Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, D-91058 Erlangen, Germany
Correspondence
Jörg Stülke
jstuelk{at}gwdg.de
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ABSTRACT |
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INTRODUCTION |
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For most bacteria, glutamine is an optimal source of nitrogen (Hu et al., 1999; Fisher & Débarbouillé, 2002
). It can easily be converted to glutamate, the major donor of nitrogen for amino acid and nucleotide biosyntheses. Thus, several mechanisms are operating to prevent the expression and activity of pathways devoted to the utilization of secondary nitrogen sources in the presence of glutamine (for a recent review, see Burkovski, 2003
). In enteric bacteria, the well-studied Ntr system controls both the biochemical activity of glutamine synthetase by adenylation/deadenylation and the expression of the corresponding gene by a two-component regulatory system, NtrB/NtrC (Merrick & Edwards, 1995
). In Gram-positive bacteria, different mechanisms of nitrogen regulation are found. In the high-G+C Gram-positive bacteria Corynebacterium glutamicum and Streptomyces coelicolor, glutamine synthetase activity is also controlled by adenylation/deadenylation (Jakoby et al., 1999
; Fink et al., 1999
). Transcription of nitrogen-responsive genes is controlled by the AmtR and GlnR regulators, respectively, in these bacteria (Jakoby et al., 2000
; Wray et al., 1991
; Fink et al., 2002
). In the low-G+C Gram-positive bacterium Bacillus subtilis, the activity of glutamine synthetase is not modulated by modification. In this organism, synthesis of glutamine synthetase is regulated by the repressor GlnR (Schreier et al., 1989
). In addition, the TnrA regulator controls expression of several genes involved in the utilization of secondary nitrogen sources as well as glnA encoding glutamine synthetase (Wray et al., 1996
; Fisher & Débarbouillé, 2002
). Interestingly, the activity of TnrA is modulated by reversible sequestration by glutamine synthetase itself (Wray et al., 2001
; Fisher et al., 2002
). In addition to glutamine synthetase, glutamate synthase encoded by the gltAB operon is essential for ammonium assimilation in B. subtilis (Fisher & Débarbouillé, 2002
). Expression of this operon is repressed by TnrA in the absence of ammonium and activated by the regulatory protein GltC in response to an as yet unknown signal (Bohannon & Sonenshein, 1989
; Belitsky et al., 2000
). Induction of the gltAB operon requires the presence of sugars that can be catabolized via glycolysis and this induction depends on the pleiotropic regulator of carbon metabolism, CcpA (Faires et al., 1999
; Blencke et al., 2003
; Wacker et al., 2003
).
An important component in signalling of nitrogen supply in both Gram-negative and Gram-positive bacteria is exemplified by the Escherichia coli PII protein, encoded by glnB. This small protein can be uridylated on a tyrosine residue under conditions of nitrogen limitation. The uridylated and non-uridylated forms of PII interact with other regulatory proteins that control either the activity of glutamine synthetase or the phosphorylation state of NtrC, thus relaying the information concerning the nitrogen status of the cell to these interaction partners (Merrick & Edwards, 1995; Arcondéguy et al., 2001
; Ninfa & Atkinson, 2000
). In cyanobacteria, PII is modified by phosphorylation on a serine residue (Forchhammer & Tandeau de Marsac, 1994
). A paralogous PII-like protein is present in E. coli and most other prokaryotes. This protein, GlnK, is usually encoded in an operon or gene cluster with an ammonium transporter, AmtB (Thomas et al., 2000b
). In E. coli, GlnK is also subject to modification by uridylation at low ammonium concentrations. At high ammonium concentrations, free GlnK binds to AmtB and prevents ammonium uptake by this transporter (Coutts et al., 2002
).
In B. subtilis, the nrgAB operon, encoding homologues of AmtB and GlnK, was identified in a search for genes that are induced by nitrogen limitation (Atkinson & Fisher, 1991). NrgA is a membrane protein, whereas NrgB is a member of the PII family (Wray et al., 1994
). In contrast to other PII proteins, NrgB does not possess one of the conserved uridylation or phosphorylation sites. Therefore, it is not known whether NrgB is covalently modified in response to the nitrogen source in B. subtilis. Moreover, there is no experimental evidence concerning the functions of the NrgA and NrgB proteins.
In this study, we analysed the functions of the two proteins encoded by the nrgAB operon. NrgA is required for the transport and utilization of ammonium at low concentrations. NrgB is localized in the membrane fraction of B. subtilis in an NrgA-dependent manner. Our data indicate that NrgB may be involved in the modulation of the activity of the transcription factor TnrA.
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METHODS |
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Transformation and characterization of the phenotype.
B. subtilis was transformed with plasmid DNA according to the two-step protocol (Kunst & Rapoport, 1995). Transformants were selected on SP plates containing kanamycin (5 µg ml-1) or chloramphenicol (5 µg ml-1). Quantitative assays of lacZ expression in B. subtilis were performed with cell extracts, using ONPG as the substrate (Kunst & Rapoport, 1995
).
Construction of reporter strains.
A translational nrgAlacZ fusion was constructed using the vector pAC7 (Weinrauch et al., 1991) which allows the introduction of the fusion into the amyE locus of B. subtilis. Briefly, the promoter region of the nrgAB operon was amplified by PCR using the oligonucleotides CD1 (5'-AAAGA ATTCT CCTTC CTTTC CATCC CTCG-3') and CD2 (5'-CATAA AAACT GGATC CCCCA TTTGC AT-3'). The PCR product was digested with EcoRI and BamHI and cloned into pAC7 linearized with the same enzymes. The resulting plasmid, pGP168, was linearized by PstI and used to transform B. subtilis 168 to give strain GP250.
Deletions of the chromosomal copies of nrgA and nrgB were obtained by cloning of a cat resistance gene encoding a chloramphenicol acetyltransferase in place of the central regions of the respective genes. The nrgA deletion was constructed as follows. A fragment containing the chromosomal region upstream of nrgA and the first 15 codons of nrgA was amplified using the primers CD26 (5'-AAAGG ATCCC TCAGT GTATT ATTTG ATGTA GTAC-3') and CD27 (5'-AAAGA ATTCG AGTAA AGCGC AAAAG AACAT AAA-3'), digested with EcoRI and BamHI and cloned into pBluescript-II SK (Stratagene) cut with the same enzymes to yield plasmid pGP179v. Then, the 3' part of nrgA was amplified using the primers CD28 (5'-AAAGA ATTCG GGCTT GACTT AACGA TGCAC GGGGA A-3') and CD29 (5'-AAAAA GCTTT TTTAC CGTCA CCTGG TGATC CCG-3'), cut with EcoRI and HindIII and cloned into pGP179v linearized with the same enzymes. The resulting plasmid was pGP179. Finally, the cat gene was amplified using the primers CATFORWARD (5'-AAAGA ATTCA AAATT GGATA AAGTG GGATA TTTTT A-3') and CATREV (5'-AAAGA ATTCC TATTA TAAAA GCCAG TCATT AGGCC T-3') and plasmid pHV33 (Primrose & Ehrlich, 1981) as the template. The PCR product was cloned into the single EcoRI site of pGP179 to give plasmid pGP184. To construct the nrgB deletion, we first amplified the region upstream of nrgB using the primers CD30 (5'-AAAGG ATCCC TTTGG CCTGC ACGGG ATCGG CGGCA C-3') and CD38 (5'-AAACA ATTGA CCGCT CATAG CGTCA CTCCT CAC-3'). This fragment was digested with MfeI (introduced with CD38) and ligated with the EcoRI-digested cat cassette. The ligation mix was used for a second round of PCR using the primers CD30 and CATREV. The region downstream of nrgB was obtained with the primer pair CD32 (5'-AAACA ATTGG GACCT GAAGC ACTTT AATAT CGGTA C-3') and CD34 (5'-CCATC GATCA TAGGC GACAA TGGCA TATTC GCTGA-3'). This fragment was cut with MfeI (introduced with CD32) and was ligated to the EcoRI-digested CD30/CATREV fragment. Finally, we performed a last round of PCR using CD30 and CD34. The resulting fragment contained the cat cassette flanked by the 5' and 3' parts of nrgB. This fragment was cut with BamHI and ClaI and cloned into pBluescript-II SK. The resulting plasmid was pGP183. The nrgAB operon deletion was constructed in a similar way. The upstream part of nrgA was amplified using the primers CD26 and CD37 (5'-AAACA ATTGG AGTAA AGCGC AAAAG AACAT AAA-3'). This fragment was digested with MfeI and ligated with the EcoRI-digested cat fragment. The ligation product was amplified using CD26 and CATREV. The nrgB downstream fragment was amplified and ligated to the CD26/CATREV fragment as described above. The obtained ligation product was amplified using CD26 and CD34 and cloned between the BamHI and ClaI sites of pBluescript-II SK to yield plasmid pGP185. To introduce the deletions into the chromosome, the plasmids were digested with ScaI and used to transform the appropriate B. subtilis strains (see Table 1
).
Overexpression and purification of NrgB.
To overexpress NrgB fused to a hexahistidine sequence at the C terminus, plasmid pGP182 was constructed as follows. A DNA fragment corresponding to the nrgB open reading frame was amplified by PCR using chromosomal DNA of B. subtilis 168 and the primer pair CD35 (5'-AAACA TATGA GCGGT CAAAT GTTCA AGGTA-3')/CD36 (5'-AAAAT GCATA GATCC ACGCG GAACA AGTGC TTCAG GTCCT TC-3'). The PCR product was digested with NdeI and NsiI, and the resulting fragment was cloned into the large fragment of the expression vector pGP807 (Schmalisch et al., 2002) cut with the same enzymes. This expression system is based on the strong T7 promoter and a lac operator.
For overexpression of the recombinant NrgB, E. coli BL21(DE3) was transformed with pGP182 and cultivated in LB at 28 °C. Expression was induced by the addition of IPTG (final concentration 1 mM) to exponentially growing cultures (OD600 of 0·8). Cells were harvested 2 h after induction, resuspended in a phosphate buffer (50 mM Na2HPO4, 300 mM NaCl, pH 6·5) and disrupted by using a French press (20 000 p.s.i., 138 000 kPa). The crude extract was passed over a Ni2+ HiTrap chelating column (Pharmacia) followed by elution with an imidazole gradient. The Bio-Rad dye-binding assay was used to determine protein concentration. BSA was used as the standard.
Preparation of membrane fractions.
Cultures of B. subtilis were harvested by centrifugation (4400 g, 10 min, 4 °C). The following steps were done as described by Coutts et al. (2002). Briefly, the cells were lysed by sonication, the cellular debris removed, and the fractions of the cell extract were separated by ultracentrifugation. The membrane pellet was washed three times and finally resuspended in phosphate buffer (50 mM Na2HPO4, 50 mM NaH2PO4, pH 6·8).
Western blot analysis.
Purified NrgBhexahistidine was used to generate rabbit polyclonal antibodies. For Western blot analysis, B. subtilis cell extracts and cellular fractions were separated on 12·5 % SDS-PAGE gels. After electrophoresis, the proteins were transferred to a PVDF membrane (Bio-Rad) by electroblotting. Antibodies were visualized by using anti-rabbit IgGalkaline phosphatase secondary antibodies (Chemikon International) and the CDP* detection system (Roche Diagnostics). For control experiments, the presence of glyceraldehyde-3-phosphate dehydrogenase was assayed using polyclonal antibodies directed against B. subtilis GapA (Meinken et al., 2003).
Assays of methylammonium transport.
B. subtilis strains were grown in SMM in the presence of glutamine (0·2 %, w/v) as the single source of nitrogen. If the culture had reached an OD600 value of 0·9, the cells were centrifuged and resuspended in SMM without any source of nitrogen. Nitrogen starvation was exerted for 210 min and transport assays were performed as described previously (Ludwig et al., 2002). Labelled [14C]methylamine hydrochloride (56 mCi mmol-1, 2·07 GBq mmol-1) was mixed with non-labelled methylamine hydrochloride and added to the cells (final concentration 7·24 µM). Samples were treated as described previously (Ludwig et al., 2002
).
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RESULTS |
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The strains were grown in minimal medium in the presence of ammonium. At a concentration of 2 mM ammonium, the wild-type and the nrgA and
nrgB mutant strains grew with virtually identical rates and reached the same final optical density. Similarly, expression of the nrgAlacZ fusion was induced at the onset of the stationary phase to a comparable extent (Fig. 1
a). Obviously, neither NrgA nor NrgB played a significant role under the conditions used in this experiment. This may result from the fact that the ammonium supplied is present in the forms of uncharged ammonia (NH3) and charged ammonium (
) in the growth medium. The actual concentration of the two species depends on two factors: the pH of the culture and the total ammonium concentration added to the medium. The minimal medium is regularly buffered with phosphate to get a neutral pH value (pH 6·9). At this pH (and 2 mM total ammonium), the concentration of uncharged ammonia is about 11·2 µM. However, ammonia may freely diffuse through the membrane (Kleiner, 1985
), thus circumventing the need for a functional ammonium transporter. We performed therefore further growth experiments at the same ammonium concentration (2 mM) and at acidic pH values at which substantially less uncharged ammonia is present. At a pH of 5·5 ([NH3]=350 nM), both the wild-type strain GP250 and the
nrgA mutant GP254 were able to grow; however, the
nrgA mutant grew more slowly (generation time of 156 min vs 93 min for the wild-type strain, see Table 2
). Thus, the availability of ammonium may be limiting for the
nrgA mutant. This assumption is further supported by the finding that the expression of the nrgAlacZ fusion is much stronger in the mutant than in the wild-type strain (see Fig. 1b
). At a pH value of 5·0 ([NH3]=110 nM), the wild-type grew, although with an increased generation time (see Table 2
). In contrast, the
nrgA mutant was not able to grow under these conditions (Fig. 1c
). The same was true for the
nrgAB double mutant, which was phenotypically very similar to the
nrgA mutant (see Table 2
).
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Studies of ammonium transport in B. subtilis
Two lines of evidence suggested that NrgA might function as an ammonium transporter: first, it is a member of the Mep/Amt family of transporters (Thomas et al., 2000a) and, second, our growth experiments indicated a need for a functional NrgA if ammonium is the single nitrogen source. To test the role of NrgA in ammonium transport, we assayed the uptake of a radioactively labelled ammonium analogue, [14C]methylammonium.
The wild-type strain GP250 showed very weak methylammonium uptake after growth in a medium containing the preferred nitrogen source, glutamine. In contrast, the transport was strongly induced after the cultures were exposed to nitrogen starvation (see Fig. 2). This result is in good agreement with the previously reported repression of the nrgAB operon by glutamine (Atkinson & Fisher, 1991
). The
nrgA mutant strain GP254 was drastically impaired in the transport of methylammonium even after nitrogen starvation. The ammonium transport of the
nrgB mutant strain GP253 was nearly identical to that of the wild-type strain. Similarly, the
nrgAB double mutant GP255 showed uptake rates of methylammonium indistinguishable from the
nrgA mutant (Fig. 2
). Taken together, these results indicate that NrgA is required for the uptake of methylammonium and, therefore, probably also of ammonium. In contrast, NrgB, encoded by the second gene of the nrgAB operon, does not seem to be involved in ammonium transport under our experimental conditions.
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First, we had to ascertain that nrgB was expressed in the nrgA mutant strain GP254. For this purpose, cell extracts were prepared from the wild-type (GP250) and
nrgA (GP254),
nrgB (GP253) and
nrgAB (GP255) mutant strains after growth in SMM supplemented with glutamate or glutamine. The extracts were subjected to Western blot analysis using polyclonal antibodies directed against B. subtilis NrgB (Fig. 3
). As expected, NrgB was detectable in the wild-type strain only after growth with the poor nitrogen source glutamate. No NrgB was detected in the
nrgB and
nrgAB mutant strains, indicating that the antibodies were specific. In the
nrgA mutant, NrgB was expressed under nitrogen-limited conditions. Thus, this mutant could be used to test the presumptive role of NrgA in membrane localization of NrgB.
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Next, we asked whether NrgB would be involved specifically in the control of the nrgAB operon or more generally in the regulation of nitrogen-controlled promoters. This was tested by studying the expression of the gltAB operon encoding glutamate synthase in the wild-type and nrgA or
nrgB mutants. Expression of the gltAB operon is repressed by the transcription regulator TnrA, whereas transcription of the nrgAB operon is activated by TnrA under conditions of nitrogen limitation. Assays of
-galactosidase expression in strains containing a fusion of the gltAB promoter to the lacZ gene were performed after growth under nitrogen-limited conditions (2 mM ammonium, pH 5·5). The expression of the gltAB operon was not affected by the
nrgB mutation (552 units of
-galactosidase in strain GP256 vs 464 units in the wild-type strain GP342). In contrast, the expression was reduced in the
nrgA mutant GP257 (212 units). This may result from the lack of internal ammonium due to the transport deficiency of the mutant. Thus, we may conclude that NrgB does not affect the regulation of all TnrA-regulated genes (see Discussion).
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DISCUSSION |
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In B. subtilis, glutamine synthesis requires the activity of glutamine synthetase since the glutamate dehydrogenase in this organism was proposed to have a catabolic rather than an anabolic function (Fisher & Débarbouillé, 2002; Belitsky & Sonenshein, 1998
). In addition, glutamate synthase and ammonium transport are required to provide the substrates for the glutamine synthetase reaction. While both the activity and expression of glutamine synthetase and glutamate synthase have been the subject of extensive previous work (reviewed by Fisher & Débarbouillé, 2002
; Belitsky, 2002
; see also Faires et al., 1999
; Wray et al., 2001
; Wacker et al., 2003
), ammonium uptake has not yet been studied in B. subtilis.
Two lines of evidence suggest that NrgA acts as the major ammonium transporter in B. subtilis. First, nrgA mutants are unable to utilize ammonium as the single source of nitrogen at low concentrations. Second, the
nrgA mutant strain is drastically impaired in the transport of methylammonium. This idea has, however, an implication regarding the transport of ammonia. Normally, ammonia and ammonium are present in equilibrium in all solutions. Only at low pH is the equilibrium shifted towards ammonium. Our findings suggest that ammonia may enter the cell by diffusion through the cytoplasmic membrane independent from the ammonium transporter NrgA. As found in this study, an active ammonium transporter (AmtB) is required for ammonium uptake at low concentrations in the nitrogen-fixing proteobacterium Azospirillum brasilense (van Dommelen et al., 1998
). Interestingly, the high-G+C Gram-positive bacterium C. glutamicum possesses two ammonium transporters: the amt gene encodes an ammonium/methylammonium transporter, whereas the AmtB protein in this organism is highly specific for ammonium. The existence in C. glutamicum of a third, as yet uncharacterized, system for ammonium uptake has been suggested (Siewe et al., 1996
; Jakoby et al., 2000
; Meier-Wagner et al., 2001
). In contrast, the AmtB transporter of E. coli was proposed to act specifically as an ammonia rather than an ammonium transporter (Soupene et al., 1998
).
In E. coli, the activity of the AmtB ammonium transporter is modulated by a small regulatory protein, GlnK. At high ammonium concentrations, the latter protein is present in its free (non-uridylated) form and binds AmtB to prevent further ammonium uptake (Coutts et al., 2002). The B. subtilis orthologue of GlnK, NrgB, lacks the conserved modification sites which are targets of either uridylation or phosphorylation (Atkinson & Ninfa, 1999
; Forchhammer & Tandeau de Marsac, 1994
; Wray et al., 1994
). However, the co-ordinated expression of the genes encoding the ammonium transporter NrgA and the presumptive regulator NrgB in the nitrogen-regulated nrgAB operon suggested that the two proteins might act in a common function. Our findings clearly indicate that NrgB is not required for ammonium transport. As observed in E. coli, NrgB is present in the membrane fraction of B. subtilis. This interaction depends on the presence of a functional NrgA, suggesting a direct physical interaction between the two proteins. In contrast to the findings in E. coli, the B. subtilis NrgB binds to the membrane in an NrgA-dependent manner even under conditions of ammonium limitation (see Fig. 5a
; Coutts et al., 2002
). Under such conditions, GlnK of E. coli is uridylated and unable to bind AmtB. Thus, the proposed permanent interaction between NrgA and NrgB is in good agreement with the hypothesis that NrgB is not subject to covalent modification in B. subtilis.
The permanent binding of NrgB to NrgA raises the question concerning the function of this interaction. Our experiments did not indicate any growth disadvantages of the nrgB mutant strains under the tested conditions. In a previous study, a role of NrgB was proposed for the utilization of nitrate (Wray et al., 1994
). Here, we observed that the extent of activity of the nrgAB operon promoter under nitrogen-limited conditions was reduced in the
nrgB mutant. As both nitrate utilization and nrgAB expression depend on transcriptional activation by TnrA we may speculate that NrgB might be involved in fine-tuning the activity of the transcription factor TnrA in response to the availability of ammonium. Moreover, NrgB might negatively affect the activity of NrgA as indicated by the reduced expression of nrgA in the
nrgB mutant while the growth and transport properties of the wild-type and
nrgB mutant strains are virtually identical. Our results with the gltAB operon did not reveal a major role of NrgB in the regulation of its transcription. This discrepancy as compared to the nrgAB operon may reflect the regulation factors by TnrA: while nrgAB expression is activated by TnrA about 1000-fold, the gltAB operon is repressed about 10-fold by TnrA (Wray et al., 1996
; Belitsky et al., 2000
). Thus, a role for NrgB may be confined to the strongly regulated genes or the regulatory effect may be too small to be detected. Clearly, more work is required to elucidate the putative regulatory role of NrgB.
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ACKNOWLEDGEMENTS |
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Received 23 May 2003;
revised 22 July 2003;
accepted 4 August 2003.
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