Department of Biology, University of
Michigan, Ann Arbor, Michigan 48109, and Department of Molecular
Biology and Biotechnology, Krebs Institute for Biomolecular
Research, University of Sheffield, Sheffield, United Kingdom
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INTRODUCTION |
Most biochemical conversions are
mediated by a single pathway (enzyme or sequence of enzymes). When
seemingly redundant pathways exist, as for some steps in the citric
acid cycle (5), detailed examination usually reveals that
the parallel paths operate under different conditions and are subject
to different controls. Thus, the ability to choose from parallel
pathways is a mechanism for control of biological function.
Most cellular nitrogen enters metabolism through glutamate. In enteric
bacteria such as Escherichia coli and in many other organisms, glutamate is synthesized by either of two primary pathways, each beginning with 2-oxoglutarate (2OG). In one, glutamate is formed
directly through reductive amination of 2OG by glutamate dehydrogenase
(GDH). Alternatively, synthesis can proceed indirectly through
amidation of glutamate to form glutamine by glutamine synthetase (GS)
followed by reductive transfer of the amide group to oxoglutarate by
glutamate synthetase (GOGAT) to give two glutamate molecules (a net
gain of one) (17). Mutants of E. coli lacking either pathway show no glutamate requirement under usual laboratory conditions; mutants lacking both require external glutamate for growth.
Thus, one or the other pathway is necessary for glutamate synthesis,
but not both.
In E. coli the two-step GOGAT pathway is known to be
important for synthesizing glutamate when the concentration of ammonium is low and for controlling the glutamine pool size (17).
Accordingly, gltBD mutants lacking GOGAT grow normally in
usual laboratory media but have a glutamate requirement when grown in
low ammonium, even though they retain GDH.
Understanding the role of GDH has been a major problem for those
studying nitrogen metabolism. Mutants lacking GDH have no obvious
growth alteration under usual laboratory conditions. However, it has
been shown recently that such gdhA mutants are impaired in
growth relative to the wild type when limited for energy (and carbon)
but ammonium and phosphate are present in excess (6). It was
hypothesized that the primary role of GDH is to form glutamate during
energy limitation because, in contrast to the GOGAT pathway, the GDH
pathway does not use ATP; the requirement for ATP in biosynthesis would
increase by nearly 20% if the GOGAT pathway were used instead of GDH
(6). Thus, the parallel pathways for glutamate synthesis may
play roles analogous to those of the sets of parallel pathways in the
respiratory chain (NADH dehydrogenases and ubiquinol oxidases) in
balancing the speed and efficiency of growth (6, 16).
At low ammonium concentration, glutamate is made primarily through GS
and GOGAT, apparently because the affinity (Km)
for ammonium of GDH is poor relative to that of GS (13, 15,
17-19). Under other conditions, the mechanisms for determining
the pathway used in glutamate synthesis remain largely unknown.
As Reitzer has pointed out (17), GS is the first step in the
GOGAT pathway, and so the relative contributions of the two pathways to
glutamate synthesis may reflect the relative activities of the two
ammonia-assimilating enzymes, GS and GDH. No specific regulation of GDH
activity is known in enteric bacteria, although it has been reported to
be subject to phosphorylation in vivo (12). Control of
transcription of its structural gene, gdhA, is not well
understood (17, 19).
GS is a highly controlled enzyme, subject to multiple forms of
regulation (13, 17). A cascade of regulatory proteins and small molecule effectors, primarily glutamine and 2-oxoglutarate, control synthesis of GS. Each GS monomer in the 12-member holoenzyme is
subject to rapid adenylylation and deadenylylation in response to the
same effectors. The adenylylated form is much less active. Partial
adenylylation of the holoenzyme sensitizes it to feedback inhibition by
a variety of small molecules that may be construed as end products of
GS activity.
The relative importance of each of the two glutamate pathways during
glucose-limited growth varies (in opposite directions) as a function of
phosphate and ammonium concentrations (6). However, the
levels of two of the three relevant enzymes, GDH and GOGAT, remain low
during glucose-limited growth in continuous culture and do not change
markedly with change in ammonium or phosphate level under conditions in
which pathway choice appears to be controlled by these compounds
(6). In the work described in this report, I looked at the
level and form (adenylylated or unadenylylated) of the third enzyme,
glutamine synthetase, to see if either varied with conditions
controlling choice of pathway for glutamate synthesis. The results show
that regulation of the form or amount of GS is also insufficient to
control the choice of pathway. Thus, concentration of substrates and/or
modulation of enzyme activity is likely to be a major determinant of
pathway choice in glutamate synthesis.
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MATERIALS AND METHODS |
Strains, media, and culture conditions.
The E. coli K-12 strains used were RH828 and RH830, isogenic except that
RH828 is gdhA1 and RH830 is gdhA+
(6). The gdhA1 mutant is devoid of GDH activity
as a result of replacement of an essential lysine (Lys-92) by glutamate
at the active site (10). RH842 is isogenic with RH830 except
that it is Arar and so can be distinguished from RH828 in
samples from competition experiments (6). Media and culture
conditions have been described previously (6, 8). The
standard medium contained 7.6 mM (NH4)2SO4, 22 mM
KH2PO4, 40.2 mM K2HPO4,
and 0.8 mM MgSO4 (6). In addition, glucose was
present at 0.0125% (0.694 mM) for continuous culture and 0.05% for
unlimited growth, and thiamine-HCl was present at 50 µg/liter. A
trace metals-iron-vitamin B12 mixture (14) was
added when the effect of these additives on growth in continuous culture was tested. Continuous cultures were grown at a dilution rate
of approximately 0.2 h
1 unless otherwise stated. In all
experiments, growth was at 30°C.
Competition experiments.
Detailed procedures have been
described previously (6, 7). In brief, the strains were
grown separately in glucose-limited continuous culture and mixed, and
during continuous growth over 30 to 40 generations, samples were
removed and plated, and frequencies of the competing genotypes were
determined by testing individual colonies. The growth rate of the
gdhA1 strain (RH828) relative to that of the
gdhA+ strain (RH842) is expressed as relative
fitness, or 1 
s (selection coefficient). Each datum
point (Fig. 2 and 5) represents the average of at least two independent
experiments.
GS measurements.
Continuous cultures (190 ml running volume)
were initiated with 100 ml of an overnight standing culture in the same
medium and grown for 12 to 25 generations. Unlimited cultures were
initiated in the continuous culture apparatus at an
A550 of 0.02 and harvested at an
A550 of 0.18 to 0.24 after exponential growth at
the maximum specific growth rate (µmax = 0.44 h1). It was shown previously that the relative fitness of
a gdhA strain did not change over (at least) the pH range
6.8 to 7.5 (6). During steady-state glucose-limited culture
in standard medium, the pH was 7.14; in 0.1× phosphate medium, the pH
was 6.84 (6). Unlimited cultures in 0.1× phosphate medium
had a pH at harvest (A550 of 0.18) of
approximately 6.7.
Harvest and assay at the isoactivity point (which was determined to be
7.02) were essentially as described previously (3). In
brief, hexadecyltrimethylammonium bromide (CTAB) was added to the
growing culture to permeabilize cells and inactivate
GS-adenylyltransferase (3), and after brief mixing 100 ml
was collected by centrifugation for GS determination by the
-glutamyl transferase assay. Both unadenylylated and total
activities were determined, and the ratio was used as an approximation
of the average proportion of active subunits (out of the total of 12 monomers per holoenzyme molecule). Specific activity is expressed as
nanomoles of product formed at 37°C per min per milligram of protein.
Each datum point corresponds to the average of at least two independent
experiments except for those taken at dilution rates between 0.3 and
0.42 h1, which represent single experiments (Fig.
1).
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FIG. 1.
Comparison of GS levels during unlimited and
glucose-limited growth. (Left) Total (upper) and unadenylylated (lower)
enzyme activity. (Right) Average number of active monomers in the
holoenzyme, as a function of dilution rate. The maximum specific growth
rate is 0.44 h1.
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RESULTS AND DISCUSSION |
Preliminary studies.
Previous work had shown that the GDH
pathway became more important to the organism as the phosphate
concentration increased (6). This was not the result of
changes in ionic strength or pH. In order to see if the effect of
phosphate might be due to trace contaminants rather than to phosphate
itself, I carried out competition experiments under standard
conditions, using medium supplemented with trace metals, iron, vitamin
B12, and additional B1. These additives did not
change the relative fitness of the gdhA strain (results not
shown). I conclude that phosphate itself is likely to control pathway
choice in glutamate synthesis. Although the rationale for control by
phosphate is unclear, it may reflect linkage between energy and
nitrogen metabolism (see reference 6).
The GDH pathway is less energy demanding than the GOGAT pathway because
it does not require ATP, so it seemed possible that gdhA
mutants forced to make glutamate via the more expensive pathway would
not survive starvation as well as the wild type (2, 4, 21,
22). In order to see if gdhA mutants were impaired in recovery from starvation, I compared survival at 30°C of
gdhA (RH828) and gdhA+ (RH842)
strains mixed after growth in equal amounts or at a 100-fold excess of
the wild type. There was no difference in survival over at least 150 days after growth in minimal glucose (0.1%) medium. Survival after
growth in tryptone broth-glucose over 3 weeks gave inconsistent
results. In several experiments the gdhA mutant showed lower
survival, but in others it showed no significant difference from that
of the gdhA+ strain. I conclude that if GDH
plays a role in survival of starvation, it is a minor one under the
conditions of the present study.
Glutamine synthetase during unlimited and glucose-limited
growth.
Earlier it was found that GDH played no essential role in
glutamate synthesis during unlimited growth but that it was important during glucose-restricted growth (6). Thus, glutamate might be made largely or entirely via the GOGAT pathway in unlimited growth,
but it is made at least in part by GDH during restricted growth. The
transition from unlimited to limited growth might be reflected in a
decrease in the amount and/or increase in adenylylation state of GS if
pathway choice were determined by the relative activities of GS and
GDH.
I determined the levels of glutamine synthetase in RH830
(gdhA+) cells harvested during unrestricted
growth and in cells harvested from glucose-limited growth at different
dilution rates to see if the enzyme level might change accordingly. As
shown in Fig. 1 (also compare Fig. 3 and 4), total enzyme was
relatively high during unrestricted growth (µmax = 0.44)
(8). (Note that the ammonium concentration is high, so the
organism should be under ammonium repression.) As growth became
restricted by a reduction in the dilution rate, total GS dropped
sharply and then stabilized at about half the specific activity of
cells in unrestricted growth. The average number of active subunits
(unadenylylated) also dropped (Fig. 1).
Together with previous results (6), this shows that under
the growth conditions employed, the levels of the three enzymes in
the pathways for glutamate synthesis drop about twofold when cells pass
from unrestricted growth to glucose-restricted growth. The drop in
levels of active enzyme presumably results in reduced synthesis of
glutamine, glutamate, and organic nitrogen. The number of active
subunits (unadenylylated) of glutamine synthetase simultaneously drops,
showing that an increase in the ratio of glutamine to 2OG follows the
growth transition (13, 17, 20). (The roles of glutamine and
2OG are separate and complex [11, 13, 17].) At the
same time, 2OG becomes channeled through both the GDH and GOGAT
pathways to form glutamate instead of substantially or solely through
the GOGAT pathway as it appears to be during steady-state unrestricted
growth. Although differences in the medium and other growth conditions
preclude a detailed comparison, Senior has shown that the change in
glutamine-2OG results from a modest drop in the glutamine pool and a
large drop in the 2OG pool as cells progress from unrestricted to
glucose-limited growth with ammonium excess (20; see
also reference 9).
The drop in the level of all three enzymes during the transition to
restricted growth presumably reflects a change in the transcription
frequencies of the structural genes (glnA, gdhA, and gltBD) in response to increasing nitrogen repression as
the cell becomes progressively stressed for a carbon and energy source (13, 17).
GS level and adenylylation state as a function of phosphate and
ammonium concentration.
Previous work had also shown that during
glucose-limited growth, the selection coefficient (disadvantage) of a
gdhA mutant increased as the ammonium or phosphate
concentration increased (6). These correlations are shown in
Fig. 2.
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FIG. 2.
Growth disadvantage of a gdhA mutant during
glucose-limited growth as a function of ammonium or phosphate
concentration. Ammonium ( ) and phosphate ( ) concentrations are
represented as fractions of those in 1× medium (see Materials and
Methods). Data are taken from reference 4.
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As noted above, the activity of GS could in principle control pathway
choice in glutamate synthesis. By this rationale the amount of
unadenylylated enzyme might be high relative to GDH at low
concentrations of ammonium or phosphate when the role of GDH is
minimized (Fig. 2) and lower as those concentrations increase. Direct
measurement showed little change in total GS with ammonium concentration, and the proportion of active subunits (unadenylylated) did not change substantially (Fig. 3).
Similarly, total enzyme and number of active subunits were little
affected by changing the phosphate concentration (Fig. 3). GS activity
in a gdhA mutant showed a similar pattern, but the total
enzyme and proportion of active subunits were slightly higher (data not
shown).
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FIG. 3.
GS during glucose-limited growth as a function of
ammonium or phosphate concentration. (Left panel) The uppermost curves
(, ) show total enzyme activity from RH830 cells, and the
lowermost curves ( ,  ) represent unadenylylated GS activity.
(Right panel) Average number of active monomers in the 12-monomer
holoenzyme. Ammonium (, ) and phosphate (, ) concentrations
are represented as fractions of those in 1× medium (see Materials and
Methods).
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For comparison, GS measurements were also made on cells from
unrestricted growth. It was already known that GDH played no essential
role under these conditions (6). Total enzyme and number of
active subunits were found to change little as the ammonium concentration changed (Fig. 4). They also
changed little with phosphate at higher phosphate concentrations, but
there was a drop in total enzyme (but not unadenylylated enzyme) as
phosphate went from 0.2 to 0.1 that of the standard medium (Fig. 4). As a result, the proportion of active subunits increased to nearly 100%
(Fig. 4). This change in the amount of adenylylated enzyme during
unrestricted growth on glucose in low-phosphate medium was not studied
further.
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FIG. 4.
GS during unrestricted growth on glucose as a function
of ammonium or phosphate concentration. (Left panel) The uppermost
curves (, ) show total enzyme activity from RH830 and the
lowermost curves (, ) represent unadenylylated GS activity.
(Right panel) Average number of active monomers in the holoenzyme.
Ammonium (, ) and phosphate (, ) concentrations are
represented as fractions of those in 1× medium.
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Confirming the importance of GDH during energy limitation.
The
hypothesis that GDH is important during energy-restricted growth was
tested by determining the relative fitness of a gdhA mutant
under conditions of increasing energy stress. The mutant organism
(RH828) was grown in competition with the wild type (RH842) on limiting
glucose as described previously (6) except with the addition
of the nonmetabolizable glucose analog 
-methylglucoside (MG)
(1). MG competes for uptake with glucose and is taken into the cell by the glucose phosphotransferase system, with an ATP
equivalent expended for every MG, but no ATP is generated from the
analog. Uptake of the glucose analog should increase the energy stress
and therefore decrease the fitness of a gdhA mutant relative
to that of the wild type, if the operating hypothesis were correct.
The results conform to expectation (Fig.
5). The data show clearly that as the
concentration of MG and, hence, energy stress increased, the
selection coefficient (1 relative fitness) of the mutant
increased. Over the range of approximately 0 to 10 mM MG (molar
ratio MG:glucose, 0 to 15), the growth rate of the mutant dropped
from 88% of that of the competing wild type to about 66% (Fig. 5). In
order to see if the amount of GS changed correspondingly, I measured
the enzyme level under the same growth conditions. The results show
that neither total GS nor the proportion of active subunits changed
significantly over the range of MG concentration in which the
selection coefficient of the gdhA mutant changed markedly
(Fig. 6).
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FIG. 5.
The growth disadvantage of glucose-limited cells lacking
GDH as a function of the concentration of the nonmetabolizable glucose
analog -methylglucoside.
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FIG. 6.
GS during glucose-restricted growth in the presence of
the nonmetabolizable glucose analog MG. (Left panel) Total (upper)
and unadenylylated (lower) enzyme activity from strain RH830. (Right
panel) average number of active monomers in the holoenzyme.
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The molecular bases for pathway determination must result from
regulation of enzyme level, enzyme activity, or availability of
substrates. As reported above, the levels of the three enzymes drop as
the organism passes from unlimited to limited growth. In contrast, no
substantial change in the amount of any of the three enzymes or in the
proportion of active subunits of GS accompanied a change in phosphate
or ammonium level, and the presence of MG did not affect the amount
or form of GS during glucose-limited growth, even though these
variables affect the relative importance of the pathways for glutamate
synthesis. The changing flux through the separate pathways for
glutamate synthesis during the latter transitions must reflect either
modulation of enzyme activity (other than by covalent modification) or
a change in substrate availability or both. These kinds of control are
expected to be exerted rapidly relative to change in amount of enzymes
and to be readily reversible. This suggests that the ability to control flux through the separate glutamate pathways is most important for
dealing with rapid fluctuation in variables such as levels of specific
metabolites rather than for long-term adaptations.
I thank J. R. Guest, M. M. Attwood, and their
colleagues at The University of Sheffield for their generous
hospitality while part of this work was done and the NIH for support as
a John E. Fogarty Senior International Fellow. I thank R. Bender and T. Goss for their criticisms.
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Abstract
Introduction
