FEMS Microbiology Letters 58 (1989) 229-232
Published by Elsevier
229
FEM 03505
Nitrogen-limited behaviour of micro-organisms growing
in the presence of large concentrations of ammonium ions
E d T. B u u r m a n , M. Joost T e i x e i r a de M a t t o s a n d O e n s e M. Neijssel
Department of Microbiology, Biotechnology Centre, University of A msterdam, Amsterdam, The Netherlands
Received 24 November 1988
Accepted 26 November 1988
Key words: Klebsiella pneumoniae; Ammonia assimilation; Glutamate dehydrogenase;
Glutamate synthase; pH; Chemostat culture
1. SUMMARY
Cells of Klebsiella pneumoniae NCTC 418
grown at low culture pH values (4.5-5) in a glucose-limited chemostat culture contained elevated
levels of glutamate synthase (EC 2.6.1.53). This
can be taken as an indication that these cells show
the physiology of nitrogen-limited cells, in spite of
the fact that high concentrations (about 80 mM)
of ammonium ions were present in the culture
extracellular fluids. This phenomenon can be explained by the rapid diffusion of ammonia (NH3)
through the cell membrane, leading to very low
cytoplasmic ammonium (NH~-) and N H 3 levels in
cells that possess an almost neutral cytoplasmic
pH value, but are growing at low culture pH
values.
2. I N T R O D U C T I O N
Ammonium salts are frequently used as a
nitrogen source in microbiological media. An in-
Correspondence to: Dr. O.M. Neijssel, Department of Microbiology, Biotechnology Centre, University of Amsterdam, Nieuwe
Achtergracht 127, 1018 WS Amsterdam, The Netherlands.
teresting problem is how ammonia (NH3) or ammonium ions (NH~-) enter the cell. Kleiner [1]
estimated that the permeability coefficient for N H 3
through the cell membrane of KlebsieUa pneumoniae is high (2 × 10 3 c m . s - l ) , which indicates
that this compound will diffuse rapidly through
the cell membrane. On the other hand it has been
shown that micro-organisms contain NH~- transport systems which are derepressed when the
medium contains nitrogen sources other than ammonium salts (for review, see [21). In addition,
there is uncertainty about which chemical species
(NH 3 or NH4~) is the substrate for the two main
assimilatory enzymes: glutamate dehydrogenase
and glutamine synthetase [3].
If N H 3 can indeed diffuse very quickly through
the cell membrane, this will lead to serious consequences when neutrophilic organisms such as K.
pneumoniae or Escherichia coli, possessing a cytoplasmic pH value of 7.5-8 [4], are growing at low
medium p H values. Since the N H 3 gradient across
the membrane will be close to zero and the extracellular N H 3 concentration will be vanishingly
small at such low extracellular pH values, the
intracellular N H 3 and NH~- concentrations cannot be large. Thus, on the basis of this hypothesis
one could predict that when Klebsiella pneumoniae
is growing in a nominally glucose-limited chemo-
0378-1097/89/$03.50 © 1989 Federation of European Microbiological Societies
230
stat culture at pH 4.5-5 with ammonium chloride
as the nitrogen source, the organisms also have to
cope with a nitrogen limitation. The experiments
described in this contribution were carried out to
test this hypothesis.
3.4. Chemicals and enzymes
Substrates, cofactors and glutamate dehydrogenase were obtained from Boehringer Mannheim.
All other chemicals were of reagent grade.
4. RESULTS
3. MATERIALS A N D M E T H O D S
3.1. Organism and culture methods
Klebsiella pneumoniae N C T C 418 was maintained on nutrient agar slopes and grown in
carbon- or nitrogen-limited chemostat culture as
described previously [5], except that the steady
state dry weight was 1.5-2.5 g.1-1; this was
achieved by an appropriate adjustment of the
medium concentration of the growth-limiting
nutrient (C limitation: glucose 27 mM; N limitation: ammonium chloride 15 mM). The dilution
rate of the culture was set at 0.4 h-a. The culture
p H value was maintained at the desired value
(_+ 0.1 unit) by automatic titration with 4N NaOH.
3.2. Enzyme analyses
Cell-free extracts were prepared according to
O'Brien et al. [6]. Glutamate dehydrogenase
( G D H , E.C. 1.4.13) and glutamate synthase
(GOGAT, E.C. 2.6.1.53) were assayed according
to [7]. Possible interference in the G O G A T assay
by ammonia, contaminating the freshly-made
glutamine solution [8] or being formed as a result
of glutaminase A [9] or B [10] activity, was checked
by an incubation with glutamate dehydrogenase
(bovine liver) and N A D H . No correction of the
G O G A T activities proved to be necessary. Protein
was assayed by the biuret method using bovine
serum albumin as a standard [11].
3.3. Calculation
The concentrations of N H 3 and NH4~ in the
steady-state extracellular fluids were calculated
(assuming a cell nitrogen content of 14%) [12]
using the formula: (NH 3 + N H ~ ) = (medium inp u t ) - - 1 0 × (dry weight (g/l)), and the culture pH
value; the p K a value of NH~- was taken to be 9.3
[3].
To investigate the contribution of both the low
and high affinity pathways to the assimilation of
N H 3 / N H ~ - , the activities of the two characteristic
enzymes, i.e. glutamate dehydrogenase ( G D H ) and
glutamate synthase (GOGAT), were determined in
cell extracts of K. pneumoniae grown in glucoselimited chemostat culture. Glucose-limited growth
at pH 8.0 resulted in significant amounts of G D H
whereas the presence of G O G A T could not be
detected in these cells. This is in agreement with
the observations of Meers et al. [6]. The cellular
levels of these enzymes, however, were found to be
highly p H dependent (Fig. 1). A decrease of the
steady state culture pH value from 8.0 to 6.0 led
to a three-fold increased G D H activity and a
derepression of G O G A T activity. Further lowering of the steady state culture pH value resulted in
a decreased G D H activity but led to an even
higher G O G A T activity.
To exclude the possibility that the observed
changes were due to a p H effect per se, cells were
120[
s 6oh
.o r
40i0
.
~
4
4 50
5
".
"",
5.50
6
Culture pH value
650
7
750
8
850
Fig. 1. Effect of the culture pH value on the cellular levels of
glutamate dehydrogenase (circles) and glutamate synthase (triangles) of Klebsiella pneumoniae growing in carbon-limited
(open symbols) or nitrogen-limited (closed symbols) chemostat
culture. Enzyme activities are expressed in nmol N A D P H
oxidized, rain - 1. mg p r o t e i n - 1. For convenience the activities
of glutamate dehydrogenase have been divided by 10.
231
Y
4(?
20
°_7
~
,
~;
,
_
,
A
_i
~
,
~
_~
,
-2
Log NH3 (M)
Fig. 2. The effect of the steady state NH 3 concentration in
glucose-limited chemostat cultures on the cellular levels of
glutamate dehydrogenase (GDH) and glutamate synthase
(GOGAT) of Klebsiella pneumoniae. (NH3) has been calculated as described in the MATERIALS AND METHODS section.
Cultures grown at a constant pH value (7.0) with different
input NH4CI concentrations: 200 raM, GDH (~), GOGAT
(4), and 30 mM, GDH (o), GOGAT (A). Cultures grown at
different culture pH values with a constant (100 mM) NH4C1
input concentration, GDH (©), GOGAT (z~). For convenience
the activities of GDH have been divided by 10.
grown C-limited at a constant culture pH value
(7.5) with varying concentrations of ammonium
chloride in the medium. Lowering of the ammonium input resulted in an increase of both
G D H and G O G A T activity. To demonstrate the
involvement of N H 3 in the regulation of the
synthesis of these enzymes, the activities that were
observed under the different growth conditions
have been plotted against log(NH3) (Fig. 2).
5. DISCUSSION
The experiments reported here clearly show
that at acidic culture pH values the levels of
G O G A T in K. pneurnoniae, growing in a nominally glucose-limited chemostat culture, were increased. This phenomenon is perfectly understandable on the basis of the results of Kleiner [1]
which indicate that N H 3 is exceedingly mobile
through the membrane of this organism.
The data provide us also with more insight into
the mechanism of adaptation of K. pneumoniae to
decreasing cytoplasmic N H 3 levels. We have
shown that first the cellular level of G D H increased and that at even lower N H 3 levels the
synthesis of this enzyme is repressed and G O G A T
synthesis is derepressed. In fact, the data in Fig. 2
predict that repression of G O G A T activity at a
culture p H value of 5 would require an extracellular NH~- concentration of 80 M! Similarly, at a
culture p H value of 8.0 full expression of G O G A T
would be obtained when the NH~- concentration
is less than 80 tzM.
If one assumes the G D H content in C-limited
cells grown at pH 8.0 to be just sufficient for
biosynthesis, one can calculate that a 6-fold increase of this enzyme would be required when the
cells grow at pH 6.0 and a 15-fold increase at pH
5.5 (assuming pH i = 7.6, (NH~-)out = 80 mM and
K m = 10 raM). Our results show that at this latter
pH value the G D H content is decreased and the
glutamine pathway becomes the most important
one. From a comparison of the G O G A T activities
in C-limited cells with those of N-limited cells one
can conclude that in C-limited cells grown at pH
values lower than 6, the major route for
NH4~ / N H 3 assimilation is the glutamine pathway.
The presence of the glutamine pathway, evolved to
increase the N H ~ - / N H 3 assimilatory capacity, is
therefore not restricted to conditions in which the
extracellular concentration of NH~- is low.
A further point is the possible involvement of
an NH~- transport system. It will be obvious that,
even if such a system were present in K. pneumoniae under the growth conditions described here,
it could not have contributed significantly to the
assimilation of the nitrogen source. Any NH~taken up via this system will generate N H 3 in the
cytoplasm in equilibrium with the pH value of this
cell compartment and this would only lead to an
efflux of N H 3 from the cytoplasm. Hence, the
presence of an NH~- transport system would induce futile cycling across the membrane (and is
therefore certainly important in terms of growth
energetics; see also [1]) but it would not have
increased the efficiency of nitrogen assimilation.
The fact that the growth yields on glucose of
cultures grown at low p H values were not significantly lower than those obtained at pH 7 (data
not shown) argues therefore against the involvement of an N H 4 transport system.
232
Finally, we wish to comment on this phenomenon in more general terms. If one accepts that the
cell membrane of K. pneumoniae is not unique, i.e.
that membranes of other microbes are also very
permeable to N H 3, one must conclude that these
experiments indicate that in many microbes
nitrogen assimilation is severely impeded when
N H 4 ~ / N H 3 serves as the sole nitrogen source and
the culture pH value is low. This leads to the
interesting question as to how acidophiles are able
to assimilate this nitrogen source. A possible explanation could be that their cytoplasmic pH value
is lower [13] or that the permeability of the membranes of these organisms to N H 3 is significantly
lower, but this latter proposal remains to be investigated.
REFERENCES
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[4] Booth, I.R. (1985) Microbiol. Rev. 49~ 359-378.
[5] Neijssel, O.M. and Tempest, D.W. (1975) Arch. Micrnbiol. 106, 251-258.
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