(Received for publication, November 27, 1995)
From the
Under nitrogen starvation conditions, Corynebacterium
glutamicum was found to take up methylammonium at a rate of 20
± 5 nmolmin
(mg dry
weight)
. The specific activity of this uptake was
10-fold lower when growing the cells under sufficient nitrogen supply,
indicating a tight regulation on the expression level. The
methylammonium uptake showed Michaelis-Menten kinetics with an K
of 44 ± 7 µM and
was completely inhibited by the addition of 10 µM ammonium. This finding and the fact that methylammonium was not
metabolized by C. glutamicum strongly suggests that the uptake
carrier actually represents an ammonium uptake system. Methylammonium
uptake was strictly dependent on the membrane potential. From the pH
optimum and the accumulation of methylammonium in equilibrium, it could
be deduced that only one net charge is transported and, thus, that
methylammonium is taken up in its protonated form via an uniport
mechanism. The amt gene encoding the (methyl)ammonium uptake
system was isolated and characterized. The predicted gene product of amt consists of 452 amino acids (M
= 47,699) and shows 26-33% identity to ammonium
transporter proteins from Saccharomyces cerevisiae and Arabidopsis thaliana. According to the hydrophobicity profile,
it is an integral membrane protein containing 10 or 11
membrane-spanning segments.
Most microorganisms use ammonium
(NH) as the preferred nitrogen source.
Although ammonia (NH
) is highly membrane-permeable,
energy-dependent transport systems for NH
have been found in many bacterial species(1) . Usually
these carriers are inhibited by glutamine, and their expression is
regulated by the availability of NH
itself(2) . At high NH
concentrations in the culture medium, i.e. when the
nitrogen supply via NH
diffusion is sufficient, the
transport systems are repressed. In general, NH
transport is catalyzed by secondary transport systems that depend
on the membrane potential as driving force. As an exception in Escherichia coli, NH
is taken up
in antiport against a potassium ion, and thus the potassium gradient is
used as energy source for uptake(3) .
Despite the large
number of carriers characterized functionally, only little information
is available concerning genes coding for NH transport proteins. Up to now, no gene for a procaryotic
NH
carrier has been isolated. By
complementation of an E. coli mutant deficient in
NH
transport, Fabiny et al.(4) identified a gene, designated amtA, that
codes for a soluble protein and which thus is discussed to represent a
cytoplasmic component of an NH
uptake
system. The first genes coding for NH
transport proteins (MEP1 and MEP2) were
isolated from Saccharomyces cerevisiae(5) (GenBank
number P41948). Expression of cDNA from Arabidopsis thaliana in a transport-deficient mutant of S. cerevisiae allowed
cloning of the gene for a high affinity NH
transporter from this plant (6) . When searching for
similarity of the yeast carrier Mep1 to other proteins, Marini et
al.(5) found that the N-terminal region of Mep1 showed
28% identity to a polypeptide of 148 amino acid residues, encoded by a
truncated open reading frame (ORF) (
)downstream of the
phosphoenolpyruvate carboxylase gene (ppc) of Corynebacterium glutamicum.
Since the ppc gene of C. glutamicum has been cloned by us(7) , the
observation of Marini et al.(5) prompted us to
isolate the complete ORF downstream of the ppc gene. In this
study, we describe the functional characterization of the
NH carrier of C. glutamicum and
the isolation and sequencing of the first procaryotic gene encoding
such a transport protein.
In order to detect transport of NH in C. glutamicum,
[
C]methylammonium as an
NH
analog was employed, since no suitable
nitrogen isotope is available. C. glutamicum cells, incubated
in nitrogen-free medium, were found to take up methylammonium at a rate
of 15 nmol
min
(mg dry
weight)
. When NH
was
added to the transport assay, uptake of methylammonium was completely
inhibited (data not shown). Inhibition was competitive, since cells
were observed to resume methylammonium uptake at uninhibited rate after
some time, i.e. when the added NH
was consumed. This suggests that the carrier analyzed is not
specific for methylammonium but accepts both NH
and methylammonium as substrate.
To test whether C.
glutamicum is able to metabolize methylammonium, cells were
incubated for 2 h in the presence of 1.7 mM methylammonium,
and the methylammonium concentration inside the cells and in the medium
was determined as a function of time by use of high pressure liquid
chromatography. No decrease of total methylammonium in the reaction
mixture was observed (data not shown), indicating that C.
glutamicum is not able to metabolize this
NH analog.
In many microorganisms, the
expression of genes encoding NH uptake
systems has been found to increase when the nitrogen source in the
medium is depleted(1) . To check whether the same pattern of
expression exists in C. glutamicum, the methylammonium
transport activity in cells transferred from complex medium to
nitrogen-free minimal medium was measured as a function of time (Fig. 1). Uptake activity was found to increase 10-fold during
the first 2-3 h of incubation in the absence of a nitrogen
source. No increase of uptake activity was observed when protein
synthesis was inhibited by chloramphenicol. For further
characterization of (methyl)ammonium uptake, cells were employed after
3 h of incubation in nitrogen-free minimal medium.
Figure 1:
Time course of
methylammonium uptake in C. glutamicum after depletion of
nitrogen sources in the culture medium. Cells were grown overnight in
complex medium and then transferred to nitrogen-free minimal medium as
described under ``Experimental Procedures.'' Uptake activity
was measured in the absence () and presence (
) of
chloramphenicol (50 µM).
To determine the driving forces of methylammonium uptake, the
effect of ionophores on transport activity was studied. Methylammonium
uptake was found to be inhibited completely when the proton-motive
force was destroyed by the protonophor carbonyl cyanide m-chlorophenylhydrazone, indicating that uptake is catalyzed
by a secondary transport system depending on components of the
proton-motive force for energization. The role of the membrane
potential () in methylammonium transport was analyzed in
detail by setting potassium diffusion potentials in the presence of
valinomycin (Fig. 2). While the external potassium concentration
had no influence on uptake activity in the absence of valinomycin,
uptake activity decreased with increasing potassium concentrations, i.e. with decreasing
, when valinomycin was present (Fig. 2A). Plotting uptake activity as a function of
the membrane potential revealed a linear dependence of transport
activity on
for membrane potentials between 30 and 150 mV (Fig. 2B).
Figure 2:
Dependence of methylammonium uptake in C. glutamicum on the membrane potential. Uptake activity was
measured as a function of the external potassium concentration in cells
preincubated for 3 min in the presence of 20 µM valinomycin () and in untreated cells (
) (A). The resulting membrane potentials were determined, and
uptake activity was plotted as a function of the membrane potential (B).
The observed dependence of
methylammonium transport could result from uniport of protonated
methylammonium or from cotransport of either unprotonated methylamine
or protonated methylammonium with cations such as H
or
Na
. Transport of unprotonated methylamine is unlikely
because methylamine is almost completely protonated at physiological pH
due to its high pK value of 10.7. When methylammonium influx
was measured as a function of the extracellular pH, the rate was found
to increase with rising pH (data not shown). Since the concentration of
the highly permeable unprotonated methylamine also increases at
alkaline pH, these data had to be corrected for methylamine influx
driven by diffusion and trapping of protonated methylammonium in the
cells due to the pH-gradient (pH
< pH
).
This diffusion-driven influx, measured as the residual influx after
carrier-mediated uptake had been inhibited by 250 µMp-chloromercuribenzoic acid, was also quantified as a
function of the external pH. The true pH dependence of carrier-mediated
methylammonium transport, obtained by subtraction of influx in the
absence and in the presence of p-chloromercuribenzoic acid,
yielded a pH optimum of 7.0, which again argues for protonated
methylammonium being the transported species.
The number of charges
transported in each transport cycle can be deduced from the
accumulation of methylammonium (c/c
) in thermodynamic
equilibrium. This could be measured in C. glutamicum, since no
degradation of methylammonium took place. As shown in Fig. 3, a
constant maximum methylammonium accumulation of 350-fold was found at
external concentrations between 6 and 60 µM. A membrane
potential of -160 mV was measured for the cells used in this
experiment. Assuming that only one charge is transported in each
transport cycle, i.e. that the carrier catalyzes uniport of
protonated methylammonium, the membrane potential of -160 mV
could be used to build up a maximum accumulation of about 460-fold,
according to the equation c
/c
= exp(-
/Z) (Z =
2.3 RT/F). The fact that the observed accumulation
was somewhat lower argues against another ion being transported
together with methylammonium since then a much higher accumulation
should be reached. In accordance with this conclusion, we observed that
the external Na
concentration had no influence on the
rate of methylammonium uptake (data not shown).
Figure 3:
Methylammonium accumulation in C.
glutamicum. Cells were incubated with
[C]methylammonium at different external
concentrations (10-500 µM) and were allowed to
accumulate this compound until equilibrium was reached. The resulting
internal and external methylammonium concentrations were determined.
The concentration ratio in equilibrium is plotted as a function of the
existing external concentration.
Figure 4: Restriction map of the chromosomal C. glutamicum 3-kb BamHI insert of pUC-dppc. Arrows represent ORFs identified after sequencing. 3`ppc is the 3` region of the ppc gene, and amt is the gene encoding the ammonium transport system. Ba, BamHI; Cl, ClaI; Ps, PstI; Sa, SalI; Sc, SacI; Sp, SphI; Xm, XmnI.
Using the amino acid sequence deduced
from the analyzed ORF, the GenBank and SwissProt data base were
searched for related proteins. As expected, a significant degree of
identity of the C. glutamicum protein to the three known
NH transporters of other organisms was
found, extending over the whole sequence. The Mep2 (GenBank entry
P41948) and Mep1 (5) proteins of S. cerevisiae show
33.4 and 28.1% identical amino acids, respectively, and the
NH
carrier of the plant A. thaliana(6) shows 25.9% identical amino acids. In addition, the C. glutamicum protein shares a high identity of 41.6% with the
NrgA protein of Bacillus subtilis, a protein of yet unknown
function(23) .
Since the Amt protein of C. glutamicum is a membrane protein, it was analyzed with respect to the distribution of hydrophobic and hydrophilic amino acid residues. The hydropathy plot (Fig. 5) shows highly hydrophobic stretches and a hydrophilic C-terminal region. Computer analysis predicts 10 or 11 membrane-spanning, helical segments. When comparing the distribution of hydrophobic regions in the corynebacterial protein with the distribution in the Mep1 and Mep2 proteins of S. cerevisiae (Fig. 4), membrane-spanning regions and hydrophilic loops were found to be located in corresponding protein stretches with the exception of helix 8 in the Mep1 protein, which is not clearly predicted for the Mep2 protein and the Amt protein of C. glutamicum (Fig. 6).
Figure 5: Hydropathy profile of the Amt protein. The average hydropathy of a moving window of 15 amino acid residues was determined by use of the SOAP program (PC/Gene, Intelligenetics, Inc.) described by Kyte and Doolittle(24) . The dotted line at -5 is the standard midpoint line that is used to divide the hydrophobic domains (above the line) from the hydrophilic domains (below the line).
Figure 6: Comparison of the deduced amino acid sequence of the C. glutamicum (C. glu.) Amt protein with sequences of ammonium transport proteins of S. cerevisiae Mep2 (S. cer.2), and Mep1 (S. cer.1) and Amt1 of A. thaliana (A. tha.). In addition, comparison of the Amt protein to the amino acid sequence of the NrgA protein of B. subtilis (B. sub.) is shown. Predicted membrane spanning segments are underlined. The dashed line indicates an additional potential helix in the C. glutamicum Amt protein. Identical amino acid residues in all (*), four of five (°), or three of five (.) of the proteins analyzed.
In this communication, we describe the functional
characterization of the NH uptake system
of C. glutamicum and the identification of the corresponding
gene. The carrier was found to be synthesized only under conditions of
nitrogen starvation. It functions as a secondary transport system. The
gene encoding the carrier protein, the first bacterial gene for an
NH
transporter available so far, is
located downstream the phosphoenolpyruvate carboxylase (ppc)
gene.
For analysis of NH transport,
methylammonium was used, a compound shown to be accepted as alternative
substrate by the NH
transporters of
numerous other organisms. NH
and
methylammonium have quite similar physical properties. Both compounds
are predominantly protonated at neutral pH due to their high pK values (9.25 for NH
, 10.7 for
methylammonium). In unprotonated, i.e. uncharged form
(NH
and methylamine), both compounds can easily diffuse
across biological membranes, while these membranes are virtually
impermeable for the protonated forms. Nevertheless, under the
conditions employed here, i.e. neutral pH and low external
substrate concentration (100 µM), diffusion can be
neglected. Several properties of methylammonium influx argue against
mediation by diffusion. (i) Uptake was saturable. (ii) Uptake rates
varied by a factor of 20 depending on the growth conditions. And (iii)
uptake resulted in significant intracellular accumulation of the
substrate.
In several methylotrophic bacteria, which can use
methylammonium as nitrogen and/or carbon source, transport systems
specific for this compound have been
described(25, 26) . This possibility can be ruled out
in case of the transport system of C. glutamicum since (i) the
organism was found not to be able to metabolize methylammonium and (ii)
methylammonium uptake was competitively inhibited by addition of
NH. In fact, inhibition by
NH
was so effective that it was impossible
to determine a K
value, indicating that the
affinity of the carrier for NH
is much
higher than for methylammonium. It can therefore be concluded that
NH
represents the natural substrate of the
carrier analyzed.
Regulation of NH uptake in C. glutamicum is similar to that in many other
organisms (2) . The carrier protein itself was found to be
inhibited by internal glutamine (data not shown). Since the
intracellular pool of this compound immediately reflects the nitrogen
supply, this regulation ensures that uptake activity corresponds to the
requirements of the organism. In addition, the expression of the uptake
carrier of C. glutamicum was repressed by high
NH
concentration in the culture medium, i.e. when supply via NH
diffusion is sufficient.
As has been pointed out by Kleiner(27) , intracellular
NH
accumulation by an uptake system may
create an energy wasting futile cycle, during which energy-dependent
uptake is counteracted by diffusion of NH
out of the cells.
Thus, repression of the carrier under conditions of sufficient nitrogen
supply ensures that energy is expended for NH
uptake only when cells are starving for nitrogen. In
enterobacteria repression of the carrier along with that of numerous
other operons encoding enzymes of nitrogen catabolism is mediated by
the ``nitrogen control'' (Ntr) system, which coordinates
carbon and nitrogen metabolism. Our results suggest that a similar
control system exists in C. glutamicum.
The
NH uptake system of C. glutamicum catalyzes uniport of NH
and uses the
membrane potential as driving force. Transport of the protonated form, i.e. the form not able to diffuse across the cytoplasmic
membrane, is rational from a physiological point of view and could be
deduced from the the pH optimum of the carrier in the neutral pH range
in which the NH
ion is the predominant
species. Analysis of the maximum accumulation of methylammonium
indicates that no other ion is cotransported with ammonium. The fact
that the accumulation ratio determined is somewhat lower than that
expected for the
-driven uptake of a monovalent cation could
result from inaccuracy of the
determination. The addition
of different external methylammonium concentrations (10 to 100
µM) led to a significant variation of the internal
concentration and thus to different diffusion rates, but it did not
result in a change of the accumulation ratio (Fig. 3).
Therefore, underestimation of the accumulation ratio due to methylamine
efflux from the cells by passive diffusion can be excluded.
In the
second part of this study, we describe the isolation and sequencing of
the amt gene encoding the characterized
NH uptake system. The isolation was based
on the observation of Marini et al.(5) that the
N-terminal region of the Mep1 protein of S. cerevisiae shows a
significant degree of identity to a polypeptide encoded by a truncated
ORF downstream of the ppc gene of C. glutamicum. In S. cerevisiae, two NH
transport
proteins Mep1 and Mep2 have been studied, which revealed functional
properties similar to the transporter of C. glutamicum. We
isolated and sequenced the 3`-region of the ppc gene and the
complete ORF downstream of it. This ORF consists of 1367 base pairs and
encodes a protein of 452 amino acids. Disruption of the ORF resulted in
20-fold reduction of methylammonium uptake activity, demonstrating that
it represents indeed the amt gene encoding the
NH
transport system characterized.
Hydrophobicity analysis of the Amt protein revealed 10 or 11 membrane-spanning segments. According to the positive inside rule of von Heijne (28) the model predicting 11 membrane spanning helices must be favored because only in this case every second interhelical loop contains a high number of arginine plus lysine residues. These positively charged amino acids have in most membrane proteins been found to be much more abundant in cytoplasmic as compared with periplasmic regions. Applying this rule to the Amt protein results in orientation of its N terminus to the outside and location of the hydrophilic C terminus in the cytoplasm.
The Amt protein of C.
gutamicum shows a significant degree of identity to the three
other known NH transport systems, i.e. Mep1 and Mep2 of S. cerevisiae(5) , and AMT1 of A. thaliana(6) . Identical stretches of amino acid
residues are spread over the whole protein. However, the Amt protein of C. glutamicum shares the highest identity with the NrgA
protein of B. subtilis, a protein of unknown
function(23) . Therefore we conclude, that the NrgA protein
probably also is an NH
uptake system. The
corresponding nrgA gene of B. subtilis is located in
an dicistronic, nitrogen-regulated operon together with nrgB.
This latter gene encodes a protein that has significant sequence
similarity with the glnB-encoded P
protein from E. coli, an important part of the nitrogen regulatory system
(Ntr). To determine whether the amt gene of C. glutamicum is also located in an operon, its downstream region was isolated
and sequenced. Analysis of a chromosomal 3-kb SacI-fragment,
containing the 3`-region of the pUCdppc-insert (1.5 kb) and the
downstream region, revealed that there is no ORF similar to nrgB (data not shown). The deduced amino acid sequence of the
downstream region showed a significant degree of identity to
procaryotic sarcosine oxidases. Since downstream of the ppc gene there is a strong stem-loop structure similar to bacterial
rho-independent terminators(7, 22) , transcription of
the amt gene most probably occurs also independent of the ppc gene.
The high similarity of all
NH transporters known so far, in
fungi(5) , plant(6) , and, as described in this study,
in bacteria, emphasizes the importance of NH
transport systems under different environmental conditions.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X93513[GenBank].