Indian Journal of Experimenta l Biology
Vol. 40, July 2002, pp. 755-764
)
Review Article
Role of rhizobial biosynthetic pathways of amino acids, nucleotide bases
and vitamins in symbiosis
Gursharn S Randhawa* & Raad Hassani§
Department of Biosciences & Biotechnology , Indian Institute of Technology, Roorkee 247 667, India
Rhi zobia require the availability of 20 amino ac ids for the establishment of effective symbiosis with legumes. Some of
these amino acids are sy nthesi zed by rhizobium, whereas the remaining are supplied by the host plant. The supply from
plant appears to be plant-type specific. Alfalfa provides arginine, cysteine, isoleucine, valine and tryptophan, and cowpea
and soybean provide histidine. The production of ornithine and anthranilic acid, the intermediates in the biosynthetic pathways of arginine and tryptophan , respectively, seems to be essential for effective symbiosis of Sinorhizobium me/iloti with
alfa lfa. The expression of ilvC gene of S. meliloti is required for induction of nodules on the roots of alfalfa plants . An undiminished metabolic flow through the rhizobial pathways for the synthesis of purines and pyrimidines and the synthesis of
biotin, nicotinic acid, riboflavin and thiamine by rhizobium appear to be requirements for normal symbiosis. To the best of
our know ledge, this is the first review a rticle on the role of rhizobial biosynthetic pathways of amino acids, nucleotide bases
and vitamins in rhizobium-legume sy mbiosis. The scientific developments of about 35 years in this field have been reviewed.
Modern agriculture relies heavily on the use of synthetic nitrogenous fertilizers, which are expensive and
cause environmental pollution. Some prokaryotic organisms, which have the ability to reduce atmospheric
nitrogen gas to ammonia, provide a better alternative
to chemical fertilizers. The process of nitrogen reduction is also known as nitrogen fixation. Some nitrogen-fixing organisms like Azotobacter fix nitrogen in
free-living state, whereas the others like rhizobia do
so in symbiotic association with plants. The rhizobial
bacteria (Rhizobium, Bradyrhizobium, Azorhizobium,
Mesorhizobium and Sinorhizobium) induce the formation of specialized organs, called nodules, on the roots
of legume plants and a non-legume plant Parasponia.
These bacteria enter the nodule cells and get converted into an endo-symbiotic form, called bacteroids,
which fixes nitrogen .
Symbiotic interactions between rhizobia and legumes involve a complex interplay of signals culminating in co-ordinated gene expression l -3. Small molecules are exchanged by symbionts both during nodule
formation and in the mature nodule. The rhizobial
bacteria depend on their host for carbon and energy
source, but very little is known about the requirement
§Present address: Department of Biology, College of Education,
University of Mosul , Mosul , Iraq.
*Correspondcnt author- Tel. No. 91-1332-85808;
Fax : 91-1332-73560;
E-mail: sharnfbs @iitr. e rne t.in;gursharn54 @lycos.co m
of other metabolites by them during symbiosis. It appears that most of the required metabolites are synthesized by rhizobia, whereas the remaining required
metabolites are available to them from the plant host.
The present review focuses on the role of rhizobial
biosynthetic pathways of amino acids, nucleotide
bases and vitamins in symbiosis. The contributions of
these pathways in rhizobium-legume interactions have
been mostly studied by symbiotic characterization of
the auxotrophic mutants of rhizobia.
Biosynthesis of amino acids and symbiosis
The studies on the contributions of the rhizobial
biosynthetic pathways of 17 amino acids in symbiosis
have been reviewed here.
A~~~e
.
The mutants of S. meliloti blocked in the later pari
of the arginine biosynthetic pathway, in either ornithine transcarbamylase or argininosuccinate synthetase, were effective in nitrogen fixation 4 . 6 . The mu tants of S. meliloti 104A14 strain blocked in the early
part of the arginine biosynthetic pathway that leads to
ornithine were ineffective6 . The symbiotic defect of
these auxotrophs was probably not related to the role
of ornithine as an arginine precursor. Ornithine is also
a precursor in polyamine synthesis? and the lack of
poJyamines may be the cause of the symbiotic defec{
The alfalfa plant host appears to provide sufficient
quantity of arginine to S. meliloti bacteria during
symbiosis.
756
INDIAN J EXP BIOL, JULY 2002
Aspartic acid
Two Tn5-induced aspartic acid mutants of S. fredii
HH303 formed nodules on soybean but these nodules
lacked the characteristic pink colour inside and were
un able to fix nitrogen . Addition of aspartic acid to the
plant growth medium did not restore symbiotic effecti veness to the mutants8. It seems that aspartic ac id
from the soybean plant host is probably not avai lab le
to S. fredii bacteri a inside the nodul es and the supplemented aspartic acid does not reach rhizobia inside
the pla nt cells.
Asparagine
Twenty one asparag ine auxotrophs of S. meliloti
104A 14 were isolated usi ng nitrous ac id mutagenesis
followed by penicillin enrichment. Seventeen
auxotrophs formed nodules on alfalfa plants but these
nodules were un able to fix nitroge n. Symbiotic properti es of th e remaining asparagine auxotrophs could
not be checked due to the probl em of reversion to prototrophy. It was concluded th at as paragine is probab ly
not ava il able to S. meliloti bacteri a in the nodul es
from th e host plant6 .
Cysteine
The cysteine auxotrophs of S. /II elitoti 2011 , R.
legulllinosarul1I 3000 and S. fredii HH 303 have been
reported to induce effecti ve nodules on alfalfa9 , peas 10
and soybeans8, respectively . The cysteine-requirin g
mutants isolated from the effective S. meliloti strain
L5-30 showed loss of effectiveness ll . In these reports
the mutated cysteine biosy ntheti c genes were not
identi fied.
By its inabi lity to grow on sul fate as the sole sul fur
source, a Tn5-induced mutant strain CTNUX8 of R.
etli was isolated and characteri zed . Sequence anal ys is
showed that Tn5 was inserted into a cysG (siroheme
sy nthetase)- homologous gene. The cysG-like gene
had a basal level expression in thi os ulfate- or cysteine-grown cells and was induced when sulfate or
methionine was used. The strain CTNUX8 induced
pink, effective (N2 fixing) nodul es on the roots of
Phaseolus vulgaris plants. It was concluded that sul fate (or sul fite) is the sul fu r source of R. elli in the
rhizosphere, while cysteine, methi oni ne, or glutathione is supplied by the root cell s to bacteria growing inside the pl ant l2.
Twenty one transposon Tn5-induced cysteine
auxotrophs of S. meliloti Rmd20 I were sulfite reductase mutants and each of these mutants had a mutati on
in cysIlcysJ gene. These auxotrophs induced full y
effective nodules on alfalfa plants. This result led to
the conclusion th at the su lfite reductase activity of S.
meliloli is not essential for effecti ve symbiosis with
alfalfa and the alfalfa plant is able to supply cysteine
and/or sulfide to S. meliloti bacteri a during symbi osis 13. All Tn5-induced cysteine auxotrophs of S.
meliloti Rmd201 were sulfite reductase mutants. Thi s
result indicates th at a hot spot for transposon Tn5 inserti on may be present in cys! or cysJ gene of S.
meliloti Rmd20I . The cysteine auxotrophs of S.
meliloti Rmd201 were ab le to grow in minimal medium l4 supplemented with methionine, homocysteine
or cystathionine l3; the latter two are the interm edi ates
in the biosynthesis of methionine. The isolation of
auxotrophs of rhi zobia which grew in mi nimal medium supplemented with cysteine or methionine has
also been reported by other workers 8. 10. IS- 17. It appears that rhizobium is able to convert methi onine,
homocysteine or cystath ion ine to cystei ne and the
biosynthesis of sulfur containing amino ac ids in
rhi zobia follow s the pathway present in Saccharol1lY.. 18 and Pseud.ol11Ol1as aerugll10sa
19.
ces cereVlswe
Glutamate
Hom et al. 20 repo rted th at five transposon Tn5induced glutamate auxotrophs of B. japonicum USDA
110 showed an altered ex pression of nitrogenase activity in free-living cultures. These workers suggested
that Klebsiella pneul1/oniae-type regul atory relati onsh ip between nitroge n metabo lism and nitrogenase
synthesis may also ex ist in B. japon icul1l .
Glutamine
Three di stinct S. meliloti loci involved in glutamine
biosy nthesis (ginA, gIn!! and glll T) have been cloned
and characteri zed. None of these loci was fo und to be
essenti al fo r symb iotic nitrogen fixation 21. Symbiotic
nitrogen fixation does not require adenyly lati on of
glutamine sy nthetase I in S. meliloti 22 .
Glycin e
Three glyc ine-dependent auxotrophic mutants of S.
meliloti 20 11 were isolated by N-methyl-N-nitro-Nnitrosog uanidine mutagenesis and pen icillin treatment. These auxotrophs were more effective in nitroge n fixation th an the parental strai n9 • Thi s is an interesting finding and furth er work should be done to find
out th e reason for the enhanced nitrogen fixat ion by
the glyci ne auxotrophs.
,
RANDHAWA & HASSANI: ROLE OF RHIZOBIAL BIOSYNTHETIC PATHWAYS IN SYMBIOSIS
,
,
757
Histidine
Histidine auxotrophs of S. meliloti ll , B. japonicum23 • 24 and R. leguminosarum bv. trifolip5 have been
isolated. The histidine auxotrophs of S. meliloti strain
L5-30 induced ineffective nodules" and the symbiotic
effectiveness was restored on supplementation of the
plant nutrient medium with histidine 26 • This result
shows that the alfalfa host is not able to provide the
sufficient quantity of histidine to S. meliloti bacteria
in the nodules. Out of four histidine auxotrophs of B.
japonicum strain USDA 122, two did not form nodules on soybean plants, whereas the other two were
symbiotically competent. Both nodulation-deficient
mutants formed effective symbioses on histidine supplementation to the plant growth medium 23 . On the
basis of these results, specific conclusion about the
role of the mutated histidine biosynthetic gene in
symbiosis cannot be made, since the same histidine
biosynthetic step appeared to be affected in all four
histidine auxotrophs. It seems that that the soybean
plant provides histidine to rhizobia in nodules but
both Nod' histidine auxotrophs were unable to utilize
plant-secreted histidine or its derivatives. Four Nod'
mutants of B. japonicllm USDA 110 were found to be
histidine auxotrophs 24 . Two Tn5-induced histidine
auxotrophs of cowpea rhizobia strain IRC256 have
been reported to induce effective nodules on cowpea
plants 27 , indicating that the cowpea plant host is able
to provide histidine to rhizobia during symbiosis.
nodules on alfalfa. The addition of ()(-ketoisovalerate
and isoleucine to the plant medium did not restore
symbiotic effectiveness to the mutant28 . Six Tn5induced ilvC mutants of S. meliloti Rmd201 were
Nod' and the Nod+ phenotype was not restored by the
supplementation of the plant nutrient medium with
isoleucine and valine, or ~- keto-~-methylvalerate
and valine29 . These results showed that the intermediate
product(s) a,~-dihydroxy- ~ -methylvalerate and/or
a, ~-dihydroxyisovalerate may have a role in nodule
formation. Another possibility is that the enzyme acetohydroxy acid isomeroreductase in S. meliloti is able
to recognize a substrate (which is not a part of isoleucine and valine pathways) and the product of this substrate is required for nodule formation . In the ilve
mutant of S. meliloti 1021 nodABC genes were not
activated by the inducer luteolin and hence Nod factor
was not formed 28 . The ilvC mutants of S. meliloti
Rmd20 I appear to synthesize, in response to plant
signal, a partially active Nod factor, since these mutants were found to induce root hair curling but not
nodule formation 29 . Recently, iivC mutants obtained
from different S. meliloti wild type strains have been
found to induce root hair deformation. Variable activation of the common nodulation genes nodABC was
observed in these mutants 30 . An ilvB/ilvG mutant of S.
I'neliloti Rmd201 did not induce root hair curling on
alfalfa plants29 . The expression of ilvBlilvG gene also
seems essential for the synthesis of the Nod factor.
Isoleucine and Valine
An isoleucine and valine auxotroph of S. meliloti
strain L5-30 was non-infective". Two transposon
Tn5-induced isoleucine and valine auxotrophs of S.
fredii HH303 formed ineffective nodules on soybean
and the addition of isoleucine and valine to the plant
nutrient medium did not restore symbiotic effectiveness to these mutants 8 . The mutated genes were not
identified in these reports; however, the results show
that a gene of isoleucine and valine biosynthetic
pathways of Sinorhizobium may have a role in symbiosis.
All ilvD mutants of S. meliloti induced effective
nodules on alfalfa plants 28 . 29 , indicating that the activity of the dihydroxy acid dehydratase enzyme is not
essential for effective symbiosis of S. meliloti with
alfalfa plant, and the alfalfa host is able to provide
both isoleucine and valine to S. meliloti bacteria during nodule formation and nitrogen fixation.
A Tn5-induced ilvC isoleucine and valine
auxotroph 1028 of S. meliloti 1021 did not induce
Leucine
All leucine auxotrophs of S. meliloti have been reported to induce ineffective nodules on alfalfa
plants 3l '34 . In all these reports, except that of Truchet
et al. 3 1, the restoration of nitrogen fixation to the
auxotrophs was not observed on supplementation of
the plant nutrient medium with leucine. Truchet
et al. 31 reported that the rhizobial bacteria were not
released from the infection threads into the host cytoplasm. Supplying the defective plant-bacterial system
with L-Ieucinc or one of its precursors, ()(-kelOisovalerate or ()(-ketoisocaproate, caused the release of
rhizobia into plant cytoplasm and restoration of nitrogen fixation. These results indicate that the lack of
nitrogen fixation in the nodules induced by S. meliloti
leucine auxotrophs was only due to the deficiency of
leucine. However, the results of the other workers
suggest that a leucine biosynthetic gene of S. meliloti
may have a role in symbiosis.
Out of three leucine auxotrophs of S. meliloti
Rmd201, one was leuC/leuD mutant and the other two
758
INDIAN J EXP BIOl, JULY 2002
were LeuB mutants. The nodules induced by LeuB mutants were structurally more advanced than the
leuCileuD mutant-induced nodules . Tn the former case
rhizobial bacteria were released from the infection
threads into pl ant cells, whereas in the latter case the
rhizobi a remained in the infection threads. These results indicated that the enzyme isopropyl malate dehydratase in S. meliloti converts an unknown substrate
into a product which is required for the release of
rhizobial bacteria from the infection threads into plant
cells, or the leucine biosynthetic pathway intermediate
~-isopropylm a l ate of S. meliloli has a role in rhizobial
release into alfalfa plant cells 34 .
The leucine auxotrophs of B. japonicul1l were
fo und to form effective nodules on soybean plants 35 .
It seems that the soybean host is ab le to provide the
required quantity of leucine to rhizobia during symbiosis.
Lysine
Two lysine auxotrophs of S. meliloti Rm41 were
isolated. However, the symbiotic properties of these
auxotrophs have not been reported 36.
Methionine
Methionine auxotrophs of rhizobia have been isolated by several workers5.6.9.lo.15.2o.36-38 . Two methionine-dependent mutants of S. meliloti 20 II strain were
obtained after mutagenesis with N-methyl-N'-nitro-Nnitrosoguanidine and penicillin treatment. These mutants induced nodule formation on lucerne and were
effective, like the parental strain, in nitrogen fixation 9.
Kerppola and Kahn 6 obtained four methionine
auxotrophs of S. meliloti 104A 14 strain by nitrous
acid mutagenesis followed by penicillin enrichment.
These mutants induced nodules on alfalfa plants but
in contrast to the above report the nodules were ineffective in nitrogen fixation. On the basis of characterization of the Tn5-induced methionine auxotroph
CTNUX23 of R. etli CE3, Tate el af. 17 concluded that
metZ gene is essential for nodulation of PhaseoLus
vuLgaris. According to these workers cysteine or glutathione, but not methionine, is supplied by the root
cells of P. vulgaris to bacteria growing inside the
pl an t.
Thirteen Tn5-induced methionine auxotrophs of S.
meliloti Rmd201 were classified as melA/metZ (10),
met E (2) and met F (1) mutants. All these mutants
formed ineffective nodules on alfalfa plants and the
supplementation of the plant nutrient medium with
methionine completely restored symbiotic effective-
ness 13. The defective symbiosis of these mutants was
most probably due to the deficiency of methi on ine.
The ineffective nodules contained reduced number of
infected nodule cells and in these nodules incomplete
transformation of bacteroids occurred 13.
Methionine auxotrophs of S. meliloti Rmd20 I did
not show any growth in minimal medium '4 supplemented with cysteine '3. ]t seems that the synthesis of
methionine from O-succinylhomocy teine and cysteine through transulfurylation pathway is highly inefficient or inactive in S. meliloti like that reported in
Pseudomonas aeruginosa l9 . Unlike th is situation, methionine auxotrophs of yeast have been reported to
grow in cysteine suppl emented minimal medium 39 .
The metE mutants of S. meliloti Rmd20 I grew in
minimal medium '4 supplemented with cyanocobalamin (vitamin BI2)I3. The isolation of 13 such mutants
of R. Leguminosarull1 Rid 1 has also been reported 38 . It
appears that the final step of methionine biosynthetic
pathway (methylation) in rhizobia resembles that present in E. coli which is cata lyzed by two distinct enzymes; one requiring only folic acid as a co factor, and
the other requiring both fol ic acid and vitamin BI2 as
cofactors.
Proline
Proline auxotrophic, catabolic and overproducing
mutants of R. leguminosarum bv. viciae strain
C 1204b were generated and used as inoculants of pea
(Pisulll sativum L. cv Alaska). Cytosolic and bacteroid levels of proline determined for the nodular ti ss ue
did not support the role of proline as a key metabolite
supplied by th e host40 . A proC mutant of B. japonicum elicited underdeveloped nodules on soybeans.
These nodules lacked nitrogen fixation activity and
plant haemoglobin . It was concluded that the proC
gene is essential for symbiosis and the mutant does
not obtain an exogenous supply of proli ne in association with soybeans sufficient to satisfy its auxotroph/I.
PhenyLalanine
A Tn5-induced phenylalanine auxotrophic mutant
RH38 of S. meliloti Rmd201 formed ineffective nodules on alfalfa plants. Histological studies revealed
that in each of these nodules the nitrogen fixation
zone was absent and degeneration of rhizo bial bacteria occurred soon after their release into plant cells42 .
It seems that the alfalfa plant is not able to provide the
required quantity of phenylalanine to S. meliloti bacteria in nodules.
RANDHA WA & HASSANI: ROLE OF RHIZOBIAL BIOSYNTHETIC PATHWAYS IN SYMBIOSIS
Tryptophan
Except the tryptophan synthetase mutants, all other
tryptophan auxotrophs of B. japonicum strain USDA
1-110 ARS did not induce nodules on soybean host
plants43 .35. It was suggested that indole glycerol phosphate, an intermediate of tryptophan biosy thetic
pathway, is necessary for nodulation in B. japonicum 35 . The trpCD deletion mutants of B. japonicum
have been reported to be symbioticall y ineffective44 .
The sequence of a symbiotically essential B. japonicum operon consisting of trpD, trpC and a moaC-like
gene has been determined 45 .
The trpE mutants of S. meliloti formed defective
nodules, containing extended invasion zones, on alfa lfa and fixed no or less nitrogen 46 .42. The tryptophan
synthetase mutants of S. meliloti showed normal symbiosis with alfalfa plants 46 .42 . These findings indicate
that the expression of trpE gene of S. meliloti is essential for normal symbiosis, and the al falfa plant host is
able to provide tryptophan to S. meliloti bacteria in
nodules. The S. meliloti trpE locus has been shown to
be a fusion between trpE and lrpG coding sequences
and has been named trpE(G)47. Barsomian el a1. 46, on
the basis of the earlier reports48 . 49 , hypothesized that
anthran ili c acid (synthesized by the expression of trpE
gene) acts as an in planta siderophore helping iron
uptake for bacterial development. S. meliloti mutants
with decreased DAHP synthase activity were sensitive to exogenous tryptophan and phenylalanine and
formed ineffective nodules. It was concl uded that the
normal tlow of metabolites through the biosynthetic
pathways of aromatic amino acids in S. meliloti is essential for the bacteroid development 5o •
The lrpB mutant CTNUX4 of R. etli strain CE3 induced ineffective nodules on Phaseolus vulgaris
plants. Under free-living conditions, the mutant strain
was not ab le to produce flavonoid-inducible Nod factors unless tryptophan was added to the growth medium. These results indicated that the lack of tryptophan biosynthesis affects the symbiotic behaviour of
R. etli51•
Tyrosine
Two tyrosine mutants of S. lI1eliloti 104A 14, obtained by nitrous acid mutagenesis, were reported to
induce ineffective nodules on alfalfa plants 6 . A Tn5induced tyrosine auxotroph of S. meliloti Rmd201
showed normal symbiosis with dlfalfa 41 •
Biosynthesis of purines and symbiosis
Several workers have reported defective symbioses
of purine auxotrophs of R. lcgulIlinosarumI0.52.54, S.
759
meliloti6,9.55.57, Rhizobium strain NGR234 58 and S.
Jredit Adenine auxotrophs of R. leguminosarum
were not able to nodulate pea plants IO ,52 ,53 ,. When
plants were grown with exogenously supplied adenine, the adenine auxotrophs of R. legul11inosarum
formed ineffective nodules 59, 1O, Purine aux otrophs of
R. legul11inosarum bv, phaseoli induced pseudo nodules on bean plants, These auxotrophs induced root
hair curling and cortical cell division but did not elicit
infection thread formation . The pseudonodules did not
contain bacteria. Addition of adenosine to the plant
growth medium had no effect on nodule phenotype
but the supplementation of 5-aminoimidazole-4carboxamide riboside (AICA-riboside), the unphosphorylated form of the purine precursor 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR),
significantly enhanced root nodule development 54 .60 .
Purine auxotrophs, each having a biochemical block
before AICAR, of R. leguminosarum bv. viciae
128C56 and S. Jredii HH303, induced poorly developed nodules which did not contain rhizobia, Addition of AICA-riboside to the plant nutrient medium
enhanced nodulation and promoted nitrogen fixation 61 • The purM and purN genes, which specify early
steps in purine biosynthesis, were found to be needed
for nodulation of peas by R, leguminosarum bv. viciae. These genes expressed at relatively high levels
even when the metabolic products of the pathway
were present62 .
A purine auxotroph of S, meliloti 2011 was ineffective 9 . Two mutants, one requiring adenine and the
other adenine with thiamine, of S. meliloti L5-30
formed ineffective nodules on lucerne plants 55 . Purine
auxotrophs of S. l11eliloti Rmd201 induced small , ineffective nodules on alfalfa, The mutants were recovered from the root nodules induced by them . External
supplementation of adenine or AICAR could not restore symbiotic effectiveness to the auxotrophs 56 .
Multiple changes in the cell surface molecules, viz.,
acidic exopolysaccharides, cellulose fibrils and ~­
(l---73)-glucans, of the purine auxotrophs of S. meliloti
Rmd20l were observed, These results showed that the
symbiotic deficiency of these purine auxotrophs was
likely to be a result of these associated changes on the
cell surface63 . It has been shown that AICA-riboside
negatively regulates the expression of fixNOQP in S,
meliloti by modulation of fixK expression 64 .
The transposon Tn5-induced purine auxotrophs of
Rhizobium strain NGR234 (ANU2861 and ANU2866)
were found to be defective for nodule formation on a
760
INDIAN J EXP SIOL, JULY 2002
wide variety of legumes and the non-legume
Parasponia. The strains ANU2861 and ANU2866
produced less acidic exopolysaccharides and cyclic B(\-72)-glucans per gram of cells than did the parent
65
strain , although they produced colony types that appeared excessively mucoid on complex agar me58
dium . On Macroptilium atropurpureum (siratro)
ANU286I rapidly induced a reaction analogous to a
hypersensitive response at the site of infection 66 .
When the plant growth medium was supplemented
with ArCA-riboside, strains AUN2861 and ANU2866
induced the formation on siratro of root outgrowths
that resembled Fix· nodules. The strain ANU2861 was
found to possess a defective lipopolysaccharide that
was corrected by the addition of ArCA-riboside to the
67
growth medium .
The above reports show that undimjnished metabolic flow through purine biosynthetic pathway, or a
particular intermediate in this pathway, is essential for
nodule morphogenesis. The requirement of adenine
synthesis by rhizobia for the formation of effective
nitrogen fixing nodules may be related to the fact that
68
the rhizobia can produce cytokinins and these plant
hormones are N-6 substituted derivatives of adenine lO •
The supplementation of guanine or guanosine to
the minimal medium did not restore growth to the Pu(
54
auxotrophs of R. leguminosarum bv. phaseoli and S.
56
melilotf . It appears that R. leguminosarum and S. ·
meliloti, unlike other bacteria69 , lack a path from guanine or guanosine to inosine monophosphate. A guaB
mutant of R. tropici had thermal sensitivity and was
defective in symbiosis with bean~. The study showed
that, in R. tropici, the production of guanine via
inosine monophosphate dehydrogenase is essential for
growth at extreme temperature and for effective nodulation 70.
Biosynthesis of pyrimidines and symbiosis
A pyrimidine auxotroph, isolated by N-methyl-Nnitro-N-nitrosoguanidine mutagenesis and penicillin
treatment, of S. meliloti 2011 strain induced ineffective nodules on alfalfa plants9 . Two out of 15
pyrimidine auxotrophs of R. leguminosarum 3000
were defecti ve in symbiosis 10. The supplementation of
uridine to the plant growth medium did not result in
the suppression of Ndv- (nodule development defective) phenotype of pyrimidine auxotroph of R. leguminosarum bv. phaseoli strain CFN42 54 . Mutations in
carbamoyl phosphate synthetase gene and the genes
controlling the steps of pyrimidine biosynthesis be-
fore orotic acid resulted in ineffective symbiosis in S.
meliloti strain 104AI46 .7 1.72. Two Tn5-induced uracilrequiring mutants of S. fredii HH303 formed ineffective nodules (Nod+ Fix-) on soybean plants. Addition
of uracil to the plant growth medium did not restore
symbiotic effectiveness to these auxotrophs 8 . The
above reports suggested that the expression of some
pyrimidine biosynthetic genes may be essential for the
effective symbiosis of rhizobia with host plants.
Twenty three Tn5-induced pyrimidine auxotrophic
mutants of S. meliloti Rmd201 were classified into
car (five), pyre (eight) and pyrE/pyrF (ten) categories. These mutants induced white, ineffective nodules
on alfalfa plants. The structural features of the nodules induced by pyre mutants were more advanced
than those of the nodules induced by car mutants, indicating that carbamoyl phosphate/carbamoyl aspartate may be involved in symbiosis. Similarly, the
structural features of the nodules induced by
pyrE/pyrF mutants were more advanced than those of
the nodules of pyre mutants, indicating that dihydroorotic acid/orotic acid may have symbiotic function(s/3.
Biosynthesis of vitamins and symbiosis
There are very few reports in which the contributions of the biosynthetic pathways of vitamins in
symbiosis have been discussed.
Biotin
Studies done with S. meliloti 102 1 showed that
both synthesis and uptake of plant-derived biotin
promote colonization of alfalfa roots. Synthesis, however, was found to be more important source of biotin
for S. meliloti 1021 because in root colonization tests
biotin auxotrophs competed very poorl y with the par74
ent strain .
Nicotinic acid
A Tn5-induced nicotinic acid auxotrophic mutant
of S. fredii HH303 produced mature nodules like
those of the parental strain, but the nodules lacked the
characteristic pink color inside and were · unable to fix
nitrogen. Addition of nicotinic acid to the plant
growth medium did not restore the symbiotic effectiveness to the mutant. These results indicated that the
legume host does not supply nicotinic acid and that
synthesis of nicotinic acid is important for effective
nodule formation 8 .
t-
RANDHAWA & HASSAN I: ROLE OF RHIZOB IAL BIOSYNTHETIC PATHWAYS IN SYMBIOSIS
761
Table I-Symbiotic characteristics of amino acid auxotrophs of rhizobia
-.
S.
No.
Auxotrophy
I.
Arginine
2.
3.
4.
Aspartic acid
Asparagine
Cysteine
5.
6.
7.
8.
9.
Glutamic acid
Glu tamine
Glycine
Histidine
Isoleucine
+ Valine
10.
II.
12.
{
Leucine
Lysine
Methionine
13.
Proline
14.
15.
Phenylal anine
Tryptophan
16.
17.
Tyrosine
Phenylalanine
+ Trptophan
+ Tyrosine
Mutated gene!
Position of
biochemical block
Name of
rhizobium
Symbiotic
phenotype
Nod+Fix+
Sinorhizobium meliloti
Nod+Fix'
-do-do5.fredii
-doS. meliloti
Nod+Fix+
-do-do-docys/lcysJ
Nod+Fix'
-doNod+Fix+
Rhizobium etli
cysG
-doR. legwninosarum
-doS· fredii
-doBradyrhizobillm japonicum
-doS. meliloti
ginA. glnll & glnT
-do-do-doB. japonicul1l
Nod'
-do-do-doNod+Fix'
S. /IIeliloti
Nod+Fix+
-doilvD
Nod'
-doilvC
-do-doilvC
-do-doilvBlilvG
-do-doNod+Fix'
S·fredii
Nod+Fix+
B. japonicum
Nod+Fix'
S. meliloti
-do-do-do-doleuClleuD & leuB
-doNod+Fix+
5. meliloti
Nod+Fix'
-do-do-dometAimetZ. metE &metF
Nod'
metZ
R. etli
Nod+Fix+
-doNod+Fix'
B. japonicum
proC
-dopheA
S. meli/oti
Nod+Fix+
trpA& trpB
-doNod+Fix'lNod+Fix+*
trpE(G)
-doNod'
8. japonicum
Nod+Fix'
trpB
R. etli
Nod+Fix+
tyrA
S. meli/oti
Nod+Fix'
-doaro
-do-doafter ornithine
before ornithine
Reference(s)
Symbiotic
phenotype after
supplementation#
NO
Nod+Fix'
NO
NO
4-6
6
8
6
9
13
II
12
lO
Nod+Fix+
NO
Nod+Fix+
Nod'
NO
Nod'
NO
-doNod+Fix+
Nod+Fix'
-do-
NO
Nod+Fix+
Nod+Fix'
NO
-doNO
-do-doNO
-do-
8
20
21
9
23
23
24
26
28, 29
28, 29
30
29
II
8
35
31
32, 33
34
31
9
6
13
17
40
41
42
46,42
46,42
43, 35
51
42
6
46, 42
The plant nutrient medium was supplemented with the auxotrophic requirement of the auxotroph.
not determined, Nod+Fix+ - formation of nodules which fix nitrogen, Nod-no
nodule formation & Noct+Fix - formation of nodules which does not fix nitrogen,
#
* Reduced ni trogen fixation. Abbreviations lIsed: NO -
762
INDI AN J EXP BIOL, JULY 2002
Riboflavin
Riboflavin has been found to be key growth factor
for alfa lfa root coloni zati on by S. meliloti 1021 74 . A
riboflavin-requiring auxotrophi c mutant of R. legu/IIil10sarUIIl bv. tnfolii strain TI, which formed ineffective or partiall y effective nodu les on some cu ltivars of
clover in the absence of riboflavin, was fully effective
when riboflavin was added externall y to the roots of
growing seedlings. This result suggested th at the
block in nodule development in vo lved a demand for
riboflav in whi ch is normally synthesized by the effective p3rent strain 75 . Further experiments indicated th at
riboflav in is essential for the co nvers ion of vegetative
bacteria of R. leglllninosarum bv. tr(folii into functional , nitrogen fixing bacteroids with in the nodules 76 .
Thiamine
Thiamine has been show n to be a key grow th factor
for alfalfa root colonization by S. lII eliloti 102 174 .
of these pathways seem to be essential for nodule
morphogenesis. The synthes is of vitam ins, viz., biotin ,
ni cotini c ac id , riboflavin and thiamine by rhi zobi um
also appears to be ne(;essary for effective sy mbiosis.
It seems th at some enzymes and intermediates of
the biosynthetic pathways of amino acids and nucleotide bases are required for the development of effecti ve sy mbiosis. However, clear evidences are lac king
and the preci se function s of these enzymes and intermediates in sy mbi osis have not been determined . Detailed information abo ut the suppl y of am ino acids,
nucleotide bases and vi tam ins, and th e intermediates
of their sy nthesis to rhizobial bacteri a by different
species of legumes is also lacki ng. Further research
work in these directions is required for better understanding of the complex rhizobium-legume sy mbiotic
re lations hip.
References
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pantothenic ac id is not required for effective symbiosis of S.Jredii with soybean plant or the soybean plant
provides th e sufficient amount of pantothenic acid to
S.ji'edii.
Conclusions
The isolation of auxotrophs of rhizobia for 17
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