Institut für Biochemie der Universität zu Köln, Zülpicher-Str. 47, D-50674 Köln, Germany1
Author for correspondence: Andreas Burkovski. Tel: +49 221 470 6472. Fax: +49 221 470 5091. e-mail: a.burkovski{at}uni-koeln.de
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: ammonium transport, Corynebacterium glutamicum, glutamine synthetase
Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone.
a Present address: Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, D-50829 Köln, Germany.
b Present address: PerkinElmer Life Sciences/Berthold, Postfach 100163, D-75312 Bad Wildbad, Germany.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In contrast to this model of an energy-dependent, membrane-potential-driven ammonium transport, proposed for a variety of ammonium uptake systems in bacteria and in plants (for reviews, see Kleiner, 1993 ; von Wirén et al., 2000
), a different concept of ammonium acquisition was suggested recently (Soupene et al., 1998
). It was proposed that AmtB/Mep proteins simply increase the rate of equilibration of uncharged ammonia across the cytoplasmic membrane rather than actively transporting and accumulating ammonium.
In Corynebacterium glutamicum, a Gram-positive soil bacterium widely used in the industrial production of amino acids, two genes encoding (putative) ammonium uptake carriers have been described. The isolation of amt (Siewe et al., 1996 ) was the first report of the sequence of a gene encoding a bacterial ammonium uptake system combined with the characterization of the corresponding protein. Recently, a second amt gene with so far unknown function was isolated. This gene was originally designated amtP, for amt paralogue (Jakoby et al., 1999
). Based on a great number of homologues cloned in different bacteria, on sequence similarity analyses and on its genetic organization, this system is now designated amtB (Jakoby et al., 2000
). The isolation of the amtB gene and the new model proposed for the mechanism of ammonium uptake (Soupene et al., 1998
) prompted us to reinvestigate (methyl)ammonium uptake in C. glutamicum.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Extraction of C. glutamicum cells and TLC.
Nitrogen-starved cells were incubated in the presence of 14C-labelled methylammonium as described above with the exceptions that a higher cell density was used (OD600 approx. 7) and that the specific activity of the approach was increased to 12·5 kBq. Filtered cells were extracted in 4 ml 0·05% SDS/hot 70% ethanol solution (1 h at room temperature) immediately after the washing steps. After 10 min incubation the ethanolic extracts were dried at room temperature under a steady stream of air and subjected to TLC. Samples were separated on cellulose plates with a mixture of n-butanol, acetic acid and water (4:1:1 by vol). The 14C-labelled products were detected either by autoradiography using Kodak X-OMAT films or BAS-MP 2025 imaging plates and a Bio-Imaging Analyzer BAS-1800 (Fuji).
Measurement of ammonium.
Decrease of ammonium in the medium was measured using an ammonia-selective electrode (Orion Research). Cells were starved of nitrogen for 3 h to induce the synthesis of Amt and AmtB and to deplete the internal nitrogen pools. Subsequently, 200 µM ammonium chloride was added to the cultures (OD600 approx. 5) and at different times cells and culture supernatant were separated by rapid filtration using glass fibre filters (Millipore). The ammonium uptake rate was calculated from the decrease of ammonia determined in the culture filtrate.
Molecular biology techniques.
For plasmid isolation, transformation and cloning, standard techniques were used (Ausubel et al., 1987 ; Sambrook et al., 1989
). Southern blotting was carried out with digoxigenin-labelled probes using the DIG-labelling and detection kit as recommended by the supplier (Roche Diagnostics).
RNA preparation and Northern hybridization analysis.
Total RNA was prepared after disruption of the C. glutamicum cells by glass beads using the RNeasy Mini Kit as recommended by the supplier (Qiagen). The RNA was either size-fractionated using agarose gels containing formaldehyde and blotted onto positively charged nylon membranes (Sambrook et al., 1989 ), or spotted directly onto nylon membranes using a Schleicher & Schuell Minifold I Dot Blotter. Hybridization of digoxigenin-labelled RNA probes was detected with a Fuji luminescent image analyser LAS1000 or Kodak X-OMAT X-ray films using alkaline phosphatase conjugated anti-digoxigenin Fab fragments and CSPD as light-emitting substrate as recommended by the supplier (Roche Diagnostics).
Construction of deletion mutants.
A chromosomal deletion of the amt gene was introduced in the C. glutamicum genome according to the protocol described by Schäfer et al. (1994) using plasmid pK19mobsacB for deletion in C. glutamicum and E. coli strain S17-1 for conjugation. For this purpose a 1080 bp AocI/KspI fragment was removed from plasmid pUCdppc. After treatment with DNA polymerase I (Klenow fragment) and re-ligation, a 2 kb BamHI fragment was isolated from plasmid pUC
dppc and ligated to BamHI-restricted and dephosphorylated pK19mobsacB DNA. The resulting plasmid, pK19mobsacB
dppc, was used for the chromosomal deletion via two independent recombination events as described by Schäfer et al. (1994)
.
To inactivate amtB, an internal fragment of this gene was removed from plasmid pUCamtB by EcoRV/BsmI restriction. After blunting of the linearized plasmid DNA by mung bean nuclease treatment and re-ligation, a DNA fragment carrying the flanking regions of the deletion was isolated from the corresponding plasmid, pUCamtB-del2, by EcoRI/BamHI restriction and used further as described above. The different deletions were verified by PCR using primers annealing at flanking regions (data not shown). In addition, chromosomal DNA of the deletion strains was amplified via PCR and sequenced using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit and an ABI 310 automated sequencer (PE Applied Biosystems) to verify that the flanking DNA regions of the different deletions were not impaired during the recombination events.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
Regulation of amt transcription depending on the medium pH
As shown recently, amt transcription is derepressed upon nitrogen starvation (Jakoby et al., 2000 ). On the assumption that C. glutamicum Amt facilitates transport of ammonia rather than catalysing ammonium transport, its expression should be increased in low pH medium, since diffusion of ammonia across the membrane would become limiting under these conditions. Northern hybridization experiments were carried out using total RNA isolated from nitrogen-starved cells incubated at pH 6·0 and 7·0. When hybridized against an amt antisense probe, RNA isolated from cells incubated at pH 6·0 showed only a faint signal as compared with RNA prepared from cells incubated at pH 7·0, whilst the amount of 16S rRNA in these samples stayed constant (Fig. 6
). These data do not indicate an increased expression of amt at low pH and are thus in accord with the model that Amt works as a (methyl)ammonium- rather than a (methyl)amine-uptake system.
|
From the measurements obtained during the comparison of wild-type and amt deletion strain MJ2-38, it was assumed that amtB might encode a low-affinity permease, since no high-affinity methylammonium uptake was observed in strain MJ2-38. In this case, methylammonium would be transported in the C. glutamicum cells via both a high-affinity uptake system, Amt, and a low-affinity carrier, AmtB, a scenario which is similar to the situation in the cyanobacterium Synechocystis PCC 6803 (Montesinos et al., 1998 ).
To investigate the basic kinetic parameters of AmtB and a possible regulation depending on the nitrogen availability, cells of strain MJ2-38 were starved for nitrogen sources and the rate of methylammonium uptake was determined using 1 mM [14C]methylammonium. However, methylammonium uptake could never be observed in strain MJ2-38 either without or with nitrogen starvation for up to 4 h (data not shown).
Deletion of the amtB gene
Since AmtB was obviously not directly involved in methylammonium transport, we speculated that this protein might have a regulatory function. Recently, a sensor function was discussed for the yeast (methyl)ammonium transporter Mep2 (Lorenz & Heitman, 1998 ). Moreover, an interaction of the different yeast Mep systems was proposed (Marini et al., 2000
). These observations led to the hypothesis that C. glutamicum AmtB might interact with Amt to regulate transport activity. However, when methylammonium uptake was determined in the amtB deletion mutant LN-1.1, no difference between wild-type and amtB deletion mutant was observed. Upon nitrogen starvation, a methylammonium uptake rate of 10·8±1·3 nmol (mg dry weight)-1 min-1 was observed in the wild-type ATCC 13032 and 11·0±1·0 nmol (mg dry weight)-1 min-1 in amtB deletion strain LN-1.1. Also, regulation of the level of Amt activity was not impaired. When glutamine was added, in both strains a decrease in transport rate of 14% was observed within 10 min (data not shown). Together, these results demonstrate that AmtB is not involved in Amt regulation.
Effect of pH on growth at different ammonium concentrations
As a more physiological approach to studying the function of Amt and AmtB and to investigate which of the different concepts for ammonium acquisition holds true for C. glutamicum, we carried out growth experiments. Based on the fact that at low pH the concentration of the unprotonated ammonia is extremely low, diffusion and uptake should become limiting if ammonia is the transported substrate species (Soupene et al., 1998 ). As a result, a difference in growth of wild-type and amt deletion mutants is expected in the case of ammonia being transported as proposed for E. coli AmtB (Soupene et al., 1998
). C. glutamicum wild-type strain ATCC 13032, amt deletion strain MJ2-38, amtB deletion LN-1.1 and amt/amtB double deletion LN-1.2 were grown at various ammonium concentrations (0·1, 0·5, 1·0 and 5·0 mM NH4Cl) in minimal medium adjusted to different pH values (pH 6·0, 6·5, 7·0, 7·5). No significant difference in the growth rate of the different C. glutamicum strains depending on pH and ammonium concentration was detected, although the final optical densities were found to depend directly on the amount of ammonium added (data not shown). This result led us to investigate whether ammonium depletion results in a decreased growth rate in C. glutamicum at all. In fact, when cells were incubated in the absence of any nitrogen source, for a short time a similar growth rate as compared to nitrogen-supplemented cells was observed before cell division ceased completely. Obviously, C. glutamicum is able to circumvent the effects of ammonium limitation on the growth rate found in other bacteria (e.g. E. coli; see Soupene et al., 1998
) by using intracellularly stored nitrogen sources like glutamate and glutamine. Up to 200 mM internal glutamate (Krämer & Lambert, 1990
) and 50 mM internal glutamine (Tesch et al., 1999
) have been determined in C. glutamicum cells grown in minimal medium. As a consequence, growth experiments seem not to be an appropriate tool to discriminate the physiological role of Amt and AmtB.
Measurement of ammonium uptake
To circumvent the problems arising from the high internal glutamate and glutamine pools in C. glutamicum, depletion of ammonium was directly determined in the culture supernatants of strains ATCC 13032, MJ2-38, LN-1.1 and LN-1.2 (Fig. 7). Ammonium chloride (200 µM) was added to nitrogen-starved cells (OD600 approx. 5) and ammonium concentrations were determined. From the linear part of the decrease, an uptake rate of approximately 11 nmol (mg dry weight)-1 min-1 was calculated for the wild-type, which perfectly correlates with the methylammonium uptake rates measured. The transport rates obtained for amt mutant strain MJ2-38 and amtB mutant LN-1.1 were found to be about 10 and 9 nmol (mg dry weight)-1 min-1, respectively, and thus not significantly lower. Interestingly, for the double mutant a biphasic decrease of ammonium in the supernatant was observed. First, ammonium decreased with a rate of approximately 8 nmol (mg dry weight)-1 min-1 until uptake ceased at a concentration of 70 µM ammonium. This result cannot be explained by diffusion alone, since diffusion should depend linearly on the transmembrane concentration difference, and therefore hints of the presence of an additional ammonium uptake system.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Whilst Amt is the main uptake system, transporting (methyl)ammonium with high affinity into the cell, AmtB obviously does not contribute to the uptake of methylammonium but is an ammonium-specific permease. Whether ammonium or ammonia is the actual substrate species transported by AmtB was not determined. A similar set of ammonium transport proteins was found in Rhodobacter sphaeroides, where an ammonium-specific uptake system together with a methylammonium/ammonium permease was found (Cordts & Gibson, 1987 ), and in Arabidopsis thaliana where AMT1 proteins transport (methyl)ammonium (Gazzarrini et al., 1999
) whilst AtAMT2 is ammonium-specific (Sohlenkamp et al., 2000
). Furthermore, the kinetic results of ammonium uptake studies indicated the presence of an additional, low-affinity ammonium transport system in C. glutamicum.
In summary, membrane-potential-driven transport of (methyl)ammonium was shown for the C. glutamicum Amt system. This type of uptake mechanism might also hold true for other bacteria. In physiological terms, moreover, energy-dependent uptake of ammonium makes sense especially for organisms living in acid environments, in which almost no ammonia is present.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1987). Current Protocols in Molecular Biology. New York: Greene Publishing Associates and Wiley Interscience.
Cordts, M. L. & Gibson, J.(1987). Ammonium and methylammonium transport in Rhodobacter sphaeroides. J Bacteriol 169, 1632-1638.[Medline]
Cremer, J., Eggeling, L. & Sahm, H.(1990). Cloning the dapA dapB cluster of the lysine-secreting bacterium Corynebacterium glutamicum. Mol Gen Genet 220, 478-480.
Gazzarrini, S., Lejay, L., Gojon, A., Ninnemann, O. & Frommer, W. B.(1999). Three functional transporters for constitutive, diurnally regulated, and starvation-induced uptake of ammonium into Arabidopsis roots. Plant Cell 11, 937-947.
Grant, S. N. G., Jessee, J., Bloom, F. R. & Hanahan, D.(1990). Differential plasmid rescue from transgenic mouse DNAs into Escherichia coli methylation-restriction mutants. Proc Natl Acad Sci USA 87, 4645-4649.[Abstract]
Jakoby, M., Tesch, M., Sahm, H., Krämer, R. & Burkovski, A.(1997). Isolation of the Corynebacterium glutamicum glnA gene encoding glutamine synthetase I. FEMS Microbiol Lett 154, 81-88.[Medline]
Jakoby, M., Krämer, R. & Burkovski, A.(1999). Nitrogen regulation in Corynebacterium glutamicum: isolation of genes involved and biochemical characterization of the corresponding proteins. FEMS Microbiol Lett 173, 303-310.[Medline]
Jakoby, M., Nolden, L., Meier-Wagner, J., Krämer, R. & Burkovski, A.(2000). AmtR, a global repressor in the nitrogen regulation system of Corynebacterium glutamicum. Mol Microbiol 37, 964-977.[Medline]
Jayakumar, A., Hong, J.-S. & Barnes, E. M.Jr(1986). Feedback inhibition of ammonium (methylammonium) ion transport in Escherichia coli by glutamine and glutamine analogs. J Bacteriol 169, 553-557.
Keilhauer, C., Eggeling, L. & Sahm, H.(1993). Isoleucine synthesis in Corynebacterium glutamicum: molecular analysis of the ilvB-ilvN-ilvC operon. J Bacteriol 175, 5595-5603.[Abstract]
Kleiner, D.(1985). Bacterial ammonium transport. FEMS Microbiol Rev 32, 87-100.
Kleiner, D.(1993). transport systems. In Alkali Cation Transport Systems in Prokaryotes , pp. 379-395. Edited by E. P. Bakker. Boca Raton, FL:CRC Press.
Kleiner, D. & Castorph, H.(1982). Inhibition of ammonium (methylammonium) transport in Klebsiella pneumoniae by glutamine and glutamine analogues. FEBS Lett 146, 201-203.[Medline]
Krämer, R. & Lambert, C.(1990). Uptake of glutamate in Corynebacterium glutamicum. 2. Evidence for a primary active transport system. Eur J Biochem 194, 937-944.[Abstract]
Krämer, R., Lambert, C., Hoischen, C. & Ebbighausen, H.(1990). Uptake of glutamate in Corynebacterium glutamicum. 1. Kinetic properties and regulation by internal pH and potassium. Eur J Biochem 194, 929-935.[Abstract]
Large, P. J.(1980). Microbial growth on methylated amines. In Microbial Growth on C1 Compounds , pp. 55-69. Edited by H. Dalton. London/Philadelphia/Rheine:Heyden.
Lorenz, M. C. & Heitman, J.(1998). The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae. EMBO J 17, 1236-1247.
Marini, A.-M., Springael, J.-Y., Frommer, W. B. & André, B.(2000). Cross-talk between ammonium transporters in yeast and interference by the soybean SAT1 protein. Mol Microbiol 35, 378-385.[Medline]
Montesinos, M. L., Muro-Pastor, A. M., Herrero, A. & Flores, E.(1998). Ammonium/methylammonium permeases of a cyanobacterium. J Biol Chem 273, 31463-31470.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schäfer, A., Tauch, A., Jäger, W., Kalinowski, J., Thierbach, G. & Pühler, A.(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.[Medline]
Siewe, R. M., Weil, B., Burkovski, A., Eikmanns, B. J., Eikmanns, M. & Krämer, R.(1996). Functional and genetic characterization of the (methyl)ammonium uptake carrier of Corynebacterium glutamicum. J Biol Chem 271, 5398-5403.
Simon, R., Priefer, U. & Pühler, A.(1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Appl Microbiol Biotechnol 1, 784-791.
Sohlenkamp, C., Shelden, M., Howitt, S. & Udvardi, M.(2000). Characterization of Arabidopsis AtAMT2, a novel ammonium transporter in plants. FEBS Lett 467, 273-278.[Medline]
Soupene, E., He, L., Yan, D. & Kustu, S.(1998). Ammonia acquisition in enteric bacteria: physiological role of the ammonium/methylammonium transport B (AmtB) protein. Proc Natl Acad Sci USA 95, 7030-7034.
Tachiki, T., Wakisaka, S., Suzuki, H., Kumagai, H. & Tochikura, T.(1981). Glutamine synthetase from Micrococcus glutamicus: effect of nitrogen sources in culture medium on enzyme formation and some properties of crystalline enzyme. Agric Biol Chem 45, 287-292.
Tachiki, T., Wakisaka, S., Suzuki, H., Kumagai, H. & Tochikura, T.(1983). Variation of Micrococcus glutamicus glutamine synthetase brought about by divalent cations. Agric Biol Chem 47, 287-292.
Tesch, M., de Graaf, A. A. & Sahm, H.(1999). In vivo fluxes in the ammonium-assimilatory pathways in Corynebacterium glutamicum studied by 15N nuclear magnetic resonance. Appl Environ Microbiol 65, 1099-1109.
Thomas, G., Coutts, G. & Merrick, M.(2000). The glnKamtB operon, a conserved gene pair in prokaryotes. Trends Genet 16, 11-14.[Medline]
von Wirén, N., Gazzarrini, S., Gojon, A. & Frommer, W. B.(2000). The molecular physiology of ammonium uptake and retrieval. Curr Opin Plant Biol 3, 254-261.[Medline]
Received 26 July 2000;
revised 25 September 2000;
accepted 4 October 2000.