FEMS Microbiology Reviews 22 (1998) 105^123
Tricarboxylic acid cycle and anaplerotic enzymes in rhizobia
Michael F. Dunn *
Departamento de Ecolog|èa Molecular, Centro de Investigacioèn sobre Fijacioèn de Nitroègeno, Universidad Nacional Autoènoma de Meèxico,
A.P. 565-A, Cuernavaca, Morelos, Mexico
Received 20 January 1998; accepted 1 June 1998
Abstract
Rhizobia are a diverse group of Gram-negative bacteria comprised of the genera Rhizobium, Bradyrhizobium,
Mesorhizobium, Sinorhizobium and Azorhizobium. A unifying characteristic of the rhizobia is their capacity to reduce (fix)
atmospheric nitrogen in symbiotic association with a compatible plant host. Symbiotic nitrogen fixation requires a substantial
input of energy from the rhizobial symbiont. This review focuses on recent studies of rhizobial carbon metabolism which have
demonstrated the importance of a functional tricarboxylic acid (TCA) cycle in allowing rhizobia to efficiently colonize the
plant host and/or develop an effective nitrogen fixing symbiosis. Several anaplerotic pathways have also been shown to
maintain TCA cycle activity under specific conditions. Biochemical and physiological characterization of carbon metabolic
mutants, along with the analysis of cloned genes and their corresponding gene products, have greatly advanced our
understanding of the function of enzymes such as citrate synthase, oxoglutarate dehydrogenase, pyruvate carboxylase and
malic enzymes. However, much remains to be learned about the control and function of these and other key metabolic enzymes
in rhizobia. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights
reserved.
Keywords : Rhizobium-legume interaction ; Carbon metabolism; Tricarboxylic acid cycle; Anaplerotic reaction; Symbiosis
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Enzymes of the TCA cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Metabolism of tricarboxylic acids via citrate synthase, aconitase and isocitrate dehydrogenase . . . . . . . . . .
2.2. Metabolism of dicarboxylic acids via 2-oxoglutarate dehydrogenase, succinyl CoA synthetase, succinate dehydrogenase, fumarase and malate dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Enzymes catalyzing anaplerotic reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Metabolism of four-carbon compounds: malic enzyme, aspartase and aspartate aminotransferase . . . . . . . .
3.2. Metabolism of 2-oxoglutarate via the Q-aminobutyrate bypass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Metabolism of three-carbon compounds: pyruvate, phosphoenolpyruvate and propionyl CoA carboxylases,
pyruvate orthophosphate dikinase and phosphoenolpyruvate carboxykinase . . . . . . . . . . . . . . . . . . . . . . . .
3.4. Metabolism of acetyl CoA via the glyoxylate bypass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
106
106
109
111
114
114
115
116
117
* Tel.: +52 (73) 13-9944; Fax: +52 (73) 17-5094; E-mail: [email protected]
0168-6445 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 4 5 ( 9 8 ) 0 0 0 1 0 - 2
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M.F. Dunn / FEMS Microbiology Reviews 22 (1998) 105^123
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Bacteria in the genera Rhizobium, Sinorhizobium,
Mesorhizobium, Bradyrhizobium and Azorhizobium
(collectively called rhizobia) are Gram-negative heterotrophs with the unique ability to reduce nitrogen
to ammonia in symbiotic association with a compatible legume host. Various aspects of carbon metabolism in this agronomically important group have
been presented in previous reviews [1^5]. Recent
studies with carbon metabolic mutants have begun
to elucidate the metabolic signi¢cance of a number
of tricarboxylic (TCA) cycle and anaplerotic enzymes in rhizobia, and this review summarizes these
¢ndings with an emphasis on the importance of these
enzymes in symbiosis.
The development of the rhizobia-legume symbiosis
has been reviewed [5^8] and what follows describes
only a few general features of the interaction. During
the formation of nitrogen-¢xing root nodules rhizobia are converted from soil-dwelling saprophytes
into intracellular symbionts, or bacteroids, which
rely on the plant host to provide them with carbon
substrates. The bacteroids are surrounded by a hostderived symbiosome membrane which mediates the
exchange of metabolites between the microsymbiont
and the plant [5,8]. In quantitative terms the major
metabolic exchange during symbiosis involves the
bacteroids receiving reduced carbon from the plant
in exchange for the nitrogen they ¢x. The dicarboxylic acids succinate and/or malate are produced in
large quantities in the plant cells of the nodule and
are the major carbon sources provided to the bacteroids [3,5,8^10]. In addition to dicarboxylic acids,
other organic or amino acids may be used by some
rhizobia during infection or by bacteroids under environmental stress [3,5,8,11^17].
Nitrogenase, the bacteroid enzyme which catalyzes
the reduction of atmospheric nitrogen, requires at
least 16 ATP and 8 reducing equivalents per mol
of ammonia produced. Because nitrogenase is oxygen-labile, a microaerobic environment is imposed
by a gas permeability barrier in the nodule subcortex
118
118
118
and the oxygen-binding protein leghemoglobin in the
infected plant cells [7^9,18]. In bacteroids, microaerobiosis triggers the synthesis of new respiratory
chain components needed for a microaerobic metabolism and energy generation by oxidative phosphorylation, as well as the induction of genes encoding
regulatory or structural proteins directly involved in
nitrogen ¢xation [7,18].
2. Enzymes of the TCA cycle
In addition to energy generation the TCA cycle
(Fig. 1) is used to produce precursors for the biosynthesis of amino acids, purines, pyrimidines and vitamins. The cycle has been intensively studied in Escherichia coli and Bacillus subtilis and a complex
network of genetic and metabolic controls have
been elucidated in these organisms [19^23]. The rhizobial oxygen-responsive regulators (FixLJ, FixK)
which induce the synthesis of electron transport
components needed for microaerobic respiration in
bacteroids [7,24] are homologs of the global regulator Fnr which performs a similar function in E. coli
during anaerobic growth [25]. However, control systems analogous to the E. coli ArcAB system, which
directs the anaerobic repression of several TCA cycle
genes [25], have not yet been encountered in rhizobia. Thus, although the details of TCA cycle regulation in rhizobia are lacking, a few broad generalizations from studies in other prokaryotes may apply,
namely (i) the TCA cycle is regulated to ensure that
energy and precursor generation match the needs
imposed by growth in a particular environment
[22], (ii) growth conditions determine which anaplerotic reactions (Section 3) are used to maintain TCA
cycle function [19] and (iii) paralogs, or products of
homologous genes which perform related but nonidentical functions in the same organism [26], exist
for several TCA cycle and anaplerotic enzymes [22].
TCA cycle enzyme activities have been measured
in bacteroids of many rhizobia (Table 1) and have
been tentatively correlated with symbiotic e¤ciency
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107
Fig. 1. Reactions of the TCA cycle. Cycle intermediates are in capital letters. Additional products shown are for the forward (clockwise)
reactions. Metabolites which are commonly used in biosynthetic reactions are also indicated.
[27,28]. Enzyme activity data (Table 1) along with
respirometric studies strongly indicate that a complete cycle is present in bacteroids [5,29,30] and in
cells in culture [1,2,5]. The TCA cycle in bacteroids
probably operates below its full aerobic potential
because the microaerobic conditions in nodules limit
the ability of the respiratory chain to oxidize reduced
nucleotides, which may inhibit the activities of citrate
synthase, isocitrate dehydrogenase (Section 2.1) and
2-oxoglutarate dehydrogenase (Section 2.2) [3,5,
9,31]. Nevertheless, studies with a variety of enzyme
mutants described here support the importance of a
complete TCA cycle in bacteroids.
Several pathways may be used to produce acetyl
CoA for entry into the TCA cycle. Under aerobic
conditions rhizobia rely mostly upon the pyruvate
dehydrogenase complex (EC 1.2.4.1) [29,32^34]
(Fig. 2). Multiple control mechanisms govern the
synthesis and activity of this enzyme in E. coli
[35,36] but little is known about its regulation in
rhizobia. In R. etli pyruvate dehydrogenase activity
is markedly decreased with low culture oxygen availability [37] and in bacteroids (Table 1), although this
may result from the down-regulation of genes encoding enzymes for the synthesis of the catalytic cofactors thiamine pyrophosphate and lipoic acid [37^39].
In contrast to R. etli, pyruvate dehydrogenase activity is detectable in bacteroids of other Rhizobium
species (Table 1).
Acetyl CoA may be derived from poly-L-hydroxybutyrate (PHB; Fig. 2), a polyester synthesized by
bacteroids of many rhizobia [40]. PHB may serve as
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M.F. Dunn / FEMS Microbiology Reviews 22 (1998) 105^123
Table 1
Speci¢c activities of pyruvate dehydrogenase and TCA cycle enzymes in bacteroidsa
Enzyme and organism
Pyruvate dehydrogenase
B. japonicum
R. etli
R. leguminosarum bv. viciae
S. meliloti
Rhizobium sp. (Cicer)
Citrate synthase
B. japonicum
R. leguminosarum bv. viciae
R. tropici
Rhizobium sp. (Cicer)
Aconitase
R. leguminosarum bv. viciae
S. meliloti
Isocitrate dehydrogenase
B. japonicum
R. etli
R. leguminosarum bv. viciae
S. meliloti
R. tropici
Rhizobium sp. (Cicer)
2-Oxoglutarate dehydrogenase
B. japonicum
R. etli
S. meliloti
R. tropici
Succinyl CoA synthetase
R. leguminosarum bv. viciae
Succinate dehydrogenase
R. leguminosarum bv. viciae
Fumarase
B. japonicum
R. leguminosarum bv. viciae
S. meliloti
Malate dehydrogenase
B. japonicum
R. etli
R. leguminosarum bv. viciae
R. tropici
Rhizobium sp. (Cicer)
a
b
Host plant
Speci¢c activity
(nmol min31 mg protein31 )
Reference
soybean
bean
pea
alfalfa
chickpea
25^57
not detected
99
44
12
[116,150]
[38]
[56]
[121]
[116]
soybean
pea
bean
chickpea
285
700
178
58
[116]
[56]
[57]
[116]
pea
alfalfa
560
31
[56]
[121]
soybean
bean
pea
alfalfa
bean
chickpea
55^480
171
520
200
596
113
[68,116,150]
unpublishedb
[56]
[121]
[57]
[116]
soybean
bean
alfalfa
bean
34^53
16
50
48
[31]
unpublishedb
[121]
unpublishedb
pea
455
[56]
pea
280
[56]
soybean
pea
alfalfa
340
960
[150]
[56]
[121]
6
soybean
bean
pea
bean
chickpea
2733^4600
2888
2400
8970
4867
[93,116,150]
unpublishedb
[56]
[57]
[116]
Bacteroids were aerobically isolated and puri¢ed by sucrose [150] or Percoll [151] density gradient centrifugation.
M. Dunn and G. Ara|èza, unpublished results.
a reserve of chemical energy [3,41,42] or as the product of an over£ow pathway which consumes excess
reductant in microaerobically respiring bacteroids
[43,44]. PHB is degraded under carbon-limited conditions in cultures and bacteroids [45^48] to produce
acetyl CoA in the reaction catalyzed by L-ketothiolase [41] (Fig. 2).
Acetyl CoA is also synthesized from acetate using
acetate kinase in combination with phosphotransacetylase [49], or by acetyl CoA synthetase [50,51] (Fig.
2). All three activities are present in B. japonicum
bacteroids and the in vitro kinetic properties of the
acetyl CoA synthetase and acetate kinase suggest
that both operate in the direction of acetyl CoA syn-
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109
Fig. 2. Possible integration of anaplerotic and bypass pathways with the TCA cycle. Intermediates of the TCA cycle are in capital letters.
Not all reaction products are shown.
thesis [51]. It is interesting to note that acetyl phosphate, the product of acetate kinase, is important in
regulating TCA cycle activity in E. coli [52], and a
possible similar role for this metabolite in S. meliloti
is currently being investigated [49].
2.1. Metabolism of tricarboxylic acids via citrate
synthase, aconitase and isocitrate dehydrogenase
The tricarboxylic acid portion of the TCA cycle
consists of a three-step conversion of oxaloacetate
plus acetyl CoA to 2-oxoglutarate (Fig. 1). In the
¢rst reaction citrate synthase (EC 4.1.3.7) condenses
the acetyl group of acetyl CoA with oxaloacetate to
form citrate (Fig. 1). Citrate synthases from Gramnegative bacteria are often feedback inhibited by 2oxoglutarate and NADH and are subject to catabo-
lite and anaerobic repression [53^55]. Little is known
about the regulation of citrate synthase in rhizobia
(see below), although its activity is high during
growth on di¡erent carbon sources [56,57] and in
bacteroids (Table 1).
Two citrate synthase paralogs are present in R.
tropici, one (pcsA) encoded on the symbiotic plasmid
and the other (ccsA) on the chromosome (Table 2).
The coding sequences of pcsA and ccsA are nearly
identical, leading to the suggestion that pcsA may
have arisen by duplication of ccsA [56]. Nodules
formed by ccsA or pcsA mutants are Fix‡ (able to
¢x nitrogen; Table 2), which is perhaps not surprising since the mutants retain 70^80% of the wild-type
citrate synthase activity in planta [58,59]. Despite the
residual activity both mutants are delayed in nodule
formation on bean plants, with the ccsA mutant
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M.F. Dunn / FEMS Microbiology Reviews 22 (1998) 105^123
Table 2
TCA cycle and anaplerotic enzymes: cloned genes and phenotypes of existing mutants
Organism and enzyme
B. japonicum
citrate synthase
aconitase
2-oxoglutarate dehydrogenase (Odh)
fumarase
malate dehydrogenase
R. etli
aspartase (Asp)
pyruvate carboxylase
Rhizobium sp. NGR234
phosphoenolpyruvate carboxykinase
succinic semialdehyde dehydrogenased
R. tropici
citrate synthase
citrate synthase
citrate synthase
pyruvate carboxylase
R. leguminosarum bv. trifolii
pyruvate carboxylase
R. leguminosarum bv. viciae
2-oxoglutarate dehydrogenase (Odh)
succinate dehydrogenase
succinyl CoA synthetase (Scs)
malate dehydrogenase
pyruvate carboxylase
phosphoenolpyruvate carboxykinase
S. meliloti
citrate synthase (Cs)
isocitrate dehydrogenase (Idh)
2-oxoglutarate dehydrogenase (Odh)
succinate dehydrogenase (Sdh)
NAD-malic enzyme
NADP-malic enzyme
NAD- and NADP-malic enzymes
pyruvate orthophosphate dikinase
phosphoenolpyruvate carboxykinase
aspartate aminotransferase
Genea
GenBank accession
gltA
acnA
sucA
fumC
mdh
U76375
U56817
U73618
M38241
Acn Ndv Fix
Odh3 Ndv‡ Fix‡
Fumred Ndv‡ Fix‡
unpublishedc
[64]
[77,78]
[89]
[80]
pyc
U51439
Aspred Ndv‡ Fix‡
Pyc3 Ndv‡ Fix‡
[112]
[123,131]
pckA
gabD
X63291
U00090
Pck3 Ndv‡ Fix3 or Fixred
[138]
[152]
pcsA
ccsA
pcsA ccsA
pyc
Z34516
L41815
as above
PcsA3 CcsA‡ Ndvþ Fix‡
CcsA3 PcsA‡ Ndvþ Fix‡
PcsA3 CcsA3 Ndvþ Fix3
Pyc3 Ndv‡ Fix‡
[59]
[58]
[58]
[123,131]
pyc
Pyc3 Ndv‡ Fix‡
[132]
sucA
sdh
sucD
mdh
pyc
pck
Odh3 Ndvþ Fix3
Sdhred Ndv‡ Fix3
Scsred Ndvþ Fix3
Pyc3 Ndv‡ Fix‡
Pck3 Ndv‡ Fix‡
[44]
[85]
[44]
[44]
[133]
[140]
Cs3 Ndv‡ Fix3
Idh3 Ndv‡ Fix3
Odhÿ Ndv‡ Fix3
Sdh3 Ndv‡ Fix3
Dme3 Tme‡ Ndv‡ Fix3
Tme3 Dme‡ Ndv‡ Fix‡
Dme3 Tme3 Ndv‡ Fix3
Pod3 Ndv‡ Fix‡
Pck3 Ndv‡ FixRed
Aatred Ndv‡ Fix3
[153]
[75]
[134]
[84,86]
[104]
[102]
[102]
[137]
[139]
[118,154]
AJ002750
gltA
icd
U75365
dme
tme
dme tme
podA
pckA
aatA
U61378
U15199
L05064
Mutant phenotypeb
red
‡
‡
Reference
a
Where no genetic designation is provided, the gene encoding the enzyme has neither been cloned nor localized by mutagenesis.
Where no phenotype is given, no mutants exist. The mutant's ability to produce the indicated enzyme is listed ¢rst, with the superscripts
signifying : 3 , no activity; ‡ , approximately wild-type activity; red , reduced activity. The symbiotic phenotypes are listed next and correspond
to: Ndv‡ , normal nodule development ; Ndvþ, delay in nodule formation and/or abnormal nodules formed; Fix3 , nodules did not ¢x
nitrogen based on plant phenotype or acetylene reduction assay; FixRed , signi¢cant reduction in nitrogen ¢xation; Fix‡ , nodules ¢xed
nitrogen at approximately wild-type levels.
c
K. LeVier and M.L. Guerinot, Dartmouth College, USA.
d
Identi¢ed only by comparison to homologs in other organisms.
b
being more severely a¡ected [58]. Double mutants
totally lacking citrate synthase activity form Fix3
(non-nitrogen ¢xing) nodules devoid of bacteroids
(Table 2), indicating a crucial role for the enzyme
early in nodule development. The ccsA and pcsA
ccsA (but not the pcsA) mutants are glutamate
auxotrophs [58], suggesting that CcsA is required
for the production of 2-oxoglutarate via the TCA
cycle [5].
In contrast to R. tropici, S. meliloti and B. japoni-
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cum appear to contain single genes encoding citrate
synthase (Table 2). The rhizobial citrate synthases
share over 70% deduced amino acid identity and
have just slightly lower homology with the enzymes
from other Gram-negative species. A S. meliloti citrate synthase mutant had the expected glutamate
auxotrophy but in addition appeared to produce altered lipopolysaccharides, which could also explain
its Fix3 phenotype (Table 2).
Citrate synthase activity in Rhizobium sp. (Cicer) is
markedly inhibited by NADH in vitro [60] and, if
the same phenomenon occurs in bacteroids, could
result in the £ow of carbon into PHB synthesis. Unfortunately, the e¡ects of citrate synthase mutation
on PHB synthesis have not been reported. The partitioning of acetyl CoA between the TCA cycle and
PHB synthesis is also determined by the metabolic
regulation of L-ketothiolase, the initial enzyme of the
later pathway (Fig. 2). Puri¢ed L-ketothiolase from
Rhizobium sp. (Cicer) [61] and B. japonicum [62] bacteroids is inhibited by CoASH, which may repress
PHB synthesis when citrate synthase activity is
high [41].
Aconitase (EC 4.2.1.3) catalyzes the reversible isomerization of citrate and isocitrate (Fig. 1) and is
encoded by acnB in E. coli, where its transcription
is repressed by anaerobiosis [53,63]. In E. coli an
aconitase paralog, AcnA, functions in protecting
the cell from oxidative stress [63].
An acnA homolog and its product have been
studied in B. japonicum (Table 2) but this aconitase
does not seem to participate in the TCA cycle. Instead, it appears that B. japonicum also encodes an
AcnB-like enzyme which ful¢ls this function. This
notion is supported by the facts that (i) acnA expression in B. japonicum [64] and E. coli [65] is induced
during aerobic growth, (ii) E. coli [65] and B. japonicum [64] acnA mutants retain substantial aconitase
activity and (iii) acnA mutants in either species are
not glutamate auxotrophs, which in the E. coli acnA
mutant results from the production of the aconitase
encoded by acnB [65]. The symbiotic properties of
the B. japonicum acnA mutant were indistinguishable
from those of the wild-type strain (Table 2),
although one might expect a true aconitase knockout
mutant to be symbiotically ine¡ective by analogy to
the rhizobial citrate synthase and isocitrate dehydrogenase (see below) mutants. Further work is needed
111
to con¢rm this and to demonstrate that B. japonicum
also encodes an acnB homolog.
Isocitrate dehydrogenase (EC 1.1.1.42) catalyzes
the oxidation of isocitrate to 2-oxoglutarate (Fig.
1). The activity of this enzyme is fairly constant during growth on di¡erent carbohydrate, amino acid or
organic acid carbon sources [56,57,66^68] and high
levels are present in bacteroids (Table 1). Rhizobia
normally contain only NADP-speci¢c isocitrate dehydrogenase [69], although an NAD-linked form occurs in some species [70,71]. The substrate kinetic
constants of NADP-isocitrate dehydrogenase from
S. meliloti [72] and B. japonicum [68] are similar to
those of the E. coli enzyme [73]. In contrast to the
E. coli isocitrate dehydrogenase, which is not allosterically regulated [19], oxoglutarate and NADPH
inhibit the S. meliloti [72] and B. japonicum [3]
enzymes, respectively. These forms of product inhibition could operate in bacteroids where the concentrations of reduced nucleotides and perhaps oxoglutarate are high [3,31,74].
The gene for the S. meliloti NADP-isocitrate dehydrogenase (Table 2) appears to encode the subunit
of a monomeric form of the enzyme [72,75] in contrast to the homodimeric isocitrate dehydrogenases
produced by E. coli and Bacillus [23]. Isocitrate dehydrogenase insertion mutants of S. meliloti were
glutamate auxotrophs and were Nod‡ but Fix3 (Table 2). This phenotype may result from the inability
of the mutant to synthesize 2-oxoglutarate, a co-substrate required for the symbiotically important reaction catalyzed by aspartate aminotransferase (Fig. 2)
[75] (Section 3.1). Glutamate auxotrophy is a common link between the citrate synthase and isocitrate
dehydrogenase mutants, although it is interesting
that S. meliloti mutants de¢cient in both isocitrate
dehydrogenase and citrate synthase are Nod3 [75], in
contrast to the Nod‡ Fix3 phenotype obtained when
only one of these enzymes is lacking (Table 2).
2.2. Metabolism of dicarboxylic acids via
2-oxoglutarate dehydrogenase, succinyl CoA
synthetase, succinate dehydrogenase, fumarase
and malate dehydrogenase
The dicarboxylic acid portion of the TCA cycle
consists of ¢ve reactions which regenerate oxaloacetate from 2-oxoglutarate. This pathway is initiated
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by the 2-oxoglutarate dehydrogenase complex (EC
1.2.4.2), which catalyzes the oxidative decarboxylation of 2-oxoglutarate to succinyl CoA (Fig. 1). Expression of 2-oxoglutarate dehydrogenase in E. coli
is repressed by anaerobiosis [25,54,76] but, based
solely on activity measurements, this does not appear
to occur in R. leguminosarum bv. viciae [56]. The low
activity observed during growth of R. etli in oxygenlimited cultures [37] and in bacteroids (Table 1) may
result from a lack of cofactor biosynthesis, as discussed for pyruvate dehydrogenase (Section 1). The
2-oxoglutarate dehydrogenase from B. japonicum is
markedly inhibited by NADH in vitro and, if this
also occurs in bacteroids, may provide 2-oxoglutarate for the synthesis and accumulation of glutamate
[31].
A 2-oxoglutarate dehydrogenase (SucA; Table 2)
mutant of B. japonicum grew surprisingly well on
malate or succinate as carbon sources and was
even able to utilize glutamate to some extent. Because this phenotype di¡ers from that reported for
most other bacterial 2-oxoglutarate dehydrogenase
mutants, it was suggested that a bypass pathway
might be circumventing the metabolic block in the
mutant [77]. However, the presence of the Q-aminobutyrate bypass (Section 3.2) or the glyoxylate bypass (Section 3.4) could not be demonstrated in the
mutant [77]. Nodulation of soybean by the B. japonicum mutant was signi¢cantly delayed (Table 2) and
infected cells contained a drastically reduced number
of bacteroids. Interestingly, those mutant bacteria
which did successfully infect and di¡erentiate into
bacteroids ¢xed nitrogen at near wild-type levels, indicating that the mutant was severely hampered during the early stages of the interaction but could generate su¤cient energy to ¢x nitrogen without 2oxoglutarate dehydrogenase activity [78], perhaps
by using a yet unidenti¢ed bypass pathway in planta
[77,78].
A R. leguminosarum bv. viciae sucA transposon
mutant formed Fix3 nodules on pea (Table 2) and
excreted elevated levels of glutamate and 2-oxoglutarate when grown in culture [44]. This later ¢nding
indirectly supports the hypothesis that glutamate accumulates in bacteroids as a result of 2-oxoglutarate
dehydrogenase inhibition [31,44,79]. The sucA mutant produced several-fold higher activities of succinyl CoA synthetase and malate dehydrogenase in vi-
tro [44], an e¡ect also observed in a chemically
induced 2-oxoglutarate dehydrogenase mutant of S.
meliloti (Table 2). In these mutants the elevated succinyl CoA synthetase activity might act in the reverse
direction to generate precursor quantities of succinyl
CoA during growth on either succinate or glucose
(Fig. 2), while the higher malate dehydrogenase activity could also serve as part of the biosynthetic
route to succinyl CoA in the case of the R. leguminosarum mutant grown on glucose [44], or to increase the synthesis of 2-oxoglutarate by operating
in the forward direction in the case of the S. meliloti
mutant grown on succinate (Table 2 and Fig. 2). An
apparent contradiction exists between the symbiotic
phenotypes of the 2-oxoglutarate dehydrogenase mutants of S. meliloti (Fix3 ) and B. japonicum (Fix‡ )
(Table 2) and the proposed existence of the Q-aminobutyrate bypass in these genera, since the bypass
appears to exist in S. meliloti but not in B. japonicum
(Section 3.2).
Of possible regulatory signi¢cance is the fact that
in R. leguminosarum and B. japonicum the genes for
2-oxoglutarate dehydrogenase (sucAB), succinylCoA synthetase (sucCD) and malate dehydrogenase
(mdh) are arranged contiguously in the order mdh
sucCDAB [44,77,80]. This di¡ers from E. coli, where
mdh is encoded elsewhere on the chromosome and,
most importantly, a cluster containing the 2-oxoglutarate dehydrogenase and citrate synthase genes
[21,76] is regulated from the succinate dehydrogenase
(sdhCDAB) promoter located directly upstream
[81,82].
The hydrolysis of succinyl CoA to succinate (Fig.
1) is catalyzed by succinyl CoA synthetase (EC
6.2.1.6). A sucD insertion mutant of R. leguminosarum bv. viciae retained substantial succinyl CoA synthetase activity (raising the possibility of a paralog)
but nevertheless formed Fix3 nodules on pea (Table
2). This symbiotic phenotype was likely due to the
polar e¡ect of the sucD insertion on sucA (recall the
sucCDAB gene order described above) which virtually eliminated 2-oxoglutarate dehydrogenase activity in the mutant [44]. The sucD mutant was also
impaired in amino acid uptake via the general amino
acid permease, possibly as a result of increased glutamate excretion via this bidirectional system [44,83].
Because of the residual succinyl CoA synthetase activity and the pleiotropic e¡ects of the sucD inser-
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tion, conclusions about the requirement for this enzyme in symbiosis cannot be made.
Succinate dehydrogenase (EC 1.3.99.1) catalyzes
the dehydrogenation of succinate to fumarate (Fig.
1) and, accordingly, nitrosoguanidine-generated mutants of S. meliloti [84] and R. leguminosarum bv.
viciae [85] de¢cient in this enzyme exhibit little or
no growth on succinate but grow well on malate or
fumarate. The S. meliloti mutant was able to infect
alfalfa but not di¡erentiate into bacteroids (Table 2).
Likewise, pea plants inoculated with the R. leguminosarum mutant formed white, Fix3 nodules (Table
2). Bacteroids isolated from these nodules had substantial succinate transport activity [85] indicating
that the ability to metabolize succinate (Fig. 1) ^ as
well as malate (Section 3.1) ^ is required for nitrogen
¢xation but not for infection [85,86]. Thus succinate
and malate are not `equivalent' substrates for bacteroid metabolism as is sometimes assumed [9], perhaps because the rhizobial succinate dehydrogenase
also presumably functions as part of the electron
transport chain [25].
Fumarase (EC 4.2.1.2) catalyzes the hydration of
fumarate to form L-malate (Fig. 1). Three distinct
fumarases (fumA, fumB and fumC) are encoded in
E. coli. FumA and FumB are termed class I fumarases and perform distinct metabolic roles in the TCA
cycle under di¡erent growth conditions [21,25].
FumC is a class II fumarase which participates not
in the TCA cycle but rather in oxidative stress protection [21,87]. Curiously, the sole fumarase encoded
by B. subtilis is a homolog of the E. coli FumC [23]
but, unlike E. coli, fumC mutants of Bacillus exhibit
defects in TCA cycle metabolism [88].
A fumarase gene cloned from B. japonicum had
signi¢cant sequence identity to class II fumarases
and was accordingly designated fumC (Table 2). A
deletion mutant retained nearly 60% of the wild-type
fumarase activity indicating that a second fumarase
was present [89]. The heat stability of the residual
activity in the mutant was indicative of a class I
(TCA cycle) fumarase, and indeed the fumC mutant
was unaltered in its growth characteristics [89] and in
symbiosis (Table 2). From this, it is reasonable to
assume that the residual activity results from a
FumA and/or FumB paralog in B. japonicum. A fumarase knockout mutant might provide a tool for
determining if the conversion of succinate to malate
113
is important in bacteroids while avoiding a direct
e¡ect on the electron transport system, as may
have occurred with the succinate dehydrogenase mutant described above.
The ¢nal step in the TCA cycle regenerates oxaloacetate from malate and is catalyzed by malate
dehydrogenase (EC 1.1.1.37; Fig. 1). In rhizobia,
changes in malate dehydrogenase activity during
growth on di¡erent substrates are modest
[57,66,67,90] and are similar to those observed in
E. coli [53,91]. In contrast to E. coli, growth under
oxygen-limited conditions results in little or no decrease in malate dehydrogenase activity in rhizobia
[37,56] and activity is very high in bacteroids (Table
1).
Like the E. coli enzyme [92], malate dehydrogenase puri¢ed from B. japonicum bacteroids is inhibited
by NADH, although its kinetic properties, subunit
composition [93] and deduced amino acid sequence
are more similar to the B. subtilis enzyme (Table 2)
[94,95]. Upstream of both the B. japonicum [80] and
R. leguminosarum (Table 2) malate dehydrogenase
genes are sequences resembling members of the
AAA gene family. AAA gene family products regulate a variety of functions, including transcription, in
diverse organisms [96]. Interestingly, bacteroids
formed by a B. japonicum mutant with a disrupted
AAA-like gene produced substantially more malate
dehydrogenase protein and activity in soybean nodules, and had up to 50% higher rates of nitrogen
¢xation compared to nodules formed by the parental
strain [80]. Although the B. japonicum malate dehydrogenase has a signi¢cantly higher a¤nity for malate as compared to malic enzyme [97,98] (Section
3.1), further increasing malate dehydrogenase activity appears to bene¢t symbiosis by generating more
oxaloacetate for the TCA cycle or biosynthesis [80].
Malate dehydrogenase mutants have not yet been
isolated from rhizobia, either because mutation is
lethal [44] or because the activities of malic enzyme
(Section 3.1) and pyruvate carboxylase (Section 3.3)
compensate for the inactivated enzyme (Fig. 2), as
occurs in B. subtilis malate dehydrogenase mutants
[23]. The apparent high £ux of plant-provided malate
through the TCA cycle almost assures that a malate
dehydrogenase mutant, when available, will be Fix3 .
This prediction is less certain for A. caulinodans,
which may also utilize lactate in planta [11^13]. Ni-
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114
M.F. Dunn / FEMS Microbiology Reviews 22 (1998) 105^123
trogen ¢xation in this organism was, however, signi¢cantly reduced when malate dehydrogenase was
selectively inhibited with exogenous 2-oxoglutarate
[13].
3. Enzymes catalyzing anaplerotic reactions
Because it is regenerated, a single molecule of oxaloacetate would be su¤cient to maintain unlimited
turns of the TCA cycle operating in a purely catabolic mode. However, rhizobia are no di¡erent from
other organisms in using TCA cycle intermediates as
anabolic precursors [19] (Fig. 1). The withdrawal of
cycle intermediates for anabolism would soon halt
the cycle if these intermediates were not replaced,
and rhizobia contain a variety of anaplerotic enzymes which function in this capacity (Fig. 2). Also
discussed here are specialized anaplerotic pathways
which may function in bypassing selected reactions
of the cycle.
3.1. Metabolism of four-carbon compounds:
malic enzyme, aspartase and aspartate
aminotransferase
Growth on dicarboxylic acids using the TCA cycle
requires that rhizobia generate both oxaloacetate
(using malate dehydrogenase; Section 2.2) and acetyl
CoA from malate [20]. The oxidative decarboxylation of malate to pyruvate is catalyzed by malic enzyme (Fig. 2). Rhizobia typically possess both an
NADP-speci¢c malic enzyme (EC 1.1.1.40) and
an NAD-malic enzyme (EC 1.1.1.39) which also
has some activity with NADP as a cofactor [57,97^
101].
Finan and coworkers have constructed and characterized NAD-malic enzyme (dme), NADP-malic
enzyme (tme) and dme tme double mutants of S.
meliloti. On alfalfa the dme and dme tme mutants
formed small, Fix3 nodules while the tme mutant
was symbiotically unimpaired (Table 2). Measurements of malic enzyme activities and gene expression
showed that only the NAD-malic enzyme was induced in bacteroids [102,103]. Because the dme mutant bacteroids were enclosed in symbiosome membranes, NAD-malic enzyme activity appears to be
important during the active phase of nitrogen ¢xa-
tion but not for infection [104]. Thus it was proposed
that the NAD-malic enzyme degrades four-carbon
dicarboxylates to supply pyruvate to the TCA cycle,
and the ¢nding that acetyl CoA selectively inhibits the enzyme in vitro suggests that this metabolite is indeed the end-product of the NAD-malic
enzyme/pyruvate dehydrogenase pathway in vivo
[103]. The NADP-malic enzyme may function mainly in generating NADPH for biosynthesis [102^
104].
In rhizobia, NADP-malic enzyme has a higher af¢nity for malate in vitro relative to the NAD form.
This di¡erence is especially notable for the malic
enzymes from B. japonicum [97,98,103,105] whose
separate activities in bacteroids are similar [97].
Thus the di¡erent a¤nities of the malic enzymes
for malate may dictate a distinct symbiotic role for
each enzyme in this organism [105]. It has been proposed that environmental factors alter the level of
malate supplied by the plant to the bacteroids, with
low malate supply favoring the provision of
NADPH to nitrogenase via the high-a¤nity
NADP-malic enzyme. Because the NAD form would
be virtually inactive under these conditions, more
malate would £ow, via malate dehydrogenase, into
the TCA cycle, consuming acetyl CoA and preventing PHB accumulation. With high malate supply,
NADH generated by the NAD-malic enzyme would
inhibit TCA cycle activity and favor PHB synthesis,
resulting in a concomitant oxidation of a portion of
the NADPH produced by the NADP-malic enzyme
and making less reductant available for nitrogenase
[105]. Although environmentally induced changes in
malate provision to the bacteroids have not been
experimentally demonstrated, £ow chamber experiments with B. japonicum bacteroids show a strong
in£uence of malate supply and catabolism on PHB
accumulation and nitrogen ¢xation [42,105]. Because
only the relative intracellular concentration of malate
in B. japonicum bacteroids is known [106], relating
the di¡erent kinetic properties of the malic enzymes
to the levels of substrate likely to be available is not
possible.
Aspartase (EC 4.3.1.1) catalyzes the reversible deamination of aspartate to yield ammonia and fumarate (Fig. 2) and may thus play an anaplerotic as well
as a biosynthetic role in prokaryotes [107]. High concentrations of aspartate are present in nodules of
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M.F. Dunn / FEMS Microbiology Reviews 22 (1998) 105^123
some rhizobia-legume combinations [74,100,106]
although whether plant-derived aspartate is available
to bacteroids is less certain. The kinetic properties of
the Bradyrhizobium sp. (Lupinus) bacteroid aspartase
would appear to favor a biosynthetic rather than
anaplerotic role [108]. In R. leguminosarum, aspartase is present only in cells grown on aspartate or
asparagine, suggesting that it functions in the degradation of one or both of these amino acids [109].
Similarly, aspartase in R. etli functions in the degradation of asparagine after its conversion to aspartate
by an inducible asparaginase [110]. High aspartase
and asparaginase activities in R. etli [109] and Bradyrhizobium sp. (Lupinus) [111] bacteroids indicate
that some degradation of asparagine may occur in
symbiosis, but whether these reactions actually function to generate aspartate for fumarate synthesis is
not known. Bean nodules formed by aspartase-de¢cient R. etli mutants were Fix‡ (Table 2), although
aspartate aminotransferase (see below) could also
catabolize aspartate and compensate for the lowered
aspartase activity [112].
Aspartate aminotransferase (EC 2.6.1.1) catalyzes
the reversible transamination of aspartate and glutamate to yield oxaloacetate (Fig. 2). These enzymes
are ubiquitous and paralogs are common [113,114].
Aspartate could potentially be catabolized by bacterial aspartate aminotransferases to produce oxaloacetate for the TCA cycle [115], as high activities
are found in bacteroids of S. meliloti and several
other rhizobia [66,100,116,117].
Several distinct genes encoding aspartate aminotransferases have been cloned from S. meliloti
although only one, the product of aatA, is substrate
speci¢c and required for nitrogen ¢xation (Table 2).
The essential role of AatA in the S. meliloti-alfalfa
symbiosis suggests that aspartate may be an important carbon source in planta [118]. Catabolism of
aspartate to oxaloacetate by aspartate aminotransferase also results in the production of glutamate
(Fig. 2), which could be metabolized via 2-oxoglutarate (Section 2.2) or via the Q-aminobutyrate bypass
(Section 3.2) [118]. Bacteroids of S. meliloti are capable of aspartate transport [119] and levels of this
amino acid in the microsymbiont are less than 1%
that in the nodule cytosol [120], indicating that if
aspartate is transported into the bacteroids it is rapidly catabolized.
115
3.2. Metabolism of 2-oxoglutarate via the
Q-aminobutyrate bypass
Q-Aminobutyrate is often present in high concentrations in nodules [74,100,106,120] or bacteroids
[74,106,121] and can be used as a sole nitrogen and
carbon source by many rhizobia in vitro
[100,122,123]. Glutamate, the precursor of Q-aminobutyrate, is synthesized from 2-oxoglutarate as
shown in Fig. 2. The Q-aminobutyrate bypass allows
E. coli to metabolize endogenous oxoglutarate during anaerobic growth when 2-oxoglutarate dehydrogenase activity is repressed [124]. A similar use of the
bypass in rhizobia was discussed in Section 2. The
lack of glutamate transport across the symbiosome
membrane [125] appears to preclude a role for the
pathway in catabolizing host-supplied glutamate.
In the ¢rst step of the bypass glutamate is decarboxylated to Q-aminobutyrate by glutamate decarboxylase (EC 4.1.1.15; Fig. 2). Several workers
have failed to detect glutamate decarboxylase activity in Bradyrhizobium [31,77,100] while others have
reported high activities in bacteroids of this genus
[126] and in S. meliloti [121], where the enzyme is
induced by glutamate in cultures [127]. The inconsistency in detecting glutamate decarboxylase in rhizobia may be due to strain di¡erences, culture growth
conditions or enzyme extraction and assay methods,
all of which have varied considerably in di¡erent
studies.
The reversible conversion of Q-aminobutyrate to
succinate semialdehyde is catalyzed by Q-aminobutyrate transaminase (EC 2.6.1.19) (Fig. 2), which has
distinct preferences for keto acid acceptors in di¡erent rhizobia. The transaminase from B. japonicum
has maximal activity with pyruvate or oxaloacetate
[126] and much lower activity with 2-oxoglutarate
[31,126], while the enzymes from cowpea Rhizobium
[100] and S. meliloti [121] have roughly the opposite
preference for keto acid acceptor. If the Q-aminobutyrate bypass does in fact function in skirting an
inhibited 2-oxoglutarate dehydrogenase reaction,
the presence of oxoglutarate resulting from this inhibition might provide a physiological bene¢t for
using it as a keto acid acceptor (Fig. 2).
The ¢nal step of the bypass is the oxidation of
succinate semialdehyde to succinate via succinate
semialdehyde dehydrogenase (EC 1.2.2.16; Fig. 2).
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M.F. Dunn / FEMS Microbiology Reviews 22 (1998) 105^123
Rhizobia contain either a single succinate semialdehyde dehydrogenase able to use either NAD or
NADP as a cofactor [100], or separate NAD- and
NADP-linked isoenzymes [128]. Perhaps the best evidence for the Q-aminobutyrate bypass in rhizobia
comes from studies with an S. meliloti mutant (presumably regulatory) having reduced levels of both
NAD- and NADP-linked succinate semialdehyde dehydrogenases. This mutant grew poorly on glutamate [128] and formed Fix3 nodules on alfalfa (Table 2). Phenotypic revertants isolated from this
mutant retained the lowered succinate semialdehyde
dehydrogenase activities but produced elevated levels
of glutamate dehydrogenase. This, in conjunction
with the residual succinate semialdehyde dehydrogenase activity present in the revertants, may account for the ability of the revertants to catabolize
glutamate (Fig. 2) and again form Fix‡ nodules
[128].
3.3. Metabolism of three-carbon compounds:
pyruvate, phosphoenolpyruvate and propionyl
CoA carboxylases, pyruvate orthophosphate
dikinase and phosphoenolpyruvate carboxykinase
Carbon dioxide is a required nutrient for rhizobia
[129] and, like other bacteria, the carboxylating enzymes pyruvate carboxylase and/or phosphoenolpyruvate carboxylase are required for growth on threecarbon substrates. These enzymes use carbon dioxide
to convert pyruvate and phosphoenolpyruvate, respectively, into oxaloacetate (Fig. 2) which is then
used for biosynthesis or energy production via the
TCA cycle [130].
Pyruvate carboxylase (EC 6.4.1.1) catalyzes the
biotin-dependent carboxylation of pyruvate to form
oxaloacetate (Fig. 2). The enzymes produced by Bacillus, Rhodobacter and Rhizobium are K4 homotetramers which are allosterically activated by acetyl
CoA and inhibited by L-aspartate [131]. Pyruvate
carboxylase-negative mutants of R. leguminosarum
bv. trifolii and bv. viciae do not grow on three-carbon substrates or carbohydrates [132,133] but form
fully functional nodules on their respective hosts
(Table 2). In R. etli pyruvate carboxylase is expressed constitutively [123] and is regulated by acetyl
CoA and L-aspartate in vitro [131]. The major factor
a¡ecting pyruvate carboxylase activity in R. etli is
biotin availability [37,131], which determines how
much constitutively produced apoenzyme is converted to the active (biotinylated) holoenzyme.
Gene transcription, but not enzyme activity, was detected in R. etli bacteroids from bean nodules, suggesting that the levels of biotin available to the microsymbiont are relative low in planta [123].
The symbiotic phenotypes of R. etli and R. tropici
pyc mutants were indistinguishable from those of the
parental strains, consistent with the results obtained
with the R. leguminosarum mutants described above
(Table 2). This shows that the carboxylation of pyruvate to form oxaloacetate has no indispensable role
in symbiosis, although oxaloacetate may be anaplerotically produced from aspartate (Section 3.1) in the
pyruvate carboxylase mutants [131]. Because pyruvate carboxylase is required for growth on a wide
variety of carbon sources [123,131] it may be of importance to rhizobia living in the soil.
While S. meliloti and R. leguminosarum bv. trifolii
have been reported to lack phosphoenolpyruvate
carboxylase (EC 4.1.1.31) activity [66,132,134], the
enzyme has been detected in B. japonicum [129], R.
etli, R. tropici [37,131] and A. caulinodans (M. Dunn,
unpublished results). In these species, phosphoenolpyruvate carboxylase might reinforce or replace pyruvate carboxylase during growth on carbohydrates
or other compounds metabolized via pyruvate [131].
Evans and coworkers demonstrated that bacteroids and free-living cells of rhizobia use the methylmalonyl CoA mutase pathway to convert exogenously supplied propionate to succinyl CoA, which
is subsequently metabolized via the TCA cycle [135]
(Fig. 2). A key enzyme in this pathway is propionyl
CoA carboxylase (EC 6.4.1.3), a biotin-dependent
enzyme which converts propionyl CoA to methylmalonyl CoA (Fig. 2). The occurrence of propionyl
CoA carboxylase in diverse rhizobia [135] and the
fact that propionyl CoA can be derived from many
sources (e.g., the catabolism of odd-chain fatty acids
or certain amino acids [136]) indicate that the enzyme could ful¢l an anaplerotic role in rhizobia.
We (M. Dunn and G. Ara|èza) are currently exploring this possibility.
Pyruvate orthophosphate dikinase (EC 2.7.9.1)
catalyzes the reversible, ATP-dependent conversion
of pyruvate to phosphoenolpyruvate (Fig. 2). In E.
coli an analogous enzyme, phosphoenolpyruvate syn-
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thase, is required for growth on three-carbon acids.
Because phosphoenolpyruvate produced by pyruvate
orthophosphate dikinase (or phosphoenolpyruvate
synthase) is both an intermediate in gluconeogenesis
and a precursor for biosynthesis, these enzymes ful¢l
both gluconeogenic and anaplerotic functions [19].
In S. meliloti phosphoenolpyruvate synthase is absent and pyruvate orthophosphate dikinase activity
is produced at very low levels. Furthermore, mutants
are una¡ected in their utilization of carbon substrates [137] or in symbiosis (Table 2) making the
function of pyruvate orthophosphate dikinase rather
mysterious, at least under the growth conditions examined so far [137].
Phosphoenolpyruvate carboxykinase (EC 4.1.1.49)
catalyzes the reversible, ATP-dependent decarboxylation of oxaloacetate to form phosphoenolpyruvate
(Fig. 2). Its role in catalyzing the ¢rst step of gluconeogenesis has been demonstrated in several rhizobia
[90,138^142] although the symbiotic phenotype of
mutants lacking the enzyme is host-dependent (Table
2). Although phosphoenolpyruvate carboxykinase in
combination with pyruvate orthophosphate dikinase
could be used to generate oxaloacetate from pyruvate [136,143,144], several lines of evidence indicate
that it plays only a gluconeogenic role in rhizobia: (i)
rhizobia contain pyruvate and/or phosphoenolpyruvate carboxylases, which function anaplerotically
during growth on three-carbon glycolytic substrates,
(ii) phosphoenolpyruvate carboxykinase gene transcription is severely repressed during growth on glucose but is markedly induced during growth on gluconeogenic substrates [138,142,145] and (iii) S.
meliloti pyruvate orthophosphate dikinase mutants
are symbiotically indistinguishable from the wildtype [137], and this enzyme would be required along
with phosphoenolpyruvate carboxykinase for the
anaplerotic synthesis of oxaloacetate from pyruvate.
3.4. Metabolism of acetyl CoA via the glyoxylate
bypass
Growth on fatty acids or acetate requires the
glyoxylate bypass to circumvent the decarboxylating
steps of the TCA cycle (Fig. 2) and allow the net
assimilation of carbon from two-carbon precursors
[21]. In rhizobia, acetate may be converted to acetyl
CoA as described in Section 2.
117
Isocitrate lyase (EC 4.1.3.1) catalyzes the cleavage
of isocitrate to form succinate and glyoxylate (Fig.
2). Activity in rhizobia is highly induced during growth on acetate or oleate [68,77,146^148] but
at most trace levels are found in bacteroids
[68,147,148]. Furthermore, bacteroids isolated from
carbon-starved nodules of Phaseolus vulgaris contained no detectable isocitrate lyase and nodule lipids
were not utilized as a reserve carbon source by these
bacteroids [148]. In contrast, isocitrate lyase induction was observed in B. japonicum bacteroids isolated
from detached nodules or from plants maintained in
darkness [48]. Thus, although free-living rhizobia utilize acetate or fatty acids via the glyoxylate bypass,
this pathway does not normally operate in bacteroids
for lack of isocitrate lyase activity.
The mechanism by which rhizobia partition isocitrate between the TCA cycle and the glyoxylate bypass has recently been addressed. In E. coli the very
low substrate a¤nity of isocitrate lyase relative to
isocitrate dehydrogenase requires that the latter enzyme be inactivated by phosphorylation in order to
shunt isocitrate into the glyoxylate bypass [21]. In
contrast, the isocitrate Km s for the B. japonicum isocitrate lyase (62 WM) and isocitrate dehydrogenase
(16 WM) di¡er by only four-fold [68], and the activity
of the latter enzyme remains relatively constant regardless of the carbon source used for growth (Section 2.1). These and other data suggest that isocitrate
is shunted to the glyoxylate cycle during growth on
acetate due to the massive induction of isocitrate
lyase and not by post-translational modi¢cation of
the enzyme. Interestingly, B. japonicum isocitrate
lyase activity is very low during growth on malate,
which could explain its near absence in bacteroids
[68].
The last reaction of the glyoxylate bypass (Fig. 2)
is catalyzed by malate synthase (EC 4.1.3.2), which
uses acetyl CoA and glyoxylate as substrates (Fig. 2).
Unlike isocitrate lyase, malate synthase activity in
rhizobia is high during growth on carbon sources
other than acetate or oleate [68,134,146^148]. Low
levels of malate synthase activity were detected in
bacteroids isolated from pea [148], alfalfa and clover
and substantially higher activities in bacteroids from
bean, cowpea and soybean [68,148]. Labeling studies
with isolated B. japonicum bacteroids showed that a
portion of exogenously supplied acetate was metab-
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M.F. Dunn / FEMS Microbiology Reviews 22 (1998) 105^123
olized via malate synthase, with the remainder being
oxidized in the TCA cycle [29,30]. If malate synthase
does produce malate in bacteroids, the origin of the
glyoxylate used in the reaction does not appear to be
isocitrate lyase (see above) but could potentially be
provided by the plant host via ureide degradation
[3,149]. McDermott et al. [3] have made the interesting observation that malate synthase activities are
signi¢cantly higher in bacteroids isolated from most
ureide-transporting plants as compared to amidetransporting species. Glyoxylate could also be produced by purine degradation in the microsymbiont
[136], a possibility which remains to be explored.
4. Conclusions
Studies with carbon metabolic mutants have provided direct evidence for the importance of several
TCA cycle and anaplerotic enzymes in rhizobia.
Most of these enzymes are required for the normal
growth of free-living rhizobia under many conditions, and several are also essential in bacteroids
for generating energy or metabolic precursors from
dicarboxylic acids. This latter group of enzymes includes citrate synthase, isocitrate dehydrogenase, 2oxoglutarate dehydrogenase, succinate dehydrogenase and NAD-malic enzyme. Still lacking are de¢nitive biochemical, physiological and mutant studies
for several key enzymes including pyruvate dehydrogenase, malate dehydrogenase and aspartase. The
apparent existence of paralogs for fumarase and aconitase has not allowed us to determine the role of
these activities using the mutant strains available,
and much remains to be learned about the distinct
metabolic functions of these and other paralogs
under di¡erent growth conditions or in symbiosis.
Another important frontier in the study of carbon
metabolism in rhizobia will be the discovery and
characterization of global regulatory systems controlling carbon metabolism and integrating it with
the rest of cellular physiology.
From a practical standpoint, the characterization
of de¢ned metabolic mutants allows us to de¢ne
which reactions are dispensable and, more importantly, which might be bene¢cially enhanced by genetic manipulation. Although the metabolic engineering approach to rhizobial strain improvement
will undoubtedly be an extremely complex undertaking (i.e., one cannot ignore the control imposed by
the plant side of the interaction), studies such as
those described here are laying the groundwork
needed for such attempts.
Acknowledgments
I am grateful to Ismael Hernaèndez-Lucas, Jaime
Mora and Esperanza Mart|ènez-Romero for helpful
comments on the manuscript. I thank D. Day, D.
Emerich, S. Encarnacioèn, T. Finan, L. Green, M.
Guerinot, T. McDermott, V. Romanov, H. Taboada, C. Tabrett and S. Tajima for discussions or for
providing data prior to publication. Work in the
author's laboratory supported was by grants from
CONACyT
(N9111-0954,
3232P-N9608
and
3309PB)
and
DGAPA-UNAM
(IN202393,
IN209697 and IN213095).
References
[1] Rawsthorne, S., Minchin, F.R., Summer¢eld, R.J., Cookson,
C. and Coombs, J. (1980) Carbon and nitrogen metabolism in
legume root nodules. Phytochemistry 19, 341^355.
[2] Stowers, M.D. (1985) Carbon metabolism in Rhizobium species. Annu. Rev. Microbiol. 39, 89^108.
[3] McDermott, T.R., Gri¤th, S.M., Vance, C.P. and Graham,
P.H. (1989) Carbon metabolism in Bradyrhizobium japonicum
bacteroids. FEMS Microbiol. Rev. 63, 327^340.
[4] O'Gara, F., Birkenhead, K., Boesten, B. and Fitzmaurice,
A.M. (1989) Carbon metabolism and catabolite repression in
Rhizobium spp. FEMS Microbiol. Rev. 63, 93^102.
[5] Streeter, J.G. (1991) Transport and metabolism of carbon and
nitrogen in legume nodules. Adv. Bot. Res. 18, 129^187.
[6] Pueppke, S.G. (1996) The genetic and biochemical basis for
nodulation of legumes by rhizobia. Crit. Rev. Biotechnol. 16,
1^51.
[7] Batut, J. and Boistard, P. (1994) Oxygen control in Rhizobium. Antonie van Leeuwenhoek 66, 129^150.
[8] Udvardi, M.K. and Day, D.A. (1997) Metabolite transport
across symbiotic membranes of legume nodules. Annu. Rev.
Plant Physiol. Plant Mol. Biol. 48, 493^523.
[9] Day, D.A. and Copeland, L. (1991) Carbon metabolism and
compartmentation in nitrogen-¢xing legume nodules. Plant
Physiol. Biochem. 29, 185^201.
[10] Vance, C.P., Miller, S.S., Driscoll, B.T., Robinson, D.L.,
Trepp, G., Gantt, J.S. and Samas, D.A. (1998) Nodule carbon
metabolism : Organic acids for N2 ¢xation. In: Biological Nitrogen Fixation for the 21st Century (Elmerich, C., Kondor-
FEMSRE 611 19-8-98
M.F. Dunn / FEMS Microbiology Reviews 22 (1998) 105^123
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
osi, A. and Newton, W.E., Eds.), pp 443^448. Kluwer Academic, Dordrecht.
Trinchant, J.C. and Rigaud, J. (1987) Acetylene reduction by
bacteroids isolated from stem nodules of Sesbania rostrata.
Speci¢c role of lactate as an energy-yielding substrate.
J. Gen. Microbiol. 133, 37^43.
Trinchant, J.C. and Rigaud, J. (1989) Alternative energyyielding substrates for bacteroids isolated from stem and
root nodules of Sesbania rostrata submitted to O2 restricted
conditions. Plant Sci. 59, 141^149.
Trinchant, J.-C. and Rigaud, J. (1990) Occurrence of two
pathways for malate oxidation in bacteroids from Sesbania
rostrata stem nodules during C2 H2 reduction. Plant Physiol.
94, 1002^1008.
Kahn, M.L., Kraus, J. and Somerville, J.E. (1985) A model of
nutrient exchange in the Rhizobium-legume symbiosis. In: Nitrogen Fixation Research Progress (Evans, H., Bottomley, P.
and Newton, W.E., Eds.), pp. 193^199. M.J. Nijho¡, New
York.
Kohl, D.H., Straub, P.F. and Shearer, G. (1994) Does proline
play a special role in bacteroid metabolism? Plant Cell Environ. 17, 1257^1262.
D'Hooghe, I., Vander Vauven, C., Michiels, J., Tricot, C., de
Wilde, P., Vanderleyden, J. and Stalon, V. (1997) The arginine
deiminase pathway in Rhizobium etli : DNA sequence analysis
and functional study of the arcABC genes. J. Bacteriol. 179,
7403^7409.
Trinchant, J.-C., Gueèrin, V. and Rigaud, J. (1994) Acetylene
reduction by symbiosomes and free bacteroids from broad
bean (Vicia faba L.) nodules. Plant Physiol. 105, 555^561.
Hennecke, H. (1998) Rhizobial respiration to support symbiotic nitrogen ¢xation. In: Biological Nitrogen Fixation for the
21st Century (Elmerich, C., Kondorosi, A. and Newton,
W.E., Eds.), pp. 429^434. Kluwer Academic, Dordrecht.
Nimmo, H.G. (1987) The tricarboxylic acid cycle and anaplerotic reactions. In: Escherichia coli and Salmonella typhimurium : Cellular and Molecular Biology (Neidhardt, F.C., Ingraham, J.L., Low, K.B., Magasanik, B., Schaechter, M. and
Umbarger, H.E., Eds.), Vol. 2, pp. 156^169. ASM Press,
Washington, DC.
Sanwal, B.D. (1970) Allosteric controls of amphibolic pathways in bacteria. Bacteriol. Rev. 34, 20^39.
Cronan, J.E. and LaPorte, D. (1996) Tricarboxylic acid cycle
and glyoxylate bypass. In: Escherichia coli and Salmonella:
Cellular and Molecular Biology (Neidhardt, F.C., Curtiss,
R., Ingraham, J.L., Lin, E.C.C., Low, K.B., Magasanik, B.
and Rezniko¡, W.S., Eds.), pp. 201^216. ASM Press, Washington, DC.
Guest, J.R. and Russell, G.C. (1992) Complexes and complexities of the citric acid cycle in Escherichia coli. Curr. Top. Cell
Reg. 33, 231^247.
Hederstedt, L. (1993) The Krebs citric acid cycle. In: Bacillus
subtilis and Other Gram-positive Bacteria (Sonnenshein, A.L.,
Hoch, J.A. and Losick, R., Eds.), pp. 181^197. ASM Press,
Washington, DC.
Fischer, H.-M. (1994) Genetic regulation of nitrogen ¢xation
in rhizobia. Microbiol. Rev. 58, 352^386.
119
[25] Spiro, S. and Guest, J.R. (1991) Adaptive responses to oxygen
limitation in Escherichia coli. Trends Biochem. Sci. 16, 310^
314.
[26] Hinton, J.C.D. (1997) The Escherichia coli genome sequence:
the end of an era or the start of the FUN ? Mol. Microbiol.
26, 417^422.
[27] Antoun, H., Bordeleau, L.M. and Sauvageau, R. (1984)
Utilization of tricarboxylic acid cycle intermediates and symbiotic e¤ciency in Rhizobium meliloti. Plant Soil 77, 29^38.
[28] Anand, R.C. and Dogra, R.C. (1997) Comparative e¤ciency
of Rhizobium/Bradyrhizobium spp. strains in nodulating Cajanus cajan in relation to characteristic metabolic enzymes. Biol.
Fertil. Soils 24, 283^287.
[29] Stovall, I. and Cole, M. (1978) Organic acid metabolism by
isolated Rhizobium japonicum bacteroids. Plant Physiol. 61,
787^790.
[30] Tajima, S., Kimura, I., Kouzai, K. and Kasai, T. (1990) Succinate degradation through the citric acid cycle in Bradyrhizobium japonicum J501 bacteroids under low oxygen concentrations. Agric. Biol. Chem. 54, 891^897.
[31] Salminen, S.O. and Streeter, J.G. (1990) Factors contributing
to the accumulation of glutamate in Bradyrhizobium japonicum bacteroids under microaerobic conditions. J. Gen. Microbiol. 136, 2119^2126.
[32] Keele, B.B., Hamilton, P.B. and Elkan, G.H. (1970) Gluconate catabolism in Rhizobium japonicum. J. Bacteriol. 101, 698^
704.
[33] Stowers, M.D. and Elkan, G.H. (1983) The transport and
metabolism of glucose in cowpea rhizobia. Can. J. Microbiol.
29, 398^406.
[34] Stowers, M.D. and Elkan, G.H. (1984) Gluconate metabolism
in cowpea rhizobia : evidence for a ketogluconate pathway.
Arch. Microbiol. 137, 3^9.
[35] Smith, M.W. and Neidhardt, F.C. (1983) 2-Oxoacid dehydrogenase complexes of Escherichia coli : cellular amounts and
patterns of synthesis. J. Bacteriol. 156, 81^88.
[36] Quail, M.A. and Guest, J.R. (1995) Puri¢cation, characterization and mode of action of PdhR, the transcriptional repressor of the pdhR-aceEF-lpd operon of Escherichia coli. Mol.
Microbiol. 15, 519^529.
[37] Encarnacioèn, S., Dunn, M., Willms, K. and Mora, J. (1995)
Fermentative and aerobic metabolism in Rhizobium etli.
J. Bacteriol. 177, 3058- 3066.
[38] Taboada, H., Encarnacioèn, S., Daèvalos, A., Leija, A., Mora,
Y., Miranda, J., Soberoèn, M. and Mora, J. (1998) Role of the
pyruvate dehydrogenase (PDH) and pyruvate formate lyase
(PFL) in Rhizobium etli symbiosis. In: Biological Nitrogen
Fixation for the 21st Century (Elmerich, C., Kondorosi, A.
and Newton, W.E., Eds.), p. 468. Kluwer Academic, Dordrecht.
[39] Tateè, R., Riccio, A., Iaccarino, M. and Patriarca, E.J. (1997)
Cloning and transcriptional analysis of the lipA (lipoic acid
synthetase) gene from Rhizobium etli. FEMS Microbiol. Lett.
149, 165^172.
[40] Copeland, L., Chohan, S.N. and Kim, S.A. (1998) Malate
metabolism and poly-3-hydroxybutyrate accumulation in bacteroids. In: Biological Nitrogen Fixation for the 21st Century
FEMSRE 611 19-8-98
120
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
[54]
M.F. Dunn / FEMS Microbiology Reviews 22 (1998) 105^123
(Elmerich, C., Kondorosi, A. and Newton, W.E., Eds.), pp
459^460. Kluwer Academic, Dordrecht.
Anderson, A.J. and Dawes, E.A. (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54, 450^472.
Bergersen, F.J. and Turner, G.L. (1990) Bacteroids from soybean root nodules : accumulation of poly-L-hydroxybutyrate
during supply of malate and succinate in relation to N2 ¢xation in £ow-chamber experiments. Proc. R. Soc. Lond B.
240, 39^59.
Mora, J., Encarnacioèn, S., Salgado, M., Mora, Y., Mendoza,
A. and Leija, A. (1993) Carbon and nitrogen metabolism in
Rhizobium. In: New Horizons in Nitrogen Fixation (Palacios,
R., Mora, J. and Newton, W.E., Eds.), pp. 513^516. Kluwer
Academic, Dordrecht.
Walshaw, D.L., Wilkinson, A., Mundy, M., Smith, M. and
Poole, P.S. (1997) Regulation of the TCA cycle and the general amino acid permease by over£ow metabolism in Rhizobium leguminosarum. Microbiology 143, 2209^2221.
Patel, J.J. and Gerson, T. (1974) Formation and utilisation of
carbon reserves by Rhizobium. Arch. Microbiol. 101, 211^
220.
Zevenhuizen, L.P.T.M. (1981) Cellular glycogen, L-1,2-glucan,
poly-L-hydroxybutyric acid and extracellular polysaccharides
in fast-growing species of Rhizobium. Antonie van Leeuwenhoek 47, 481^497.
Stam, H., van Verseveld, H.W., de Vries, W. and Stouthamer,
A.H. (1986) Utilization of poly-L-hydroxybutyrate in free-living cultures of Rhizobium ORS571. FEMS Microbiol. Lett.
35, 215^220.
Wong, P.P. and Evans, H.J. (1971) Poly-L-hydroxybutyrate
utilization by soybean (Glycine max Merr.) nodules and assessment of its role in maintenance of nitrogenase activity.
Plant Physiol. 47, 750^755.
Summers, M.L. and McDermott, T.R. (1998) Rhizobium meliloti genes coding for phosphotransacetylase and acetate kinase are in an operon under PhoB regulation. Abstracts of the
16th North American Conference on Nitrogen Fixation, p.
I.12.
Preston, G.G., Wall, J.D. and Emerich, D.W. (1990) Puri¢cation and properties of acetyl-CoA synthetase from Bradyrhizobium japonicum bacteroids. Biochem. J. 267, 179^183.
Preston, G.G., Zeiher, C., Wall, J.D. and Emerich, D.W.
(1989) Acetate-activating enzymes of Bradyrhizobium japonicum bacteroids. Appl. Environ. Microbiol. 55, 165^170.
Nystroëm, T. (1994) The glucose-starvation stimulon of Escherichia coli: induced and repressed synthesis of enzymes of
central metabolic pathways and role of acetyl phosphate in
gene expression and starvation survival. Mol. Microbiol. 12,
833^843.
Gray, C.T., Wimpenny, J.W.T. and Mossman, M.R. (1966)
Regulation of metabolism in facultative bacteria. II. E¡ects of
aerobiosis, anaerobiosis and nutrition on the formation of
Krebs cycle enzymes in Escherichia coli. Biochim. Biophys.
Acta 117, 33^41.
Iuchi, S. and Lin, E.C.C. (1988) arcA (dye), a global regulatory gene in Escherichia coli mediating repression of enzymes
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
in aerobic pathways. Proc. Natl. Acad. Sci. USA 85, 1888^
1892.
Park, S.-J., McCabe, J., Turna, J. and Gunsalus, R.P. (1994)
Regulation of the citrate synthase (gltA) gene of Escherichia
coli in response to anaerobiosis and carbon supply : role of the
arcA gene product. J. Bacteriol. 176, 5085^5092.
McKay, I.A., Dilworth, M.J. and Glenn, A.R. (1989) Chemostat studies of carbon catabolism in Rhizobium leguminosarum
MNF3841. Arch. Microbiol. 152, 606^610.
Romanov, V.I., Hernaèndez-Lucas, I. and Mart|ènez-Romero,
E. (1994) Carbon metabolism enzymes of Rhizobium tropici
cultures and bacteroids. Appl. Environ. Microbiol. 60, 2339^
2342.
Hernaèndez-Lucas, I., Pardo, M.A., Segovia, L., Miranda, J.
and Mart|ènez-Romero, E. (1995) Rhizobium tropici chromosomal citrate synthase gene. Appl. Environ. Microbiol. 61,
3992^3997.
Pardo, M.A., Lagunez, J., Miranda, J. and Mart|ènez, E.
(1994) Nodulating ability of Rhizobium tropici is conditioned
by a plasmid-encoded citrate synthase. Mol. Microbiol. 11,
315^321.
Tabrett, C.A. and Copeland, L. (1998) Inhibition of citrate
synthase from chickpea nodulating bacteria. In: Biological
Nitrogen Fixation for the 21st Century (Elmerich, C., Kondorosi, A. and Newton, W.E., Eds.), p. 474. Kluwer Academic, Dordrecht.
Kim, S.A. and Copeland, L. (1997) Acetyl coenzyme A acetyltransferase of Rhizobium sp. (Cicer) strain CC 1192. Appl.
Environ. Microbiol. 63, 3432^3437.
Suzuki, F., Zahler, W.L. and Emerich, D.W. (1987) Acetoacetyl-CoA thiolase of Bradyrhizobium japonicum bacteroids:
puri¢cation and properties. Arch. Biochem. Biophys. 254,
272^281.
Gruer, M.J., Artymiuk, P.J. and Guest, J.R. (1997) The aconitase family: three structural variations on a common theme.
Trends Biochem. Sci. 22, 3^6.
Thoëny-Meyer, L. and Kuënzler, P. (1996) The Bradyrhizobium
japonicum aconitase gene (acnA) is important for free-living
growth but not for an e¡ective root nodule symbiosis. J. Bacteriol. 178, 6166^6172.
Gruer, M.J., Bradbury, A.J. and Guest, J.R. (1997) Construction and properties of aconitase mutants of Escherichia coli.
Microbiology 143, 1837^1846.
Irigoyen, J.J., Sanchez-Diaz, M. and Emerich, D.W. (1990)
Carbon metabolism enzymes of Rhizobium meliloti cultures
and bacteroids and their distribution within alfalfa nodules.
Appl. Environ. Microbiol. 56, 2587^2589.
Mandal, N.C. and Chakrabartty, P.K. (1993) Succinate-mediated catabolite repression of enzymes of glucose metabolism
in root-nodule bacteria. Curr. Microbiol. 26, 247^251.
Green, L.S., Karr, D.B. and Emerich, D.W. (1998) Isocitrate
dehydrogenase and glyoxylate cycle enzyme activities in Bradyrhizobium japonicum under various growth conditions.
Arch. Microbiol. 169, 445^451.
Mulongoy, K. and Elkan, G.H. (1977) Glucose catabolism in
two derivatives of a Rhizobium japonicum strain di¡ering in
nitrogen-¢xing e¤ciency. J. Bacteriol. 131, 179^187.
FEMSRE 611 19-8-98
M.F. Dunn / FEMS Microbiology Reviews 22 (1998) 105^123
[70] Moustafa, E. and Leong, C.K. (1975) E¡ect of adenine nucleotides on NAD-dependent isocitrate dehydrogenases in rhizobia and bacteroids of legume root nodules. Biochim. Biophys. Acta 391, 9^14.
[71] Henson, C.A., Collins, M. and Duke, S.H. (1982) Subcellular
localization of enzymes of carbon and nitrogen metabolism
in nodules of Medicago sativa. Plant Cell Physiol. 23, 227^
235.
[72] Nambiar, P.T.C. and Shethna, Y.I. (1976) Puri¢cation and
properties of an NADP‡ -speci¢c isocitrate dehydrogenase
from Rhizobium meliloti. Antonie van Leeuwenhoek 42, 471^
482.
[73] Walsh, K. and Koshland, D.E. (1984) Determination of £ux
through the branch point of two metabolic cycles. The tricarboxylic acid cycle and the glyoxylate shunt. J. Biol. Chem.
259, 9646^9654.
[74] Ta, T.-C., Faris, M.A. and Macdowall, F.D.H. (1986) Pathways of nitrogen metabolism in nodules of alfalfa (Medicago
sativa L.). Plant Physiol. 80, 1002^1005.
[75] McDermott, T.R. and Kahn, M.L. (1992) Cloning and mutagenesis of the Rhizobium meliloti isocitrate dehydrogenase
gene. J. Bacteriol. 174, 4790^4797.
[76] Park, S.-J., Chao, G. and Gunsalus, R.P. (1997) Aerobic regulation of the sucABCD genes of Escherichia coli, which encode K-ketoglutarate dehydrogenase and succinyl coenzyme A
synthetase: Roles of ArcA, Fnr, and the upstream sdhCDAB
promoter. J. Bacteriol. 179, 4138^4142.
[77] Green, L.S. and Emerich, D.W. (1997) Bradyrhizobium japonicum does not require K-ketoglutarate dehydrogenase for
growth on succinate or malate. J. Bacteriol. 179, 194^201.
[78] Green, L.S. and Emerich, D.W. (1997) The formation of nitrogen-¢xing bacteroids is delayed but not abolished in soybean infected by an K-ketoglutarate dehydrogenase-de¢cient
mutant of Bradyrhizobium japonicum. Plant Physiol. 114,
1359^1368.
[79] Salminen, S.O. and Streeter, J.G. (1992) Labeling of carbon
pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv. viciae bacteroids following incubation of intact nodules with 14 CO2 . Plant Physiol. 100, 597^604.
[80] Birke, S.R., Green, L.S., Purcell, L.C. and Emerich, D.W.
(1998) Insertional mutagenesis of an AAA-like gene in Bradyrhizobium japonicum leads to increased levels of malate dehydrogenase and increased acetylene reduction activity by soybean nodules. In: Biological Nitrogen Fixation for the 21st
Century (Elmerich, C., Kondorosi, A. and Newton, W.E.,
Eds.), pp. 461^462. Kluwer Academic, Dordrecht.
[81] Suzuki, M., Sahara, T., Tsuruha, J.-I., Takada, Y. and Fukunaga, N. (1995) Di¡erential expression in Escherichia coli of
the Vibrio sp. strain ABE-1 icdI and icdII genes encoding
structurally di¡erent isocitrate dehydrogenase isozymes.
J. Bacteriol. 177, 2138^2142.
[82] Shen, J. and Gunsalus, R.P. (1997) Role of multiple ArcA
recognition sites in anaerobic regulation of succinate dehydrogenase (sdhCDAB) gene expression in Escherichia coli. Mol.
Microbiol. 26, 223^236.
[83] Walshaw, D.L. and Poole, P.S. (1996) The general L-amino
acid permease of Rhizobium leguminosarum is an ABC uptake
121
system that in£uences e¥ux of solutes. Mol. Microbiol. 21,
1239^1252.
[84] Gardiol, A., Arias, A., Cervenansky, C. and Mart|ènez-Derets,
G. (1982) Succinate dehydrogenase mutant of Rhizobium meliloti. J. Bacteriol. 151, 1621^1623.
[85] Finan, T.M., Wood, J.M. and Jordan, D.C. (1981) Succinate
transport in Rhizobium leguminosarum. J. Bacteriol. 148, 193^
202.
[86] Gardiol, A.E., Truchet, G.L. and Dazzo, F.B. (1987) Requirement of succinate dehydrogenase activity for symbiotic bacteroid di¡erentiation of Rhizobium meliloti in alfalfa nodules.
Appl. Environ. Microbiol. 53, 1947^1950.
[87] Liochev, S.I. and Fridovich, I. (1992) Fumarase C, the stable
fumarase of Escherichia coli, is controlled by the soxRS regulon. Proc. Natl. Acad. Sci. USA 89, 5892^5896.
[88] Carls, R.A. and Hanson, R.S. (1971) Isolation and characterization of tricarboxylic acid cycle mutants of Bacillus subtilis.
J. Bacteriol. 106, 848^855.
[89] Acunìa, G., Ebeling, S. and Hennecke, H. (1991) Cloning,
sequencing, and mutational analysis of the Bradyrhizobium
japonicum fumC-like gene: evidence for the existence of two
di¡erent fumarases. J. Gen. Microbiol. 137, 991^1000.
[90] Finan, T.M., Oresnik, I. and Bottacin, A. (1988) Mutants of
Rhizobium meliloti defective in succinate metabolism. J. Bacteriol. 170, 3396^3403.
[91] Park, S.-J., Cotter, P.A. and Gunsalus, R.P. (1995) Regulation of malate dehydrogenase (mdh) gene expression in Escherichia coli in response to oxygen, carbon, and heme availability. J. Bacteriol. 177, 6652^6656.
[92] Sanwal, B.D. (1969) Regulatory mechanisms involving nicotinamide adenine nucleotides as allosteric e¡ectors. I. Control
characteristics of malate dehydrogenase. J. Biol. Chem. 244,
1831^1837.
[93] Waters, J.K., Karr, D.B. and Emerich, D.W. (1985) Malate
dehydrogenase from Rhizobium japonicum 3I1B-143 bacteroids and Glycine max root-nodule mitochondria. Biochem.
24, 6479^6486.
[94] Yoshida, A. (1965) Puri¢cation and chemical characterization
of malate dehydrogenase of Bacillus subtilis. J. Biol. Chem.
240, 1113^1117.
[95] Yoshida, A. (1965) Enzymic properties of malate dehydrogenase of Bacillus subtilis. J. Biol. Chem. 240, 1118^1124.
[96] Confalonieri, F. and Duguet, M. (1995) A 200-amino acid
ATPase module in search of a basic function. BioEssays 17,
639^650.
[97] Copeland, L., Quinnell, R.G. and Day, D.A. (1989) Malic
enzyme activity in bacteroids from soybean nodules. J. Gen.
Microbiol. 135, 2005^2011.
[98] Tomaszewska, B. and Werner, D. (1995) Puri¢cation and
properties of NAD- and NADP-dependent malic enzymes
from Bradyrhizobium japonicum bacteroids. J. Plant Physiol.
146, 591^595.
[99] McKay, I.A., Dilworth, M.J. and Glenn, A.R. (1988) C4 -dicarboxylate metabolism in free-living and bacteroid forms of
Rhizobium leguminosarum MNF3841. J. Gen. Microbiol. 134,
1433^1440.
[100] Jin, H.N., Dilworth, M.J. and Glenn, A.R. (1990) 4-Amino-
FEMSRE 611 19-8-98
122
[101]
[102]
[103]
[104]
[105]
[106]
[107]
[108]
[109]
[110]
[111]
[112]
[113]
[114]
[115]
[116]
M.F. Dunn / FEMS Microbiology Reviews 22 (1998) 105^123
butyrate is not available to bacteroids of cowpea Rhizobium
MNF2030 in snake bean nodules. Arch. Microbiol. 153, 455^
462.
Chen, F., Okabe, Osano, K. and Tajima, S. (1997) Puri¢cation and characterization of the NADP-malic enzyme from
Bradyrhizobium japonicum A1017. Biosci. Biotechnol. Biochem. 61, 384^386.
Driscoll, B.T. and Finan, T.M. (1996) NADP‡ -dependent
malic enzyme of Rhizobium meliloti. J. Bacteriol. 178,
2224^2231.
Driscoll, B.T. and Finan, T.M. (1997) Properties of NAD‡ and NADP‡ -dependent malic enzymes of Rhizobium (Sinorhizobium) meliloti and di¡erential expression of their genes
in nitrogen-¢xing bacteroids. Microbiology 143, 489^498.
Driscoll, B.T. and Finan, T.M. (1993) NAD‡ -dependent
malic enzyme of Rhizobium meliloti is required for symbiotic
nitrogen ¢xation. Mol. Microbiol. 7, 865^873.
Day, D.A., Quinnell, R.G. and Bergersen, F.J. (1994) An
hypothesis for the role of malic enzyme in symbiotic nitrogen
¢xation in soybean nodules. In: Symbiotic Nitrogen Fixation
(Graham, P.H., Sadowsky, M.J. and Vance, C.P., Eds.)
pp. 159^164. Kluwer Academic, Dordrecht.
Streeter, J.G. (1987) Carbohydrate, organic acid, and amino
acid composition of bacteroids and cytosol from soybean
nodules. Plant Physiol. 85, 768^773.
Hubert, J.-C. and Wurtz, B. (1975) Regulation and physiological signi¢cance of aspartate-ammonium lyase (aspartase)
of Pseudomonas £uorescens type R. Arch. Microbiol. 102,
35^39.
Kretovich, W.L., Kariakina, T.I., Weinova, M.K., Sidelnikova, L.I. and Kazakova, O.W. (1981) The synthesis of aspartic
acid in Rhizobium lupini bacteroids. Plant Soil 61, 145^156.
Poole, P.S., Dilworth, M.J. and Glenn, A.R. (1984) Acquisition of aspartase activity in Rhizobium leguminosarum
WU235. J. Gen. Microbiol. 130, 881^886.
Huerta-Zepeda, A., Duraèn, S., Du Pont, G. and Calderoèn, J.
(1996) Asparagine degradation in Rhizobium etli. Microbiology 142, 1071^1076.
Kretovich, V.L., Sidel'nikova, L.I., Ivanushkin, A.G. and
Karayakina, T.I. (1984) Localization of aspartase, asparaginase, and glutaminase in intact bacteroids of Rhizobium lupini.
Prikl. Biokhim. Mikrobiol. 20, 445^447.
Huerta-Zepeda, A., Ortunìo, L., Du Pont, G., Duraèn, S.,
Lloret, A., Merchant-Larios, H. and Calderoèn, J. (1997) Isolation and characterization of Rhizobium etli mutants altered
in degradation of asparagine. J. Bacteriol. 179, 2068^2072.
Cooper, A.J.L. and Meister, A. (1989) An appreciation of
professor Alexander E. Braunstein. The discovery and scope
of enzymatic transamination. Biochimie 71, 387^404.
Wine¢eld, C.S., Farnden, K.J.F., Reynolds, P.H.S. and Marshall, C.J. (1995) Evolutionary analysis of aspartate aminotransferases. J. Mol. Evol. 40, 455^463.
Alfano, J.R. and Kahn, M.L. (1993) Isolation and characterization of a gene coding for a novel aspartate aminotransferase from Rhizobium meliloti. J. Bacteriol. 175, 4186^4196.
Kim, S.A. and Copeland, L. (1996) Enzymes of poly-L-hy-
[117]
[118]
[119]
[120]
[121]
[122]
[123]
[124]
[125]
[126]
[127]
[128]
[129]
[130]
[131]
[132]
droxybutyrate metabolism in soybean and chickpea bacteroids. Appl. Environ. Microbiol. 62, 4186^4190.
Streeter, J.G. and Salminen, S.O. (1990) Periplasmic metabolism of glutamate and aspartate by intact Bradyrhizobium
japonicum bacteroids. Biochim. Biophys. Acta 1035, 257^265.
Rastogi, V.K. and Watson, R.J. (1991) Aspartate aminotransferase activity is required for aspartate catabolism and
symbiotic nitrogen ¢xation in Rhizobium meliloti. J. Bacteriol. 173, 2879^2887.
McRae, D.G., Miller, R.W., Berndt, W.B. and Joy, K.
(1989) Transport of C4 -dicarboxylates and amino acids by
Rhizobium meliloti bacteroids. Mol. Plant-Microbe Interact.
2, 273^278.
Fougeère, F., Le Rudulier, D. and Streeter, J.G. (1991) E¡ects
of salt stress on amino acid, organic acid, and carbohydrate
composition of roots, bacteroids, and cytosol of alfalfa
(Medicago sativa L.). Plant Physiol. 96, 1228^1236.
Miller, R.W., McRae, D.G. and Joy, K. (1991) Glutamate
and Q-aminobutyrate metabolism in isolated Rhizobium meliloti bacteroids. Mol. Plant-Microbe Interact. 4, 37^45.
Labidi, M., Lalande, R., Laberge, S. and Antoun, H. (1996)
E¡ect of glutamate transport and catabolism on symbiotic
e¡ectiveness in Rhizobium leguminosarum bv. phaseoli. Plant
Soil 182, 51^58.
Dunn, M.F., Ara|èza, G., Cevallos, M.A. and Mora, J. (1997)
Regulation of pyruvate carboxylase in Rhizobium etli. FEMS
Microbiol. Lett. 157, 301^306.
Rosenqvist, H., Kasula, H., Reunanen, O. and Nurmikko, V.
(1973) The 4-aminobutyrate pathway and 2-oxoglutarate dehydrogenase in Escherichia coli. Acta Chem. Scand. 27,
3091^3100.
Udvardi, M.K., Salom, C.L. and Day, D.A. (1988) Transport of L-glutamate across the bacteroid membrane but not
the peribacteroid membrane from soybean root nodules.
Mol. Plant-Microbe Interact. 1, 250^254.
Kouchi, H., Fukai, D. and Kihara, A. (1991) Metabolism of
glutamate and aspartate in bacteroids isolated from soybean
root nodules. J. Gen. Microbiol. 137, 2901^2910.
Fitzmaurice, A.M. and O'Gara, F. (1991) Glutamate catabolism in Rhizobium meliloti. Arch. Microbiol. 155, 422^427.
Fitzmaurice, A.M. and O'Gara, F. (1993) A Rhizobium meliloti mutant, lacking a functional Q-aminobutyrate (GABA)
bypass, is defective in glutamate catabolism and symbiotic
nitrogen ¢xation. FEMS Microbiol. Lett. 109, 195^202.
Lowe, R.H. and Evans, H.J. (1962) Carbon dioxide requirement for growth of legume nodule bacteria. Soil Sci. 94, 351^
356.
Scrutton, M.C. (1978) Fine control of the conversion of pyruvate (phosphoenolpyruvate) to oxaloacetate in various species. FEBS Lett. 89, 1^9.
Dunn, M.F., Encarnacioèn, S., Ara|èza, G., Vargas, M.C.,
Daèvalos, A., Peralta, H., Mora, Y. and Mora, J. (1996)
Pyruvate carboxylase from Rhizobium etli: Mutant characterization, nucleotide sequence, and physiological role.
J. Bacteriol. 178, 5960^5970.
Ronson, C.W. and Primrose, S.B. (1979) Carbohydrate me-
FEMSRE 611 19-8-98
M.F. Dunn / FEMS Microbiology Reviews 22 (1998) 105^123
[133]
[134]
[135]
[136]
[137]
[138]
[139]
[140]
[141]
[142]
[143]
tabolism in Rhizobium trifolii: Identi¢cation and symbiotic
properties of mutants. J. Gen. Microbiol. 112, 77^88.
Arwas, R., Glenn, A.R., McKay, I.A. and Dilworth, M.J.
(1986) Properties of double mutants of Rhizobium leguminosarum which are defective in the utilization of dicarboxylic
acids and sugars. J. Gen. Microbiol. 132, 2743^2747.
Duncan, M.J. and Fraenkel, D.G. (1979) K-Ketoglutarate
dehydrogenase mutant of Rhizobium meliloti. J. Bacteriol.
37, 415^419.
De Hertogh, A.A., Meyeux, P.A. and Evans, H.J. (1964) The
relationship of cobalt requirement to propionate metabolism
in Rhizobium. J. Biol. Chem. 239, 2446^2453.
Gottschalk, G. (1986) Bacterial Metabolism, 2nd edn., 359
pp. Springer-Verlag, New York.
Òsteraîs, M., Driscoll, B.T. and Finan, T.M. (1997) Increased
pyruvate orthophosphate dikinase activity results in an alternative gluconeogenic pathway in Rhizobium (Sinorhizobium)
meliloti. Microbiology 143, 1639^1648.
Òsteraîs, M., Finan, T.M. and Stanley, J. (1991) Site-directed
mutagenesis and DNA sequence of pckA of Rhizobium
NGR234, encoding phosphoenolpyruvate carboxykinase:
gluconeogenesis and host-dependent symbiotic phenotype.
Mol. Gen. Genet. 230, 257^269.
Finan, T.M., McWhinnie, E., Driscoll, B. and Watson, R.J.
(1991) Complex symbiotic phenotypes result from gluconeogenic mutations in Rhizobium meliloti. Mol. Plant-Microbe
Interact. 4, 386^392.
Arwas, R., McKay, I.A., Rowney, F.R.P., Dilworth, M.J.
and Glenn, A.R. (1985) Properties of organic acid utilization
mutants of Rhizobium leguminosarum strain 300. J. Gen. Microbiol. 131, 2059^2066.
McKay, I.A., Glenn, A.R. and Dilworth, M.J. (1985) Gluconeogenesis in Rhizobium leguminosarum MNF3841. J. Gen.
Microbiol. 131, 2067^2073.
Òsteraîs, M., Driscoll, B.T. and Finan, T.M. (1995) Molecular and expression analysis of the Rhizobium meliloti phosphoenolpyruvate carboxykinase (pckA) gene. J. Bacteriol.
177, 1452^1460.
Schobert, P. and Bowien, B. (1984) Unusual C3 and C4 metabolism in the chemoautotroph Alcaligenes eutrophus.
J. Bacteriol. 159, 167^172.
123
[144] Laivenieks, M., Vieille, C. and Zeikus, J.G. (1977) Cloning,
sequencing, and overexpression of the Anaerobiospirillum
succiniciproducens
phosphoenolpyruvate
carboxykinase
(pckA) gene. Appl. Environ. Microbiol. 63, 2273^2280.
[145] Òsteraîs, M., O'Brien, S.A. and Finan, T.M. (1997) Genetic
analysis of mutations a¡ecting pckA regulation in Rhizobium
(Sinorhizobium) meliloti. Genetics 147, 1521^1531.
[146] Mandal, N.C. and Chakrabartty, P.K. (1992) Regulation of
enzymes of glyoxylate pathway in root-nodule bacteria.
J. Gen. Appl. Microbiol. 38, 417^427.
[147] Hernaèndez-Lucas, I. (1996) Ph.D. Thesis, National University of Mexico.
[148] Johnson, G.V., Evans, H.J. and Ching, T. (1965) Enzymes of
the glyoxylate cycle in rhizobia and nodules of legumes.
Plant Physiol. 41, 1330^1336.
[149] Schubert, K.R. (1986) Products of biological nitrogen ¢xation in higher plants: Synthesis, transport, and metabolism.
Annu. Rev. Plant Physiol. 37, 539^574.
[150] Karr, D.B., Waters, J.K., Suzuki, F. and Emerich, D.W.
(1984) Enzymes of the poly-L-hydroxybutyrate and citric
acid cycles of Rhizobium japonicum bacteroids. Plant Physiol.
75, 1158^1152.
[151] Reibach, P.H., Mask, P.L. and Streeter, J.G. (1981) A rapid
one-step method for the isolation of bacteroids from root
nodules of soybean plants, utilizing self-generating Percoll
gradients. Can. J. Microbiol. 27, 491^495.
[152] Freiberg, C., Fellay, R., Bairoch, A., Broughton, W.J.,
Rosenthal, A. and Parret, X. (1997) Molecular basis of symbiosis between Rhizobium and legumes. Nature 387, 394^
401.
[153] Kahn, M.L., Mortimer, M., Park, K.S. and Zhang, W.
(1995) Carbon metabolism in the Rhizobium-legume symbiosis. In: Nitrogen Fixation: Fundamentals and Applications
(Tikhonovich, I.A., Provorov, N.A., Romanov, V.I. and
Newton, W.E., Eds.), pp. 525^532. Kluwer Academic, Dordrecht.
[154] Watson, R.J. and Rastogi, V.K. (1993) Cloning and nucleotide sequencing of Rhizobium meliloti aminotransferase genes: an aspartate aminotransferase required for
symbiotic nitrogen ¢xation is atypical. J. Bacteriol. 175,
1919^1928.
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