JOURNAL OF BACTERIOLOGY, July 1993, p. 4186-4196 Vol. 175, No.

13
0021-9193/93/134186-11$02.00/0
Copyright X 1993, American Society for Microbiology

Isolation and Characterization of a Gene Coding for a Novel

Aspartate Aminotransferase from Rhizobium meliloti
JAMES R. ALFANO AND MICHAEL L. KAHN*
Institute of Biological Chemistry and Department of Microbiology, Washington State University,
Pullman, Washington 99164-6340
Received 2 November 1992/Accepted 21 April 1993

Aspartate aminotransferase (AAT) is an important enzyme in aspartate catabolism and biosynthesis and, by
converting tricarboxylic acid cycle intermediates to amino acids, AAT is also significant in linking carbon
metabolism with nitrogen metabolism. To examine the role of AAT in symbiotic nitrogen fixation further,
plasmids encoding three different aminotransferases from Rhizobium meliloti 104A14 were isolated by
complementation of an Escherichia coli auxotroph that lacks three aminotransferases. pJA10 contained a gene,
aatB, that coded for a previously undescribed AAT, AatB. pJA30 encoded an aromatic aminotransferase,
TatA, that had significant AAT activity, and pJA20 encoded a branched-chain aminotransferase designated
BatA. Genes for the latter two enzymes, WttA and batA, were previously isolated from R. meliloti. aatB is distinct
from but hybridizes to aaL4, which codes for AatA, a protein required for symbiotic nitrogen fixation. The DNA
sequence of aatB contained an open reading frame that could encode a protein 410 amino acids long and with
a monomer molecular mass of 45,100 Da. The amino acid sequence of aatB is unusual, and AatB appears to
be a member of a newly described class of AATs. AatB expressed in E. coli has a Km for aspartate of 5.3 mM
and a Km for 2-oxoglutarate of 0.87 mM. Its pH optimum is between 8.0 and 8.5. Mutations were constructed
in aatB and hztA and transferred to the genome ofR. melioti 104A14. Both mutants were prototrophs and were
able to carry out symbiotic nitrogen fixation.

Bacteria of the genus Rhizobium fix nitrogen in a symbi-
otic association with leguminous plants. Carbon compounds
produced by the plant are fed to the bacteria and metabo-
lized to supply the energy and reductant needed for nitrogen
fixation. Identifying which carbon compounds are trans-
ferred to the bacteria and how these compounds are metab-
olized is important in understanding the physiology of the
symbiosis. Although sucrose is the major carbon source
transported from the shoot to the nodule (44), most data
suggest that it is not an important source of reductant for
bacteroids (58) and that dicarboxylic acids are more likely to
be their main carbon and energy sources (8, 13, 56). Succi-
nate and malate are found in high concentrations in nodules
(20, 49, 57) and bacteroids (15, 29, 45, 51) and are actively
transported across the symbiosome membrane (29, 62).
Bacterial mutants that lack dicarboxylate transport protein
DctA are Fix- (5, 17, 48), demonstrating that the acquisition
of dicarboxylic acids is important to the bacteria during
symbiosis.
Kahn et al. (32) proposed that the carbon compounds
supplied to bacteroids include amino acids. The amino acid
aspartate is a dicarboxylic acid, like succinate and malate,
and the dicarboxylate transport system of Rhizobium meli-
loti is required for aspartate to be used as a carbon source
(65). Growth on aspartate as a sole carbon and nitrogen
source is also blocked by mutations in aspartate aminotrans-
ferase (AAT) (43) and glutamate dehydrogenase (36). AAT,
which catalyzes the reversible conversion of aspartate and
2-oxoglutarate to glutamate and oxaloacetate (OAA), is
generally important in aspartate synthesis and catabolism, in
the coupling of cell carbon metabolism to nitrogen metabo-
lism, and in the metabolism of tricarboxylic acid cycle
intermediates (10). Cells often have more than one AAT.
*
Corresponding author.
4186
Recently, an AAT mutant of R. meliloti was isolated; in
addition to its defect in catabolism, this mutant was Fix-
(43).
The concentration of AAT is high in both the plant and the
bacterial compartments of nodules relative to those of other
enzymes needed for assimilation (12, 24, 28, 47, 52, 59).
Alfalfa cDNAs encoding two different forms of AAT, AAT-1
and AAT-2, have been cloned, and mRNA analysis shows
that both forms are induced in alfalfa nodules (21, 61).
Although AAT is generally considered to be important in
the conversion of ammonia produced by nitrogen fixation
into asparagine for transport to the rest of the plant (64), one
role for plant nodule AATs could be to produce aspartate for
bacterial catabolism. This catabolism could be direct or part
of a more complicated pathway, such as the malate-aspartate
shuttle found in mitochondria (1, 32). The two soluble
enzymes of this shuttle, AAT and malate dehydrogenase (1),
are present in high levels in both the plant cytoplasm and
bacteroid fractions in pea root nodules (49), as are their
substrates. For such a shuttle to function in nodules, the
carbon compounds involved must be transported across the
symbiosome membrane and the bacteroid membrane. By
using an in vitro assay, Udvardi et al. (63) found that
glutamate was not taken up in physiologically relevant
concentrations by isolated soybean symbiosomes. However,
an R. meliloti isocitrate dehydrogenase mutant that is a
glutamate auxotroph (40) grew to normal concentrations in
alfalfa nodules, suggesting that in alfalfa nodules glutamate is
available to bacteroids.
We are interested in the role of aspartate in the Rhizobi-
um-alfalfa symbiosis. We chose to investigate bacterial
AATs since they are central to aspartate biosynthesis and
catabolism. Three aminotransferase genes from R. meliloti
were isolated by complementing an Eschenchia coli auxo-
troph defective in three aminotransferases with plasmids
containing R. meliloti DNA. Extracts from strains contain-
VOL. 175, 1993 R. MELILOTI AAT 4187

TABLE 1. Genetic materials

Bacterium, plasmid, or Relevant properties Reference
bacteriophage or source
Bacteria
E. coli
DL39(Mucts) aspC13 ilvE12 tyrB507 (Mucts) 2
XL1-Blue lacq lacZAM15 Stratagene
C-2324 P2 1g cc 33
S17-1 pro RK2 tra+ 53
C-2110 poL4 31
R. melioti
104A14 Wild type 55
WSU600 104A14 tatA This work
WSU601 104A14 aatB This work
WSU602 104A14 tatA aatB This work
JJlC10 Wild type 43
4R3 JJC1O aatA 43

Plasmids
pBluescript KS+/- Phagemid Stratagene
pMK409 tetA sacB sacR P4cos 67
pMK411 tetA sacB sacR P4cos with pUC18 homology This work
pMK413 tetA sacB sacR RK2mob 40
TnS-B61 Genr Tn5 derivative 54
pJA10 R. meliloti aatB in pUC18 This work
pJA12 3.7-kb BamHI-PstI fragment carrying aatB in pUC18 This work
pJA14 2.2-kb BamHI-PstI fragment carrying aatB in pUC18 This work
pJA15 Genr aatB mutant of pJA14 This work
pJA16 pJA15 cloned into pMK413 This work
pJA20 R- melloti batA gene in pUC18 This work
pJA30 R. meliloti tatA gene in pUC18 This work
pJA31 Kan' tatA::TnS mutant of pJA30 This work
pJA32 pJA31 recombined with pMK411 This work

Bacteriophages
P2 virl Clear plaques; immunity sensitive 33
X467 X b221 rex::TnS cI857 Oam29 Pam8O 9
VCSM13 Interference-resistant helper phage Stratagene

ing two of these plasmids had significant AAT activity, and
one of these enzymes is a new AAT, AatB. To begin
assessing the role(s) of these enzymes in nitrogen fixation,
we generated mutants with mutations in both AATs. In
contrast to the AatA mutant recently isolated (43), both
mutants were Fix'. The DNA sequence of aatB, the gene
that encodes AatB, predicts a protein that is different from
the well known and highly conserved AATs from eubacteria
and eukaryotes. Instead, it is similar to a recently discovered
group of AATs that includes AATs from archaebacteria
and thermophiles (7). aatB is also similar to the R meliloti
aatA gene, which is necessary for symbiotic nitrogen fixa-
tion.
MATERIALS AND METHODS
Bacterial strains, plasmids, and media. Bacterial strains,
plasmids, and bacteriophages are listed in Table 1. R
meliloti was grown at 30'C on yeast mannitol broth (YMB)
(55) or minimal mannitol (55) medium supplemented with
0.05% NH4Cl or 0.1% aspartate. For growth studies with
aspartate as a sole carbon source, minimal salts medium was
supplemented with 0.4% aspartate and 0.01% yeast extract.
Antibiotics used for R. meliloti were tetracycline (10 mg/
liter), kanamycin (200 mg/liter), and gentamicin (30 mg/liter).
YMB was supplemented with 5% sucrose for selection
against plasmids that contained the Bacillus subtilis levan-
sucrase genes. E. coli strains were grown at 370C on LB
medium (55), except for DL39(Mucts) (2), which was grown
at 30'C. DL39(Mucts) is heat sensitive and will not grow at
370C. For selection for the rescue of DL39(Mucts) by the R.
meliloti gene library, DL39(Mucts) was grown on minimal
medium lacking aspartate, tyrosine and phenylalanine, or
leucine; the minimal medium used was modified from that of
Kuramitsu et al. (38) and has been described elsewhere (61).
Antibiotics used for E. coli were penicillin (200 mg/liter),
tetracycline (25 mg/liter), kanamycin (75 mg/liter), and gen-
tamicin (20 mg/liter).
DNA manipulations. Restriction enzymes and T4 DNA
ligase were purchased from New England Biolabs and used
in accordance with the manufacturer's instructions. Stan-
dard recombinant DNA manipulations were done as de-
scribed by Sambrook et al. (50). The R. meliloti gene library
used in this study was constructed in pUC18 and has been
described elsewhere (40).
Construction of marker exchange vehicle pMK411.
pMK409 (67) is an RK2-derived cosmid that contains the
bacteriophage P4 cos sequence, a Tetr gene, and a levansu-
4188 ALFANO AND KAHN
crase gene (sacB) from B. subtilis that confers sucrose
sensitivity (Sucs) to gram-negative bacteria. To provide a
region of homology for recombination between this plasmid
and plasmids from the pUC18-derived gene library, pUC18
and pMK409 were digested with BamHI and HindIII, ligated
together, and transformed into C-2110 (31), a strain that does
not permit the replication of pUC18. Tetr Penr transformants
were screened for Suc'. Only one type of plasmid was
expected to have all of these properties, and examination of
the plasmid DNAs in these transformants confirmed the
construction of pMK411.
Mutagenesis and marker exchange. X467::TnS (9) was used
to introduce TnS mutations into pJA30, and one plasmid that
was Kanr and unable to complement DL39(Mucts) was
named pJA31. pMK411 was introduced by transformation
and selection for both Tetr and Kanr, and P2 phage lysates
were made from these colonies. The P2 lysates were ex-
pected to contain pJA32, a recombinant between pJA31 and
pMK411 that would carry the antibiotic resistance genes of
both plasmids. The titers of the lysates were determined by
infecting a P2-immune E. coli strain, C-2324 (33), and a
lysate containing approximately 5 x 107 transducing parti-
cles per ml was used to infect R. meliloti 104A14 as de-
scribed previously (55). The infected cells were plated on
YMB-kanamycin agar, and colonies that grew were trans-
ferred to YMB-tetracycline agar to confirm the presence of
both markers. Cells that were both Kanr and Tetr were
transferred to YMB-kanamycin agar to allow the loss of
pJA32 and then to YMB-kanamycin-sucrose plates to select
for the loss of pJA32 and the retention of TnS.
To construct an insertion mutation in aatB, a 3-kb BamHI
fragment from TnS-B61 that confers gentamicin resistance
(54) was inserted into the unique BglII site in pJA14 near the
5' end of the aatB gene. The resulting plasmid, pJA15, was
subcloned into the BamHI site of pMK413 (40) and conju-
gated into R. meliloti by use of the E. coli helper strain S17-1
(53). The loss of the plasmid and marker exchange were
selected for by using the method described by McDermott
and Kahn (40).
Southern hybridizations. R. melioti genomic DNA was
prepared as described previously (55). DNA was digested
with restriction enzymes, separated by electrophoresis in
0.8% agarose gels, and transferred to nylon membranes
(GeneScreen Plus; Dupont) by capillary blotting. DNA
probes were labeled with [a-32P]dCTP (3,000 Ci/mmol) by
random priming (16). Hybridization and washing of the
membranes were done in accordance with the manufactur-
er's instructions. All hybridizations and washes were done at
a high stringency (0.1x SSC [lx SSC is 0.15 M NaCl plus
0.015 M sodium citrate]-1% sodium dodecyl sulfate [SDS],
65°C), except for the aatA-aatB hybridization, which was
done at a lower stringency (lx SSC-1% SDS, 60°C).
Enzyme assays. Cells were grown to the late log phase,
collected by centrifugation, washed in phosphate buffer (100
mM KH2PO4 [pH 7.5], 0.1 mM pyridoxal phosphate), cen-
trifuged again, and resuspended in 1 ml of phosphate buffer.
The cells were subjected to three cycles of freezing and
thawing and sonicated three times for 30 s each time. Cell
debris was removed by centrifugation for 10 min at 15,000 x
g. The resulting supernatants were assayed for AAT activ-
ity, aromatic aminotransferase (AroAT) activity, and
branched-chain aminotransferase (BcAT) activity. The total
protein concentration was measured by the method of Brad-
ford (6) with bovine serum albumin as the standard.
Aminotransferase assays. (i) AAT-catalyzed aspartate for-
mation. When the clones carrying the aminotransferase
J. BACTERIOL.
activity were initially isolated, an assay that enabled us to
measure aspartate production from the keto acid OAA was
used (14). The assay measures the conversion of [4-14C]OAA
into products stable in the presence of Cu2", such as
aspartate or malate. 14C-labeled OAA was made enzymati-
cally by use of phosphoenolpyruvate carboxylase and 14C02
(26). Assays were conducted with microtiter plates at 250C.
Each well contained cell extracts (2 to 10 Al), 100 Al of a 10
mM amino acid substrate, and a 100 mM Tris (pH 7.5)
solution, and the reaction was initiated by the addition of 25
pAl of a 5 mM [4-14C]OAA solution (0.125 mmol of OAA;
approximately 2,500 dpm). After 10 min, the reaction was
stopped by the addition of 25 Al of 5% perchloric acid, and
the samples were transferred to scintillation vials. A freshly
made 0.08 M CuSO4-0.12 M KH2PO4 solution (200 pAl) was
added to each vial, and the vials were baked under vacuum
at 80'C for 20 min. Heating the samples degrades
[4-14C]OAA to pyruvate and releases 14C02. Nonvolatile
radioactivity was measured in an aqueous counting scintil-
lant (Amersham) with a liquid scintillation counter. Amino-
transferase activity was expressed as nanomoles of product
formed per minute per milligram of protein.
(ii) AAT-catalyzed OAA formation. AAT activity was
measured at pH 8.0 and 220C as described by Yagi et al. (68).
In this assay, AAT activity was coupled to malate dehydro-
genase activity such that the OAA formed from the AAT-
catalyzed reaction was reduced to malate in a stoichiometric
fashion by malate dehydrogenase, with the concomitant
oxidation of NADH. The oxidation of NADH was monitored
spectrophotometrically at 340 nm. AAT activity was ex-
pressed as micromoles of OAA formed per minute per
milligram of protein.
AAT activity staining of nondenaturing polyacrylamide
gels. Nondenaturing (native) polyacrylamide gels were used
to separate aminotransferases. The nondenaturing gel sys-
tem used has been described elsewhere (40). Native gels
contained 10% acrylamide in the resolving phase and 5%
acrylamide in the stacking phase. Approximately 100 pug of
cell protein extract was added to each lane. After electro-
phoresis, the native gels were incubated in 50 mM Tris (pH
8.0) containing 5 mM aspartate, 1 mM 2-oxoglutarate, and
2.4 mM lithium hydroxide for 30 min at 370C. Fast blue BB
salt (Sigma) (1 mg/ml) was added to the solution, and
incubation was continued in the dark at 370C. Blue bands
appeared after approximately 15 to 30 min as the result of a
reaction between the dye and the OAA produced by AAT.
Preincubation of the native gels in substrates was necessary
because AatB was inactivated by the fast blue stain (data not
shown).
DNA sequencing. DNA was subcloned in the phagemid
pBluescript KS+/1- (Stratagene, La Jolla, Calif.), and single-
stranded DNA was isolated in accordance with the manu-
facturer's instructions, using helper phage VCSM13. The
DNA sequence was determined by the dideoxynucleotide
termination method with a Sequenase kit from U.S. Bio-
chemicals and [a-35S]dATP from NEN Research Products.
The sequence was determined for both strands and across all
restriction sites used in subcloning. Polyacrylamide gels that
contained 40% formamide were used to determine sequences
in DNA regions that had compressions.
Sequence analysis and comparisons. Sequence analysis and
data base searching were done with software from the
Genetics Computer Group (11) and with the assistance of the
Visualization and Design in the Molecular Sciences
(VADMS) Center at Washington State University. The data
bases accessed for sequence comparisons were GenBank (3)
VOL. 175, 1993
and EMBL (25). For GAP comparisons, the penalty to open
a gap was 3, and that to extend a gap was 0.1. The sequences
shown in Fig. 4 were aligned with the program PILEUP,
with the exception of one gap corresponding to Arg-385 of
AatB, which was inserted manually in alfalfa leaf, rat, and
mouse cytosolic, rat and mouse mitochondrial, and E. coli
AAT sequences. This was done because in other sequence
comparisons, these Arg residues were aligned (4, 60, 66),
and they are conserved among known AATs, AroATs, and
histidinol-phosphate aminotransferases (41).
Plant tests. Alfalfa seedlings (Medicago sativa, Ladak
variety) were grown by the method described by McDermott
and Kahn (40) or Kerppola and Kahn (34), and acetylene
reduction and plant weights were measured.
Nucleotide sequence accession number. The nucleotide
sequence data reported in this paper have been submitted to
GenBank and assigned accession number L12149.
RESULTS
Cloning of genes encoding aminotransferase activity. To
better understand the function of aminotransferases in R
meliloti, particularly AAT, we attempted to isolate their
corresponding genes. The strategy used to clone R meliloti
aminotransferases was to complement the E. coli amino-
transferase auxotroph DL39(Mucts). DL39(Mucts) lacks
functional AAT (aspC), AroAT (tyrB), and BcAT (ilvE)
activities and is an auxotroph for aspartate, tyrosine, phen-
ylalanine, leucine, and isoleucine (2). We transformed
DL39(Mucts) with an R meliloti gene library and plated the
transformants on minimal medium lacking aspartate, phen-
ylalanine and tyrosine, or leucine. Restriction digests were
analyzed, and plasmids that contained similar fragments
were grouped. When aspartate was left out of the minimal
medium, we isolated predominantly one class of clones
represented by pJA10 and, at a much lower frequency,
another class represented by pJA30. When the minimal
medium did not contain leucine, we isolated a different class
of clones represented by pJA20. When transformants were
placed on minimal medium lacking tyrosine and phenylala-
nine, pJA30 was again isolated, but this time at a higher
frequency. Unlike the results obtained on minimal medium
lacking aspartate, pJA30 was the only plasmid isolated in the
absence of aromatic amino acids.
pJA10, pJA20, and pJA30 were tested for the ability to
complement different amino acid auxotrophies by plating
plasmid-containing derivatives of DL39(Mucts) on minimal
medium lacking aspartate, tyrosine, phenylalanine, or
leucine. pJA10 could only rescue aspartate auxotrophy, but
pJA20 was able to rescue leucine and phenylalanine auxotro-
phies and pJA30 was able to complement tyrosine, phenyl-
alanine, and aspartate auxotrophies. Similar overlapping
specificities of aminotransferases occur for E. coli ami-
notransferases (2).
Characterization of the aminotransferase activity of each
clone. To characterize OAA conversion to aspartate by the
aminotransferases encoded by the plasmids, we assayed
extracts from DL39(Mucts) containing pJA10, pJA20, or
pJA30 by using an assay that measured the conversion of
[14C]OAA into aspartate. The activities of the extracts when
glutamate, tyrosine, phenylalanine, or leucine was used to
donate an amino group to OAA are shown in Table 2. pJA1O
encodes an aminotransferase that had highest activity with
glutamate, suggesting that pJA10 encodes an AAT. Signifi-
cant AAT activity was also measured in DL39(Mucts)
(pJA30) extracts. Extracts expressing pJA10 had lower
R MELILOTI AAT 4189
TABLE 2. Aminotransferase specific activities in
complemented strains
Plasmid in Aminotransferase sp act' with the following
DL39(Mucts) amino donor:
extract' Glutamate Tyrosine Phenylalanine Leucine
None 0.0 0.89 0.06 3.84
pJA10 7.74 0.14 0.37 0.90
pJA20 0.28 0.26 3.42 5.94
pJA30 5.60 15.13 53.10 3.17
aDL39(Mucts) containing different plasmids was grown on LB medium
with penicillin selection.
b Expressed as nanomoles of [O4CJOAA converted to aspartate per minute
per milligram of protein with the indicated cosubstrate.
activities with leucine and tyrosine as substrates than did
DL39(Mucts) without a plasmid, suggesting that the AAT
encoded on pJA10 represses another aminotransferase
present in DL39(Mucts).
When the aromatic amino acids tyrosine and phenylala-
nine were used with OAA, only DL39(Mucts)(pJA30) ex-
tracts had high aminotransferase activity, suggesting that
pJA30 encodes an AroAT. DL39(Mucts)(pJA20) extracts
also had some aminotransferase activity when phenylalanine
was used as the amino donor.
DL39(Mucts)(pJA20) extracts had highest activity when
leucine was used as the amino donor. The specific activities
in the DL39(Mucts)(pJA20) extracts were somewhat low,
and the DL39(Mucts) and DL39(Mucts)(pJA30) extracts also
showed significant BcAT activity. Our interpretation of the
high background activity seen in the DL39(Mucts) extracts is
that it was probably due to an unknown enzymets), possibly
another BcAT in DL39(Mucts), converting the [ 4C]OAA to
a heat-stable product. Since pJA20 rescued leucine auxotro-
phy and extracts containing this plasmid showed BcAT
activity when 2-oxoglutarate was used as the amino group
acceptor in a coupled glutamate dehydrogenase assay (46)
(data not shown), the aminotransferase gene carried on
pJA20 probably encodes a BcAT. This BcAT does not
appear to use OAA efficiently as an amino acceptor and
therefore does not produce significant amounts of aspartate.
This idea is consistent with data from Gonzalez-Gonzalez et
al. (22), who showed that R meliloti extracts have high
BcAT activity when branched-chain amino acids are used as
amino donors with 2-oxoglutarate as the amino acceptor.
pJA20 hybridized strongly in Southern analysis (data not
shown) with an aminotransferase gene designated aat2 by
Kittell et al. (35) on the basis of the AroAT activity of its
gene product expressed in R. leguminosarum. Kittell et al.
(35) were unable to show AroAT activity when E. coli
extracts were stained for AroAT activity on nondenaturing
polyacrylamide gels, and complementation of an E. coli
auxotroph lacking AAT and AroAT activities with aat2 for
tyrosine and phenylalanine auxotrophy was quite weak. Our
activity results are consistent with those of Kittell et al. (35),
but our complementation results suggest that branched-
chain amino acids are a more important substrate for the
enzyme produced by this gene. The E. coli BcAT encoded
by UivE can use phenylalanine as a substrate and is a
precedent for suggesting this type of substrate specificity in
the aminotransferase encoded by pJA20. In addition, when
Kittell et al. (35) mutated Aat2 in R meliloti 102F34, there
was no decrease in AroAT activity, suggesting that AroAT
may not be the primary activity of Aat2.
The aminotransferase genes carried on pJA10, pJA20, and
4190 ALFANO AND KAHN
AFIG 1H 2 3 I 5 6 7 a B D t D from
23.1I1 ** III*II
wild-type R. mel.loti 104A14 and JJ1C1O and their mutants. Lanes:
1 and 2, total DNA from wild-type 104A14; 3 and 4, total DNA from
104A14 mutant WSU601; 5 and 6, total DNA from wild-type JJ1C1O;
7 and 8, total DNA from JJ1C1O mutant 4R3 (43). In lanes 1, 3, 5,
and 7, DNA was digested with EcoRI; in lanes 2, 4, 6, and 8, DNA
was digested with HindIII. (A) Membrane probed with the BamHI-
PstI fragment of pJA14, which carries aatB from 104A14. (B) The
same membrane probed with pVR8 (43), which carries aatA from
JJ1C1O. 4R3 is an aatAf point mutant of JJC1,.
pJA30 were designated aatB, batA, and tatA, respectively.
On the basis of the results discussed above, aatB encodes an
AAT, batA encodes a BcAT that also has AroAT activity
when phenylalanine is the amino donor, and tatA encodes an
AroAT that also has significant AAT activity.
aatB is distinct from but hybridizes to aat4, a gene that is
needed for symbiotic nitrogen fixation. Recently, Rastogi and
Watson (43) isolated a gene called aat4 from R. meliloti
JJ1C1O; this gene encodes an AAT. They constructed an
aatA mutant and found that plants inoculated with the
mutant were unable to fix nitrogen (43). We were interested
in the relationship between our AAT and theirs. Hybridiza-
tion of aatB and aatA DNAs to total DNAs from wild-type
R. meliloti 104A14 and JJlC10 and their mutants indicated
that the two genes were different (Fig. 1). The BamHI-PstI
fragment of pJA14, which carries aatB, hybridized to two
bands of approximately 16 and 3.9 kb present on a HindIII
digest of 104A14 total DNA (Fig. 1A). When DNA from
WSU601, a 104A14 mutant that contains a Genr gene within
aatB, was probed with the BamHI-PstI fragment of pJA14,
the 3.9-kb fragment was disrupted to yield 6- and 0.4-kb
fragments. When these strains were hybridized to pVR8,
which carries aatA, there was no difference between wild-
type 104A14 and WSU601 (Fig. 1B), suggesting that the aatA
gene carried on pVR8 was different from aatB. The BamHI-
PstI fragment of pJA14 hybridized to a 9-kb fragment in
EcoRI-digested total DNA from JJ1C10 (Fig. 1A), but pVR8
hybridized to two bands, approximately 15 and 1.3 kb in
size, in DNA from JJ1C10 (Fig. 1B). At lower stringencies
(lx SSC-1% SDS, 600C), both probes hybridized to all of
the aat fragments in each strain of R meliloti (data not
shown), but the amount of hybridization to the bands that
appear at the lower stringency was lower. These results
suggest that aatB is distinct from aatA, that both strains of
R. meliloti carry both aatA and aatB, and that the two genes
are related to each other. Analysis of Southern blots ob-
tained with various restriction enzymes showed that the
genes were not closely linked to each other (data not shown).
Southern analysis was done to compare batA and tatA
with aminotransferase genes cloned from R. meliloti by
J. BACTERIOL.
FIG. 2. Electrophoretic separation of AAT activities in extracts
from R. meliloti and E. coli. Extracts were prepared as described in
Materials and Methods and fractionated by electrophoresis with
nondenaturing polyacrylamide gels. Lanes: a, E. coli DL39(Mucts)
without a plasmid; b, DL39(Mucts)(pJA10); c, DL39(Mucts)
(pJA30); d, DL39(Mucts)(pVR56); e, wild-type R. meliloti 104A14;
f, 104A14 mutant WSU600; g, 104A14 mutant WSU601; h, 104A14
mutant WSU602; i, wild-type R. meliloti JJlC10; j, JJ1C1O mutant
4R3 (43). The arrowhead indicates AatB.
Kittell et al. (35) and Rastogi and Watson (43). pJA20, which
carries batA, hybridized with a clone isolated by Kittell et al.
and designated aat2. pJA20 and aat2 yielded identical pat-
terns when they were hybridized to R. meliloti 104A14,
102F34, and JJlC1O (data not shown). The gene, here
designated tatA, for the AroAT encoded by pJA30 hybrid-
ized with aatl isolated by Kittell et al. (35) and tatA isolated
by Rastogi and Watson (43) (data not shown).
AAT activity in R. meliloti. For distinguishing the AAT
enzymes in R. meliloti, nondenaturing polyacrylamide gels
were stained with fast blue. The results for a typical exper-
iment are shown in Fig. 2. R. meliloti 104A14 extracts
showed three bands on native gels that were stained for AAT
activity (Fig. 2, lane e). The DL39(Mucts)(pJA30) extract
(Fig. 2, lane c) showed a band that migrated with the top
band in R. meliloti extracts (Fig. 2, lane e) and was missing
in extracts from WSU600 (Fig. 2, lane f). The other two
bands in 104A14 were probably both due to the aminotrans-
ferase encoded by aatA. Rastogi and Watson (43) found two
bands when aatA was transferred to Agrobacterium strains
and when extracts of the Agrobactenium strains that con-
tained aatA were fractionated on native gels and stained for
AAT activity. DL39(Mucts)(pJA10) contained an AAT band
that migrated more slowly on native gels than the AAT
bands in R. meliloti extracts (Fig. 2, lane b). This band
stained weakly with fast blue, and the stain was not quanti-
tative. For example, a direct assay of the AAT activity
loaded on the gel in Fig. 2 indicated that DL39(Mucts)
(pJA10) had 10 times the activity of DL39(Mucts)(pJA30)
(data not shown). AatB encoded by pJA10 may be more
unstable during electrophoresis than TatA encoded by
pJA30. In addition, AatB is sensitive to fast blue stain (data
not shown). Nonetheless, these results showed that the top
band in R. meliloti corresponded to the AroAT encoded by
tatA and failed to reveal an AAT enzyme in R. meliloti
extracts that corresponded to AatB expressed in E. coli.
Isolation and characterization of AAT-negative mutants.
aatB and tatA were mutagenized by insertion, and the
mutant alleles were recombined into R. meliloti 104A14 as
described above to generate mutants WSU600 and WSU601,
respectively. A double mutant lacking both of these AATs,
VOL. 175, 1993 R MELILOTI AAT 4191

WSU602, was also constructed. Replacement of the wild-
type allele was confirmed in all mutant constructions by
Southern analysis (data not shown).
WSU600, WSU601, and WSU602 were able to grow on
minimal medium containing mannitol and ammonia as the
sole carbon and nitrogen sources, respectively, indicating
that aatB and tatA encode aminotransferases that are not
solely responsible for the synthesis of aspartate or the
aromatic amino acid tyrosine or phenylalanine in 104A14.
The mutants were able to use aspartate as their sole nitrogen
source, with growth rates equal to that of wild-type 104A14.
In addition, the mutants were tested for their ability to utilize
other carbon sources that may be used as substrates in
transaminase reactions. The mutants grew as well as wild-

1 tggaaggcctatagtgcacgcgccacaaatacggtgaactattccaagctctgccgctgg 60
61 cgcagggcgtctctttcgacatcgaataatcgaacaggatcggccATGACCATCAATGCA 120
M T I N A
121 ACGGTCAAGGAGGCGGGCTTCCGGCCCGCCTCGCGGATTTCTTCGATCGGCGTTTCGGAA 180
T V K E A G F R P A S R I S S I G V S E
181 ATCCTGAAGATCGGCGCCCGCGCCGCCGCCATGAAGCGCGAAGGCAAGCCGGTGATCATT 240
I L K I G A R A A A M K R E G K P V I I
241 CTCGGCGCCGGCGAGCCGGATTTCGACACGCCGGACCACGTGAAGCAAGCCGCCTCGGAC 300
L G A G E P D F D T P D H V K Q A A S D
301 GCGATCCATCGCGGCGAGACGAAATATACCGCACTCGACGGCACGCCGGAATTGAAGAAG 360
A I H R G E T K Y T A L D G T P E L K K
361 GCGATACGCGAAAAATTCCAGCGTGAGAACGGGCTAGCCTACGAGCTCGACGAAATCACG 420
A I R E K F Q R E N G L A Y E L D E I T
421 GTTGCCACCGGCGCCAAACAGATCCTGTTCAATGCCATGATGGCGTCGCTCGATCCCGGC 480
V A T G A K Q I L F N A M M A S L D P G
481 GACGAGGTGGTCATTCCGACGCCCTATTGGACCTCCTATTCGGATATCGTCCAGATCTGC 540
D E V V I P T P Y W T S Y S D I V Q I C
541 GAGGGCAAGCCGATCCTGATTGCATGCGACGCATCTTCCGGCTTCCGCCTGACGGCGCAA 600
E G K P I L I A C D A S S G F R L T A Q
601 AAGCTGGAAGCGGCGATCACGCCACGGACGAGGTGGGTGCTCCTGAACTCGCCCTCGAAC 660
K L E A A I T P R T R W V L L N S P S N
661 CCCTCCGGCGCCGCCTACAGCGCGGCCGACTACCGGCCTCTGCTCGACGTGCTGCTCAAG 720
P S G A A Y S A A D Y R P L L D V L L K
721 CACCCGCATGTCTGGCTGCTCGTCGATGATATGTACGAGCACATCGTCTATGACGCTTTC 780
H P H V W L L V D D M Y E H I V Y D A F
781 CGCTTCGTCACGCCGGCTCGGCTCGAGCCGGGCCTCAAGGATCGGACGCTCACCGTCAAC 840
R F V T P A R L E P G L K D R T L T V N
841 GGCGTCTCGAAGGCCTATGCGATGACGGGCTGGCGGATCGGCTATGCCGGGGGCCCGCGC 900
G V S K A Y A M T G W R I G Y A G G P R
901 GCGCTCATCAAGGCGATGGCCGTCGTCCAGAGCCAGGCGACCTCCTGTCCCTCTTCGGTA 960
A L I K A M A V V Q S Q A T S C P S S V
961 AGCCAGGCGGCTTCGGTCGCGGCGCTCAACGGACCGCAGGACTTTCTGAAAGAACGGACC 1020
S Q A A S V A A L N G P Q D F L K E R T
1021 GAAAGCTTCCAACGCCGCCGCAATCTCGTGGTCAACGGGCTGAATGCGATCGAAGGGCTC 1080
E S F Q R R R N L V V N G L N A I E G L
1081 GACTGCCGCGTTCCCGAAGGCGCCTTCTACACCTTCTCCGGCTGTGCCGGCGTCGCTCGG 1140
D C R V P E G A F Y T F S G C A G V A R
1141 AGGGTGACGCCATCAGGCAAAAGGATCGAGTCGGACACGGACTTCTGCGCCTACCTTCTG 1200
R V T P S G K R I E S D T D F C A Y L L
1201 GAAGACTCCCACGTCGCCGTCGTCCCGGGCTCGGCCTTCGGCCTCTCCCCTTATTTCCGC 1260
E D S H V A V V P G S A F G L S P Y F R

1261 ATCTCCTATGCGACCTCGGAGGCGGAACTGAAGGAAGCGCTTGAACGCATTTCGGCAGCA 1320

I S Y A T S E A E L K E A L E R I S A A
1321 TGCAAGCGGCTGTCAtaagtctacaccgcgtttaagaggactcgggcggcgcggctccta 1380
C K R L S
1381 tccgatgcccggtgacttgaaacgacccctcatccggcccggcgcgtctgggacgattca 1440
1441 cacc 1444
FIG. 3. Nucleotide sequence of the K meliloti aatB gene and deduced amino acid sequence of the AatB protein. The nucleotide sequence
is numbered on each side. The deduced AatB protein is 410 amino acids long and has a monomer molecular mass of 45,100 Da. The predicted
pyridoxal phosphate attachment site motif of the protein is underlined.
4192 ALFANO AND KAHN J. BACTERIOL.

TABLE 3. Amino acid sequence identity between bacterial and eukaryotic AATsa
% Sequence identity to:
Enzyme
AATB AATA BY SS RTAT HlvE TyrB AspC MC AL MM RM
AATB 100
AATA 58.2 100
BY 41.0 45.2 100
Ss 29.7 31.3 35.5 100
RTAT 24.3 24.9 26.4 27.1 100
i1vE 20.8 19.7 18.5 16.3 14.7 100
TyrB 21.6 17.3 21.2 23.9 18.7 16.4 100
AspC 18.8 20.3 19.7 22.6 17.3 16.4 42.6 100
MC 17.7 17.9 18.0 18.6 18.7 18.0 39.2 41.1 100
AL 17.4 20.0 20.5 18.8 17.6 14.0 39.4 45.3 52.1 100
MM 15.0 21.1 19.4 20.8 16.5 15.8 41.3 42.1 48.1 52.7 100
RM 14.8 20.5 19.1 20.6 15.7 15.5 41.1 42.1 48.1 52.9 98.4 100
a
Comparisons were made by use of the GAP program of the Genetics Computer Group Sequence Analysis Software Package, version 7.1 (11). Abbreviations
and sources of data are as follows: AATA, R. meliloti AatA protein (66); AATB, R meliloti AatB protein (this work); AspC, E. coli AspC protein (19); TyrB,
E. coli TyrB protein (19); IlvE, E. coli HlvE protein (37); SS, S. solfataricus AAT (7); BY, Bacillus sp. strain YM-2 AAT (60); AL, alfalfa leaf AAT-1 cDNA (61);
MC, mouse cytosolic AAT (42); MM, mouse mitochondrial AAT (42); RTAT, rat tyrosine aminotransferase (23); RM, rat mitochondrial AAT (30). Boldfacing
indicates families of AATs as described in the text.

type 104A14 on tyrosine, phenylalanine, leucine, 1-alanine,
y-aminobutyric acid, and spermidine (data not shown).
Growth on aspartate as the only carbon and nitrogen
source was more complex. When 104A14 or WSU601 was
grown on aspartate, spontaneous mutants that could grow
more rapidly were easily isolated. Such mutants were not
seen when tatA mutants WSU600 and WSU602 were grown
in a similar way. The addition of 0.01% yeast extract or
Casamino Acids to the medium allowed all of the strains to
grow better, and the faster-growing mutants were not seen.
WSU600 (Fig. 2, lane f) and WSU602 (Fig. 2, lane h)
lacked the top band observed in 104A14 extracts. This band
corresponded to a band found in extracts of DL39(Mucts)
(pJA30) and confirmed that recombination of the tatA::Tn5
mutant allele into R. meliloti 104A14 inactivated this AroAT.
Extracts of aatB mutant WSU601 were also stained for
AAT activity and had the same banding pattern as wild-type
extracts (Fig. 2, lane g). These results probably indicate that
aatB is not expressed in R. meliloti under the conditions
tested. However, because AatB is sensitive to the staining
procedure, it is also possible that when aatB is present as a
single copy, AatB is present but cannot be detected. We
have occasionally seen a faint band in extracts from
WSU600 that migrates near the band expressed in DL39
(Mucts)(pJA1O).
Since AatA has been shown to be important to nitrogen-
fixing symbiosis with alfalfa (43), we tested the nitrogen
fixation phenotypes of WSU600, WSU601, and WSU602.
Alfalfa plants inoculated with the mutants had the same
acetylene reduction rates and dry weights as those inocu-
lated with wild-type R. meliloti 104A14 (data not shown).
Therefore, tatA and aatB do not encode aminotransferases
that are critical to symbiotic nitrogen fixation.
Sequence analysis of the aatB gene. The nucleotide se-
quence of aatB, together with its deduced amino acid
sequence, is shown in Fig. 3. The DNA sequence contains
an open reading frame of 1,230 nucleotides. If translation
begins with the first ATG codon in the open reading frame,
the predicted AAT would be 410 amino acids long and have
a molecular mass of 45,100 Da. A computer-aided search of
the GenBank and EMBL sequence data bases showed that
the predicted AatB protein was 41% identical to an AAT
from a thermophilic Bacillus strain, YM-2, 29.7% identical to
an AAT from Sulfolobus solfataricus, and 24.3% identical to
an AroAT from a rat (Table 3). In addition, the aatA gene
from R. mehfloi JJ1C1O was recently sequenced (66); AatA
is 60% identical to AatB. In GAP comparisons, low identity
was seen between AatB and other well-known AATs in the
data bases. Particularly striking was the low identity score
between AatB and the E. coli AAT (18.8%) and AAT
sequences from eukaryotes. When these AATs were com-
pared against each other, they showed about 40% amino acid
identity (61).
Although AatB does not appear to be closely related to
conventional AATs, it contains most of the highly conserved
amino acids in regions that are important for catalytic
activity. A pyridoxal phosphate attachment site motif, al-
though slightly different from that in other AATs (61), is
underlined in Fig. 3. The lysine residue that covalently binds
pyridoxal phosphate in AatB is probably Lys-249. This
lysine aligns with Lys-241 of the S. solfataricus AAT, which
has been shown to bind pyridoxal phosphate (4). Mehta et al.
(41) have shown that members of a subclass of pyridoxal
5'-phosphate-dependent enzymes that includes mesophilic
AATs, tyrosine aminotransferases, and histidinol-phosphate
aminotransferases share 12 amino acids responsible for
catalytic activity. An alignment of amino acids of AATs from
the two groups showed that even though AatB is not closely
related to these AATs, in this alignment, the 12 conserved
amino acids can be identified (Fig. 4). Using the Genetics
Computer Group program PHYLIP, we constructed an
unrooted phylogenic tree (Fig. 5). AatB and AatA are
members of a novel group of enzymes that contain archae-
bacterial and eubacterial AATs and eukaryotic tyrosine
aminotransferases.
AatB has kinetics similar to those of other AATs. We asked
whether the kinetic properties of AatB were similar to those
of more conventional AATs. Extracts from DL39(Mucts)
(pJA10) contained an AAT with a Km for aspartate of 5.3
mM and a Km for 2-oxoglutarate of 0.87 mM (Fig. 6). The
Vm for the forward reaction (that producing OAA) was 2.4
,umol of OAA produced per min per mg of protein. The pH
optimum for the reaction was between 8.0 and 8.5 (data not
shown). These properties are comparable to those of AATs
from other organisms (61), suggesting that the AAT encoded
by aatB, although from a different lineage than the E. coli
and eukaryotic AATs, has a similar metabolic role.
VOL. 175, 1993

AATB
AATA
BY
S8
..........
..........

Rtat MDSYVIQTDV
AL ..........
RM . . . . . . . . . .
xC ..........
RC . . . . . . . . .
Ml .
AspC .
Il1v

AATA
.

AATB LGIP.
.
.

.a..
.
.

LGEP. . . .
BY LGIP
SS FIOQP....
Rtat LIODPSVFG
.
.
.
.. . . . . . . .
.
.
.
.
.
.
.
.
.
.
:..EALL.E.SGR
DDS LSSVLDV
. .

.EALLES.SR*
. .

DFDPEDE VQ AASDAIHRR.
DPDP D EIK
DFN EQNIED AAIDOE QO
. . ..

TXIHYIa
.. .. .. .
I L aSG A A A F

P*LKKAIR3K FQRZflGLAYZ
L PALKQAIIRK FKRDIQLZYK
DLPPTKRIUD AAK3AL QO. FPFETSAFEI DELRZKIAQY LETRYOTDVX
E..
AVTI
AFLADA
AKRBAGFR
.NT!
LLLADN...

...E..T
TIT
PASRISSIGa
.... LSRVKP
. .K
VNI BGRNSV QGRKKGRKAR WDVRPSDXSN KTFNP RAKT
....
V LaEAAAFH
...
AR
SZILKEGARA
SATIAUSQKA
RVKTLTP STTLALTAKA
VSLLD .....FNGSQVTG ZTTLLYKRIA

EA PPSFFAQVPQ APPVL FlKLI

PGaL A A A ASAR A S 8W 1TNXV B XG1aP P DP LGaV T
K K AD IY
.EX
U
FZNEITA APADP LaLA
E
N
....
...

EN I T P S P T A S S D S V F A H L V R A P B D P L G V T
.

PaLAAAASAR ASSEWTHVIE
E...........
GPPDPV LaVT
A PPSVFAQVPQ APPVL FlKLT

E V R W Z D A K E V ES

3TflT3LDflT
R MELILOTI AAT

AAE. ... KRZa

RZL ... KAKO
K RN ... KAQG
REV..ZRKTKK
DNXKVQPNPN
VAY N KDP S P I
FAFKRDTNSK
ADFRDDPDPR
ADFRDDPDPR
Z A F KR D TNsK
DLFRADBRPa
N ALN YOTS

LD....IT
PE . II:
KR....VI
1
. .
PVI I 45
ID
VI
IDVI
IKII l
T VI
*LNL
X L
EVNL
9VNL
EENL
INL

PPRPR
VKRTK
DRKLR
37
63
32
....V 36

VT -104
4193

35
36
39
75
47
63
37

110
100
2101
ELPPDPBV PQ AMXDAL sG0 YNG A SI Y LsSRZZVASY YHCH A.PLB A . VI L T 8C 144
AL V!YRPTaXK PLVLDVVR V RQLLEN ESR EXZ I IV L A FNXLSAXL IFOA SPAIQ BNRVTTV OL aOS V 122
RN V Y R D DK PY LEVRAV BAQ.IA KEL DXB L I0 L A FCKASABL ALGOSBVLK TVI
SRFVTV 3131. 137
EC V YRTD38Q PV; VLV
VV VRAV 3QKIAE ESL EZl L IL L A FRSCA8RL VLDa SPAIR ZERVGGV SL 3181.l 112
RC V YRTDDSQ PWVLVEVVV BQXIAE ROL El1 L IL L A FRSCASQL VLaD SPALR FENVooV SL o 112
M
AupC I
V YRDDNOK
YKDTPaK
PYVLE SVR A ZAQ.IAAXEL DIX L I0 L A FCXA8ABL ALOE*NVL
TPVLTSVK A B.QYLLEENBT TXN LGID I P FaRCTQZL LFGKSALIN
8aRFVTV TXI
DIRARTA TP ESV.
@|Zu137
106
I vz F I RCYD R K OGPVVFRE R HEQRLHSAAK IYR PVSQ I D LEIACRDV IRKEN... LT SAYIRPLIFV AOD 107
AATB KQILIENA A 8L PODF .. .VI T Y OKPILIA.BD . .ASSFRLT
SYSDIVQIC3 A. T P R $ R 176
.:QZR
A I
AaTa X Q I L F N A K A T L P a D R ... *VI A Y V C TPVFVP.TR . .QNEEFXLK
SYP3EVAL T P K T x 16 6
A. .3DR I
BY X XV LY T LQ V I LX a D F . . . EI I Y SYPBQVXL
V OVPVYIB.AT . .SQNYKXIT RXEN I
A. .ZT ED X T 167
S8XP A LF L V I L YIIPSD * .. . EIL D S SYABVVKL L GXPIYAN.LK W8R
Y F8ID 8 X R $ X 172
V. .D iQSI
Rtat S QA I E LC AV LA PO QE . . . *LI R a 8 LYRT ... LU OGIIVL*I LLPKX WID L. .K S8LI D Z xL 210
AL L R VGaoFFA K N YX . Q R I . . . YLL T NETKVFNL L.TVXTYR Y APATR LDFQ aLL GOSaP
RK L R VGOA8 ~Q R FF X F 8RD .. . FL S 0 NETPIFRD
K X.QLQGYR Y DPKTC FDF8 GALE SKIP Q 8 V 206
RC R WYOPTDKNPT T 3 EENAVFSA ENAP 2 F a I .186
RC LRIOADF
P YV 8 FXDIRPYC W DAIKR LDLQ OFLN
LR IaAD PAR 1 Y TDNK1T
E P YVSS T N1ENEaVFA FXDIRSYR W DARKR LDLQ aFLN E AP FF8 I .186
MM LRVASP QR FFKFSRD ... .EEK
5L 0 EHPPIFRD .QLQGYR Y DPXTC FDFS GALBZ SIP Q 8 V 206
AspC LRVAAD DA NTSVKR E . . .EVJIE S P NEKSVFNS L. VRRYA Y DAREETLDFD ALIE NFQ * O D V .174
I1vz . . . . . . . . . I O VEPPAOYS8 DIIAPP0 AYL OARALZQ OIDAEVSS8 E RAAPETIPTA A ... 0a aGN YL8 167
U .
AATB WV L S P
AATA WF F E B P S
AAYSAADY
AAYS iEL
RP LD LLXE P V L L VE DE
P EV VVLT D *
R 11 V
RE TPYT DFR YJA F R
FATPV.RVZP
IVTPE.ELZP OLKDRTLTVN
OLYTRTLTXN
avMAP 240
av
250
BY AVVI N B P 9 Gl V Y T RXL IDEAK ALZU .N I L I V S RI 3K LYEOAZ ps5IA. Q I 5S RVXAQTIVIN
a IF aIE8T 243
a 240
S8 XI N N P IT L F 8 P DV KK VD ORDN .X I ILL 8GI I AD NEDV Y5G K E RSTLR ...DS DWRDFLIYVN
Rtat CL V N N P C SVFSKREL Q QLA A R Q C V P I L AER I O VP D C K YRPLA .NLST NV. PILSCa LA ER 281
AL V L K A C A VDPTL QW EQA: 8u L D ADAQ V LFV ADG ZLLVAQ 8 Y A N 264
RML RACA QW KX AA VKKK .B11LPFAFF XA
D LD
V 5F I RQO INVCLCQ 8 Y A SN 280
MC F V N A C A
RC F V N A C A
M L L 8 AC A
V
TDPT
GT D P T Zs 11
w
VD P R P Q W
AspC V LX O C Cel~9I I D P T LQ W
I lvZ 8 LV . . . .. .X ASARREaY
DPRPUPPQ KQSK A
AARV
KQRAAEQXRR
Q Z I1DV
QRR
VK
IKATE DEX SFLLPF
.

.GVLPLF
.

PA
Q A
Q
Q
Q
A
D
AS
KDAV
L Z KDAWVI YFV
Q AS D L R KDAWVIA YFV
DO D KDAV VSFI
EDAitL AFA
18 G ALRa RNLF3V....
.NIAR
.
SRaFRLFCAQ
SRGFRLFCPQ
RQGINVCLCQ
AMEKRLIVAS
N
8F N 260
F
SYA N 280
SY
260
EN 247
D 205
.
XIRDGL . . . . . . . . . . .

AATB Y A T W I. YAO..GPRAL
IVR AQ AV VQ SQA TECEa LN.GPQDFLK RRTRSFQ. R R :L 314
AATA Y A TV I . YAA. .OPLEL
IQGQQ P.O A VI A WfA V R LE.GPQDFIG O RR L 304
BYES A ETWI. YAA. .GNADI
I NA TD LASIS T T TA AAIR
I YN.GPQDSVR KERKAF.. 8 RL T 304
SSFS TEWVLE. YIV..AXR I
LAANVYTA T 8IV AV
VK FD.TFDI.VN QMVSLF f.. XRR V 306
Rtat WL G W L. WILIEDRRDI FGNZIRDGLV KLSQRILOAC TIV L 8I LQRTPQRPYR DTLBFL .. s NEA L 352
ALMG1Y Z1V1A LS IVSK SADV 88RVZ8Q KL VIIP K Y 8 P AILKDRDLYN DETIRLA. AXA R 334
RX M GY R V A IPTVVCXDARR AKRVR8Q XI LIUP L YNE P L RIA TILTSPDLRK QWLQZV U. OXA R 350
EC G YE V E LTVVaXFSDS IVE IPTV N P .A RIV ATLSDPELFK VKOENV U. TXA sR 330
RC P G YENRZV N LTVVGXKEDS VLRVLSQE IV IPTESNP RK .AER I V TTLSENPLFK ZWXGNV .. TXA aR 330
MMX4 E YER V A FTVVCKDA*I AKRVSQE KI LI P L YNE P L R IA TILTSPDLRK QWLQZV. OE A R 350
AupC F G Y N Z V A CTLVAADSRT VDRAISQ KA Al A N YNE P 8 V VE TIL8NDALRA DXR
8 LLA
IV3QRLT. R 317
IlvZ G V F T P 8P A
S SALPOITRDA IIKLAKR GI RV^ QVLSRR L YLLDZVFXM SGTAARITPV RSVD aIQVaZ GRC P 280

AATBMVNGLNAIEG
NVSMLNQAKG
AATA
BY MYPKLSAIPG
.SCPTPZ|3A
. FYTFSGCA
DCRVPE A FYVYP8CA
KVVKPQIA FYLLPDVS
V
L
A
ARRVTPSGKR
I OKX'P 8 G V ITD zDF8
AQ
IUSDTDFCAY
FASVDFASA
|LLEESE
LT
AVV
T OAV
AN
V
AVI
PGSAFGLS.
HGSAGOLO
PO00FOAP .8 T I
.PYP
.PNF
385
375
370
SS YDZLTKVKG .EVSKPNEEA IYEFPNVSKI LITP FDV..LAIK 13X KG VTI POGVIPLNIa KX F L 374
Rtat CYGALADLPG .QPVRPSEA EYLEVGIEXZ .. . E.I. FPE . IEEDVE FP T Z R I QA RCL PATCJFYP. .N FF 417
AL IN RQQLFD A RAROTPED WVSIIKQI N1 FTF LNPEQ VS....... I TI YE YL T 8D a . . . . . . . ...z391
RE
MC
RC
mm
IS RTQLVS
LT
LT
IS
RSILRA
RSZLRA
RTQLV
R
R aOSSENERN WSEITZQQIM
NE EALKTPT
ZALKTP
KKEGSS
WVRIT
WQEITDQI
E FCFI
QEITDQI FNFSFP
FCPC
T
W
QI F
LKPQ V
LNPKQ
LN PEPKQ
LKPEQ
V
.

V ....... Y
V ....... R
R
....... Y
TI F5 YET
VE
VE
TI
KR
YLE
KE
PS
YXT
YLL
KD a . . . .
P50.
P8 a . . . .
XD a . . . .
.

.
.

.
.

.
...a387
. 407
407
387

AspC QR RQLFVN T QXo AERD FSFIIKQN M FIIS LTKZQ VL R R PG YAV A8 a . . . . . . . 374

I1vW TIRIQQAPF G FTGZTEIK WV WLDQVNQ* .......... .......... .......... . . . . . . . . . . ..... . . 309

AATB YE TSEAEL KRA. . .LIRI SAACKRLS .4.....

410 .

AATA Y TSEALL R3ACRRIQRI CAACR. .400...

oo
BY Y1 TS L L I RRAIZRIDRF . . . . .392 .
SS E V11 EVI KEG IQKIREF AZQXNESR. 402
Rtat aj ITVPEVMX LEACSRIQQF CIQIYECDRG SQ iEC D X454
AL I'AG vT 8OGN L8 KT VPELADAIEA VVTRVA. 417
RN YG VGYLAEAIEQ VTI'. : : :'. '430 .
NC |KtCGOL T T x LDYVATSIlE AVTK IQ . . .. .413 .
RC EXCOLTTKE LDYVATSINE AVTKFQ^ 413
94 _ V AGVTSGN VGYLAHAIEQ VTI'. :: :: ' '430 .
A-pC 5
I
° T PDN EAPLCEAIVA VL^ 396
IlvE. . . . . . . . . . . . . . . . . . . . . : : :'. .309 .

FIG. 4. Alignment of the AatB amino acid sequence with the sequences of other aminotransferases. The first four aminotransferases listed
are those that share homology with AatB. The remaining sequences selected show low homology to AatB and represent the more
conventional AATs present in eukaryotes and eubacteria, with the exception of IlvE. The 12 amino acids conserved among AATs, tyrosine
aminotransferases, and histidinol-phosphate aminotransferases (41) are marked with closed circles. Abbreviations and sources of data are as
follows: AATB, R meliloti AatB (this work); AATA, R. meliloti AatA (66); BY, Bacillus sp. strain YM-2 AAT (60); SS, S. solfatancus AAT
(7); Rtat, rat tyrosine aminotransferase (23); AL, alfalfa leaf AAT-1 (61); RM, rat mitochondrial AAT (30); MC, mouse cytosolic AAT (42);
RC, rat cytosolic AAT (27); MM, mouse mitochondrial AAT (42); AspC, E. coli AAT (19); IlvE, E. coli BcAT (37). Amino acids are shown
shaded when 7 of 12 sequences were identical. Black indicates identity; the darker the shading, the more similar the amino acids.
4194 ALFANO AND KAHN
RM
MM
AATB
AATA
BY
RTAT
\lvE
FIG. 5. Phylogenetic relationship between AatB and other ami-
notransferases. An unrooted tree was constructed by use of the
Genetics Computer Group program PHYLIP. Abbreviations and
sources of data are as follows: AATB, R. meliloti AatB (this work);
AATA, R. meliloti AatA (66); BY, Bacillus sp. strain YM-2 AAT
(60); SS, S. solfataricus AAT (7); RTAT, rat tyrosine aminotrans-
ferase (23); MC, mouse cytosolic AAT (42); RC, rat cytosolic AAT
(27); AL, alfalfa leaf AAT-1 (61); RM, rat mitochondrial AAT (30);
MM, mouse mitochondrial AAT (42); AspC, E. coli AAT (19); IlvE,
E. coli BcAT (37).
DISCUSSION
We have cloned three aminotransferase genes from R.
meliloti 104A14 by complementing an E. coli strain defective
in AAT, AroAT, and BcAT. R meliloti mutants that lacked
AAT activities were constructed. The mutants were proto-
trophs and had growth characteristics similar to those of the
wild type, except that the tatA mutant and the aatB tatA
double mutant grew more slowly than wild-type 104A14 on
minimal aspartate medium. The slower growth rates exhib-
ited by these mutants were corrected by the addition of small
amounts of yeast extract, indicating that it was the absence
of a specific component, not the inability to catabolize
aspartate, that limited their growth. In addition, the mutants
were tested for their ability to grow on other carbon sources
that may participate in aminotransferase reactions. The
mutants were able to grow on tyrosine, phenylalanine,
leucine, ,-alanine, -y-aminobutyric acid, and spermidine at
rates similar to those of wild-type R. meliloti.
A comparison of the deduced amino acid sequence of
AatB with those of other aminotransferases revealed signif-
icant similarity with an AAT from S. solfataricus, an AAT
from a thermophilic Bacillus species, a rat AroAT, and AatA
from R. meliloti (Table 3). On the basis of sequence com-
parisons, AatB appears to belong to a new subclass of
aminotransferases that is distinct from conventional AATs.
Because the atypical AATs are present in each of the three
primary phyla, this AAT family probably predates the diver-
gence of these groups and may be older than that of the
conventional AATs, which does not yet have a representa-
tive in the archaebacteria.
AatB and other members of its subclass showed low levels
of similarity to all other aminotransferases in the data bases.
Despite these low levels of similarity, the 12 invariant amino
acids conserved among AATs, AroATs, and histidinol-
phosphate aminotransferases and observed by Mehta et al.
(41) also appeared to be conserved in AatB (Fig. 4). Most of
J. BACT1ERIOL.
E~ 202
0
C ii) 1-1
01
-1 0 1 2 3 4
0 20 40 6 80
[aspartate] (mM)
3
B
- 2-
O- /S
.5C
E
FIG. 6. Kinetics of AatB expressed in E. ccli DL39(Mucts)
(pJA10). AAT activity is expressed as micromoles of OAA formed
per minute per milligram of protein. Data in panel A were obtained
at a saturating concentration of 2-oxoglutarate (10 mM). Similarly,
for the experiment shown in panel B, the aspartate concentration
was saturating at 50 mM. AAT activity in DL39(Mucts) controls was
negligible. The insets are the Lineweaver-Burk plots of the data (r2
= 1.00 in both cases).
these conserved amino acids interact with the pyridoxal
phosphate coenzyme or the substrates (18). Two arginine
residues important in enzyme catalysis in conventional
AATs are Arg-386 and Arg-292 (numbered on the basis of the
pig cytosolic AAT). Arg-386 had to be manually aligned with
AatB. This alignment seemed appropriate since, in other
alignments with similar AATs, Arg-386 was conserved.
Arg-386 is important in binding of the oc-carboxylate group of
the external aldimine intermediate (18). Arg-292, which
binds the distal carboxylate of the dicarboxylate substrates,
is not found in AatB, cannot easily be manually aligned, and
is not conserved in other AATs belonging to this subclass.
This result suggests that these AATs may have substrate
specificities different from those of the AATs belonging to
the larger group. AatB expressed in E. ccli had similar
kinetic properties with the substrates aspartate and 2-oxo-
glutarate. However, Marino et al. (39) have observed differ-
ences in kinetic properties for an AAT from S. solfatancus
and conventional AATs. In particular, the S. solfatancus
AAT is fairly active when alanine is used as a substrate (4).
It has been suggested that the atypical AATs may be
specific for thermophiles (4), but this appears not to be the
VOL. 175, 1993
case, since representatives of this group are present in
mesophilic Rhizobium spp. and in eukaryotes. However, the
thermophilic AATs of S. solfataricus and Bacillus sp. strain
YM-2 contain no cysteine residues, a characteristic that may
increase their thermophilic stability (60). AatA and AatB
from R. meliloti both contain cysteines; this may be a factor
in the instability of AatB in crude cell extracts at tempera-
tures above 50'C (data not shown).
It is unclear whether aatB is expressed in R. meliloti. We
could not see a band of AAT activity in R. meliloti extracts
fractionated on polyacrylamide gels that corresponded to the
AAT activity band in E. coli extracts containing AatB.
However, AatB produces only a weak band despite the
presence of substantial AAT activity, and the lack of an
AatB band in R. meliloti may have been due to a failure of
the assay. We have been unable to detect a phenotype for
aatB mutants and do not believe that AatB is important in
aspartate catabolism and synthesis in free-living R meliloti.
It is possible that other AATs compensate for the absence of
AatB and that, in the absence of these AATs, a more
significant phenotype becomes apparent. Construction of an
aatA aatB tatA mutant is being attempted in collaboration
with R. J. Watson. The regulation of AatB and AatA under
both symbiotic and free-living conditions is important to
understanding how these enzymes function in R. meliloti,
and experiments have been initiated to explore this question.
ACKNOWLEDGMENTS
We thank Robert Watson and Vipin Rastogi for providing their
aatA sequence prior to publication, for supplying R. meliloti JJ1C10
and mutant 4R3, and for very helpful discussions. We thank Barbara
Kittell, Donald Helinski, and Gary Ditta for supplying R meliloti
102F34, the AroAT mutants, and the plasmids containing the R
meliloti 102F34 AroAT. We thank Barry McGurl and Tim McDer-
mott for help and advice and Steve Thompson of the VADMS Center,
WSU, for assistance with the sequence analysis and comparisons.
This work was supported by grants 88-37120-3890 and 92-03461
from the USDA Competitive Research Grants Office and by funds
provided by the Graduate School and the Agriculture Research
Center at Washington State University.
REFERENCES
1. Appels, M. A., and H. Haaker. 1991. Glutamate oxaloacetate
transaminase in pea root nodules: participation in a malate/
aspartate shuttle between plant and bacteroid. Plant Physiol.
95:740-747.
2. Berg, C. M., M. Wang, N. B. Vartak, and L. Liu. 1988.
Acquisition of new metabolic capabilities: multicopy suppres-
sion by cloned transaminase genes in Escherichia coli K-12.
Gene 65:195-202.
3. Bilofsky, H. S., C. Burks, J. W. Fickett, W. B. Goad, F. 1.
Lewitter, W. P. Rindone, C. D. Swindell, and C. S. Tung. 1986.
The GenBankTm genetic sequence data bank. Nucleic Acids
Res. 14:1-4.
4. Birolo, L., M. I. Arnone, M. V. Cubellis, G. Andreotti, G. Nitti,
G. Marino, and G. Sannia. 1991. The active site of Sulfolobus
solfataricus aspartate aminotransferase. Biochim. Biophys.
Acta 1080:198-204.
5. Bolton, E., B. Higgisson, A. Harrington, and F. O'Gara. 1986.
Dicarboxylic acid transport in Rhizobium meliloti: isolation of
mutants and cloning of dicarboxylic acid transport genes. Arch.
Microbiol. 144:142-146.
6. Bradford, M. M. 1976. A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem. 72:248-254.
7. Cooper, A. J. L., and A. Meister. 1985. Metabolic significance of
transamination, p. 533-563. In P. Christen and D. E. Metzler
(ed.), Transaminases. John Wiley & Sons, Inc., New York.
8. Cubellis, M. V., C. Rozzo, G. Nitti, M. I. Arnone, G. Marino, and
R MELILOTI AAT 4195
G. Sannia. 1989. Cloning and sequencing of the gene coding for
aspartate aminotransferase from the thermoacidophilic archaebac-
terium Sulfolobus solfataricus. Eur. J. Biochem. 186:375-381.
9. Day, D. A., and L. Copeland. 1991. Carbon metabolism and
compartmentation in nitrogen-fixing legume nodules. Plant
Physiol. Biochem. 29:185-201.
10. de Bruijn, F. J., and J. R. Lupski. 1984. The use of transposon
TnS mutagenesis in the rapid generation of correlated physical
and genetic maps of DNA segments cloned into multicopy
plasmids-a review. Gene 27:131-149.
11. Devereux, J., P. Haeberli, and 0. Smithies. 1984. A comprehen-
sive set of sequence analysis programs for the VAX. Nucleic
Acids Res. 12:387-395.
12. DeVries, G. E., P. int'Veld, and J. W. KlJne. 1980. Production of
organic acids in Pisum sativum root nodules as a result of
oxygen stress. Plant Sci. Lett. 13:115-133.
13. Dilworth, M., and A. Glenn. 1984. How does a legume nodule
work? Trends Biochem. Sci. 9:519-523.
14. Ebbighausen, H., C. Jia, and H. W. Heldt. 1985. Oxaloacetate
translocator in plant mitochondria. Biochim. Biophys. Acta
810:184-199.
15. Engelke, T. H., M. N. Jagadish, and A. Puihler. 1987. Biochem-
ical and genetical analysis of Rhizobium meliloti mutants defec-
tive in C4-dicarboxylic acid transport. J. Gen. Microbiol. 133:
3019-3029.
16. Feinberg, A. P., and B. Vogelstein. 1983. A technique for
radiolabeling DNA restriction endonuclease fragments to high
specific activity. Anal. Biochem. 132:6-13.
17. Finan, T. M., J. M. Wood, and D. C. Jordon. 1983. Symbiotic
properties of C4-dicarboxylic acid transport mutants of Rhizo-
bium leguminosarum. J. Bacteriol. 154:1403-1413.
18. Ford, G. C., G. Eichele, and J. N. Jansonius. 1980. Three-
dimensional structure of a pyridoxal-phosphate-dependent en-
zyme, mitochondrial aspartate aminotransferase. Proc. Natl.
Acad. Sci. USA 77:2559-2563.
19. Fotheringham, I. G., S. A. Dacey, P. P. Taylor, T. J. Smith,
M. G. Hunter, M. E. Finlay, S. B. Primrose, D. M. Parker, and
R. M. Edwards. 1986. The cloning and sequence analysis of the
aspC and tyrB genes from Eschenichia coli K12-comparison of
the primary structures of the aspartate aminotransferase and
aromatic aminotransferase of E. coli with those of the pig
aspartate aminotransferase. Biochem. J. 234:593-604.
20. Fougere, F., D. Le Rudulier, and J. G. Streeter. 1991. Effects of
salt stress on amino acid, organic acid, and carbohydrate
composition of roots, bacteroids, and cytosol of alfalfa (Medi-
cago sativa L.). Plant Physiol. 96:1228-1236.
21. Gantt, J. S., R J. Larson, M. W. Farnham, S. M. Pathirana,
S. S. Miller, and C. P. Vance. 1992. Aspartate aminotransferase
in effective and ineffective alfalfa nodules: cloning of a cDNA
and determination of enzyme activity, protein, and mRNA
levels. Plant Physiol. 98:868-878.
22. Gonzalez-Gonzalez, R., J. L. Botsford, and T. Lewis. 1990.
Osmoregulation in Rhizobium meliloti: characterization of en-
zymes involved in glutamate synthesis. Can. J. Microbiol.
36:469-474.
23. Grange, T., C. Guenet, J. B. Dietrich, S. Chasserot, M. Fromont,
N. Befort, J. Jami, G. Beck, and R. Pictet. 1985. Complete
complementary DNA of rat tyrosine aminotransferase messen-
ger RNA: deduction of the primary structure of the enzyme. J.
Mol. Biol. 184:347-350.
24. Griffith, S. M., and C. P. Vance. 1989. Aspartate aminotrans-
ferase in alfalfa root nodules. I. Purification and partial charac-
terization. Plant Physiol. 90:1622-1629.
25. Hamm, G. H., and G. N. Cameron. 1986. The EMBL data
library. Nucleic Acids Res. 14:5-10.
26. Hatch, M. D., and H. W. Heldt. 1985. Synthesis, storage, and
stability of [4-l4C]oxaloacetic acid. Anal. Biochem. 145:393-397.
27. Hayashi, H., Y. Horio, T. Tanaka, M. Taketoshi, and H. Wada.
1987. Rat cytosolic aspartate aminotransferase; molecular clon-
ing of cDNA and expression in E. coli, p. 39-42. In T. Korpela
and P. Christen (ed.), Biochemistry of vitamin B6: Proceedings
of the 7th International Congress on Chemical and Biological
Aspects of Vitamin B6 Catalysis. Birkhauser Verlag, Basel.
4196 ALFANO AND KAHN
28. Henson, C. A., M. Collins, and S. H. Duke. 1982. Subcellular
localization of enzymes of carbon and nitrogen metabolism in
nodules of Medicago sativa. Plant Cell Physiol. 23:227-235.
29. Herrada, G., A. Puppo, and J. Rigaud. 1989. Uptake of metab-
olites by bacteroid-containing vesicles and by free bacteroids
from French bean nodules. J. Gen. Microbiol. 135:3165-3171.
30. Huynh, Q. K., R. Sakakibara, T. Watanabe, and H. Wada. 1981.
The complete amino acid sequence of mitochondrial glutamic
oxaloacetic transaminase from rat liver. J. Biochem. 90:863-
875.
31. Kahn, M. L., and P. Hanawalt. 1979. Size distribution of DNA
replicative intermediates in bacteriophage P4 and E. coli. J.
Mol. Biol. 128:501-525.
32. Kahn, M. L., J. Kraus, and J. E. Somerville. 1985. A model of
nutrient exchange in the Rhizobium-legume symbiosis, p. 193-
199. In H. Evans, P. Bottomley, and W. E. Newton (ed.),
Nitrogen fixation research progress. M. J. Nijhoff, New York.
33. Kahn, M. L., R. Ziermann, G. Deho, D. W. Ow, M. G. Sunshine,
and R. Calendar. 1991. Use of bacteriophages P2 and P4.
Methods Enzymol. 204:264-280.
34. Kerppola, T. K., and M. L. Kahn. 1988. Symbiotic phenotypes
of auxotrophic mutants of Rhizobium meliloti 104A14. J. Gen.
Microbiol. 134:913-919.
35. Kittell, B. L., D. R. Helinski, and G. S. Ditta. 1989. Aromatic
aminotransferase activity and indoleacetic acid production in
Rhizobium meliloti. J. Bacteriol. 171:5458-5466.
36. Kraus, J. 1987. Nutrient exchange in the Rhizobium-legume
symbiosis: glutamate catabolism by Rhizobium meliloti Ph.D.
thesis. Washington State University, Pullman.
37. Kuramitsu, S., T. Ogawa, H. Ogawa, and H. Kagamiyama. 1985.
Branched-chain amino acid aminotransferase of Escherichia
coli: nucleotide sequence of the ilvE gene and the deduced
amino acid sequence. J. Biochem. 97:993-999.
38. Kuramitsu, S., S. Okuno, T. Ogawa, H. Ogawa, and H. Kag-
amiyama. 1985. Aspartate aminotransferase of Escherichia coli:
nucleotide sequence of the aspC gene. J. Biochem. 97:1259-
1262.
39. Marino, G., G. Nitti, M. I. Arnone, G. Sannia, A. Gambacorta,
and M. De Rosa. 1988. Purification and characterization of
aspartate aminotransferase from the thermoacidophilic archae-
bacterium Sulfolobus solfataricus. J. Biol. Chem. 263:12305-
12309.
40. McDermott, T. R., and M. L. Kahn. 1992. Cloning and muta-
genesis of the Rhizobium meliloti isocitrate dehydrogenase
gene. J. Bacteriol. 174:4790-4797.
41. Mehta, P. K., T. I. Hale, and P. Christen. 1989. Evolutionary
relationships among aminotransferases: tyrosine aminotransfer-
ase, histidinol-phosphate aminotransferase, and aspartate ami-
notransferase are homologous proteins. Eur. J. Biochem. 186:
249-253.
42. Obaru, K., H. Nomiyama, K. Shimada, F. Nagashima, and Y.
Morino. 1986. Cloning and sequence analysis of mRNA for
mouse aspartate aminotransferase isoenzymes. J. Biol. Chem.
261:16976-16983.
43. Rastogi, V. K., and R. J. Watson. 1991. Aspartate aminotrans-
ferase activity is required for aspartate catabolism and symbi-
otic nitrogen fixation in Rhizobium meliloti. J. Bacteriol. 173:
2879-2887.
44. Reibach, P. H., and J. G. Streeter. 1983. Metabolism of "'C-
labeled photosynthate and distribution of enzymes of glucose
metabolism in soybean nodules. Plant Physiol. 72:634-640.
45. Reibach, P. H., and J. G. Streeter. 1984. Evaluation of active
versus passive uptake of metabolites by Rhizobium japonicum
bacteroids. J. Bacteriol. 159:47-52.
46. Rej, R. 1982. A convenient continuous-rate spectrophotometric
method for determination of amino acid substrate specificity of
aminotransferases: application to isoenzymes of aspartate ami-
notransferase. Anal. Biochem. 119:205-210.
47. Reynolds, P. H. S., and K. Farnden. 1979. The involvement of
aspartate aminotransferase in ammonium assimilation in lupin
nodules. Phytochemistry 18:1625-1630.
48. Ronson, C. W., P. Lyttleton, and J. G. Robertson. 1981. C4-
J. BACT1ERIOL.
dicarboxylate transport mutants of Rhizobium tnifolii form inef-
fective nodules on Tnfolium repens. Proc. Natl. Acad. Sci.
USA 78:4284-4288.
49. Rosendahl, L., C. P. Vance, and W. B. Pedersen. 1990. Products
of dark CO2 fixation in pea root nodules support bacteroid
metabolism. Plant Physiol. 93:12-19.
50. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular
cloning: a laboratory manual. Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y.
51. San Francisco, M. J. D., and G. R. Jacobson. 1985. Uptake of
succinate and malate in cultured cells and bacteroids of two
slow-growing species of Rhizobium. J. Gen. Microbiol. 131:
1441-1448.
52. Saroso, S., M. J. Dilworth, and A. R. Glenn. 1986. The use of
activities of carbon catabolic enzymes as a probe for the carbon
nutrition of snakebean nodule bacteroids. J. Gen. Microbiol.
132:243-249.
53. Simon, R., U. Priefer, and A. Pfihler. 1983. A broad host range
mobilization system for in vivo genetic engineering: transposon
mutagenesis in gram negative bacteria. Bio/Technology 1:784-
791.
54. Simon, R., J. Quandt, and W. Klipp. 1989. New derivatives of
transposon Tn5 suitable for mobilization of replicons, genera-
tion of operon fusions and induction of genes in gram-negative
bacteria. Gene 80:161-169.
55. Somerville, J. E., and M. L. Kahn. 1983. Cloning of the
glutamine synthetase I gene from Rhizobium meliloti. J. Bacte-
riol. 156:168-176.
56. Stowers, M. D. 1985. Carbon metabolism in Rhizobium species.
Annu. Rev. Microbiol. 39:89-108.
57. Streeter, J. G. 1987. Carbohydrate, organic acid, and amino acid
composition of bacteroids and cytosol from soybean nodules.
Plant Physiol. 85:768-773.
58. Streeter, J. G. 1991. Transport and metabolism of carbon and
nitrogen in legume nodules. Adv. Bot. Res. 18:129-187.
59. Stripf, R., and D. Werner. 1978. Differentiation of Rhizobium
japonicum. II. Enzymatic activities in bacteroids and plant
cytoplasm during the development of nodules of Glycine max.
Z. Naturforsch. Teil C 33:373-381.
60. Sung, M., K. Tanizawa, H. Tanaka, S. Kuramitsu, H. Kag-
amiyama, K. Hirotsu, A. Okamoto, T. Higuchi, and K. Soda.
1991. Thermostable aspartate aminotransferase from a thermo-
philic Bacillus species. J. Biol. Chem. 266:2567-2572.
61. Udvardi, M. K., and M. L. Kahn. 1991. Isolation and analysis of
a cDNA clone that encodes an alfalfa (Medicago sativa) aspar-
tate aminotransferase. Mol. Gen. Genet. 231:97-105.
62. Udvardi, M. K., G. D. Price, P. M. Gresshoff, and D. A. Day.
1988. A dicarboxylate transporter on the peribacteroid mem-
brane of soybean nodules. FEBS Lett. 231:36-40.
63. Udvardi, M. K., C. L. Salom, and D. A. Day. 1988. Transport of
L-glutamate across the bacteroid membrane but not the perib-
acteroid membrane from soybean root nodules. Mol. Plant
Microbe Interact. 1:250-254.
64. Vance, C. P., and G. H. Heichel. 1991. Carbon in N2 fixation:
limitation or exquisite adaption. Annu. Rev. Plant Physiol. Plant
Mol. Biol. 42:373-392.
65. Watson, R. J. 1990. Analysis of the C4-dicarboxylate transport
genes of Rhizobium meliloti: nucleotide sequence and deduced
products of dctA, dctB and dctD. Mol. Plant Microbe Interact.
3:174-181.
66. Watson, R. J., and V. K. Rastogi. 1993. Cloning and nucleotide
sequencing of Rhizobium meliloti aminotransferase genes: an
aspartate aminotransferase required for symbiotic nitrogen fix-
ation is atypical. J. Bacteriol. 175:1919-1928.
67. Xu, J., M. E. Olson, M. L. Kahn, and R. E. Hurlbert. 1991.
Characterization of TnS-induced mutants of Xenorhabdus nem-
atophilus ATCC 19061. Appl. Environ. Microbiol. 57:1173-
1180.
68. Yagi, T., H. Kagamiyama, M. Nozaki, and K. Soda. 1985.
Glutamate-aspartate transaminase from microorganisms. Meth-
ods Enzymol. 113:83-89.