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1 Department of Molecular and Cell Biology, University of Cape Town, Rondebosch 7701, South Africa
2 Department of Food and Agricultural Sciences, Cape Technikon, Cape Town 8001, South Africa
Correspondence
Sharon J. Reid
Shez.Reid{at}uct.ac.za
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the gltAB sequence of Clostridium saccharobutylicum is AF082880.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Schubert, 2006), but the economic viability of any such process is dependent on product yield. The closely related saccharolytic Clostridium species, Clostridium saccharobutylicum NCP262 and Clostridium beijerinckii NCIMB 8052 (both formerly known as Clostridium acetobutylicum; Keis et al., 1995), are obligately anaerobic endospore-forming bacteria that produce acetone and butanol as fermentation end-products (Jones & Woods, 1986). C. saccharobutylicum NCP262 has long been recognized as one of the best industrial strains for the production of solvents, but many aspects of the fundamental metabolism of the solvent-producing clostridia remain poorly characterized. Several studies have demonstrated that solvent yields are significantly affected by the nitrogen source (Long et al., 1984; Monot & Engasser, 1983), while C. saccharobutylicum NCP262 did not produce solvents in ammonia-limited cultures (Gottschal & Morris, 1981; Long et al., 1984).
Most bacteria possess two primary pathways for the assimilation of ammonia, the energy-dependent glutamine synthetase (GS)/glutamate synthase (GOGAT) pathway and the energy-conserving glutamate dehydrogenase (GDH) pathway (Merrick & Edwards, 1995). The tightly coupled GS/GOGAT pathway catalyses the synthesis of glutamine and glutamate, effectively cycling these key metabolites from which nearly all other cellular nitrogen-containing compounds are derived: GS catalyses the amidation of endogenous glutamate to form glutamine, and GOGAT catalyses the transamidation of the amide group from glutamine to 2-oxoglutarate to form glutamate. GDH produces glutamate from 2-oxoglutarate. In some organisms, including Bacillus subtilis (Fisher & Sonenshein, 1991), Clostridium pasteurianum (Dainty, 1972) and Clostridium thermoautotrophicum (Bogdahn & Kleiner, 1986), the GS/GOGAT pathway is solely responsible for the assimilation of ammonia into organic compounds. Bacteria growing with preferred nitrogen sources, i.e. those that support the fastest cell growth rate, generally contain low levels of GS and GOGAT, whereas high levels of these enzymes are present during growth under nitrogen-limited conditions to ensure adequate supplies of glutamine and glutamate.
The global regulation of these nitrogen-assimilatory enzyme activities is generally tightly controlled in response to nitrogen and energy conditions. The arrangement of nitrogen metabolism genes varies in different species and there are no reports to date in which the genes encoding GS and GOGAT, glnA and gltAB respectively, have been found to be physically linked on the genome. In the Enterobacteriaceae, the glnA gene forms an operon with the ntrB and ntrC genes which control the regulation of GS activity (Merrick & Edwards, 1995). In B. subtilis, three independent regulatory proteins, GlnR, TnrA and CodY, function under different nutritional conditions to control nitrogen assimilation (Fisher, 1999; Belitsky, 2002). In Streptomyces coelicolor and Corynebacterium, the glnA transcription unit is monocistronic, and is not adjacent to regulatory genes on the genome (Fisher, 1992; Schulz et al., 2001), although AmtR has been identified as a global regulator in Corynebacterium glutamicum (Jakoby et al., 2000).
The C. saccharobutylicum glnA gene, encoding GS, was isolated by functional complementation of the GS-deficient Escherichia coli strain YMC11 (Usdin et al., 1986), and a second gene, nitR, encoding a putative response regulator protein, was subsequently identified downstream (Woods & Reid, 1995). The nitR gene encodes an antiterminator protein, which would allow RNA polymerase to read through the terminator-like structures in the promoter region of the glnA gene (Woods & Reid, 1995). Antisense RNA (AS-RNA), complementary to the 5' end of the glnA mRNA, has been implicated in the post-transcriptional inhibition of glnA translation under nitrogen-rich conditions (Fierro-Monti et al., 1992). A gene encoding a putative GOGAT enzyme has been isolated from C. saccharobutylicum and was annotated gltX, because the deduced protein showed significant sequence identity (44 %) to the E. coli GOGAT β-subunit (Stutz & Reid, 2004). However, RNA studies showed that gltX expression was not regulated by nitrogen, and it is therefore unlikely to encode a functional GOGAT enzyme.
In order to facilitate metabolic engineering of the ammonium assimilation pathways for improved solvent yields, we undertook a study of the nitrogen growth requirements of C. saccharobutylicum NCP262 and of the regulation of nitrogen assimilation. In this study, we characterize the chromosomal regions flanking the glnA gene and show that the structural genes encoding the GS and GOGAT enzymes are clustered on the genome. This is believed to be the first report of a functional gene cluster encoding both key enzymes of nitrogen assimilation. The genes are expressed under the same nitrogen-limiting conditions, and we suggest a model for their co-ordinated regulation by an antitermination mechanism as well as by AS-RNA.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), or glucose-mineral salts-biotin minimal medium (GSMM; Holdeman et al., 1977) containing various concentrations of glucose, Casamino acids (Difco), glutamine or monosodium glutamate (MSG), and inorganic nitrogen (ammonium acetate). Cell-line cultures represented cells grown to OD600 0.3 in CBM, washed and used at a 5 % (v/v) inoculum, and spore-line cultures (10 ml) were grown directly from 5 µl spore stock heat-shocked at 70 °C for 5 min. E. coli strains JM105 and JM109 were grown in Luria–Bertani (LB) medium (Sambrook & Russell, 2001). E. coli MX3004 (thi-1 gdh-1 pro+ hutC gltD227 : : MudIIPR13) (Castano et al., 1992) was grown on NN minimal medium (Covarrubias et al., 1980).
General molecular techniques.
C. saccharobutylicum genomic DNA was prepared according to Zappe et al. (1986). DNA manipulations were carried out as described by Sambrook & Russell (2001). Southern hybridization was performed using the non-radioactive DIG DNA labelling and detection protocol (Roche). DNA sequencing was performed using an ALFexpress automated DNA sequencer (Pharmacia). Sequence data were analysed using DNAMAN (version 4.13) and the NCBI databases (http://www.ncbi.nlm.nih.gov). The C. saccharobutylicum gltAB region was assigned GenBank accession number AF082880.
Cloning of regions adjacent to glnA-nitR in the C. saccharobutylicum genome.
The C. saccharobutylicum glnA gene had been previously cloned on plasmid pHZ200 (Fig. 1) (Usdin et al., 1986). The Bluescript pSK vector (Stratagene) was used for subcloning and sequencing. Plasmids pHS4 and pHS5 were isolated consecutively by chromosome walking, using a 0.38 kb HindIII probe from pHZ200 to identify pHS4, and a 0.67 kb PvuII–XbaI pHS4 fragment to detect the 3.5 kb PvuII fragment cloned in pHS5. For complementation of GOGAT activity in E. coli, the 3.5 kb PvuII fragment from pHS5 encoding the gltB gene was subcloned into pSK and pEcoR251 (Zappe et al., 1986) to give pHS6 and pHS7. Plasmid pHS9 was constructed by first reconstituting the gltA gene by cloning the 3.8 kb XbaI fragment from pHS4 into the XbaI site of pHZ200, and then subcloning the entire gene on a EcoRV–NotI fragment into the unique EcoRI site of pACYC184 (Rose, 1988) after blunting the NotI and EcoRI sites (Fig. 1).
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. Northern and dot blots were performed (Sambrook & Russell, 2001) using the DIG Labelling and Detection kit (Roche). The internal probes from the glnA and nitR genes were prepared as above, and probes for gltA and gltB were the 0.79 kb EcoRV–XbaI and 1.0 kb internal XbaI fragments respectively (Fig. 1
). RNA dotblots were quantified using a densitometer and the software program GelTrak (D. Maeder, University of Cape Town). The detection and hybridization data for each probe were collected in triplicate.
GOGAT, GDH and GS enzyme activities.
Cell-free extracts were prepared under strictly anaerobic conditions. Cells (
300 mg) were washed in 60 ml 20 mM KH2PO4/K2HPO4 buffer, pH 6.5, resuspended in 6 ml buffer and disrupted in a French pressure cell. The lysate was collected in a sealed Hungate tube on ice, and transferred back into the anaerobic cabinet for clarification and assays. Protein concentration was determined by the Bio-Rad assay system. GOGAT activity was determined spectrophotometrically by measuring the rate of NAD(P)H oxidation as described by Meister (1985), and was monitored in the anaerobic glove cabinet as it was highly sensitive to oxygen. Specific activity was expressed as µmol NADH oxidized per min per mg protein. Assimilatory GDH assays were essentially as in the GOGAT assay, with 100 mM NH4Cl and 10 mM 2-oxoglutarate replacing glutamine. GS activities were measured by the 
-glutamyl transferase assay described by Shapiro & Stadtman (1968) and specific activity was expressed as µmol -glutamyl hydroxamate produced per min per mg protein.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), with growth in 0.2 % (w/v) Casamino acids being equivalent to that in complete medium (CBM) culture (results not shown). Casamino acids could not be substituted by glutamine or glutamate, unlike in B. subtilis, where glutamine is a preferred source of nitrogen (Fisher & Sonenshein, 1991). Growth with 0.2 % ammonium acetate as the sole nitrogen source was considerably inhibited and was equivalent to that with 0.01 % Casamino acids (data not shown). Furthermore, the presence of ammonium acetate had a retarding effect on the germination and growth of the spore line cultures in the presence of 0.2 % (w/v) Casamino acids (Fig. 2b). This effect was also observed with alternative sources of ammonia including NH4Cl, NH4NO3 and (NH4)2SO4 (data not shown).
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), confirmed that 0.2 % (w/v) Casamino acids is representative of nitrogen-rich growth conditions, with the most repressed levels of GS activity (referred to as non-inducing media). While the presence of inorganic nitrogen (ammonium acetate) had little effect on the levels of GS in the cells, GS activity was unexpectedly elevated early in the growth cycle in the presence of its product glutamine (Table 1). Thus, 0.025 % (w/v) Casamino acids containing 0.15 % (w/v) glutamine was selected to represent nitrogen-limiting conditions since GS activity was significantly induced in this medium (inducing media). These induced and non-induced trends were most pronounced at early exponential growth phase (OD600 0.3) with levels differing as much as 9.2-fold (Table 1, Fig. 3b). Interestingly, growth of C. saccharobutylicum in either the inducing or non-inducing media with various concentrations of glutamate (0.1 %, 0.5 % or 1 %) had no effect on either GOGAT or GS activity (data not shown).
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). At early exponential growth phase (OD600 0.3), there was a 5.6-fold difference in GOGAT activity between the inducing [111 µmol NADH oxidized min–1 (mg protein)–1] and non-inducing growth media [20 µmol NADH oxidized min–1 (mg protein)–1]. Similarly, a 6.2-fold increase was measured in GS activity in inducing media [1.2 µmol -glutamyl hydroxymate produced min–1 (mg protein)–1] compared to non-inducing media [0.2 µmol -glutamyl hydroxymate produced min–1 (mg protein)–1]. Both induced enzyme activities decreased towards late exponential phase, with GS levels dropping significantly (3.5-fold) between OD600 0.6 and 0.8. This trend has also been observed in B. subtilis (Schreier, 1993), and suggests that neither enzyme is required at high levels during late stationary phase or sporulation. Neither of the enzyme activity levels fluctuated much throughout growth in non-inducing medium (Fig. 3). Assimilatory GDH activity could not be detected from either early or late-exponential phase cultures grown in either CBM, inducing or non-inducing media or in cultures restricted in carbon source (0.5 and 0.25 %, w/v, glucose) and supplemented with ammonia (100 mM NH4Cl).
Identification of genes flanking the glnA and nitR genes in C. saccharobutylicum
The cloning of the glnA-nitR genes on a 7.9 kb genomic fragment of C. saccharobutylicum was described previously (Usdin et al., 1986). DNA sequence analysis of the region upstream from the glnA gene indicated a truncated aspartokinase gene. Downstream of the glnA-nitR genes, in the same orientation, are two genes encoding the large or 
(gltA, 4554 bp) and small or β (gltB, 1473 bp) subunits of GOGAT. The initiation codon for gltA is separated by 108 bp from the nitR stop codon. A truncated ORF was identified downstream from gltB encoding the amino-terminal region of isocitrate dehydrogenase, which catalyses the oxidative decarboxylation of isocitrate to form 2-oxoglutarate, the substrate for GOGAT.
The deduced amino acid sequences for C. saccharobutylicum GltA and GltB (1530 and 490 aa respectively) contain the highly conserved domains primarily identified by analysis of the Azospirillum brasilense enzyme subunits (Vanoni & Curti, 2005). The glutamine-binding and catalysis domain of PurF-type glutamine amidotransferases is conserved in the N-terminal domain of GltA, as is the cysteine cluster, characteristic of [3Fe-4S] containing enzymes. The NAD(P)H-binding domain in the β subunit contains the conserved residues, GXGXXG, characteristic of NADH binding (Vanoni & Curti, 2005). This is consistent with our GOGAT activity assays, where NADH, rather than NADPH, is the required coenzyme for C. saccharobutylicum GOGAT activity. This is unusual for bacterial GOGAT enzymes as the majority use NADPH, whereas use of NADH is typical of the eukaryotic enzymes (Vanoni & Curti, 2005).
Functional complementation of an E. coli gltB null mutant
In order to determine whether the C. saccharobutylicum gltB gene could functionally complement the mutation in E. coli MX3004 (a glutamate auxotroph lacking the GOGAT β subunit), the gltB gene was cloned downstream of the lambda and lacZ promoters in pEcoR251 and pSK, yielding constructs pHS7 and pHS6, respectively (Fig. 1). Expression of gltB in E. coli MX3004, from either pHS6 or pHS7, failed to complement the E. coli subunit, and did not restore growth of MX3004 on minimal medium containing ammonia as the sole source of nitrogen. However, co-transformation of pHS7 with pHS9, containing the C. saccharobutylicum gltA gene, did result in weak growth of the GOGAT mutant under anaerobic conditions only. RNA and protein analysis confirmed that both the C. saccharolyticum gltA and gltB genes were effectively expressed in E. coli (data not shown).
Regulation of glnA, nitR and gltAB transcription
RNA was extracted from C. saccharobutylicum cultures grown to early and late exponential phase under the specified inducing and non-inducing conditions. Hybridization experiments showed that for all four genes, glnA, nitR, gltA and gltB, pronounced induction occurred under inducing conditions (Fig. 4, lanes 3 and 4) and repression was observed under non-inducing conditions (Fig. 4, lanes 1 and 2). The glnA and nitR probes detected a single RNA band of approximately 2.3 kb, resulting from the co-transcription of glnA-nitR under inducing conditions. Similarly, gltA and gltB form an operon, as the gene-specific probes hybridized to a common 6.2 kb band. Levels of mRNA were quantified from dot blots (results not shown). Signal intensities revealed that, at early exponential phase (OD600 0.30), there was a seven- to eightfold difference in the signals generated between the specified nitrogen-limiting vs nitrogen-rich growth media for both the glnA and nitR genes. The difference in the expression levels recorded for the gltA and gltB genes was also similar (5.5–6.0 fold), indicating co-transcription under all conditions. By OD600 0.7 there was a drop in the induced expression levels of glnA and nitR of approximately 1.6- and 1.7-fold respectively, whereas expression of the gltA and gltB genes remained relatively constant.
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). The DNA sequence is identical to the recently released genome sequence of the same strain of C. beijerinckii (NZAALO01000002). The regions upstream of the nitrogen assimilation genes from C. saccharobutylicum and C. beijerinckii were aligned (Fig. 5), and show that these genes share common regulatory features. The two regions differ only in that C. saccharobutylicum has a longer intergenic region between glnA and nitR, while C. beijerinckii has an extra 66 bp between the nitR and gltA genes. The transcriptional initiation site upstream of the glnA gene, T1, and a putative promoter sequence with similarity to the clostridial extended consensus promoter sequences (Young et al., 1989), has been identified previously in C. beijerinckii (Quixley, 1999). A number of regions of dyad symmetry with the potential to form stable stem–loop structures were identified in this leader region, IR1 (
G)=–14.4 kcal mol–1; –60.2 kJ mol–1 and IR2 (G=–20.4 kcal mol–1; –85.4 kJ mol–1; Fig. 5a). A similar stem–loop (G=–12.1 kcal mol–1; –50.6 kJ mol–1) was identified between the putative gltA promoter sequence and its structural gene (Fig. 5c). It is also noteworthy that the AS-RNA, which was shown to regulate glnA expression in C. saccharobutylicum by binding to a complementary region spanning the ribosome-binding site (RBS) and ATG start codons of the glnA gene with G=–33.5 kcal mol–1 (–140.2 kJ mol–1) (Fierro-Monti et al., 1992; Fig. 5a), shares significant complementarity with the corresponding region of gltA (G=–28.9 kcal mol–1; –120.9 kJ mol–1) (Fig. 5c). Furthermore, the identical conserved clostridial RBS (Young et al., 1989), 5'-AGGGGG-3', is present 7–8 bp upstream of the glnA and gltA initiation codons, respectively (Fig. 5a, c). The first two bases of the RBS for the gltB gene overlap the TAG termination codon for the gltA gene, suggesting that these two genes are translationally coupled. The presence of a transcriptional terminator (G=–23.7 kcal mol–1; –99.2 kJ mol–1), located 18–62 bp downstream of gltB (data not shown) is consistent with co-transcription of the gltA and gltB genes.
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and β subunits respectively. In contrast, they showed only 49–53 % identity to the predicted proteins from the other solvent-producing strain, C. acetobutylicum ATCC 824 (NCBI: NC003030). Phylogenetic analyses revealed that the GltA gene products from these two clostridia were most closely related to those from the lactic acid bacteria Lactobacillus casei, Streptococcus mutans and Lactococcus lactis (46–53 % identity), while the nearest relatives of the deduced GltB proteins were from Desulfotomaculum reducens and Synechocystis (59 % identity).
The nitR gene from C. saccharobutylicum encodes a response regulator (RR) protein belonging to the family pfam03861. All of these proteins have the conserved ANTAR domain, the RNA-binding domain found in transcription-antitermination regulatory proteins (Shu & Zhulin, 2002) and the REC domain, or signal-receiver domain, which is the phosphoaccepter region present in response regulators such as CheY (Galperin, 2006). The deduced NitR protein has significant similarity over its entire length to uncharacterized response regulators from Streptomyces coelicolor (NCBI: T35758) and Mycobacterium tuberculosis (NCBI: H70558), and to the NasT protein from Azotobacter vinelandii (Gutierrez et al., 1995). NasT has been shown to be a positive regulator of the assimilatory nitrate/nitrite reductase operon, nasAB. In addition, the C-terminus of the predicted NitR protein shares similarity with C-terminal domains of several antiterminator regulatory proteins such as the aliphatic amidase regulator, AmiR, from Pseudomonas aeruginosa (Wilson et al., 1993) and NasR, the positive regulator of the nitrite/nitrate reductase operon from Klebsiella oxytoca (Chai & Stewart, 1998).
Analysis of the genomic context of the close homologues of the Clostridium glnA and gltAB genes failed to identify other operons exactly like those of C. saccharobutylicum and C. beijerinckii. C. acetobutylicum 824 has no glnA gene, only a glnN, encoding a GSIII enzyme. The protein subunits of the GSIII family are substantially larger than those encoded by glnA genes, and the amino acid sequence shows only 9 % sequence identity to the GSI family (van Rooyen et al., 2006). In Streptococcus mutans the glnA gene lies adjacent to the gltAB genes but they are associated with a regulator gene of the MerR family (Brown et al., 2003). In addition, three other Firmicutes show clustering of nitrogen assimilation genes: Carboxydothermus hydrogenoformans, Desulfotomaculum reducens and Moorella thermoacetica. In all three of these gene clusters, the glnA gene lies directly downstream of a regulator gene, which encodes a RR with both REC and ANTAR domains (Fig. 6). While D. reducens has the most similar gene arrangement to the clostridia, the other two species do not possess typical gltAB genes, and instead have various combinations of gltS- and gltB-like genes, which may be involved in glutamate synthesis.
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DISCUSSION |
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; Castano et al., 1988). In C. pasteurianum, an increase in the glutamine pool levels correlated with increased GOGAT activity (Kleiner & Fitzke 1979). There was no GDH activity detectable in C. saccharobutylicum, indicating that the energy-dependent GS/GOGAT pathway is the primary route for ammonia assimilation in the saccharolytic clostridia, as in C. acetobutylicum ATCC 824 (Amine et al., 1990).
The genes encoding the and β subunits of GOGAT are situated downstream of glnA in C. saccharobutylicum, and their function was confirmed by complementation of an E. coli GOGAT mutant, where the expression of both Clostridium subunit genes together enabled weak growth on minimal medium under anaerobic conditions only. The Clostridium β subunit alone was not able to functionally complement the E. coli subunit, suggesting subunit incompatibility. Recent evidence has indicated that the and β subunits of GOGAT enzymes form a tight complex which is stabilized by the [4Fe-4S] clusters at the interface of the two subunits (Vanoni & Curti, 2005). This specific interaction may not take place efficiently in E. coli. The native C. saccharobutylicum GOGAT enzyme was particularly sensitive to oxygen and requires a different co-factor to that from E. coli, factors which may contribute to the inefficient functioning of the Clostridium enzyme in this heterologous host.
The expression of the glnA, gltA and gltB genes showed that they were induced by nitrogen-limiting conditions and repressed by nitrogen-rich conditions. The glnA and nitR genes were co-transcribed as an operon on the same RNA transcript, as were gltA and gltB. Clearly, the changes in glnA and gltAB mRNA levels in relation to the nitrogen source were reflected in the corresponding levels of GS and GOGAT activities (Fig. 3), leading to the conclusion that these enzymes are similarly regulated by the same nitrogen conditions primarily at the level of transcription.
Several features of the nitrogen assimilation operons from C. saccharobutylicum and C. beijerinckii suggest that they are primarily controlled by an antitermination mechanism.
The position of the nitR gene between the glnA and the gltAB genes suggests that it has a key role in the regulation of the operon. NitR represents a response regulator with the RNA-binding capability found in transcription antitermination regulatory proteins (Shu & Zhulin, 2002; Galperin, 2006). The REC or signal-receiver domain would sense environmental nitrogen levels, while the ANTAR domain is proposed to act by binding to a stem–loop structure in the leader region of the RNA transcript, allowing transcriptional read-through into the structural genes. Molecular analysis of the nitrogen operons of C. beijerinckii and C. saccharobutylicum revealed a long leader mRNA transcript of 200 bp upstream of the glnA genes (Quixley, 1999; Janssen et al., 1990). The presence of several inverted repeat sequences with the potential to form intrinsic transcriptional terminators in these leader regions suggests that these may be the targets of the anti-terminator proteins. The identification of a transcriptional terminator between the putative gltA promoter and its structural gene suggests that the gltAB genes have a similar regulatory mechanism.
Based on these findings, we have proposed a model for nitrogen regulation in C. saccharobutylicum and C. beijerinckii. Under nitrogen-limiting conditions, the putative response regulator, NitR, is activated by a signal transduction mechanism and binds to a region of dyad symmetry present between the transcriptional start site and the glnA initiation codon, thus positively controlling glnA transcription via an antitermination mechanism. The glnA and nitR genes are transcribed together, with the transcript terminating at the inverted repeat sequence downstream of nitR. The activated NitR protein would regulate transcriptional read-through of this terminator as well so that both the glnA-nitR and the gltAB operons are simultaneously expressed.
Furthermore, under nitrogen-rich conditions, a 43 bp glnA AS-RNA (Fig. 5b), has been implicated in the downregulation of GS expression by binding to a complementary sequence spanning the Shine–Dalgarno and start codons of the C. saccharobutylicum glnA mRNA (Fig. 5a) (Fierro-Monti et al., 1992). The AS-RNA also shows complementarity to a region spanning the Shine–Dalgarno and ATG start codon of gltA (Fig. 5c), providing the mechanism to downregulate the translation of both mRNA transcripts under the same conditions. The involvement of a post-transcriptional regulatory system is supported by the result that, at late exponential phase, the decrease in GS activity (3.5-fold) in nitrogen-limiting media was not reflected in the decrease in mRNA levels (1.6-fold).
The clostridia show great diversity in the genes involved in nitrogen assimilation. The clustering of the genes encoding GS and GOGAT, which is seen in C. saccharobutylicum and C. beijerinckii, is not found in C. acetobutylicum ATCC 824, where only glnN, encoding a GSIII enzyme, occurs. It is also not found in Clostridium perfringens (GenBank accession number NC003366) or Clostridium tetani (GenBank accession number NC004557), where the glnA genes are not adjacent to other nitrogen genes on the genomes. It is interesting that the nitrogen cluster in S. mutans, a member of the class Bacilli of the Firmicutes, includes the glnA and gltAB genes and is similar in arrangement to that of C. saccharobutylicum and C. beijerinckii (Fig. 6). This gene arrangement is not seen in the other streptococci. In addition, the regulator which is associated with the S. mutans glnA gene is very different to NitR and belongs to the MerR family. This suggests that the S. mutans glnA-gltAB gene cluster may have been acquired by horizontal gene transfer from the clostridia or vice versa, but that the regulator gene was sequestered from another source. The other species that show similar gene clustering to these clostridia also have response regulator proteins associated with them and belong to the Clostridia class of the Firmicutes. In these cases, the different gene arrangements could represent divergence from a common ancestor, with the acquisition of genes or modules at different times. The clustering of the genes encoding functional GS and GOGAT enzymes on the genomes of C. saccharobutylicum and C. beijerinckii and the co-ordinated regulation of these genes would appear to be the most simple and efficient means of ensuring that both enzymes are available at the same time. It may, however, also imply less metabolic flexibility and it will be interesting to know whether this novel regulatory model is also observed in other Gram-positive anaerobic bacteria.
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ACKNOWLEDGEMENTS |
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Edited by: W. H. Schwarz
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REFERENCES |
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Received 3 January 2007;
revised 8 May 2007;
accepted 9 May 2007.
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), 0.1 % (
), 0.05 % (x), 0.01 % (
) and none (
). (b) Growth profiles for spore-germinated cultures in GSMM containing 2 % (w/v) glucose, 0.2 % (w/v) Casamino acids, and various concentrations of ammonium acetate (w/v): 0.4 % (
