that also in the presence of NodA,
NodS acts immediately after NodB, and NodA after NodS.
The formation
of LCOs in the strain DH5(pUCNABCS) suggests a substrate-product
relation for the NodBCS-NodABCS metabolites. A large pool of the NodBCS
products, however, was not converted even though Nod factor synthesis
was still proceeding. A possible explanation is that the NodC
metabolites remain linked, covalently or noncovalently, to the NodC
protein while the other proteins (NodB, NodS, and NodA) modify the
protruding nonreducing end. Accumulation of NodBC or NodBCS metabolites
in the strains DH5
(pUCNABC) or DH5
(pUCNABCS), respectively,
would then be the outcome of insufficient NodA protein or acyl donor,
resulting in the release from NodC in the cytoplasm where they cannot
be acylated anymore. This would also explain why in vitro acylation of deacetylated chitooligosaccharides was unsuccessful
in our hands and equally explains the inefficient in vitro acylation reported by Atkinson et al.(7) (only
0.1% of the substrate was converted).
The proposed mechanism
requires oligosaccharide elongation from the nonreducing end toward the
reducing end, in agreement with a model concerning the action of
-glycosyl transferases(22) . The chain length of Nod
factors would be determined by the length of the sugar chain at the
moment that the nonreducing end becomes accessible for the other
proteins. A consequence of this model is that modifications on the
reducing end of the Nod factor core have to be introduced after the
synthesis of the core is completed, while modifications at the
nonreducing end can be introduced either before or after acylation.
This fits with data of Schultze et al. (23) showing
that the sulfate group at the reducing end of R. meliloti Nod
factors is introduced after the core synthesis. The acetyl group
present at the nonreducing end of the Nod factors of R.
leguminosarum bv. viciae is introduced after
deacetylation by NodB and before acylation by NodA(24) . Also,
our results concerning methylation by NodS ( (10) and this
work) are consistent with this.
The E. coli strain
expressing the nodABC genes provides a basis to analyze the
functions of other nod genes in Nod factor modifications.
Moreover, the strains DH5(pUCNC) and DH5
(pUCNBC) can be used
to determine where in the biochemical pathway a modification is
introduced. The feasibility of such a strategy was shown in this work
for the NodS protein. The role of NodU in carbamoylation of Nod factors
was also confirmed making use of this approach.