Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr. 5, D-91058 Erlangen, Germany
Correspondence
Jörg Stülke
jstuelke{at}biologie.uni-erlangen.de
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ABSTRACT |
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These authors contributed equally to this work.
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INTRODUCTION |
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The global control of carbon catabolism in B. subtilis is exerted by a pleiotropic regulatory protein, CcpA. In the presence of glucose, CcpA can interact with regulatory sites in the control regions of regulated operons to either repress or activate transcription. DNA-binding activity of CcpA is triggered by interaction with a protein of the phosphotransferase system (PTS), HPr or its regulatory paralogue Crh. In the presence of glucose, HPr and Crh are phosphorylated by a HPr kinase/phosphorylase (HPrK/P) on a regulatory seryl residue. HPr(Ser-P) and Crh(Ser-P) act as cofactors for CcpA (Deutscher et al., 1995, 2002
; Galinier et al., 1997
; Henkin, 1996
; Stülke & Hillen, 2000
). Recent proteome and transcriptome studies have demonstrated that about 250 and 85 genes are subject to CcpA-dependent repression and activation, respectively. Among the genes repressed by CcpA are those encoding enzymes required for the utilization of secondary carbon sources, but also genes of the Krebs citric acid cycle. The genes activated by CcpA include those required for overflow metabolism, glycolysis and the biosynthesis of certain amino acids (Blencke et al., 2003
; Moreno et al., 2001
; Tobisch et al., 1999
; Yoshida et al., 2001
).
Nitrogen metabolism is controlled by another transcription regulator, TnrA. In the absence of glutamine or ammonium, this protein activates transcription of genes encoding enzymes to utilize secondary nitrogen sources and represses expression of the glnA gene and the gltAB operon encoding enzymes of glutamine biosynthesis (Belitsky, 2002; Fisher & Débarbouillé, 2002
). In the presence of repressing nitrogen sources, i.e. glutamine or ammonium, the glutamine synthetase binds and thereby inactivates TnrA (Wray et al., 2001
).
The two regulatory systems allow the bacteria to utilize sequentially the available sources of carbon and nitrogen, thus enabling them to optimize their metabolism. There are, however, also interactions between carbon and nitrogen regulation. Glycolysis is induced by glucose, but full induction occurs only if amino acids are available as well. Similarly, the Krebs citric acid cycle is synergistically repressed by glucose and glutamate (Ludwig et al., 2001; Rosenkrantz et al., 1985
; Sonenshein, 2002
). The molecular mechanisms that allow control of gene expression by both carbon and nitrogen sources have not yet been elucidated in B. subtilis.
B. subtilis ccpA mutants are defective in carbon catabolite repression. This is, however, not their only phenotype: they also exhibit a severe growth defect on minimal media (Lindner et al., 1994; Martin et al., 1989
). ccpA mutants are also unable to grow with glucose and ammonium as single sources of carbon and nitrogen, respectively (Faires et al., 1999
; Miwa et al., 1994
; Wray et al., 1994
). The observation that ccpA mutants require glutamate (or a source of it) as a source of nitrogen led us to propose that CcpA might be involved in the control of glutamate biosynthesis (Faires et al., 1999
). However, while ccpA mutants grow with glucose, ammonium and glutamate, growth is still slower than observed with wild-type bacteria. This can be circumvented by the addition of methionine and the branched-chain amino acids to the growth medium. The ilvleu operon encoding enzymes of branched-chain amino acid biosynthesis is not fully expressed in ccpA mutants. The reason for the methionine requirement has not yet been elucidated (Ludwig et al., 2002a
).
CcpA can regulate transcription in different ways. As mentioned above, many genes are controlled by CcpA by binding to a catabolite-responsive element (cre) in the control region. However, there are many CcpA-dependent genes which do not possess any detectable cre target sites (Blencke et al., 2003; Moreno et al., 2001
; Yoshida et al., 2001
). A novel mechanism of gene regulation was recently discovered for the gapA operon, encoding enzymes of the triose phosphate interconversion part of glycolysis. This operon is induced by glucose and other sugars, but induction by glucose cannot occur in a ccpA mutant. The genetic evidence suggested that this effect is exerted in an indirect way (Fillinger et al., 2000
; Ludwig et al., 2001
). Detailed analyses revealed that ccpA mutants are defective in the transport of sugars by the PTS. This defect results from a strongly increased phosphorylation of HPr on the regulatory Ser-46 in the ccpA mutant which prevents participation of HPr in sugar transport and phosphorylation (Ludwig et al., 2002b
). Mutations that prevent phosphorylation of HPr on Ser-46 result in the restoration of sugar transport and gapA operon expression. Thus, due to the defective transport of PTS sugars in ccpA mutants, the internal inducer of the operon cannot accumulate, resulting in lack of induction of the gapA operon. This novel mode of control of gene expression by CcpA was defined as class II regulation (Ludwig et al., 2002b
).
Ammonium assimilation involves two enzymes in B. subtilis: the glutamine synthetase catalyses the formation of glutamine from glutamate and ammonium, and the glutamate synthase converts 2-oxoglutarate and glutamine to two molecules of glutamate. One of these molecules of glutamate can be recycled to glutamine while the second molecule is now available for anabolism. This reaction is the main link between carbon metabolism in the Krebs citric acid cycle and nitrogen metabolism since glutamate is the universal donor of amino groups (Belitsky, 2002). In contrast to Escherichia coli and other bacteria, the glutamate dehydrogenase does not contribute to glutamate biosynthesis in B. subtilis. It was proposed that this enzyme has a catabolic role in B. subtilis (Belitsky & Sonenshein, 1998
). Thus, the glutamate synthase encoded by the gltAB operon is essential for glutamate biosynthesis in this bacterium. Expression of the gltAB operon is repressed in the absence of ammonium by the pleiotropic regulator TnrA (Belitsky et al., 2000
). In addition, the operon is under positive control of the transcriptional activator GltC (Bohannon & Sonenshein, 1989
). The signal to which GltC responds is currently unknown. In addition, the gltAB operon is subject to control by the pleiotropic regulator CcpA. This control may link ammonium assimilation to the carbon and energy state of the cell (Faires et al., 1999
).
In this study, we investigated the mechanism by which CcpA controls expression of the gltAB operon and the relation between expression of gltAB and the growth defect of ccpA mutants. As observed for the glycolytic gapA operon, gltAB belongs to the recently discovered class II of CcpA-dependent genes. Expression of the operon requires induction by any of a variety of sugars. Induction by PTS sugars is prevented in ccpA mutants, consistent with the idea that the PTS is inactive in these mutants (Ludwig et al., 2002b). The formation of an internal inducer may thus result from transport and metabolism of the sugars. Mutations that allow expression of the gltAB operon independent of CcpA result in a suppression of the growth defect of the ccpA mutants. Thus, the inefficient expression of the gltAB operon is the major bottleneck that limits growth of ccpA mutants.
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METHODS |
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Transformation and characterization of the phenotype.
B. subtilis was transformed with plasmid and chromosomal DNA according to the two-step protocol (Kunst & Rapoport, 1995). Transformants were selected on SP plates containing chloramphenicol (5 µg ml-1), spectinomycin (100 µg ml-1), kanamycin (5 µg ml-1), or erythromycin plus lincomycin (1 and 10 µg ml-1, respectively). Quantitative assays of lacZ expression in B. subtilis were performed with cell extracts using ONPG as the substrate (Kunst & Rapoport, 1995
).
Plasmid constructions.
A translational fusion of the gltAB promoter to a promoterless lacZ gene was constructed using the vector pAC7 (Weinrauch et al., 1991), which allows the introduction of the fusion into the amyE locus of B. subtilis. The 245 bp gltA promoter fragment (216 bp upstream to 29 bp downstream of the translational start codon) was amplified by PCR using primer pair IW1 (5'AAAGAATTCGATCAGCGGCTTCTGAAACGTG3')/IW2 (5'AAAGGATCCTGAGCTTTTGGCATTTGATTGTACGTC3'). The PCR product was digested by EcoRI and BamHI (the sites were introduced with the PCR primers; they are underlined in the sequences) and ligated with pAC7 linearized with the same enzymes. The identity of the cloned insert was verified by sequencing and the resulting plasmid was pGP526.
Construction of a strain allowing expression of the gltAB operon under control of the pxylA promoter.
To express the gltAB operon under a controllable promoter plasmid pGP724 was constructed as follows (see Fig. 1). A 520 bp PCR fragment (extending from 53 bp upstream to 467 bp downstream relative to the translational start of gltA) was generated by PCR using primers HMB55 (5'AACGCGGATCCGTTGTTAGATTTTATGACCGG3') and HMB56 (5'AACGCGGATCCAACTTGCGCCGATAAATACC3'). The resulting PCR product was digested with BamHI and ligated into plasmid pX2 (Mogk et al., 1997
) linearized with BamHI (see Fig. 1a
). The identity of the cloned insert was verified by sequencing. B. subtilis 168 was transformed with the resulting plasmid pGP724 and transformants were selected on SP plates containing chloramphenichol and xylose (1·5 %, w/v). The resulting strain GP222 was able to grow in C-Glc minimal medium only in the presence of xylose (1·5 %, w/v) or glutamate (0·8 %, w/v). Strain GP223, carrying the ccpA : : Tn917 insertion in addition to the xylose-inducible gltAB operon, was constructed by transformation of GP222 with chromosomal DNA of BGW2. The chromosomal arrangement of the gltAB operon of these strains is shown in Fig. 1(b)
.
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RESULTS |
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The specific loss of induction by PTS sugars in the CcpA mutant suggested that the lack of PTS activity was the reason for the absence of expression of the gltAB operon. It had already been demonstrated that the reduced PTS activity in ccpA mutants is due to an excessive kinase activity of HPrK/P, which interferes with phosphorylation of HPr by Enzyme I of the PTS (Ludwig et al., 2002b). If this were the reason for the loss of glucose induction of gltAB in the ccpA mutant, we would expect that a mutation that prevents phosphorylation of HPr by the HPr kinase would restore sugar transport and concomitant induction of the gltAB operon. Such a mutation is the ptsH1 mutation, which affects the regulatory phosphorylation site in HPr. The ccpA ptsH1 double mutant strain is indeed capable of transporting glucose (Ludwig et al., 2002b
). To address the involvement of CcpA and the PTS in the regulation of gltAB, we analysed the transcription of the operon in a wild-type strain (B. subtilis 168) and in its isogenic derivatives GP302 (ccpA) and GP335 (ccpA ptsH1). RNA was extracted from cells of the three strains grown in CSE minimal medium in the absence or presence of glucose and subjected to Northern blot analysis using a riboprobe specific for gltA (Fig. 2
). In the wild-type strain, a 6·1 kb transcript was detected for glucose-grown cells. This mRNA size corresponds to the bicistronic gltAB operon encoding the two subunits of glutamate synthase. As observed with the lacZ fusion, no gltAB expression was detectable after growth of B. subtilis 168 in CSE medium without glucose. In the ccpA mutant, glucose did not induce the transcription of the gltAB operon. In contrast, induction was restored in the ccpA ptsH1 double mutant strain GP335. This finding is in good agreement with an analysis at the transcriptome level (Blencke et al., 2003
) and with the idea that the reduced sugar transport by the PTS is the limiting factor for induction of the gltAB operon in the ccpA mutant, and suggests that gltAB is the second recognized member of class II of CcpA-regulated genes (see Discussion).
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DISCUSSION |
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Two alternative explanations have been proposed to explain the glutamate auxotrophy of ccpA mutants: while Faires et al. (1999) argued in favour of an insufficient expression of the gltAB operon, Belitsky (2002)
suggested that the glutamate pool would be low in ccpA mutants due to the loss of carbon catabolite repression of rocG, encoding glutamate dehydrogenase (Belitsky & Sonenshein, 1998
, 1999
). Three lines of evidence presented in this work are clearly in agreement with the former idea. First, the gltAB operon is expressed in the ccpA mutant if non-PTS carbohydrates are present. Expression of the operon correlates with the growth of the ccpA mutant in the presence of these substrates. Second, the ptsH1 mutation suppresses both the loss of gltAB expression and the growth deficiency without restoring carbon catabolite repression (Blencke et al., 2003
). Thus, loss of catabolite repression of rocG cannot be the cause of glutamate auxotrophy of the ccpA mutant. Finally, independent expression of the gltAB operon in a ccpA mutant is sufficient to suppress the growth defect. We were not able to get this result in a previous study (Faires et al., 1999
). Detailed analyses revealed that the construct used in that analysis was not fully inducible (our unpublished results). However, the pxylA system used for artificial induction in this work has already proven to be very useful in previous studies (Ludwig et al., 2002a
; Mogk et al., 1997
). Thus, expression of the gltAB operon is necessary and sufficient to allow the ccpA mutant strain to grow on minimal media with glucose and ammonium.
Expression of the gltAB operon is controlled by three factors. First, in the absence of ammonium, the operon is repressed by the pleiotropic transcriptional regulator of nitrogen metabolism in B. subtilis, TnrA (Belitsky et al., 2000). Second, glutamate causes a mild repression of the gltAB operon. Third, expression of the gltAB operon is induced by the presence of carbohydrates such as glucose, glycerol and glucitol. Catabolism of all these carbohydrates involves glyceraldehyde 3-phosphate, which can then be further catabolized via the lower part of glycolysis. It is, however, unknown by which mechanism(s) repression and induction by glutamate and sugars, respectively, are exerted. The positive regulator of the gltAB operon, GltC, is a candidate for either control (Bohannon & Sonenshein, 1989
).
Induction of the operon by glucose and fructose but not by glucitol and glycerol requires a functional CcpA protein. A similar induction pattern was observed for the glycolytic gapA operon of B. subtilis (Ludwig et al., 2002b). Moreover, we were not able to detect a potential cre site in the control region of the gltAB operon that might serve as a target for regulation by CcpA. We considered, therefore, that CcpA might play a similar role in induction of gltAB as recently demonstrated for the gapA operon: ccpA mutants are impaired in PTS sugar transport and phosphorylation due to an excessive kinase activity of HPr kinase/phosphatase. Therefore, internal inducers derived from the catabolism of these sugars cannot accumulate in ccpA mutants, resulting in loss of induction of gene expression. The PTS defect of ccpA mutants can be suppressed by a ptsH1 mutation, which prevents HPr phosphorylation by the HPr kinase. Indeed, the ptsH1 mutation did also suppress both the deficient expression of the gltAB operon and the growth defect of the ccpA mutant. Thus, the gltAB operon is the second recognized member of the class II of CcpA-responsive genes in addition to the glycolytic gapA operon. In a previous study, we isolated spontaneous mutants that exhibited suppression of the growth defect of ccpA mutants. One of these mutations, sgd-1, restored expression of the gltAB operon (Faires et al., 1999
). However, the sgd-1 mutation has so far not been identified. With the finding that ptsH1 causes exactly the same phenotype, this latter mutation can be regarded as the first genetically defined sgd mutation understood at the molecular level.
For efficient growth of bacteria, the different branches of metabolism need to be tightly coordinated. Recent work demonstrates that there are several regulatory interrelationships between carbon and nitrogen metabolism in B. subtilis. As shown in this work, ammonium assimilation is strongly dependent on the carbohydrate supply. Moreover, syntheses of methionine and the branched-chain amino acids also depend on a functional CcpA, although to a lower degree (Ludwig et al., 2002a). In turn, several important steps of carbon metabolism are co-regulated by signals from carbohydrate and amino acid metabolism: expression of the glycolytic gapA operon is only fully induced if the cells are provided with both a sugar and a good source of amino acids (Ludwig et al., 2001
); on the other hand, several genes encoding enzymes of the Krebs citric acid cycle such as citZ and citB are synergistically repressed by glucose and glutamate (Rosenkrantz et al., 1985
). Interestingly, the specific regulators of citB and gltAB, CcpC and GltC, respectively, are both members of the LysR family of transcriptional regulators (Bohannon & Sonenshein, 1989
; Jourlin-Castelli et al., 2000
; Schell, 1993
).
To better understand both the regulation of the gltAB operon and the interdependence between carbon and nitrogen metabolism it will be necessary to study the molecular details of the induction process by sugars. Work with this aim is in progress in our laboratory.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 11 May 2003;
revised 2 July 2003;
accepted 2 July 2003.