Assessment of CcpA-mediated catabolite control of gene expression in Bacillus cereusATCC 14579
© van der Voort et al; licensee BioMed Central Ltd. 2008
Received: 19 October 2007
Accepted: 16 April 2008
Published: 16 April 2008
The catabolite control protein CcpA is a transcriptional regulator conserved in many Gram-positives, controlling the efficiency of glucose metabolism. Here we studied the role of Bacillus cereus ATCC 14579 CcpA in regulation of metabolic pathways and expression of enterotoxin genes by comparative transcriptome analysis of the wild-type and a ccpA-deletion strain.
Comparative analysis revealed the growth performance and glucose consumption rates to be lower in the B. cereus ATCC 14579 ccpA deletion strain than in the wild-type. In exponentially grown cells, the expression of glycolytic genes, including a non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase that mediates conversion of D-glyceraldehyde 3-phosphate to 3-phospho-D-glycerate in one single step, was down-regulated and expression of gluconeogenic genes and genes encoding the citric acid cycle was up-regulated in the B. cereus ccpA deletion strain. Furthermore, putative CRE-sites, that act as binding sites for CcpA, were identified to be present for these genes. These results indicate CcpA to be involved in the regulation of glucose metabolism, thereby optimizing the efficiency of glucose catabolism. Other genes of which the expression was affected by ccpA deletion and for which putative CRE-sites could be identified, included genes with an annotated function in the catabolism of ribose, histidine and possibly fucose/arabinose and aspartate. Notably, expression of the operons encoding non-hemolytic enterotoxin (Nhe) and hemolytic enterotoxin (Hbl) was affected by ccpA deletion, and putative CRE-sites were identified, which suggests catabolite repression of the enterotoxin operons to be CcpA-dependent.
The catabolite control protein CcpA in B. cereus ATCC 14579 is involved in optimizing the catabolism of glucose with concomitant repression of gluconeogenesis and alternative metabolic pathways. Furthermore, the results point to metabolic control of enterotoxin gene expression and suggest that CcpA-mediated glucose sensing provides an additional mode of control in moderating the expression of the nhe and hbl operons in B. cereus ATCC 14579.
Bacillus cereus is an important Gram-positive, spore-forming food-borne pathogen. Many strains cause either an emetic or a diarrhoeal type of disease. The production of emetic toxin in foods, also referred to as cereulide, may cause nausea and vomiting. The diarrhoeal type of disease is associated with the production of enterotoxins in the intestines and may involve Nhe, Hbl and CytK [1–3]. Food-borne disease caused by B. cereus is generally characterized by mild symptoms. However, recently more severe cases with a lethal outcome have been described [4, 5]. B. cereus can also be the causative agent of other diseases, such as periodontitis, fulminant endophthalmitis, and meningitis in immuno-compromised patients [1, 6–8]. B. cereus is ubiquitously found in the environment, including in soil. Therefore, the transfer to food is not surprising and causes many problems . In nutrient-rich environments, such as food, B. cereus shows low generation times putatively gaining advantage from its capacity to use various carbohydrates and proteinaceous substrates . The regulation of gene expression plays an important role in the efficient selection of the preferred carbon and energy source for growth. Annotation of the genome of B. cereus ATCC 14579 predicted the regulation of gene expression to be highly complex involving over two hundred transcriptional regulators managing its 5370 open reading frames (ORFs) [9, 10]. One of these putative regulators is the catabolite control protein CcpA, which is a member of the LacI-family of transcriptional regulators. CcpA and the regulatory mechanism of the catabolite repression are highly conserved in low-GC Gram-positives . B. cereus ATCC 14579 CcpA shows 77% identity with B. subtilis CcpA. Furthermore, CcpA in B. subtilis has been shown to have a role in optimizing glucose metabolism and the underlying regulatory mechanisms have recently been reviewed [12–14]. Regulation of gene expression by CcpA is mediated by its binding to DNA at a specific cis-binding sequence, the Catabolite Responsive Element (CRE) [14–16].
In recent years the regulon of B. subtilis CcpA has been studied extensively by transcriptome analyses, revealing genes and operons under direct and indirect control of CcpA [17–20]. Furthermore, Moreno et al.  showed a clear correlation between the glucose-repressed genes and the presence of predicted CRE-sites. Moreover, they showed CcpA-mediated glucose-independent regulation of expression . Other organisms for which the role of CcpA in carbon metabolism was established are Lactobacillus acidophilus  and Lactococcus lactis . Recently, a role for CcpA in the control of virulence of Staphylococcus aureus , Streptococcus pneumoniae , and Clostridium perfringens was reported  and reviewed .
Notably, comparative genomics of the different species of the B. cereus group revealed reduced capacity to metabolize carbohydrates and increased potential for protein metabolism as compared to B. subtilis [28, 29]. Here we report on the role of CcpA in regulation of metabolism and virulence in B. cereus ATCC 14579.
Results and Discussion
Growth and glucose utilization of the ccpAdeletion strain compared to the wild-type
Overview of the transcriptome data
Expression ratios of six randomly chosen genes with significantly altered expression in the ccpA deletion strain (cggR, acoR, gapB, ymfC, fruR and odhA) were quantified using qPCR. Expression ratios obtained by microarray analysis were compared to ratios obtained by qPCR. The tested genes showed the same trend in expression, with differential expression only slightly more pronounced in qPCR experiments, a feature observed before in such comparisons . This indicates the microarray platform to be suited for gene expression analysis (for qPCR data see Additional file 1).
Identification of the B. cereus CRE-site consensus and in silicoanalysis
Analysis of genes differentially expressed in the ccpAdeletion strain
Genes involved in B. cereus ATCC14579 glucose metabolism and their putative CRE-sites
PTS system, glucose-specific IIABC component
Phosphocarrier protein HPr
Central glycolytic genes regulator
NADP-dependent glyceraldehyde-3-P dehydrogenase
Pyruvate dehydrogenase (acetyl-transferring)
Pyruvate dehydrogenase (acetyl-transferring)
Acetolactate synthase large subunit
Citric acid cycle
Isocitrate dehydrogenase [NADP]
Succinate-CoA ligase (ADP-forming)
Succinate-CoA ligase (ADP-forming)
Phosphoenolpyruvate carboxykinase (ATP)
One of the glycolytic genes found to be expressed lower in the ccpA deletion strain was yfmT. This gene encodes a non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN), for which 2 putative CRE-sites could be identified. We propose to rename yfmT as gapN, since this gene shows similarity with gapN of the other members of the B. cereus group, and with gapN of B. halodurans, Streptococci and Clostridia . Notably, the gene is lacking in B. subtilis [29, 33]. Next to the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase, two phosphorylating glyceraldehyde-3-phosphate dehydrogenases encoding genes (gapA and gapB) are present on the B. cereus ATCC 14579 genome. The function of GAPN in microbial metabolism is yet unclear. Recently, Asanuma and Hino (2006) showed a role for CcpA in expression control of gapN in Streptococcus bovis and proposed that NADPH is provided by GAPN activity for NADPH-dependent biosynthetic reactions, thereby maintaining an optimal redox balance at the same time. However, in hyperthermophilic archaea GAPN has been shown to play a role in accelerating glycolysis as it can produce 3-phospho-D-glycerate from D-glyceraldehyde 3-phosphate in a one-step reaction, instead of two steps. As a putative drawback, ATP is not produced in this one-step reaction. The exact role of GAPN in B. cereus metabolism remains to be elucidated.
The gluconeogenic genes expressed higher in the ccpA deletion strain included ywjI and gapB. The ywjI gene is part of the putative murA2-ywjI operon and is annotated as a fructose-1,6-bisphosphatase. The ywjI gene of B. subtilis, that shows similarity to ywjI of B. cereus, is annotated to be a fructose-1,6-bisphosphatase, as a member of the glpX-family. The other annotated fructose-1,6-bisphosphatase gene for B. subtilis, fbp, which is no member of the glpX-family, is not regulated by CcpA and deletion of this gene seems to have no effect on gluconeogenesis . A corresponding fbp homologue appears to be absent in B. cereus. However, on the B. cereus genome two members of the glpX-family of fructose-1,6-bisphosphatases are present, of which ywjI expression was found to be affected by ccpA deletion, accordingly a putative CRE-site was identified in the promoter region of its operon. The gapB gene encodes a NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase. Three putative CRE-sites were identified for gapB in B. cereus (Table 1). In contrast, no CRE-sites were identified for gapB in B. subtilis, and its expression was shown to be only indirectly affected by ccpA deletion . Furthermore, gluconeogenesis in B. subtilis was shown to be regulated by the transcriptional regulator CcpN [37, 38]. CcpN is also present on the genome of B. cereus ATCC 14579 and putative CcpN binding sites can be found in front of the gluconeogenic genes encoding GapB and PckA . Combined with our results this would point to dual control of the expression of gluconeogenic genes in B. cereus by CcpN and CcpA.
Glutamate is a product of protein and amino acid catabolism and acts as link between nitrogen and carbon metabolism, mediated by glutamate synthase and glutamate dehydrogenase, as described for B. subtilis [46, 47]. The B. subtilis genome contains a glutamate synthase (gltAB) and two glutamate dehydrogenases (rocG and gudB) of which the GudB dehydrogenase appears to be inactive . Glutamate biosynthesis in B. subtilis has recently been shown to be tightly regulated by interaction of RocG and GltC , but these genes are not encoded on the B. cereus genome. The microbial glutamate synthases generally belong to the NADPH-GltS family of glutamate synthases and are encoded by two genes . In the glutamate synthase reaction L-glutamate is produced from L-glutamine and 2-oxoglutarate, with the latter compound derived from the citric acid cycle . The gltAB operon of B. subtilis is suggested to be regulated by CcpA [46, 50]. On the genome of B. cereus ATCC 14579 only one glutamate synthase gene (gltA) is present and its expression at various phases of growth is similar in the wild-type and its ccpA deletion strain, indicating that its expression is not regulated by CcpA. Moreover, it is unclear to which family of glutamate synthases GltA of B. cereus belongs. Interestingly, gudB was the only gene present on the genome of B. cereus encoding a glutamate dehydrogenase, and its expression was observed to be clearly higher in the ccpA deletion strain (Table 1). Glutamate dehydrogenase is responsible for the reaction from L-glutamate to 2-oxoglutarate linking L-glutamate to the citric acid cycle . Higher expression of gudB in the ccpA deletion strain compared to the wild-type, together with the fact that it is the only glutamate dehydrogenase annotated on the genome of B. cereus suggests that GudB is the active glutamate dehydrogenase for B. cereus. This is supported by the fact that the 9-bp sequence, encoding the 3 amino acids causing the inactivity of GudB in B. subtilis , are absent in the gudB sequence of B. cereus ATCC 14579. Two putative CRE-sites in front of and one within gudB were identified pointing to CcpA-controlled expression in B. cereus.
B. subtilis contains a large number of carbohydrate catabolic pathways , whereas the number of these pathways is limited in B. cereus. The observed deficiency of the B. subtilis ccpA deletion strain in growth with ammonium as the sole nitrogen-source, has been attributed to the regulation of the gltAB genes by CcpA [46, 50] and the read-through transcription of the rocG gene . The lack of regulation of gltA in B. cereus by CcpA, together with a similarity to the B. subtilis gltA of only 46%, offers an explanation for the observation that the ccpA deletion strain of B. cereus is able to grow with ammonium as the sole nitrogen source (data not shown), conceivably with GudB as the active glutamate dehydrogenase.
The role of CcpA in optimisation of glucose metabolism is apparent, since glycolytic enzymes were expressed lower and expression of genes encoding citric acid cycle enzymes was shown to be higher in the ccpA deletion strain compared to the wild-type. Up-regulation in the ccpA deletion strain was found for ~30 genes encoding enzymes involved in protein, peptide and amino acid metabolism (see Additional file 1). This is seemingly in contrast with the proposed preferred use of proteinaceous substrates for growth of B. cereus, a hypothesis put forward by Ivanova et al. . This hypothesis was supported by the annotation of a large number of genes encoding proteolytic enzymes, a multiplicity of peptide and amino acid transporters, and a large variety of amino acid degradation pathways. However, under the conditions tested glucose is used as the carbon and energy source (Fig. 1) and genes encoding enzymes involved in protein, peptide and amino acid catabolism appear to be subject to catabolite repression, indicating that under the conditions tested, glycolysis rather than protein catabolism is the preferred energy generation route of B. cereus ATCC 14579.
CcpA-mediated catabolite control of enterotoxin gene expression in B. cereus
B. cereus ATCC 14579 catabolite control protein CcpA is involved in optimizing the catabolism of glucose with concomitant repression of a range of metabolic pathways. Furthermore, CcpA-mediated glucose sensing is shown to provide an additional mode of control in moderating the expression of the nhe- and hbl-operon in this human pathogen.
Bacterial strains, culture media, growth conditions, and genetic methods
B. cereus ATCC 14579 and its ccpA deletion strain FM1403 were cultured in brain heart infusion broth (BHI, Becton and Dickinson, The Netherlands) medium at 30°C, with shaking at 200 rpm. The growth of the culture was monitored by measurement of the optical density at OD600. D-glucose concentrations were measured by use of a D-glucose measuring kit (Boehringer). Growth experiments and glucose measurements were performed in three fold. Plasmid DNAs were purified from E. coli with a Qiaprep Spin Miniprep kit (Westburg, Leusden, The Netherlands). Pwo polymerase (Roche Diagnostics, Almere, The Netherlands) was used for PCR generated fragments that were used in cloning and Taq polymerase (Fermentas, Amersfoort, The Netherlands) was used in control PCRs. E. coli HB101/pRK24  was used as the donor host in conjugation experiments. The antibiotics used were ampicillin (Sigma, Zwijndrecht, The Netherlands) at a concentration of 50 μg/ml, kanamycin (Sigma) at a concentration of 70 μg/ml, erythromycin (Sigma) at a concentration of 150 μg/ml (for E. coli) or 5 μg/ml (for B. cereus), spectinomycin (Sigma) at a concentration of 100 μg/ml, and polymyxin B (VWR, Amsterdam, The Netherlands) at a concentration of 50 μg/ml for counter-selection against E. coli upon conjugation.
Construction of ccpAdeletion strain
To construct a double cross-over deletion strain of ccpA, an ~3.5-kb PCR product, comprising ccpA and 1-kb flanking regions was obtained by use of forward primer ccpAKOsacIforw (TCgagctcAGATTACGTTGATGTTATTC) and reverse primer ccpAKOxbaIrev (TGtctagaAGAAGAAGAAAAAGAGGAAGAAAT). This PCR product was cloned into pGemT-easy (Promega, Leiden, The Netherlands) according to the manufacturer's protocol resulting in pGemTccpA. Subsequently, an erythromycin-resistance cassette amplified from pUC18ERY  with forward primer ErycasFBsrGI (TCtgtacaGTCCGCAAAAGAAAAACG) and reverse primer ErycasRClaI (TCatcgatCATACCTAATAATTTATCTAC) was cloned into pGemTccpA after digesting both with Bsp1407I (BsrGI) and Bsu15I (ClaI) (Fermentas). The insert of the resulting plasmid, comprising the 1-kb flanking regions and the erythromycin-resistance cassette was cloned into the conjugal vector pATΔS28  by digestion of the insert and the vector with XbaI and SacI (Fermentas). The resulting plasmid pATΔCcpAery was isolated from DH5α and transformed into E. coli HB101/pRK24. The resulting strain was used in a conjugation experiment with B. cereus ATCC 14579 following established procedures . Transconjugants were obtained by selection for spectinomycin sensitivity and erythromycin resistance and one was analysed in comparison with the wild-type strain. PCR and Southern Blot analysis confirmed the deletion of ccpA by double homologous recombination (data not shown). The B. cereus ccpA deletion strain was designated B. cereus FM1403.
RNA was extracted from both the ccpA deletion strain and the wild-type at four time points in the growth curve at OD600 of 0.2, 0.8, 4 and 8 which corresponds to early-exponential, mid-exponential, transition and stationary phase of growth from two independent cultures per phase by using RNAwiz (Ambion, Huntingdon, United Kingdom) according to the manufacturers protocol. Residual DNA from the RNA preparations was enzymatically removed by using TURBO DNA-free (Ambion). Extracted RNA samples were stored in 70% EtOH, 0.3 M sodium acetate buffer (pH 5.2) at -80°C.
Microarray construction and transcriptome analysis
Amplicon based DNA-microarrays were constructed for B. cereus ATCC 14579 as described for L. lactis IL1403 [64, 65] with modifications as detailed below. Amplicons were designed on 5199 genes selected from the 5311 annotated genes (ORFs smaller than 80-bp were omitted) on the genome of B. cereus ATCC 14579 . To reduce cross-hybridization between probe and target DNA sequences the amplicons had sizes of 70 – 700-bp (depending on gene sizes) and comprised the most unique part of a gene. The amplicons were synthesized by EuroGentec (Seraing, Belgium) in two amplification steps. In the first amplification step, primers were used with a unique tag-sequence for B. cereus ATCC 14579 (forward primers were extended with the sequence: 5'-TCGGGCAGCTGCTCC-3'; and reverse primers with the sequence: 5'-TGGCGCCCCTAGATG-3'). Two copies of each amplicon were present per array, resulting in microarrays comprising 10398 spots.
Normalized expression data (Feature Extract, Agilent) for each spot was used in a statistical analysis. The biological replicate experiments were merged with the web-supported VAMPIRE microarray suite, based on a Bayesian frame work. Furthermore, VAMPIRE calculated p-values for individual spots and subsequently used this p-value to identify statistically differentially expressed spots between compared growth conditions by use of a false discovery rate (FDR) of 0.05 as a threshold [66, 67]. In addition, only ORFs of which both individual spots passed the FDR based threshold were considered to be putatively differentially regulated. Expression ratios per ORF were established by calculating the average of the log-values of individual spots. This value (R) was then used to calculate the average expression ratio (10R) per ORF. Finally, only ORFs that showed a change in expression of at least 2-fold (up/down) were considered to be differentially expressed. Microarray data are submitted to the GEO database with accession number: GSE7843.
To determine gene similarity, homology and gene context NCBI BLAST and the ERGO database were used , while KEGG  was used for assessment of metabolic functions and pathways. Whether succeeding genes were part of one operon was determined according to operon prediction as performed by .
Definition and identification of B. cereusCRE-site
The 350-bp upstream and 150-bp downstream sequences of the translation start of genes identified by the array experiments to be significantly higher expressed in the ccpA deletion strain compared to the wild-type strain for early- and mid-exponential growth were analyzed with AlignACE 3.1 , which searches the input sequences for stretches of nucleotides which align between the different input sequences. The 13-bp CRE-sites identified by AlignACE were aligned with MUSCLE 3.6  and a Hidden Markov Model (HMM) was constructed with the HMMER package . The HMM was used to search CRE-sites in the complete genome sequence of B. cereus ATCC 14579 , and not only the 350-bp upstream and 150-bp downstream sequences significantly higher expressed genes in the ccpA deletion strain. Subsequently, to analyse whether the B. cereus consensus was longer than 13-bp, the obtained sites after the HMM search were extended to 18-bp, as has been the reported length for B. subtilis . The resulting extended CRE-sites were again aligned using MUSCLE 3.6 . This alignment was subsequently visualized with WebLogo  and a iteration HMM search was performed on the B. cereus ATCC 14579 sequence  to identify all putative CRE-sites confirming to the new alignment in the B. cereus ATCC 14579 genome.
Financial support was received from the IOP Genomics Program of Senter Novem (grant IGE1018). The authors would like to thank Mark de Been and Christof Francke (Centre for Molecular and Biomolecular Informatics, Nijmegen, The Netherlands) for their bioinformatics input, and Anne de Jong for his efforts in construction of the DNA microarrays.
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