Department of Biological Chemistry, University of Copenhagen, DK-1307 Copenhagen K, Denmark1
Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark2
Department of Genetics, Trinity College, Dublin 2, Ireland3
Author for correspondence: Per Nygaard. Tel: +45 35322005. Fax: +45 35322040. e-mail: nygaard{at}mermaid.molbio.ku.dk
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ABSTRACT |
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Keywords: guanine deaminase, purine catabolism, nitrogen metabolism
Abbreviations: GDEase, guanine deaminase; XDHase, xanthine dehydrogenase
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INTRODUCTION |
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The three regulatory proteins, GlnA, GlnR and TnrA, are important for the regulation of expression of nitrogen metabolism genes of B. subtilis (Schreier, 1993 ; Wray et al., 1996
; Fisher, 1999
). GlnA, glutamine synthetase, is suggested to generate a metabolic signal that during excess nitrogen conditions, as when glutamine or ammonia serve as the nitrogen source, activates GlnR and inhibits TnrA activity. During nitrogen-limiting conditions, as when glutamate or allantoin serve as the nitrogen source, the signal is not generated. As a result of this, GlnR is inhibited and TnrA activated. GlnR is a repressor of the glnRA operon and during excess nitrogen conditions glnRA expression is low. During nitrogen-limiting conditions, glnRA expression increases due to the relief from repression by GlnR (Wray et al., 1996
). Under excess nitrogen conditions GlnR also represses tnrA and ureABC expression (Wray et al., 1997
; Fisher, 1999
). TnrA is a transcription factor (Wray et al., 2000
) required for the activation of several genes that encode transport proteins and enzymes involved in the uptake and degradation of nitrogen-containing compounds such as nitrate, urea, asparagine and
-aminobutyrate. TnrA positively regulates its own synthesis during nitrogen limitation (Wray et al., 1996
, 1997
; Fisher, 1999
). TnrA-dependent genes are constitutively expressed in a glnA genetic background, which requires glutamine for growth (Wray et al., 1996
). GlnR and TnrA share significant amino acid sequence similarity in the amino-terminal part, which encodes the DNA-binding motif, and they both recognize the same operator sequence (5'-TGTNA-N7TNACA-3').
This study was carried out to analyse, and to obtain better understanding of, how B. subtilis directs the channeling of purines into either salvage or degradation pathways. Guanine deaminase, on which we report here, catalyses the deamination of guanine to xanthine, which is the first step in guanine degradation. The level of activity is increased when cells are grown on a poor nitrogen source and is induced by purines in the medium.
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METHODS |
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Enzyme assays.
Cells were harvested in mid-exponential growth phase and homogenized by sonication in 30 mM phosphate buffer (pH 7·5), 1 mM EDTA and 1 mM DTT. Cell debris was removed by centrifugation. Guanine deaminase was measured by the following procedure: an assay mixture composed of 4 µl 1 mM guanine, 0·5 µl 2 mM 14C-labelled guanine (2 GBq mmol-1), 35·4 µl 100 mM phosphate buffer (pH 7·2) and 0·1 µl bovine xanthine oxidase (Boehringer Mannheim) was mixed at 37 °C. Ten microlitres of cell extract was added and after 1, 2, 4 and 8 min 10 µl samples were removed and spotted on a polyethyleneimine-impregnated thin-layer chromatography plate (Merck). The plate was dried and developed in methanol (3 cm) and to the top in water to separate guanine from the degradation products formed in the assay mixture. The plate was dried again and the radioactivity in the different spots (guanine, xanthine, uric acid and allantoin) were determined in an Instant Imager (Packard). ß-Galactosidase and XDHase activity were determined as described previously (Christiansen et al., 1997 ). Enzyme activity is given as nmol product min-1 (=1 unit). Values given are the means of three to five different experiments±SD. Total protein was determined by the Lowry method.
Construction of a transcriptional gdelacZ fusion.
A 196 bp region of the B. subtilis chromosome containing the gde promoter was amplified by PCR. Two primers were used: primer-1, 5'-GCCGGAATTC-G1382763GTTTTTTTCTATAATACAGCC-3' and primer-2, 5'-GCGGGATCC-C1382567ACTCCTTCACATGCG-3' (Kunst et al., 1997 ). Primer-1 was fitted with an EcoRI restriction site and primer-2 with a BamHI site (underlined). The DNA fragment was digested with EcoRI and BamHI and ligated into pDG268neo digested with the same enzymes. The ligation mixture was transformed into E. coli, selecting for ampicillin resistance. A plasmid with the cloned and correct fragment was linearized by digestion with KpnI and transformed into B. subtilis 168, selecting for neomycin resistance. The resulting strain (HH353) was amylase-negative, indicating the correct integration of the plasmid into the amyE locus.
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RESULTS |
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Our results indicate that high levels of gde expression in B. subtilis require not only nitrogen-limiting growth conditions, but also the presence of a purine catabolic-pathway-specific induction signal. All compounds tested that can be degraded to allantoic acid act as inducers. This indicates that allantoic acid, and probably also allantoin, are inducer molecules. We observed that addition of purines and purine degradative compounds to glutamate-grown cultures had a significant growth-stimulating effect. Culture doubling time in glutamate was 151 min; however, in the presence of both glutamate and allantoin the doubling time was reduced to 62 min. When ammonia served as the nitrogen source, the doubling time was 66 min. It appears that we have a situation in which the cells, sensing nitrogen limitation, induce a degradation pathway that slowly liberates ammonia and as a result of this the growth rate is increased. We therefore define a new term, semi-excess nitrogen, to describe this nitrogen situation.
Regulators involved in the control of gde expression
As outlined in the Introduction, GlnA (glutamine synthetase), GlnR (glnRA repressor) and TnrA (nitrogen catabolism activator) are the key proteins responsible for the nitrogen catabolite control of gene expression in B. subtilis. The role of GlnA, GlnR and TnrA in the control of gde expression were examined by measuring the ß-galactosidase levels produced from the transcriptional gdelacZ fusion in mutant strains grown under various nitrogen conditions.
As shown in Table 4, gde expression in the glnA strain (ED427), which requires glutamine for growth, was increased 24-fold relative to the level in the wild-type grown with glutamine (Table 3
). Whilst the induction by allantoin in the wild-type strain is repressed during growth on glutamine (Table 3
), gde expression in the glnA strain was induced 20-fold by allantoin. This 20-fold induction equals that by allantoin observed in the wild-type strain grown with glutamate as the nitrogen source (Table 3
). Due to the loss of inhibition of TnrA activity by GlnA, TnrA-activated genes are expressed at a high constitutive level in a glnA genetic background (Wray et al., 1996
). To test whether the increased level of gde expression in the glnA strain was mediated through TnrA, gde expression was analysed in a glnA tnrA double mutant (ED432). As shown in Table 4
, gde expression in glutamine-grown cells was reduced to the basal level, 4 units (mg protein)-1, and addition of allantoin increased the expression to only 23 units (mg protein)-1 compared with 1748 units (mg protein)-1 in the glnA strain. In a tnrA single mutant (ED429, Table 4
), the level on glutamate was low, 36 units (mg protein)-1, and induction of gde expression by allantoin was also significantly reduced (a threefold induction compared with a tenfold induction in the wild-type). We therefore conclude that induction of gde expression during nitrogen-limiting conditions in the presence of allantoin is TnrA dependent. Surprisingly, the level of gde expression in a glnR mutant was low, 33 units (mg protein)-1, and induction by allantoin was also affected (a sixfold induction in strain ED428 compared with a tenfold increase in the wild-type, Table 3
). Induction by allantoin was also decreased in glnA glnR (ED433) and tnrA glnR (ED431) double mutants and in the glnA tnrA glnR triple mutant (ED434). Therefore, the GlnR regulator seems to have a positive effect on gde expression, although not as strong as TnrA. In summary, we conclude that gde expression is subjected to global nitrogen catabolite repression mediated through the GlnATnrA signalling pathway and that the purine catabolic-pathway-specific induction requires TnrA and GlnR. Since there is no TnrA/GlnR-binding site located in the promoter region upstream of gde, the TnrA and GlnR effects are most likely indirect.
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Are the levels of GDEase and XDHase co-regulated?
Both enzymes are part of the purine degradation pathway (Fig. 1). Previously we found that the level of XDHase is high, 26 units (mg protein)-1, in a GlnA mutant and <0·3 units (mg protein)-1 in a wild-type cell grown on glutamine. When grown on hypoxanthine as the sole nitrogen source, the level was 24 units (mg protein)-1 (Christiansen et al., 1997
). To test whether the synthesis of XDHase was co-regulated with the expression of the gde gene, we determined the level of XDHase in strain HH353 grown with glutamate and with glutamate plus allantoin or hypoxanthine. On glutamate and on glutamate plus hypoxanthine the level was high, 16 units (mg protein)-1 and 9 units (mg protein)-1, respectively. When both glutamate and allantoin were present, the expression was low, 0·4 units (mg protein)-1. gde expression and the level of XDHase are therefore not co-regulated. A likely explanation is that under the semi-excess-nitrogen situation, in the presence of allantoin, where XDHase is not required, the cell saves energy by not synthesizing XDHase and the molybdenum enzymes required for the synthesis of XDHase (Leimkühler et al., 1998
). However, when hypoxanthine serves as the nitrogen source, XDHase is needed and is synthesized.
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DISCUSSION |
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Interestingly, the doubling time of a B. subtilis wild-type culture grown in the presence of glutamate plus allantoin (62 min) is more similar to the doubling time of ammonia-grown cells (66 min) than to the doubling time of glutamate-grown cells (151 min). We defined the term semi-excess-nitrogen growth state to describe the conditions in glutamate- plus allantoin-grown cells and raise the question whether growth of glutamate- plus allantoin-supplemented cells actually is nitrogen limited. We present data that indicate that the semi-excess-nitrogen growth state, which is also seen when purines and intermediary compounds of purine degradation are added, represents a situation during which factors associated with excess nitrogen conditions (GlnR) and limiting nitrogen conditions (TnrA) act simultaneously. The current model for nitrogen catabolite repression states that TnrA is only activated in slow-growing nitrogen-limited cells. We observed that TnrA-dependent expression of gde occurs in cells growing with a fast growth rate (semi-excess-nitrogen growth conditions). The model for nitrogen catabolite repression in B. subtilis furthermore predicts that GlnR is active only during excess nitrogen conditions; however, we observed a small but significant effect of GlnR on gde expression during semi-excess-nitrogen growth conditions.
The available information on the regulation of expression of genes in B. subtilis encoding enzymes involved in the degradation of nitrogen containing compounds as reviewed by Fisher (1999) has some common traits, but, also shows some major differences. For genes encoding enzymes involved in purine degradation, the expression of the ade gene, encoding adenine deaminase, is not regulated by the nitrogen source in the growth medium (Nygaard et al., 1996
). The expression of the yet unknown gene encoding XDHase, as well as the ureABC operon (Wray et al., 1997
) and the gde gene, all are subjected to a regulatory control exerted by the GlnA protein (Wray et al., 1996
). Whilst the expression of the ureABC operon is directly regulated by the TnrA protein, the gde gene most likely is indirectly controlled by both the TnrA and the GlnR proteins. The expression of the ureABC operon, in addition, is directly controlled by GlnR and affected by the CodY protein, but not by urea and compounds that can give rise to urea. In contrast, the gde gene is induced by purines and intermediary compounds of the purine catabolic pathway. The expression of the gene encoding XDHase is also high under semi-excess-nitrogen conditions, but only in the presence of purine bases.
This study led us to propose a model for the channeling of guanine in B. subtilis. When nitrogen sources are abundant, GDEase is not synthesized and guanine when present is converted to GMP. Under these conditions, induced synthesis of GMP reductase is observed (Nygaard, 1993 ) and as a result of this the cell can now convert GMP to AMP via IMP (Fig. 1
). During limiting nitrogen conditions (glutamate), GDEase and XDHase are synthesized and guanine can now be deaminated to xanthine, which can be metabolized by two routes, either converted to XMP, catalysed by xanthine phosphoribosyltransferase, and further to GMP, or oxidized by XDHase to uric acid. When glutamate plus purines serve as the nitrogen source, the GDEase level is increased, the level of XDHase is still high and the level of xanthine phosphoribosyltransferase is reduced (Christiansen et al., 1997
). This situation favours purine degradation. In agreement with the different roles of GDEase in bacteria and mammals is the fact that the amino acid sequence of the bacterial and mammalian enzyme shows no homology, indicating different ancestor molecules.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Ashihara, H. & Crozier, A. (1999). Biosynthesis and metabolism of caffeine and related purine alkaloids in plants. Adv Bot Res 30, 118-205.
Atkinson, M. R. & Fisher, S. (1991). Identification of genes and gene products whose expression is activated during nitrogen-limited growth in Bacillus subtilis. J Bacteriol 173, 23-27.[Medline]
Bairoch, A., Bucher, P. & Hofman, K. (1997). The PROSITE database, its status in 1997. Nucleic Acid Res 25, 217-221.
Bhattacharya, S., Navaratnam, N., Morrison, J. R. & Scott, J. (1994). Cytosine nucleoside/nucleotide deaminases and apolipoprotein B mRNA editing. Trends Biochem Sci 19, 105-106.[Medline]
Bongaerts, G. P. A., Uitzetter, J., Brouns, R. & Vogels, G. D. (1978). Uricase of Bacillus fastidiosus, properties and regulation of synthesis. Biochim Biophys Acta 527, 348-358.[Medline]
Brown, S. W. & Sonenshein, A. B. (1996). Autogenous regulation of the Bacillus subtilis glnRAoperon. J Bacteriol 178, 2450-2454.[Abstract]
Christiansen, L. C., Schou, S., Nygaard, P. & Saxild, H. H. (1997). Xanthine metabolism in Bacillus subtilis: characterization of the xpt-pbuX operon and evidence for purine- and nitrogen-controlled expression of genes involved in xanthine salvage and catabolism. J Bacteriol 179, 2540-2550.[Abstract]
Cruz-Ramos, H., Glaser, P., Wray, L. V. & Fisher, S. H. (1997). The Bacillus subtilis ureABC operon. J Bacteriol 179, 3371-3373.[Abstract]
DeMoll, E. & Auffenberg, T. (1993). Purine metabolism in Methanococcus vannielii. J Bacteriol 175, 5754-5761.[Abstract]
Erbs, P., Exinger, F. & Jund, R. (1997). Characterization of the Saccharomyces cerevisiae FCY1 gene encoding cytosine deaminase and its homologue FCA1 of Candida albicans. Curr Genet 31, 1-6.[Medline]
Fisher, S. (1999). Regulation of nitrogen metabolism in Bacillus subtilis: vive la différence! Mol Microbiol 32, 223-232.[Medline]
Krug, E. C., Marr, J. J. & Berens, R. L. (1989). Purine metabolism in Toxoplasma gondii. J Biol Chem 264, 10601-10607.
Kunst, F., Ogasawara, N., Moszer, I. & 148 other authors (1997). The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390, 249256.[Medline]
Leimkühler, S., Kern, M., Solomon, P. S., McEwan, A. G., Schwarz, G., Mendel, R. R. & Klipp, W. (1998). XDHase from the phototrophic purple bacterium Rhodobacter capsulatus is more similar to its eukaryotic counterparts than to prokaryotic enzymes. Mol Microbiol 27, 853-869.[Medline]
Magill, C. W., Sabina, R. L., Garber, T. L. & Magill, J. M. (1982). Guanine uptake and metabolism in Neurospora crassa. J Bacteriol 149, 941-947.[Medline]
Nygaard, P. (1983). Utilization of preformed purine bases and nucleosides. In Metabolism of Nucleotides, Nucleosides and Nucleobases in Microorganisms , pp. 27-93. Edited by A. Munch-Petersen. New York:Academic Press.
Nygaard, P. (1993). Purine and pyrimidine salvage pathways. In Bacillus subtilis and Other Gram-positive Bacteria , pp. 359-378. Edited by A. L. Sonenshein, J. A. Hoch & R. Losick. Washington, DC:American Society for Microbiology.
Nygaard, P., Duckert, P. & Saxild, H. H. (1996). Role of adenine deaminase in purine salvage and nitrogen metabolism and characterization of the ade gene in Bacillus subtilis. J Bacteriol 178, 846-853.[Abstract]
Saxild, H. H. & Nygaard, P. (1987). Genetic and physiological characterization of Bacillus subtilis mutants resistant to purine analogs. J Bacteriol 169, 2977-2983.[Medline]
Saxild, H. H., Jacobsen, J. H. & Nygaard, P. (1995). Functional analysis of the Bacillus subtilis purT gene encoding formate-dependent glycinamide ribonucleotide transformylase. Microbiology 141, 2211-2218.[Abstract]
Schreier, H. J. (1993). Biosynthesis of glutamine and glutamate and the assimilation of ammonia. In Bacillus subtilis and Other Gram-positive Bacteria , pp. 281-298. Edited by A. L. Sonenshein, J. A. Hoch & R. Losick. Washington, DC:American Society for Microbiology.
Stuer-Lauridsen, B. & Nygaard, P. (1998). Purine salvage in two halophilic archaea: characterization of salvage pathways and isolation of mutants resistant to purine analogs. J Bacteriol 180, 457-463.
Vagner, V., Dervyn, E. & Ehrlich, D. (1998). A vector for systematic gene inactivation in Bacillus subtilis. Microbiology 144, 3097-3104.[Abstract]
Vogels, G. D. & van der Drift, C. (1976). Degradation of purines and pyrimidines by microorganisms. Bacteriol Rev 40, 403-468.[Medline]
Wipat, A. & Harwood, C. (1999). The Bacillus subtilis genome sequence: the molecular blueprint of a soil bacterium. FEMS Microbiol Ecol 28, 1-9.
Wray, L. V., Ferson, A. E., Rohrer, K. & Fisher, S. H. (1996). TnrA, a transcription factor required for global nitrogen regulation in Bacillus subtilis. Proc Natl Acad Sci USA 93, 8841-8845.
Wray, L. V.Jr, Ferson, A. E. & Fisher, S. H. (1997). Expression of the Bacillus subtilis ureABC operon is controlled by multiple regulatory factors including CodY, GlnR, TnrA, and Spo0H. J Bacteriol 179, 5494-5501.[Abstract]
Wray, L. V.Jr, Zalieckas, J. M. & Fisher, S. H. (2000). Purification and in vitro activities of the Bacillus subtilis TnrA transcription factor. J Mol Biol 300, 29-40.[Medline]
Yuan, G., Bin, J. C., McKay, D. J. & Snyder, F. F. (1999). Cloning and characterization of human guanine deaminase. J Biol Chem 274, 8175-8180.
Received 13 June 2000;
revised 7 August 2000;
accepted 25 August 2000.