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Journal of Bacteriology, August 1998, p. 4278-4286, Vol. 180, No. 16
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Arginine Catabolism and the Arginine
Succinyltransferase Pathway in Escherichia coli
Barbara L.
Schneider,
Alexandros K.
Kiupakis, and
Lawrence J.
Reitzer*
Department of Molecular and Cell Biology, The
University of Texas at Dallas, Richardson, Texas 75083-0688
Received 11 September 1997/Accepted 3 June 1998
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ABSTRACT |
Arginine catabolism produces ammonia without transferring nitrogen
to another compound, yet the only known pathway of arginine catabolism
in Escherichia coli (through arginine decarboxylase) does
not produce ammonia. Our aims were to find the ammonia-producing pathway of arginine catabolism in E. coli and to examine
its function. We showed that the only previously described pathway of
arginine catabolism, which does not produce ammonia, accounted for only 3% of the arginine consumed. A search for another arginine catabolic pathway led to discovery of the ammonia-producing arginine
succinyltransferase (AST) pathway in E. coli. Nitrogen
limitation induced this pathway in both E. coli and
Klebsiella aerogenes, but the mechanisms of activation
clearly differed in these two organisms. We identified the E. coli gene for succinylornithine aminotransferase, the third enzyme of the AST pathway, which appears to be the first of an astCADBE operon. Its disruption prevented arginine
catabolism, impaired ornithine utilization, and affected the synthesis
of all the enzymes of the AST pathway. Disruption of astB
eliminated succinylarginine dihydrolase activity and prevented arginine
utilization but did not impair ornithine catabolism. Overproduction of
AST enzymes resulted in faster growth with arginine and aspartate. We
conclude that the AST pathway is necessary for aerobic arginine catabolism in E. coli and that at least one enzyme of this
pathway contributes to ornithine catabolism.
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INTRODUCTION |
In a defined minimal medium,
Escherichia coli and Klebsiella aerogenes can use
several amino acids as sole nitrogen sources (reviewed in reference
24). Such catabolism must result in assimilation of
nitrogen into glutamate and glutamine, which provide nitrogen for the
synthesis of virtually all nitrogen-containing compounds. Because
glutamine synthesis requires ammonia, amino acid degradation must
produce ammonia, whose concentration and generation limit growth. A low
level of ammonia results in a low level of glutamine, which
induces proteins that transport, degrade, and assimilate nitrogen-containing compounds. This response, which in effect is a
response to ammonia limitation, is called the nitrogen-regulated (Ntr)
response (see references 24 and
25 for reviews).
Despite the importance of reactions that produce ammonia, such
reactions have not been identified for the catabolism of
-aminobutyrate, aspartate, arginine, glutamate, proline, putrescine,
and other compounds (24). The obvious approach to analyze
such catabolism, i.e., to isolate mutants, has not succeeded for
aspartate and arginine catabolism. Therefore, we adopted an alternate
approach: we characterized 15N-aspartate catabolism by a
variety of methods, including nuclear magnetic resonance analysis of
cellular extracts (11). When E. coli was
incubated with 14N-arginine (in the form of a rapidly
metabolized dipeptide) and 15N-aspartate, all of the
ammonia was 14N, which implied that arginine catabolism
produced ammonia. Alanine, aspartate, and glutamate were heavily
labeled, which implied that direct deamination of these amino acids did
not produce ammonia. These results were unexpected since the only known
pathway of arginine catabolism does not generate ammonia.
It has been proposed that E. coli degrades arginine via a
pathway that is initiated with arginine decarboxylation (30,
31). The agmatine produced from this reaction is metabolized to
putrescine, which is eventually degraded to glutamate and succinate
(see Fig. 1). A catabolic function for this pathway is consistent with
the induction by nitrogen limitation of all the enzymes of this
pathway, except for arginine decarboxylase (ADC) itself, which is
constitutive (31). Furthermore, mutants deficient in ADC,
agmatine ureohydrolase, or putrescine transaminase grow more slowly
than the corresponding wild-type strain with arginine as the sole
nitrogen source. Nonetheless, we suspected another pathway because this
pathway does not produce ammonia and because all mutants with
deficiencies in the ADC pathway still grow reasonably well with
arginine; for example, a speB (agmatine
ureohydrolase-encoding) mutant has a 300-min doubling time, whereas the
isogenic wild-type strain has a 160-min doubling time (30).
In the present study, we show that the ADC pathway does not
significantly contribute to arginine degradation. Instead, we find that
E. coli possesses the ammonia-producing arginine succinyltransferase (AST) pathway. We present evidence that the AST
pathway is necessary for arginine degradation during nitrogen-limited growth and that it contributes to the degradation of other amino acids.
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MATERIALS AND METHODS |
Strains and plasmids.
All E. coli K-12 strains
used for enzyme assays and growth experiments were derivatives of
W3110, which was considered a wild-type strain (29). Strain
LR1 also contains glnG10::Tn5, and AKK1 contains a disruption of astB, the fourth gene of the
ast operon. AKK1 was constructed by ligating the 2-kb
SmaI/NsiI fragment of pLC3-11, which contains the
fourth gene of the ast operon, into pUC18, which had been
digested with SmaI and PstI. Removing a 300-bp
NruI/BssHII fragment from the insert and
replacing it with a kanamycin resistance gene resulted in disruption of
the cloned gene. A 4.2-kb linear DNA fragment with the disrupted gene
was electroporated into strain K4633 (recD), and integrants
were selected on kanamycin-containing plates. The disruption was
transduced into W3110 with P1 vir. K4633 (recD)
was obtained from A. Ninfa (University of Michigan). The K. aerogenes strains were KC2668 (wild type) and KC2738
(glnG2::Tn5-131), which were obtained
from R. Bender (University of Michigan). C. Fraley and A. C. Matin (Stanford University) disrupted the astC gene, which codes
for succinylornithine transaminase, as part of an independent study. They provided us with a strain containing the disruption, and we
transferred the astC::kan into W3110 by
P1 transduction. The E. coli Genetic Stock Center provided
the Carbon-Clarke plasmid pLC3-11 (3, 26). Plasmid
p
LC3-11 is derived from pLC3-11 by deletion of two adjacent
PstI fragments, which were replaced by a kanamycin
resistance-encoding cassette from pUC-4-KAPA (Pharmacia). The altered
plasmid contains chromosomal DNA from osmE to
xthA.
Cell growth.
The minimal growth medium contained W salts
(27), 0.4% of the carbon source and 0.2% of each nitrogen
source, unless otherwise indicated. Cells were grown and harvested as
described previously (27); whole-cell pellets were frozen
until needed.
Enzyme assays.
Cell pellets from a 15-ml culture were
resuspended in 1 ml of 50 mM K-PO4 buffer (pH 7.5) (mixture
of K2HPO4 and KH2PO4 at pH 7.5) with 1 mM 
-mercaptoethanol and sonicated three times for
5 s. Cell debris was removed by centrifugation for 10 min at 4°C
in a microfuge.
Syntheses for substrates of enzymes of the AST pathway, i.e.,
N-succinyl derivatives of arginine, ornithine, and
glutamate, have been described (40). The assays for AST
pathway enzymes were performed essentially as described previously
(14, 40). In brief, AST was assayed by measuring the
decrease in succinyl coenzyme A at 232 nm. The activity of
succinylarginine dihydrolase was assessed by determination of the
amount of ammonia produced, which was coupled enzymatically by
glutamate dehydrogenase to the consumption of NADH. Succinylglutamate
desuccinylase was analyzed by measuring the liberation of glutamate,
which was coupled to the glutamate dehydrogenase-dependent production
of NADH, which absorbs at 340 nm. Succinylornithine transaminase was
assayed by measuring the product succinylglutamic semialdehyde, which is hydrolyzed to glutamic semialdehyde. Glutamic semialdehyde spontaneously cyclizes to '-pyrroline-5-carboxylic acid, which reacts with o-aminobenzaldehyde to yield a product that
absorbs at 440 nm. Succinylglutamic semialdehyde dehydrogenase was
measured by monitoring NAD reduction at 340 nm. The substrate for this reaction was synthesized during the reaction by adding
succinylornithine and 
-ketoglutarate, together with
succinylornithine transaminase, which had been purified from E. coli W3110. For all assays and for purification of the
transaminase, protein was determined as described earlier
(18).
Purification and partial sequencing of succinylornithine
transaminase.
The procedure for this enzyme purification differed
substantially from that previously described (6). The
transaminase was produced from cells of W3110 containing plasmid
pLC3-11 that had been grown in glucose-arginine minimal medium. The
cell extract was prepared by sonication in a pH 7.5 buffer containing
50 mM KPO4, 0.1 mM pyridoxal phosphate, 2.5 mM
-ketoglutarate, 0.1 mM EDTA, and 1 mM dithiothreitol. The protein
was precipitated with 60%
(NH4)2SO4, redissolved, applied to
a DEAE-Sephadex column, and eluted with a KCl gradient (peak activity
eluted at 200 mM KCl). The protein was subjected to gel filtration on a
G-100 column and applied to a phenyl-Sepharose column, with peak
activity eluting at 500 mM
(NH4)2SO4. The protein was dialyzed
before use. The Harvard Microchemistry Facility sequenced two peptides
of the transaminase after proteolytic digestion.
Arginine and urea determination.
Cells were grown at 30°C
in media described in Results. Samples of 1.5 ml were centrifuged, and
the cell-free culture medium was assayed for arginine and urea.
Arginine was converted to urea and ornithine by arginase, and the urea
produced was measured. Arginine and urea were added to growth medium
and processed as described below to generate standard curves. Arginine
reacted with the urea reagent; therefore, it was necessary to
physically separate arginine from urea. Separation was achieved by
modifying a previously described method (5). Pasteur
pipettes containing 2 ml of Dowex 50×4 resin (400 mesh), which had
been prepared as described previously (5), were washed with
2 ml of 1 N NaOH and then equilibrated with 2 ml of fresh culture
medium, which had been adjusted to pH 2.5 with HCl. A 0.5-ml sample was
adjusted to pH 2.5 with HCl, applied to the column, and eluted with 4.5 ml of 0.1 M Na citrate (pH 3.1). The 5 ml of solution that came through
was then assayed for urea. Arginine was eluted with 8 ml of a solution
that contained 0.5 M Na citrate and 0.35 M acetic acid that had been
adjusted to pH 9.5 with NaOH. (The resulting high ion concentration was
deliberate.)
Urea was measured essentially as described earlier (
22).
Samples of 0.35 ml were mixed with 0.25 ml of an acid mixture
(H
2SO
4-H
3PO
4-H
2O,
1:3:1 [vol/vol/vol]) and 0.04 ml of 4%
-isonitrosopropiophenone
in 95% ethanol. The mixtures were boiled in the dark for 1 h and
then cooled to room temperature; their absorbance at 540 nm was
then
determined.
For the arginine determination, 0.1 ml of sample was mixed with 0.05 ml
of arginase (30 U/ml) in 50 mM maleic acid (pH 7.0)
with 50 mM
MnSO
4 (
41) and incubated for 30 min at 37°C;
the
reaction was stopped with 0.15 ml of the acid mix. A portion of
this solution (0.2 ml) was mixed with 0.15 ml of the acid mix,
0.25 ml
of H
2O, and 0.04 ml of 4%
-isonitrosopropiophenone in
95% ethanol. This mixture was then treated as described above
for the
urea determination.
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RESULTS |
Role of ADC in arginine degradation in E. coli.
ADC
initiates the only known pathway of arginine degradation in E. coli (Fig. 1; references
24, 30, and 31). The sum of the
complete reaction sequence is shown below:
To quantify this pathway's contribution to arginine catabolism,
we compared arginine utilization and urea production.
E. coli does not metabolize urea, which is rapidly excreted into
the
medium (
21); therefore, we measured urea from the medium.
When arginine was used as the sole nitrogen source, less than
3% of
the arginine consumed resulted in urea (Table
1). To validate
the assay for arginine,
we assessed arginine utilization from
ammonia-containing medium.
Ammonia and the subsequent elevation
of the glutamine pool prevent
activation of Ntr genes, such as
the genes of arginine transport, and
should therefore diminish
arginine utilization (
24). We
observed the expected result:
there was five times less total arginine
consumed despite almost
twice the cell growth. Unlike growth with
arginine as a nitrogen
source, the ADC pathway metabolized 36% of the
arginine consumed.
It was expected that the ADC pathway would become
the major route
of putrescine synthesis in ammonia-containing medium,
since arginine
inhibits the only other route of putrescine synthesis
via ornithine
decarboxylase and represses the enzymes of ornithine
synthesis.
To further validate our measurements, we assayed urea
production
from cells which contain an unusually high level of ADC,
i.e.,
cells grown anaerobically in a complex acidic medium (
4,
35,
36). It has been proposed that ADC helps raise the pH by
releasing
acidic carboxyl groups as CO
2 (
9). As
expected, the urea produced,
when normalized to cell mass, was greater
than that from any other
medium. In summary, these results show that
the ADC pathway does
not contribute significantly to the degradation of
arginine as
the sole nitrogen source. Therefore, there must be another
pathway
for arginine degradation in
E. coli.
AST pathway in E. coli.
Bacteria contain a surprising
variety of pathways for arginine degradation (reviewed in references
4 and 24). We assumed that
E. coli contained a pathway that had previously been
characterized in another organism. Therefore, we sought a pathway that
produces ammonia but not urea. Two well-studied pathways fit these
criteria: the arginine deiminase pathway and the AST pathway
(4). The arginine deiminase pathway involves three enzymes,
arginine deiminase, a catabolic ornithine transcarbamoylase, and
carbamate kinase, which catalyze the reactions of arginine to
citrulline, of citrulline and phosphate to ornithine and carbamoyl
phosphate, and of carbamoyl phosphate and ADP to CO2,
NH3, and ATP, respectively. Despite biochemical searches
for these enzymes in E. coli and K. aerogenes and
careful genetic searches in E. coli, enzymes of this pathway have not been found (8, 15, 16, 20). On the other hand, the
AST pathway has been found in K. aerogenes, which is closely related to E. coli (33). Therefore, we focused
our attention on the AST pathway. The reactions of this pathway are
shown in Fig. 2 and their sum is shown
below:
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As described in more detail below, we successfully assayed all
five enzymes of the pathway from wild-type
E. coli.
Induction of the AST pathway by nitrogen limitation.
To
initially analyze the function of the AST pathway, we examined the
regulation of its enzymes. The degradation of arginine as the sole
nitrogen source limits growth and requires the transcriptional activator nitrogen regulator I (NRI, also called NtrC), the
product of glnG (or ntrC) (23).
Therefore, we tested whether nitrogen limitation induced enzymes of the
AST pathway and, if so, whether such induction required
NRI. Nitrogen-limited E. coli had much more
succinylarginine dihydrolase and succinylglutamate desuccinylase than
cells grown in the corresponding nitrogen-rich (ammonia-containing) medium (compare lines 1 and 3 to lines 2 and 4 in Table
2). Furthermore, the levels of these
enzymes were low in glnG mutants (compare lines 3 and 5 in
Table 2). By these two criteria, these AST enzymes in E. coli are under nitrogen control.
The results for succinylornithine transaminase, the third enzyme of the
pathway, appeared to suggest that this enzyme was
not under nitrogen
control. However, it is likely that this assay
measures the activities
of two different enzymes. This suspicion
is based on the fact that a
mutation in
argD, which codes for
an arginine-repressible
acetylornithine transaminase, an enzyme
of arginine synthesis, can be
suppressed by a mutation in
argM,
an arginine-inducible
acetylornithine transaminase (
10). ArgM
was subsequently
shown to prefer succinylornithine as a substrate
(
4,
10).
Since both ArgD and ArgM can use acetylornithine
as a substrate, we
assumed that both can also use succinylornithine.
Therefore, to
minimize the effects of ArgD, we assayed succinylornithine
transaminase
activity from cells grown in two different arginine-containing
media,
one nitrogen limited (glucose-arginine) and the other nitrogen
rich
(glucose-arginine-ammonia). Succinylornithine transaminase
activity was
10 times higher in the nitrogen-limited medium (lines
1 and 2 in Table
2), which indicates that succinylornithine transaminase
is also under
nitrogen control.
We could assay the other two enzymes of the AST pathway, arginine
succinyltransferase and succinylglutamic semialdehyde dehydrogenase,
from wild-type cells grown with arginine as the sole nitrogen
source
(which induces optimally) but not in any other medium (
28).
However, in strains with genes of a putative five-gene
ast
operon
on a high-copy-number plasmid, all five enzymes could be
measured,
and nitrogen limitation induced all of them (see below). In
summary,
all five enzymes of the AST pathway are nitrogen regulated.
Specific induction of the AST pathway by other amino acids.
All growth-limiting nitrogen sources induce the well-studied
glnALG operon equally well (29). However, this
was clearly not the case for enzymes of the AST pathway (lines 1 and 3 in Table 2). Therefore, we examined the effect of various amino acids
on the induction of the AST pathway (Table
3). Arginine was the best inducer, but
other amino acids could also induce, albeit to a lesser extent. For
example, aspartate induced to 50% of the maximal induction (Table 3).
One possible explanation for this regulation is that arginine is the
true inducer and the other amino acids induce to the
extent that they
affect arginine levels. Such an explanation can
account for induction
by aspartate, which is a substrate in the
penultimate step of arginine
synthesis, i.e., the reaction catalyzed
by argininosuccinate
synthetase. We tried to test this hypothesis
by preventing assimilation
of aspartate's nitrogen into arginine
and then examining whether
aspartate could still induce. We used
an
argG strain which
is deficient in argininosuccinate synthetase,
the enzyme that
assimilates aspartate's nitrogen. Because this
strain is an arginine
auxotroph, the proposed experiment requires
finding a concentration of
arginine that satisfies the auxotrophy
but does not induce the AST
pathway. Unfortunately, such a concentration
does not exist: arginine
concentrations that limited growth twofold
fully induced the AST
pathway (
28). Further examination of the
mechanism of the
apparent arginine-dependent induction must await
characterization of
the transcription of these genes.
Catabolite repression of the AST pathway.
Prior to the results
presented here, the AST pathway had been found only in those organisms
that degrade arginine as a carbon source, an ability which E. coli lacks (4, 33, 34). Utilization of carbon sources
other than glucose frequently involves regulation by catabolite
repression. Therefore, we examined whether a residual catabolite
repression controlled the AST enzymes in E. coli. With aspartate, succinate, or glycerol as carbon and energy sources, the
amounts of succinylarginine dihydrolase were 2- to 10-fold higher and
the amounts of succinylglutamate desuccinylase were 9- to 70-fold
higher than those with glucose (Table 4).
We also examined the expression of succinylarginine dihydrolase and
succinylglutamate desuccinylase from cells grown in Luria-Bertani
broth
and found that these enzymes were induced to 15 to 20% of
the level
found from cells grown in glucose-arginine minimal medium.
This
induction was completely abolished by glucose in the Luria-Bertani
broth but was not affected by ammonia (
28). These results
suggest
control by catabolite repression.
Nitrogen source utilization and the AST pathway in K. aerogenes.
Unlike E. coli, K. aerogenes can
utilize arginine as the sole carbon, energy, and nitrogen source
(8, 33). Such growth induced the AST enzymes to much higher
levels than those observed in E. coli (line 6 in Table 2).
However, like E. coli, K. aerogenes grown in two
nitrogen-limited media, glucose-arginine and glucose-glutamine, produced higher levels of succinylarginine dihydrolase and
succinylglutamate desuccinylase than cells grown in the corresponding
nitrogen-rich (ammonia-containing) medium (compare lines 7 and 9 to
lines 8 and 10 in Table 2). Unexpectedly, their induction in K. aerogenes did not require NRI in glucose-arginine
medium (lines 7 and 12 in Table 2). This result explains why this
glnG mutant of K. aerogenes can still utilize
arginine as a nitrogen source (1, 28). However, other
results suggest that these two enzymes are under Ntr control in
glucose-glutamine minimal medium: their levels were lower in a
glnG mutant (lines 9 and 11 in Table 2).
Transaminase activity was unexpectedly higher in the
glnG
mutant compared to that in the wild-type strain when grown in
glucose-glutamine
medium (compare lines 9 and 11 in Table
2). Such
regulation was
not observed in
E. coli (see lines 3 and 5 in
Table
2). Succinylornithine
transaminase is probably regulated in
parallel with other enzymes
of the AST pathway, which were at very low
levels in the
glnG mutant (line 11 in Table
2). This implies
that another transaminase,
probably the biosynthetic acetylornithine
transaminase, is repressed
by NR
I. The function of such
control is not apparent.
Identification of the E. coli gene for
succinylornithine transaminase.
To identify the genes of the AST
pathway, we tried to isolate mutants that could not utilize arginine
but which could use ammonium sulfate as the sole nitrogen source. We
assayed just over 100 mutants, and none of them were defective in AST
enzymes. (The reason for this failure is not obvious since, as shown
below, AST mutants cannot utilize arginine as a nitrogen source.)
Therefore, we tried a reverse genetic strategy to isolate mutants
deficient in AST enzymes. We focused on analyzing succinylornithine
transaminase because its purification was required for assay of the
fourth enzyme of the AST pathway, succinylglutamic semialdehyde
dehydrogenase. This transaminase, called the product of
argM, had been previously characterized because high levels
of this enzyme suppressed a mutation in argD, which codes
for the biosynthetic acetylornithine transaminase (26). It
was subsequently shown to have a higher affinity for succinylornithine
than for acetylornithine (4, 10).
Sequencing of two internal peptides gave the following
se- quences: VLELINTPEMLNGVK and (Q)(P)ITRENF-(E) (W)MIPVYAP(A).
(The
parentheses indicate probable assignments, while the dash
indicates
that an assignment was not possible.) These peptides show a
perfect
match with the deduced product of a gene (called b1748 in the
latest annotation of the genome) that starts at bp 1830006 of
the
E. coli genome (about minute 39.3) (Fig.
3). The gene predicts
a 406-residue
protein and a subunit mass of 43,665 Da, which agrees
with the
subunit size of the purified transaminase. (This protein
was
previously reported to have a subunit mass of 30 kDa [see
reference
6]. The discrepancy results from
proteolysis, which
readily occurs if precautions against cleavage are
not taken [
28].)
The gene for the catabolic
succinylornithine transaminase is 60%
identical to
argD,
which codes for the arginine-repressible acetylornithine
transaminase.
Because this enzyme codes for the third enzyme of
the AST pathway, we
propose that its gene should be designated
as
astC instead
of
argM.
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FIG. 3.
Deduced amino acid sequence of succinylornithine
transaminase. The underlined residues correspond to those determined by
direct protein sequencing.
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Preliminary characterization of an astCADBE
operon.
Computer analysis of the regulatory region and four open
reading frames (ORFs) downstream from astC suggests that it
is the first of a five-gene, nitrogen-regulated astCADBE
operon. (The complete sequence can be accessed in GenBank under
ECAE000269.) Binding sites for 
54-RNA polymerase
and NRI would be expected for Ntr genes, and such sites are
readily identifiable. A probable 54-RNA
polymerase-binding site, TGGCACN5CTGCA, is
located 72 bases upstream from the initiating methionine codon. It
differs from the consensus binding site, TGGCACN5TTGC(A/T),
by only one base (19). Two possible NRI-binding
sites are located between 195 and 241 bases upstream from the upstream
edge of the RNA polymerase site. While these sites are farther
from the RNA polymerase site than is normal for activators of
54-dependent promoters, there is precedent for such
spacing (12). The four genes downstream from astC
are transcribed in the same direction, and an apparent rho-independent
terminator follows the last gene. The end of each gene overlaps with
the beginning of the next; there is a 4-bp overlap for the first three
junctions and an 8-bp overlap for the fourth.
Homology searches suggest that the four downstream genes code for
enzymes of the AST pathway. The second ORF (numeric identifier
b1747),
which we designate as
astA, codes for a 344-residue protein
that appears to be AST. This enzyme, the first of the AST pathway,
has
been purified from
Pseudomonas aeruginosa and has been shown
to have two different subunits (
38). Only the first few
amino
acids of each subunit have been determined, but these residues
are homologous to each other and to the deduced sequence of the
N
terminus coded by the second ORF (Table
5). The third ORF (numeric
identifier
b1746) apparently codes for a protein of 492 residues.
It is 32%
identical to
gabD, which specifies succinic semialdehyde
dehydrogenase. This gene probably codes for the fourth enzyme
of the
AST pathway, succinylglutamic semialdehyde dehydrogenase;
hence, we
designate this gene as
astD. The fourth ORF (numeric
identifier b1745) codes for a 447-residue protein that is homologous
to
nothing in the databases. Such a result would be expected for
succinylarginine dihydrolase, the second enzyme of the AST
pathway,
because it catalyzes an unusual reaction, the complete
degradation
of the guanidino group of succinylarginine. Disruption of
this
ORF results in selective loss of dihydrolase activity (described
below), therefore, we designate this gene as
astB. The fifth
ORF
(numeric identifier b1744) appears to code for a 322-residue
protein.
The gene product is homologous to human aspartoacylase, which
deacetylates
N-acetylaspartate. Aspartoacylase catalyzes a
reaction
very similar to that of succinylglutamate desuccinylase, the
last
enzyme of the AST pathway; therefore, we designate the gene as
astE.
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TABLE 5.
Comparison of the N termini of AST from P. aeruginosa and the protein from the second gene of the putative
ast operon from E. coli
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Phenotypes of mutants with disruption of ast
genes.
Table 6 shows growth rates
for strains with disruptions of astB and astC.
These disruptions had no effect on the utilization of ammonium sulfate,
glutamine, alanine, proline, and aspartate as nitrogen sources.
However, either of these disruptions completely prevented arginine
utilization (there was no growth after 1 week). Curiously, a disruption
of astC, but not of astB, severely impaired ornithine catabolism. (In the Discussion, we will propose that succinylornithine transaminase, but not other enzymes of the AST pathway, contributes to ornithine degradation.)
Experiments to analyze aspartate catabolism in an
argG
mutant, which is an arginine auxotroph, led to the unexpected
observation
that certain mixtures of amino acids support significantly
faster
growth than that with either amino acid alone (
28).
Furthermore,
a trace amount of the second amino acid, i.e., 0.01%,
instead
of the 0.2% used for amino acids as sole nitrogen source, was
sufficient for this effect. For example, 0.2% aspartate and 0.2%
arginine supported generation times of about 330 and 540 min,
respectively, while a mixture of 0.2% aspartate with 0.01% arginine
or, conversely, of 0.2% arginine with 0.01% aspartate both sustained
generation times of 105 min. We tested whether this synergism
requires
the AST pathway when arginine is one component of this
mixture. When
arginine is the major nitrogen source, the low level
of aspartate does
not restore growth in either mutant (Table
6).
However, when aspartate
is the major nitrogen source and arginine
is a trace nitrogen source,
the effect is not as dramatic but
growth is still impaired (Table
6).
Therefore, arginine catabolism
requires the AST pathway when arginine
is one component of a synergistic
binary mixture.
We also examined the disruptions' effects on AST pathway enzymes.
Because these mutants could not utilize arginine, cells
were grown in
medium with 0.2% of glutamine (which induces somewhat)
and 0.1%
arginine (which induces best). Despite the presence of
arginine, the
level of induction in the wild-type strain was virtually
the same as
that found in wild-type cells grown without the arginine.
As a result,
we could not detect AST, but we could detect the
other four AST pathway
enzymes from a wild-type strain (Fig.
4).
Disruption of
astC, the first gene of the operon, severely
affected
the levels of the remaining enzymes, except for transaminase
activity,
which was reduced only twofold (Fig.
4). This latter result
presumably
reflects assay of the biosynthetic acetylornithine
aminotransferase,
which may not be completely repressed by the arginine
in the medium.
Nevertheless, these results provide strong evidence for
an
ast operon. Disruption of the fourth gene most severely
affected succinylarginine
dihydrolase, had no effect on transaminase
activity, and reduced
dehydrogenase and desuccinylase activity twofold
(Fig.
4). The
effects on transaminase and dehydrogenase activities were
expected,
since these enzymes appear to be encoded by genes upstream
from
the insertion. The twofold reduction in desuccinylase activity
was
unexpected, since the gene for this enzyme is downstream from
the
insertion. For reasons that are not clear, this disruption
appears to
be less polar than the first. Nonetheless, the effects
of this
disruption shows that the fourth gene codes for succinylarginine
dihydrolase.
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|
FIG. 4.
AST enzyme activities in mutants with disruptions in
astC and astB. Activity in W3110 (wild type)
(black bars), in the astB derivative (white bars), and in
the astC derivative (cross-hatched bars) is shown. The
values given are the averages from at least five cultures. Activities
are given in micromoles of product per hour per milligram of total
protein; the error bars show the standard error of the mean. SOT,
succinylornithine transaminase; SGSDH, succinylglutamic semialdehyde
dehydrogenase; SAD, succinylarginine dihydrolase; SGDS,
succinylglutamate desuccinylase.
|
|
Plasmid pLC3-11 and AST enzyme overproduction in E. coli.
Plasmid pLC3-11 contains chromosomal DNA from min 39 of the
E. coli chromosome. A map of restriction endonuclease sites
verified that this plasmid contained astC (28).
It had been proposed that pLC3-11 contained astC (previously
called argM), because Riley and Glansdorff showed that it
was one of two plasmids that could complement a mutation in
argD (26). (The other complementing plasmid,
pLC2-28, contained argD itself.)
Plasmid pLC3-11 caused an approximately fivefold increase in all of the
enzymes of the AST pathway (
28). We deleted 8 kb
from the
19-kb chromosomal insert in pLC3-11, which produced plasmid
p
LC3-11.
The remaining insert contained the five genes of the
putative
ast operon and very little else. Cells with this plasmid
had
a 6- to 12-fold increase in all five AST pathway enzymes (Table
7). Such cells also grew faster with
arginine and aspartate as
nitrogen sources, reducing the doubling time
from 296 to 166 min
with arginine and from 418 to 245 min with
aspartate (Table
7).
Deletion into the most upstream or downstream
region of these
five genes on the plasmid resulted in loss of
expression of all
five gene on the plasmid, i.e., we could detect only
activities
that resulted from expression of the chromosomal genes.
| |
DISCUSSION |
E. coli has the AST pathway.
We examined arginine
catabolism in E. coli because an analysis of amino acid
catabolism suggested that arginine catabolism produces ammonia but that
direct deamination of alanine, aspartate, and glutamate does not
(11). The previously described ADC pathway does not produce
ammonia (Fig. 1), and we showed that it could account for only about
3% of the arginine consumed (Table 1). Friedrich and Magasanik, based
on similar evidence, also concluded that the ADC pathway does not
degrade arginine in K. aerogenes (8). We sought
an ammonia-generating pathway of arginine catabolism and found
activities for all five enzymes of the AST pathway, which is initiated
by the succinylation of arginine's amino group.
We identified an
astCADBE operon, which codes for the five
enzymes of the AST pathway. The gene for succinylornithine transaminase
was found based on the identity of the protein sequence and the
deduced
gene product. We have designated this gene
astC, and it
appears to be the first gene of the operon. Homology searches
of the
second, third, and fifth ORFs of this operon suggest that
they code for
AST, succinylglutamic semialdehyde dehydrogenase,
and succinylglutamate
desuccinylase, the first, fourth, and fifth
enzymes of the AST pathway,
respectively. A homology search did
not suggest a function for the
fourth ORF, but its disruption
specifically eliminated the activity of
the second enzyme of the
pathway, succinylarginine dihydrolase. The
observation that each
gene overlaps with the one that follows it is
consistent with
an operon organization and suggests that translation of
a polycistronic
mRNA may require just one ribosome. Disruption of the
first gene
impairs synthesis of all the AST enzymes, a finding which is
also
consistent with an operon structure. Finally, these observations,
together with the fact that cells with plasmid p
LC3-11, which
contains this putative operon, have elevated levels of all five
enzymes, provide further evidence for an
astCADBE operon.
Functions of the AST pathway.
Mutants deficient in the AST
pathway fail to utilize arginine, which shows that the AST pathway is
necessary for catabolism of arginine as the sole nitrogen source during
aerobic exponential growth. Other evidence also suggests a catabolic
function. The presence of the astCADBE operon on plasmid
pLC3-11 resulted in high levels of all of the AST enzymes and caused
faster growth with arginine. Furthermore, the regulation of AST enzymes
is consistent with a catabolic function, i.e., the levels are highest
when arginine is the sole nitrogen source.
The AST pathway also appears to contribute to ornithine degradation.
Disruption of
astC, which codes for succinylornithine
aminotransferase, significantly impaired ornithine degradation.
It has
previously been proposed that ornithine is degraded via
putrescine and
-aminobutyrate as indicated in Fig.
1 (
30).
However, the
phenotype of the
astC::
kan mutant
suggests that the
AST pathway contributes to ornithine catabolism.
There is precedent
for involvement of the AST pathway in ornithine
catabolism (
39).
In
P. aeruginosa, succinylation
of ornithine by a multispecific
heteromeric succinyltransferase
initiates such catabolism, and
the resulting succinylornithine is then
degraded by the last three
enzymes of the AST pathway (
38,
39). However, such a pathway
may not function in
E. coli, since disruption of
astB, which reduces
the
expression of
astE twofold (Fig.
4), does not impair
ornithine
catabolism. Furthermore, the
E. coli
succinyltransferase appears
to be homomeric (more accurately, there is
no evidence for a second
homologous subunit) and may not have the
capacity to succinylate
ornithine. It is possible that the only enzyme
of the AST pathway
that contributes to ornithine catabolism is the
transaminase,
the product of
astC, which has been shown to
utilize ornithine
as a substrate (
2). Such a reaction would
produce glutamic
semialdehyde, which cyclyzes to form
'-pyrroline-5-carboxylate,
the immediate precursor for proline
synthesis. It could be hypothesized
that
'-pyrroline-5-carboxylate
is directly metabolized to glutamate.
However, the PutA protein
catalyzes this reaction, and its induction
requires proline. Therefore,
we propose that ornithine degradation
requires its conversion to
proline. A similar pathway has been
proposed for ornithine catabolism
in
Pseudomonas putida (
37).
Some evidence also suggests that the AST pathway contributes to the
degradation of aspartate. First, cells with p
LC3-11 grew
faster with
aspartate as the nitrogen source (Table
7). Second,
aspartate induces
the AST pathway (Table
3). Third, glutamate
and aspartate accumulate in
an
argG mutant when aspartate is the
sole nitrogen source
(
11). If arginine were an intermediate
in aspartate
degradation, then this result would be expected because
the last two
enzymes of arginine synthesis, argininosuccinate
synthetase and
argininosuccinase (products of
argG and
argH,
respectively),
would then be required for aspartate degradation.
However, mutants
with low levels of AST enzymes grew normally with
aspartate as
nitrogen source. There is an unidentified
NR
I-independent pathway
of aspartate degradation that also
contributes to aspartate catabolism
(
23). It is possible
that this pathway compensates for loss
of the AST pathway. Despite its
potential to contribute to aspartate
catabolism (as suggested by the
faster growth that accompanies
elevated levels of AST pathway enzymes),
the evidence is inadequate
to demonstrate that the AST pathway
contributes to aspartate catabolism.
The regulation of the AST pathway by conditions other than nitrogen
limitation suggests additional functions. The AST pathway
is induced by
growth in broth (
28). The AST pathway also appears
to be
induced upon entry into stationary-phase growth (
7).
The
function of the AST pathway under such conditions is not clear.
Unlike
all other organisms that contain the AST pathway,
E. coli does not degrade arginine as a carbon source (
33,
34).
E. coli may fail to utilize arginine as a carbon source
because the
ast genes may lack an appropriately controlled
promoter or because
of inadequate transport during carbon-limited
growth.
Functions of ADC in E. coli.
If the AST pathway degrades
arginine, then the question arises as to what is the function of ADC.
E. coli has two ADC isozymes. The first, encoded by
speA, is constitutively synthesized and its product is
referred to as the biosynthetic ADC. It probably synthesizes putrescine
when the major route of putrescine synthesis via ornithine
decarboxylase is blocked by arginine, which represses the enzymes of
ornithine synthesis, i.e., the first five enzymes of arginine synthesis
(36) (see Fig. 1). Tabor and Tabor have suggested
that a biosynthetic ADC is present in E. coli only because of the absence of a mechanism to degrade arginine to ornithine (36). A role for the biosynthetic ADC in putrescine
synthesis accounts for why mutants deficient in this isozyme grew
slowly with arginine as the sole nitrogen source (30, 31).
It also explains why 36% of arginine utilized is metabolized via ADC
when ammonia is in the medium (Table 1). In this situation, ammonia supplies the cell with nitrogen, as indicated by the fact that arginine
is not efficiently catabolized (Table 1), and ADC must initiate the
synthesis of putrescine to the extent that arginine represses the
enzymes of ornithine synthesis.
The second ADC isozyme is specified by the
adi gene and is
generally referred to as the biodegradative ADC (
17,
35).
This
enzyme is required for arginine-dependent acid resistance for
E. coli grown anaerobically in complex medium
(
17). During such
growth, this ADC can become a few percent
cellular protein (reference
35 and references
therein), and there is significant urea production
(Table
1). However,
it is not clear how arginine degradation
promotes acid resistance,
since it is known that ammonia generation
does not contribute to
survival under these conditions (
17).
In summary, arginine is an energy-rich amino acid that various bacteria
can use for nitrogen, carbon, or energy and for other
functions, such
as survival in an acidic environment.
E. coli appears to
contain only one pathway of arginine catabolism. In
contrast,
P. aeruginosa has at least four pathways of arginine
catabolism with
overlapping functions (
13,
37). The limited
number of such
pathways in
E. coli undoubtedly reflects its ability
to grow
in a much narrower ecological niche.
| |
ACKNOWLEDGMENTS |
This work was supported by National Institute of General Medical
Sciences grant GM47965 and by National Science Foundation grant
MCB-9723003.
We are grateful to Catherine Bailey for the preparation of figures, to
Warren Goux (Department of Chemistry, University of Texas at Dallas)
for assistance in synthesis of the N-succinylated substrates
required for the enzyme assays, to Cres Fraley and A. C. Matin
(Stanford University) for providing a strain with a disruption of
astC, and to A. Ninfa and R. Bender (University of Michigan)
for the strains. This work is presented as partial fulfillment for the
requirements for the Ph.D. degree at the University of Texas at Dallas
for B. L. Schneider and A. K. Kiupakis.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular and Cell Biology, Mail Station FO 3.1, The University of
Texas at Dallas, P.O. Box 830688, Richardson, TX 75083-0688. Phone: (972) 883-2523. Fax: (972) 883-2409. E-mail:
reitzer{at}utdallas.edu.
Present address: Department of Biology, University of California at
San Diego, La Jolla, CA 92093-0116.
| |
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Pioszak, A. A., Ninfa, A. J.
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185: 1299-1315
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Schneider, B. L., Ruback, S., Kiupakis, A. K., Kasbarian, H., Pybus, C., Reitzer, L.
(2002). The Escherichia coli gabDTPC Operon: Specific {gamma}-Aminobutyrate Catabolism and Nonspecific Induction. J. Bacteriol.
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Atkinson, M. R., Blauwkamp, T. A., Bondarenko, V., Studitsky, V., Ninfa, A. J.
(2002). Activation of the glnA, glnK, and nac Promoters as Escherichia coli Undergoes the Transition from Nitrogen Excess Growth to Nitrogen Starvation. J. Bacteriol.
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Atkinson, M. R., Blauwkamp, T. A., Ninfa, A. J.
(2002). Context-Dependent Functions of the PII and GlnK Signal Transduction Proteins in Escherichia coli. J. Bacteriol.
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Stancik, L. M., Stancik, D. M., Schmidt, B., Barnhart, D. M., Yoncheva, Y. N., Slonczewski, J. L.
(2002). pH-Dependent Expression of Periplasmic Proteins and Amino Acid Catabolism in Escherichia coli. J. Bacteriol.
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Kiupakis, A. K., Reitzer, L.
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Soupene, E., Lee, H., Kustu, S.
(2002). Ammonium/methylammonium transport (Amt) proteins facilitate diffusion of NH3 bidirectionally. Proc. Natl. Acad. Sci. USA
99: 3926-3931
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Reitzer, L., Schneider, B. L.
(2001). Metabolic Context and Possible Physiological Themes of {sigma}54-Dependent Genes in Escherichia coli. Microbiol. Mol. Biol. Rev.
65: 422-444
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Bochner, B. R., Gadzinski, P., Panomitros, E.
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Zimmer, D. P., Soupene, E., Lee, H. L., Wendisch, V. F., Khodursky, A. B., Peter, B. J., Bender, R. A., Kustu, S.
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Xi, H., Schneider, B. L., Reitzer, L.
(2000). Purine Catabolism in Escherichia coli and Function of Xanthine Dehydrogenase in Purine Salvage. J. Bacteriol.
182: 5332-5341
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Quintero, M. J., Muro-Pastor, A. M., Herrero, A., Flores, E.
(2000). Arginine Catabolism in the Cyanobacterium Synechocystis sp. Strain PCC 6803 Involves the Urea Cycle and Arginase Pathway. J. Bacteriol.
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Baca-DeLancey, R. R., South, M. M. T., Ding, X., Rather, P. N.
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Lu, C.-D., Abdelal, A. T.
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Schneider, B. L., Reitzer, L. J.
(1998). Salmonella typhimurium nit Is nadE: Defective Nitrogen Utilization and Ammonia-Dependent NAD Synthetase. J. Bacteriol.
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Fraley, C. D., Kim, J. H., McCann, M. P., Matin, A.
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Abstract
Introduction
