Institut für Mikrobiologie und Molekularbiologie der Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 26-32, D-35392 Giessen, Germany
Correspondence
Karl Forchhammer
Karl.Forchhammer{at}mikro.bio.uni-giessen.de
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Both PII proteins are covalently modified by uridylylation at a conserved tyrosyl residue (Tyr-51) (Son & Rhee, 1987) that is located at the tip of a solvent-exposed loop (T-loop) (Jaggi et al., 1996
). Uridylylation and deuridylylation are catalysed by a bifunctional uridylyltransferase/uridylylremovase enzyme (UTase/UR), the glnD product, which responds to the cellular glutamine level (Adler et al., 1975
). In the presence of high intracellular glutamine levels, signalling nitrogen-excess conditions, uridylylremovase activity of GlnD dominates, causing demodification of uridylylated PII proteins. Low glutamine levels, signalling nitrogen deficiency, promote the uridylyltransferase activity, leading to uridylylation of PII (Jiang et al., 1998a
).
The interaction of the PII proteins with signal receptors is modulated by uridylylation of the T-loop. One receptor of PII signalling is the adenylyltransferase/removase enzyme GlnE (ATase), which regulates the activity of GS by reversible adenylylation. The default mode of ATase, which is stimulated by glutamine and by non-uridylylated PII, is adenylylation of GS. Upon uridylylation of PII, a different complex is formed, which switches the activity of ATase towards deadenylylation and thereby activation of GS (Rhee et al., 1985; Jiang et al., 1998c
; Jaggi et al., 1997
). Non-modified GlnB forms a complex with NtrB, which dephosphorylates the response regulator NtrC, thus switching off transcription of glnA (Jiang & Ninfa, 1999
; Pioszak et al., 2000
) and of other NtrC-dependent genes like glnK (Atkinson et al., 2002a
). By contrast, GlnB-UMP is not able to form a complex with NtrB. In this state, the histidine kinase activity of NtrB dominates, and by phosphoryl transfer, NtrC is phosphorylated and is now transcriptionally active (Jiang & Ninfa, 1999
). In addition to uridylylation, GlnB is regulated allosterically by the effector molecule 2-oxoglutarate as shown by Ninfa and colleagues in a series of in vitro analyses (Jiang et al., 1998a
, b
, c
). Of the three binding sites for 2-oxoglutarate within the trimeric GlnB protein, only one site is occupied with high affinity, whereas affinity of the second and third sites is decreased by negative cooperativity. Only at high 2-oxoglutarate concentrations does complete occupation of GlnB with this effector molecule occur. This state impairs complex formation of non-uridylylated GlnB with NtrB, which finally results in phosphorylation and, thus, activation of NtrC (Jiang & Ninfa, 1999
). From these biochemical properties, it was proposed that GlnB integrates signals of the nitrogen status (by uridylylation) with a signal of central carbon metabolism (2-oxoglutarate) to co-ordinate the expression of key enzymes of nitrogen assimilation in response to the nitrogen and carbon state. However, transduction of carbon signals by PII proteins in E. coli has not yet been demonstrated in vivo.
Another link between carbon and nitrogen regulation has been demonstrated for the regulation of expression of the glnALG operon. Its transcription is initiated from tandem promoters (glnAp1 and glnAp2), which are under carbon and nitrogen control (reviewed by Reitzer, 1996, and by Magasanik, 1996
). The activity of the weak,
70-dependent glnAp1 promoter requires binding of the CRPcAMP complex to an upstream activating sequence. The CRPcAMP complex is formed when cells are grown with a carbon source that is not transported by the phosphotransferase system (PTS). The utilization of PTS sugars like glucose leads to a decrease in cellular cAMP levels and dissociation of the complex, thereby causing catabolite repression of a large number of genes (reviewed by Saier et al., 1996
). Located 116 bp downstream of glnAp1 is a strong
54-dependent promoter, glnAp2, which is activated by NtrC-phosphate. Concomitant with the activation of glnAp2, NtrC-phosphate blocks transcription from the weak glnAp1 promoter. Thus, when NtrC-phosphate accumulates during nitrogen-limited growth, the glnALG operon is almost exclusively transcribed to high levels from glnAp2 (Magasanik, 1996
). A recent investigation showed that transcription from glnAp2 also responds to catabolite repression. Elevated levels of cAMP, through formation of the CRPcAMP complex, caused a 21-fold downregulation of transcription from glnAp2 (Tian et al., 2001
). However, the mechanism by which the CRPcAMP complex affects transcription from glnAp2 remains unclear.
To elucidate the mechanism whereby carbon signals affect NtrC-dependent gene expression, we studied the in vivo response of the PII signalling system to different carbon sources. Expression of the NtrC-dependent genes glnA and glnK was monitored and compared to the uridylylation status of the PII proteins. All responses of glnA and glnK expression to carbon or cAMP signals were in accord with alterations in the uridylylation status of the PII proteins. We conclude that the effect of carbon source on NtrC-dependent gene expression is mediated through cAMP-dependent glutamine uptake, thereby affecting the glutamine-sensitive UTasePII signalling system.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Determination of the modification status of GlnB and GlnK.
The modification status of the PII proteins GlnB and GlnK was determined by non-denaturing polyacrylamide gel electrophoresis (Forchhammer et al., 1999) followed by immunodetection of the PII proteins using appropriate antibodies. The frozen cells (see above) were thawed and lysed in a buffer containing 50 mM Tris/HCl pH 7·4, 4 mM EDTA, 1 mM DTT, 0·5 mM benzamidine and 0·5 mM PMSF by grinding the cells with glass beads (0·11 mm; Sigma-Aldrich) in a Hybaid Ribolyser (Hybaid). Cell debris and glass beads were removed by centrifugation and the protein concentration in the supernatant was estimated according to the method of Bradford (1976)
. A total of 2·5 µg protein was loaded per lane for non-denaturing PAGE. Following electrophoresis, the proteins were blotted onto nitrocellulose membranes (BioTrace NT, Pall Corporation) and the blots were probed with specific anti-GlnB (see below) or anti-GlnK antibodies (Forchhammer et al., 1999
), which were subsequently visualized using peroxidase-conjugated secondary antibodies (Sigma-Aldrich) and a chemoluminescent substrate (Roche Diagnostics). GlnB-specific antibodies were raised in rabbits (Pineda Antibody Service, Berlin, Germany) against purified native GlnB protein that was produced in E. coli cells of strain HS10 overexpressing the glnB gene from plasmid pMP2 (Forchhammer et al., 1999
). The GlnB protein was purified essentially as described for the purification of GlnK (Forchhammer et al., 1999
). At a dilution of 1 : 50 000 (final concentration), the anti-GlnB serum did not show any cross-reactivity against GlnK, and, vice versa, at a dilution of 1 : 10 000, the anti-GlnK serum did not cross-react with GlnB under our assay conditions.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Accumulation of the GlnK protein (Fig. 2B) showed a similar pattern as observed with GSt activity in wild-type cells: glycerol as carbon source or the presence of cAMP repressed GlnK accumulation. In contrast to the constitutive high GSt activities in the glnB mutant, synthesis of GlnK was still repressed by cAMP or glycerol in this mutant. This might suggest that the GlnK protein, in the absence of GlnB, is sufficient to regulate its own synthesis although it is unable to regulate glnA expression properly. This conclusion is in accord with recent studies by Atkinson et al. (2002a)
, where they showed that the expression of glnK requires higher levels of activated NtrC than that of glnA. Therefore, a partial inhibition of NtrC activity by GlnK might be sufficient to decrease glnK expression although it might be insufficient to affect glnA expression. As expected, glnK was not expressed in the glnD mutant, in which GlnB is permanently unmodified.
The carbon/cAMP effect on GlnB signal transduction depends on extracellular glutamine
The above experiment showed that uridylylation of the GlnB signalling protein responds to the carbon source or to the cAMP levels and it strongly suggested that the GlnB signalling protein is required to transmit the carbon/cAMP signal towards NtrC-dependent gene expression. These conclusions imply that the signal of the carbon source is perceived through the GlnD enzyme (UTase/UR), which is known to respond to the cellular glutamine levels. Therefore, we speculated whether the observed carbon/cAMP effects might result from different permeability of the cells to the exogenous nitrogen source glutamine when growing with different carbon sources or in the presence of cAMP. To examine this possibility, the nitrogen source glutamine was replaced by limiting amounts of ammonium, which were adjusted such that they caused nitrogen-poor conditions. Preliminary experiments revealed that addition of 2 mM ammonium chloride led to derepression of glnA expression in glucose-supplemented minimal medium when cells were harvested at the mid-exponential phase of growth (OD600 approx. 0·4), which is also in accord with the results from Atkinson et al. (2002a). Under those conditions, using either glucose or glycerol as carbon source, the four strains (wild-type, glnB, glnD and glnK mutants) were grown in the presence or absence of 2 mM cAMP. From cells harvested at the mid-exponential phase of growth, GSt activity was determined (Table 4
) and the GlnB and GlnK proteins were analysed as above (Fig. 3
). Clearly, GSt activity in wild-type cells was not decreased by the addition of cAMP and was only slightly affected by the presence of glycerol. Similarly, the glnK mutant, which displayed a strong response to cAMP and to the carbon source in the presence of glutamine, no longer responded to the addition of cAMP. Its GSt activity in the presence of glucose was twofold elevated compared to the wild-type and was similar to the wild-type in the presence of glycerol, regardless of the presence of exogenous cAMP. The glnB mutant again exhibited constitutively high GSt activity and the glnD mutant showed low levels of GSt activity.
|
|
In the presence of cAMP, extracellular glutamine elicits a nitrogen-excess signal
Next, an experiment was performed to test whether exogenously added glutamine directly elicits a nitrogen-excess signal in the presence of cAMP. The cellular nitrogen status was monitored through determination of the adenylylation status of GS. Adenylylation of GS responds to the intracellular nitrogen status, determined by the cellular glutamine level, in a dual way: adenylylation is stimulated by non-uridylylated PII (which accumulates in the presence of elevated glutamine levels) and also responds directly to elevated glutamine levels (Jaggi et al., 1997; Jiang et al., 1998c
). Therefore, increased cellular glutamine pools are reflected by increased GS adenylylation. Cells of wild-type and of the glnB and glnK mutants were grown in ammonium-limited, glucose-supplemented medium, in the absence or presence of 2 mM cAMP. At the mid-exponential phase of growth, glutamine was added to the cultures (14 mM final concentration), and over a 20 min time period, samples were removed and the adenylylation state of GS was assayed. As shown in Fig. 4
(A), in wild-type cells, the adenylylation state of GS rapidly and permanently increased upon the addition of glutamine, but only when the cells had been grown in the presence of cAMP. By contrast, in the absence of cAMP, addition of glutamine caused only a slight and transient increase in GS adenylylation. In the GlnK-deficient strain, a similar cAMP-dependent GS adenylylation was observed (Fig. 4C
). In the glnB mutant, GS was already highly adenylylated at the beginning of the experiment (Fig. 4B
), since GlnB is required for the deadenylylation of GS under nitrogen-limiting conditions (Jiang et al., 1998c
; Forchhammer et al., 1999
). Thus, in the absence of GlnB, GS is highly adenylylated even under nitrogen-poor conditions, and no further increase can be observed upon the addition of glutamine.
|
|
|
Outlook
Analysing the carbon effect on the PII-signalling system and NtrC-dependent gene expression in the presence of glutamine as nitrogen source revealed an indirect effect of the carbon source on nitrogen regulation. This occurs by catabolite repression influencing glutamine uptake. If we wish to address the question concerning a direct integration of carbon and nitrogen signals by the PII protein, in vivo conditions have to be chosen that avoid the use of glutamine as nitrogen source. Limiting amounts of ammonium seem to be appropriate for this purpose. Under such conditions, however, glnA and glnK expression did not respond significantly to the nature of the carbon source, as shown in this study. Therefore, it remains to be demonstrated whether GlnB, in addition to signalling the nitrogen status by uridylylation, integrates signals from the carbon status of the cells in vivo.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arcondeguy, T. Jack R. & Merrick, M. (2001). PII signal transduction proteins: pivotal players in microbial nitrogen control. Microbiol Mol Biol Rev 65, 80105.
Atkinson, M. R. & Ninfa, A. J. (1998). Role of the GlnK signal transduction protein in the regulation of nitrogen assimilation in Escherichia coli. Mol Microbiol 29, 431447.[CrossRef][Medline]
Atkinson, M. A., Blauwkamp, T. A., Bondarenko, V., Studistky, V. & Ninfa, A. J. (2002a). Activation of the glnA, glnK and nac promoters as Escherichia coli undergoes the transition from nitrogen excess growth to nitrogen starvation. J Bacteriol 184, 53585363.
Atkinson, M. A., Blauwkamp, T. A. & Ninfa, A. J. (2002b). Context-dependent functions of the PII and GlnK signal transduction proteins in Escherichia coli. J Bacteriol 184, 53645375.
Backman, K., Chen, Y.-M. & Magasanik, B. (1981). Physical and genetic characterization of the glnA-glnG region of the Escherichia coli chromosome. Proc Natl Acad Sci U S A 78, 37433747.[Abstract]
Blauwkamp, T. A. & Ninfa, A. J. (2002a). Nac-mediated repression of the serA promoter of Escherichia coli. Mol Microbiol 45, 351363.[CrossRef][Medline]
Blauwkamp, T. A. & Ninfa, A. J. (2002b). Physiological role of the GlnK signal transduction protein of Escherichia coli: survival of nitrogen starvation. Mol Microbiol 46, 203214.[CrossRef][Medline]
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248254.[CrossRef][Medline]
Bueno, R., Pahel, G. & Magasanik, B. (1985). Role of glnB and glnD gene products in the regulation of the glnALG operon of Escherichia coli. J Bacteriol 164, 816822.[Medline]
Claverie-Martin, F. & Magasanik, B. (1991). Role of the integration host factor in the regulation of the glnHp2 promoter of Escherichia coli. Proc Natl Acad Sci U S A 88, 16311635.[Abstract]
Forchhammer, K., Hedler, A., Strobel, H. & Weiss, V. (1999). Heterotrimerization of PII-like signaling proteins: implications for PII-mediated signal transduction systems. Mol Microbiol 33, 338349.[CrossRef][Medline]
Jaggi, R., Ybarlucea, W., Chea, E., Carr, P. D., Edwards, K. J., Ollis, D. L. & Vasudevan, S. G. (1996). The role of the T-loop of the signal transduction protein PII from Escherichia coli. FEBS Lett 391, 223228.[CrossRef][Medline]
Jaggi, R., van Heeswijk, W. C., Westerhoff, H. V., Ollis, D. L. & Vasudevan, S. G. (1997). The two opposing activities of adenylyltransferase reside in distinct homologous domains, with intramolecular signal transduction. EMBO J 16, 55625571.
Jiang, P. & Ninfa, A. J. (1999). Regulation of the autophosphorylation of Escherichia coli nitrogen regulator II by the PII signal transduction protein. J Bacteriol 181, 19061911.
Jiang, P., Peliska, J. A. & Ninfa, A. J. (1998a). Enzymological characterization of the signal-transducing uridylyltransferase/uridylyl-removing enzyme (EC 2.7.7.59) of Escherichia coli and its interaction with the PII protein. Biochemistry 37, 1278212794.[CrossRef][Medline]
Jiang, P., Peliska, J. A. & Ninfa, A. J. (1998b). Reconstitution of the signal-transduction bicyclic cascade responsible for the regulation of Ntr gene transcription in Escherichia coli. Biochemistry 37, 1279512801.[CrossRef][Medline]
Jiang, P., Peliska, J. A. & Ninfa, A. J. (1998c). The regulation of Escherichia coli glutamine synthetase revisited: role of 2-ketoglutarate in the regulation of glutamine synthetase adenylylation state. Biochemistry 37, 1280212810.[CrossRef][Medline]
Liu, J. & Magasanik, B. (1993). The glnB region of the Escherichia coli chromosome. J Bacteriol 175, 74417449.[Abstract]
Magasanik, B. (1996). Regulation of nitrogen utilization. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, pp. 13441356. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
McFall, E. & Newman, E. B. (1996). Amino acids as carbon sources. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, pp. 358379. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Ninfa, A. J. & Atkinson, M. R. (2000). PII signal transduction proteins. Trends Microbiol 8, 172179.[CrossRef][Medline]
Pahel, G., Rothstein, D. M. & Magasanik, B. (1982). Complex glnA-glnL-glnG operon of Escherichia coli. Proc Natl Acad Sci U S A 76, 45444548.
Pioszak, A. A., Jiang, P. & Ninfa, A. J. (2000). The Escherichia coli PII signal transduction protein regulates the activities of the two-component system transmitter protein NRII by direct interaction with the kinase domain of the transmitter mode. Biochemistry 39, 1345013461.[CrossRef][Medline]
Reitzer, L. J. (1996). Ammonia assimilation and the biosynthesis of glutamine, glutamate, aspartate, asparagine, L-alanine and D-alanine. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, pp. 391407. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Reitzer, L. J. & Magasanik, B. (1985). Expression of glnA in Escherichia coli is regulated by tandem promoters. Proc Natl Acad Sci U S A 82, 19791983.[Abstract]
Rhee, S. G., Chock, P. B. & Stadtman, E. R. (1985). Glutamine synthetase from Escherichia coli. Methods Enzymol 113, 213241.[Medline]
Rippka, R. (1988). Isolation and purification of cyanobacteria. Methods Enzymol 167, 327.[Medline]
Saier, M. H., Ramseier, T. M. & Reizer, J. (1996). Regulation of carbon utilization. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, pp. 13251343. Edited by F. C. Neidhardt and others. Washington, DC: American Society for Microbiology.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Shapiro, B. M. & Stadtman, E. R. (1968). 5'-Adenylyl-O-tyrosine: the novel phosphodiester residue of adenylylated glutamine synthetase from Escherichia coli. J Biol Chem 243, 37693771.
Son, H. S. & Rhee, S. G. (1987). Cascade control of Escherichia coli glutamine synthetase. J Biol Chem 262, 86908695.
Stadtman, E. R. (1990). Discovery of glutamine synthetase cascade. Methods Enzymol 182, 793809.[Medline]
Stadtman, E. R., Smyrniotis, P. Z., Davis, N. J. & Wittenberger, M. E. (1979). Enzymatic procedures for determining the average state of adenylylation of Escherichia coli glutamine synthetase. Anal Biochem 95, 275285.[Medline]
Strobel, H. (1998). Die Rolle von GlnY, PII und GlnK bei der Stimulierung der regulierten Dephosphorylierung von NtrC-P und Kontrolle der Glutaminsynthetase Aktivität. PhD thesis, Universität Konstanz.
Tian, Z.-X., Li, Q.-S., Buck, M., Kolb, A. & Wang, Y.-P. (2001). The CRP-cAMP complex and downregulation of the glnAp2 promoter provides a novel regulatory linkage between carbon metabolism and nitrogen assimilation in Escherichia coli. Mol Microbiol 41, 911924.[CrossRef][Medline]
van Heeswijk, W. C., Hoving, S., Molenaar, D., Stegeman, B., Kahn, D. & Westerhoff, H. V. (1996). An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli. Mol Microbiol 21, 133146.[Medline]
Weiner, J. H. & Heppel, L. A. (1971). A binding protein for glutamine and its relation to active transport in Escherichia coli. J Biol Chem 246, 69336941.
Willis, R. C. & Furlong, C. E. (1975). Interactions of a glutamate-aspartate binding protein with the glutamate transport system of Escherichia coli. J Biol Chem 250, 25812586.[Abstract]
Willis, R. C., Iwata, K. K. & Furlong, C. E. (1975). Regulation of glutamine transport in Escherichia coli. J Bacteriol 122, 10321027.[Medline]
Received 30 April 2003;
accepted 6 May 2003.