JOURNAL OF BACTERIOLOGY, Apr. 2008, p. 2611–2614 0021-9193/08/$08.00⫹0 doi:10.1128/JB.01896-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 190, No. 7
Dissection of Ammonium Uptake Systems in Corynebacterium glutamicum: Mechanism of Action and Energetics of AmtA and AmtB䌤 Britta Walter,1 Melanie Ku ¨spert,1 Daniel Ansorge,2 Reinhard Kra¨mer,2* and Andreas Burkovski1* Lehrstuhl fu ¨r Mikrobiologie, Friedrich-Alexander-Universita ¨t Erlangen-Nu ¨rnberg, Staudtstr. 5, 91058 Erlangen, Germany,1 and Institut fu ¨r Biochemie der Universita ¨t zu Ko ¨ln, Zu ¨lpicher-Str. 47, 50674 Ko ¨ln, Germany2 Received 4 December 2007/Accepted 18 January 2008
Corynebacterium glutamicum has two different Amt-type proteins. While AmtB has a low substrate affinity and is not saturable up to 3 mM methylammonium, AmtA has a high substrate affinity and mediates saturable, membrane potential-dependent transport, resulting in a high steady-state accumulation of methylammonium, even in the absence of metabolic trapping. nium/ammonium uptake via Amt proteins, glnA2, coding for a GS without known function (15), and gdh, coding for glutamate dehydrogenase (GDH)—in addition to glnA, encoding the only active GS in C. glutamicum—were deleted as described previously (17). The triple-deletion strain DA-2 (⌬glnA ⌬glnA2 ⌬gdh) was generated based on strain TM⌬gdh⌬glnA (13) using plasmid pK18⌬glnA2 (15), which carries an internal glnA2 deletion. Plasmid pK⌬amtB, carrying an internal amtB deletion (14), was used to generate a glnA glnA2 gdh amtB quadruple-mutant strain, MeK-1, based on strain DA-2. The resulting strains were tested with respect to ammonium assimilation and nitrogen control (data not shown). Transcription and Western blot analyses were carried out using probes and antisera directed against GS and GDH. As expected, the deletion strains DA-2 and MeK-1 lacked the corresponding mRNAs and proteins, and neither GS nor GDH activity was detectable (data not shown). Furthermore, polar effects of the introduced mutations were excluded by complementation assays and transcription analyses (data not shown). To investigate the effect of the absence of methylammonium/ammonium assimilation in strain DA-2, cell extracts were prepared from cells incubated with methylammonium and subjected to thin-layer chromatography (12, 19). While in the wild-type ATCC 13032 (1) methylammonium was completely converted by GS to an unidentified assimilation product, most likely methylglutamine, as discussed previously (12), only free methylammonium was detectable in the glnA glnA2 gdh mutant DA-2 (Fig. 1). Differentiation of methylammonium fluxes. To discriminate the three possible methylammonium uptake pathways, i.e., uptake via AmtA and AmtB, respectively, as well as influx by passive diffusion, the uptake rates depending on the external methylammonium concentration for the wild-type ATCC 13032 (1), the amtB mutant LN-1.1 (12), the amtA mutant MJ2-38 (12), and the amtA amtB mutant JS-1 (20) were measured as described previously (12, 18) (Fig. 2). As indicated by the comparison of the wild type, LN-1.1, and MJ2-38, AmtA is the major uptake system for methylammonium in C. glutamicum, while AmtB contributes significantly to the total uptake only at very high external substrate concentrations. This is also the reason why in previous publications, where uptake was measured at methylammonium concentrations not exceeding
Ammonium uptake systems have been described in many bacteria, although its uncharged form, ammonia (NH3), is supposed to be highly membrane permeable. Until 1998, exclusively energy-dependent, membrane potential-driven ammonium transport was proposed, when Soupene and coworkers argued that AmtB proteins in enteric bacteria increase the rate of equilibration of uncharged ammonia across the cytoplasmic membrane rather than actively transporting and accumulating ammonium (19). According to this concept, transport is driven by metabolic trapping, i.e., intracellular assimilation of ammonium by glutamine synthetase (GS). This model was supported by crystallographic data for AmtB from Escherichia coli and Archaeoglobus fulgidus (2, 7, 21), which indicated the presence of a hydrophobic channel responsible for ammonia transport, and by methylammonium uptake measurements (5). In Corynebacterium glutamicum, two different ammonium transporters are present. AmtA exhibits a relatively high affinity of 44 ⫾ 7 M for methylammonium, an ammonium analogue commonly used for transport measurements (5, 12, 18, 19), and a maximal velocity of 25 ⫾ 5 nmol mg (dry weight)⫺1 min⫺1. Uptake is dependent on the membrane potential and can be abolished by the addition of protonophores, like carbonyl cyanide m-chlorophenylhydrazone (CCCP) (18). In the presence of 50 M methylammonium, 10 M of ammonium is sufficient for a half-maximal inhibition of methylammonium uptake, indicating a high affinity of AmtA for this solute (12). In contrast to AmtA, no methylammonium uptake activity was detected for AmtB until now (12). The aim of this study was to investigate the contributions of AmtA and AmtB to methylammonium/ammonium transport and to address the question of the transport mechanism(s) and energetic coupling. Construction of mutant strains. To investigate whether metabolic trapping by GS is a major driving force for methylammo-
* Corresponding author. Mailing address for Reinhard Kra¨mer: Institut fu ¨r Biochemie, Universita¨t zu Ko ¨ln, Zu ¨lpicher-Str. 47, 50674 Ko ¨ln, Germany. Phone: 49 221 470 6461. Fax: 49 221 470 5091. E-mail: r.kraemer@uni-koeln .de. Mailing address for Andreas Burkovski: Lehrstuhl fu ¨r Mikrobiologie, Friedrich-Alexander-Universita¨t Erlangen-Nu ¨rnberg, Staudtstr. 5, 91058 Erlangen, Germany. Phone: 49 9131 85 28086. Fax: 49 9131 85 28082. E-mail:
[email protected]. 䌤 Published ahead of print on 1 February 2008. 2611
2612
NOTES
J. BACTERIOL.
FIG. 1. Metabolization of methylammonium. Thin-layer chromatography of nitrogen-starved C. glutamicum wild type (lane 2) and the deletion strain DA-2 (lane 3) incubated with [14C]methylammonium. Lane 1, [14C]methylammonium as a control.
100 M, AmtB-dependent transport was not observed (12). Within the range of methylammonium concentrations tested here, AmtB-mediated uptake was linearly dependent on the external substrate concentration and was not saturable up to 3 mM of external methylammonium. In the amtA amtB mutant JS-1, methylammonium transport was negligible. It should be taken into consideration that JS-1 still harbors the ammoniumassimilating enzymes, which means that metabolic trapping is in principle fully functional. Consequently, the contribution of passive diffusion to the total methylammonium flux in the substrate concentration range used for kinetic measurements presented in the following experiments is less than 1%. Energetics of methylammonium uptake. Metabolic trapping of ammonium or methylammonium by GS activity was always a major argument in connection with ammonium uptake in bacteria (5, 19). When methylammonium uptake rates mediated by AmtA depending on the substrate concentration in
FIG. 2. Discrimination of methylammonium fluxes in different mutant strains of C. glutamicum. [14C]methylammonium uptake was determined for the wild-type ATCC 13032 (F), the amtB deletion strain LN-1.1 (), the amtA deletion strain MJ2-38 (Œ), and the amtA amtB double-deletion strain JS-1 (f). To induce expression of the amount genes, cells were incubated for 1.0 h in minimal medium without a nitrogen source (12, 18). Uptake was started by the addition of [14C] methylammonium to the cells at an optical density at 600 nm of approximately 4, and the uptake rate was determined by filtration of samples at 0.5, 1, 2, and 4 min. dw, dry weight. The error bars represent standard deviations.
FIG. 3. Uptake of methylammonium depending on the external substrate concentration in the absence of metabolic trapping. Uptake was investigated in the deletion strain MeK-1 (⌬glnA ⌬glnA2 ⌬gdh ⌬amtB) (f). For comparison, the uptake rates of the amtB deletion strain LN-1.1 (F) are shown. The experimental conditions were as described in the legend to Fig. 2. The error bars represent standard deviations.
strain MeK-1, which lacks GS, GDH, and AmtB, were measured in comparison with those in strain LN-1.1, which still carries the ammonium-processing enzymes, the two strains were not significantly different in terms of uptake kinetics (Fig. 3). Consequently, the mechanism of metabolic trapping is not valid for ammonium/methylammonium uptake by AmtA. As an alternative driving force, we tested the electrochemical potential across the plasma membrane. The influence of the membrane potential can be quantitated with respect to both kinetic (uptake rates) and thermodynamic (accumulation ratio) values. First, we measured the methylammonium uptake kinetics within 3 min after addition of the labeled substrate (Fig. 4). In order to identify a possible dependence of this uptake rate on the membrane potential, we decreased the electrical gradient by increasing the power of uncoupling reagents, using the addition of a low concentration of CCCP (3 M), leading to partial uncoupling only, as well as a high concentration of CCCP (50 M). As a control, the same experiment was carried out using the amtA amtB deletion strain JS-1. Background values for cell and filter binding were subtracted. These were determined by carrying out experiments in the presence of 0.1% (wt/vol) of the cationic detergent cetyltrimethylammonium bromide (CTAB), which permeabilizes the cell membrane without fully disrupting the cells (16). The initial uptake rates calculated from Fig. 4 result in about 10, 4, and 2 nmol mg (dry weight)⫺1 min⫺1 for the different conditions applied to the deletion strain MeK-1 (no addition, 3 M, and 50 M CCCP, respectively). These results demonstrate that a gradual decrease in the membrane potential by uncoupling strongly decreases the initial rate of methylammonium uptake. Again, no or negligible uptake was observed in the double-deletion strain JS-1. Since substrate accumulation, which, in contrast to uptake
VOL. 190, 2008
FIG. 4. Methylammonium uptake under different energetic conditions. Uptake was determined in C. glutamicum strain MeK-1 without the addition of uncouplers (f), with 3 M CCCP (F), and with 50 M CCCP (Œ). Strain JS-1 was used as a control (). All other conditions were as described in the legend to Fig. 2. The error bars represent standard deviations.
rates, is a thermodynamic value, might be more convincing for defining energetic aspects of transport, experiments were carried out to determine the chemical gradient (accumulation ratio) of methylammonium (internal/external concentration) under steady-state conditions, i.e., after long-term uptake. These experiments were only possible based on the availability of strain MeK-1, because the putative driving force (membrane potential) is present while metabolic trapping is absent and, equally important, because the passive diffusion of methylammonium is negligibly low in C. glutamicum under the experimental conditions used. For a correct calculation of accumulation ratios, the supernatant (external methylammonium) and cells (internal methylammonium) were harvested separately and the substrate concentration was quantified (Fig. 5). The cells accumulated methylammonium to different extents, and a situation of approximate steady state was reached after about 10 min of uptake. Untreated cells of strain MeK-1, i.e., devoid of metabolic trapping and lacking the second uptake system, AmtB, achieved a steady-state accumulation ratio of up to 23,000 (internal/external), whereas the steady-state accumulation was significantly decreased by the addition of uncouplers. Strain JS-1, devoid of both uptake systems but equipped with enzymes for ammonium metabolism and thus capable of metabolic trapping, was characterized by very low methylammonium accumulation, most probably due to adsorption. As a control, we also measured the methylammonium accumulation in cells permeabilized by low concentrations of CTAB, which indicated a residual amount of adsorption of transport substrate. Concluding remarks. The dissection of methylammonium fluxes achieved in this work in C. glutamicum cells demonstrates that (i) the function of AmtA is fundamentally different from that of AmtB, (ii) AmtA is the predominant ammonium uptake system in C. glutamicum, (iii) uptake of methylammonium via AmtA is independent of the possible driving force of metabolic trapping and thus represents an energy-driven uptake mechanism, and (iv)
NOTES
2613
FIG. 5. Methylammonium accumulation under different conditions. Methylammonium uptake in C. glutamicum strain MeK-1 was measured without further addition (f) and in the presence of 3 M CCCP (F), 50 M CCCP (Œ), and 0.1% (wt/wt) CTAB (}), as well as in strain JS-1 () without any further addition. The internal concentrations of methylamine were calculated on the basis of a cytoplasmic volume of 1.7 l/mg cell dry mass (3). In independent kinetic experiments, the external concentrations of methylammonium were measured at identical time points in the uptake experiment, leading to the accumulation ratios shown. The error bars represent standard deviations.
uptake of methylammonium is dependent on the presence of a membrane potential in terms of both the uptake rate and steadystate accumulation. The dependence of uptake rates on the membrane potential has been observed before in several cells, including C. glutamicum (references 6, 12, and 18 and references therein). In the experiments on steady-state methylammonium accumulation, we observed extremely high values, which, however, were substantiated by appropriate controls using cells devoid of ammonium uptake systems, as well as permeabilized cells. Furthermore, the observed high steady-state accumulation of methylammonium in the cytoplasm in the absence of any significant pH gradient again argues for the membrane potential being the driving force for this energy-consuming process. This conclusion directly argues for a net positive charge(s) being moved in the course of methylammonium uptake in C. glutamicum, which is not in line with transport of uncharged ammonia. Very recently, Fong and coworkers (4) provided experimental evidence that, in contrast to their previous hypothesis (19), in fact the charged methylammonium seemed to be the species transported by E. coli AmtB. In principle, this is in line with the experimental data presented here. However, there is an obvious difference between the two investigations, due to the low permeability of C. glutamicum membranes to methylammonium. We were able to quantify the passive (background) flux of methylammonium by using a strain devoid of both the amtA and the amtB genes but still harboring the metabolizing enzymes and thus the system of metabolic trapping. The high accumulation ratio of up to 23,000 (internal/external methylammonium concentration) observed in our experiments using recombinant C. glutamicum strains is surprising. It is not in line with a basic conception of the methylammonium cation being the sole transport substrate of C. glutamicum
2614
NOTES
J. BACTERIOL.
AmtA. Steady-state accumulation values higher than 500, which would equilibrate the typcial membrane potential of 160 mV measured in C. glutamicum under the growth conditions used (unpublished results), indicate the transfer of more than one charge in the course of substrate translocation by AmtA. This will be investigated in more detail in future studies. In view of its very low activity compared to AmtA, AmtB does not seem to play a significant role in C. glutamicum under physiological conditions. The mechanism of transport by AmtB is less clear, but it is different from that of AmtA. The low accumulation of methylammonium by AmtB in the absence of metabolic trapping would in principle be in agreement with a pore-like mechanism accepting the uncharged species as the substrate. The affinity of AmtB for methylammonium seems to be extremely low, since we did not achieve saturation within a concentration range of up to 3 mM. Consequently, characterization of AmtB in the AmtA deletion mutant was hampered by this fact, which leads to very low transport activity at reasonable concentrations of labeled substrate. In any case, the observed results do not argue for an electrophoretic mechanism of AmtB, nor are they in obvious agreement with the kinetic behavior of AmtB proteins in other organisms. It was shown earlier by Ludewig, Mayer, and coworkers that Amt proteins in plants seem to function as ammonium transporters (8, 10, 11), while the closely related animal Rh proteins function as ammonia channels. This was taken as an indication that related proteins with high structural similarity can function according to different mechanisms (9). In fact, C. glutamicum AmtA has a very high degree of similarity to other bacterial Amt proteins, including AmtB, with 68% identical amino acids. The two strategic phenylalanine residues at the entry into the substrate pathway are present in AmtA from C. glutamicum, as well as the highly conserved two histidine residues in the central part of the pathway (6). Consequently, based on the primary structure, there is no indication of a different function for C. glutamicum AmtA compared to other Amt proteins in bacteria. We thank H. Sticht (Erlangen) for bioinformatic analyses of Amt proteins. This work was supported by the Bundesministerium fu ¨r Forschung und Technologie.
3.
4.
5.
6.
7.
8.
9.
10. 11.
12.
13.
14. 15.
16.
17.
18.
19.
20.
REFERENCES 1. Abe, S., K. Takayama, and S. Kinoshita. 1967. Taxonomical studies on glutamic acid-producing bacteria. J. Gen. Microbiol. 13:279–301. 2. Andrade, S. L. A., A. Dickmanns, R. Ficner, and O. Einsle. 2005. Crystal
21.
structure of the archaeal ammonium transporter Amt-1 from Archaeoglobus fulgidus. Proc. Natl. Acad. Sci. USA 102:14994–14999. Botzenhardt, J., S. Morbach, and R. Kra ¨mer. 2004. Activity regulation of the betaine transporter BetP of Corynebacterium glutamicum in response to osmotic compensation. Biochim. Biophys. Acta 1667:229–240. Fong, R. N., K.-S. Kim, C. Yoshihara, W. B. Inwood, and S. Kustu. 2007. The W148L substitution in the Escherichia coli ammonium channel AmtB increases flux and indicates that the substrate is an ion. Proc. Natl. Acad. Sci. USA 104:18706–18711. Javelle, A., G. Thomas, A. M. Marini, R. Kra ¨mer, and M. Merrick. 2005. In vivo functional characterization of the Escherichia coli ammonium channel AmtB: evidence for metabolic coupling of AmtB to glutamine synthetase. Biochem. J. 390:215–222. Javelle, A., L. D. Lupo, X.-D. Li, M. Merrick, M. Chami, P. Ripoche, and F. K. Winkler. 2007. Structural and mechanistic aspects of Amt/Rh proteins. J. Struct. Biol. 158:472–481. Khademi. S., J. O’Connell III, J. Remis, Y. Robles-Colmenares, L.-J. W. Miercke, and R. M. Stroud. 2004. Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 Å. Science 305:1587–1594. Ludewig U., N. von Wiren, and W. B. Frommer. 2002. Uniport of NH4⫹ by the root hair plasma membrane ammonium transporter LeAMT1;1. J. Biol. Chem. 277:13548–13555. Ludewig, U., B. Neuha ¨user, and M. Dynowski. 2007. Molecular mechanisms of ammonium transport and accumulation in plants. FEBS Lett. 581:2301– 2308. Mayer, M., M. Dynowski, and U. Ludewig. 2006. Ammonium ion transport by the AMT/Rh homologue LeAMT1;1. Biochem. J. 396:431–437. Mayer, M., G. Schaaf, I. Mouro, C. Lopez, Y. Colin, P. Neumann, J. P. Cartron, and U. Ludewig. 2006. Different transport mechanisms in plants and human AMT/Rh-type ammonium transporters. J. Gen. Physiol. 127: 133–144. Meier-Wagner, J., L. Nolden, M. Jakoby, R. M. Siewe, R. Kra ¨mer, and A. Burkovski. 2001. Multiplicity of ammonium uptake systems in Corynebacterium glutamicum: role of Amt and AmtB. Microbiology 147:135–143. Mu ¨ller, T., J. Stro ¨sser, S. Buchinger, L. Nolden, A. Wirtz, R. Kra ¨mer, and A. Burkovski. 2006. Mutation-induced metabolite pool alterations in Corynebacterium glutamicum: towards the identification of nitrogen control signals. J. Biotechnol. 126:440–453. Nolden, L. 2001. Ph.D. thesis. University of Cologne, Cologne, Germany. Nolden, L., M. Farwick, R. Kra ¨mer, and A. Burkovski. 2001. Glutamine synthetases in Corynebacterium glutamicum: transcriptional control and regulation of activity. FEMS Microbiol. Lett. 201:91–98. Nolden, L., C.-E. Ngouoto-Nkili, A. K. Bendt, R. Kra ¨mer, and A. Burkovski. 2001. Sensing nitrogen limitation in Corynebacterium glutamicum: The role of glnK and glnD. Mol. Microbiol. 42:1281–1295. Scha ¨fer, A., A. Tauch, W. Ja ¨ger, J. Kalinowski, G. Thierbach, and A. Pu ¨hler. 1994. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73. Siewe, R. M., B. Weil, A. Burkovski, B. J. Eikmanns, M. Eikmanns, and R. Kra ¨mer. 1996. Functional and genetic characterization of the (methyl)ammonium uptake carrier of Corynebacterium glutamicum. J. Biol. Chem. 271: 5398–5403. Soupene, E., L. He, D. Yan, and S. Kustu. 1998. Ammonia acquisition in enteric bacteria: physiological role of the ammonium/methylammonium transport B (AmtB) protein. Proc. Natl. Acad. Sci. USA 95:7030–7034. Stro ¨sser, J., A. Lu ¨dke, S. Schaffer, R. Kra ¨mer, and A. Burkovski. 2004. Regulation of GlnK activity: modification, membrane sequestration, and proteolysis as regulatory principles in the network of nitrogen control in Corynebacterium glutamicum. Mol. Microbiol. 54:132–147. Zheng, L., D. Kostrewa, S. Berneche, F. K. Winkler, and X.-D. Li. 2004. The mechanism of ammonia transport based on the crystal structure of AmtB of Escherichia coli. Proc. Natl. Acad. Sci. USA 101:17090–17095.
