Ren et al. BMC Genomics 2012, 13:349 http://www.biomedcentral.com/1471-2164/13/349 RESEARCH ARTICLE Open Access Pleiotropic functions of catabolite control protein CcpA in Butanol-producing Clostridium acetobutylicum Cong Ren1†, Yang Gu1†, Yan Wu1, Weiwen Zhang2, Chen Yang1, Sheng Yang1,3 and Weihong Jiang1* Abstract Background: Clostridium acetobutylicum has been used to produce butanol in industry. Catabolite control protein A (CcpA), known to mediate carbon catabolite repression (CCR) in low GC gram-positive bacteria, has been identified and characterized in C. acetobutylicum by our previous work (Ren, C. et al. 2010, Metab Eng 12:446–54). To further dissect its regulatory function in C. acetobutylicum, CcpA was investigated using DNA microarray followed by phenotypic, genetic and biochemical validation. Results: CcpA controls not only genes in carbon metabolism, but also those genes in solvent production and sporulation of the life cycle in C. acetobutylicum: i) CcpA directly repressed transcription of genes related to transport and metabolism of non-preferred carbon sources such as D-xylose and L-arabinose, and activated expression of genes responsible for D-glucose PTS system; ii) CcpA is involved in positive regulation of the key solventogenic operon sol (adhE1-ctfA-ctfB) and negative regulation of acidogenic gene bukII; and iii) transcriptional alterations were observed for several sporulation-related genes upon ccpA inactivation, which may account for the lower sporulation efficiency in the mutant, suggesting CcpA may be necessary for efficient sporulation of C. acetobutylicum, an important trait adversely affecting the solvent productivity. Conclusions: This study provided insights to the pleiotropic functions that CcpA displayed in butanol-producing C. acetobutylicum. The information could be valuable for further dissecting its pleiotropic regulatory mechanism in C. acetobutylicum, and for genetic modification in order to obtain more effective butanol-producing Clostridium strains. Keywords: CcpA, Pleiotropic regulator, Acidogenesis and solventogenesis, Sporulation Background Clostridium species, CcpA was reported to be involved CcpA is a conserved regulator protein in gram-positive in various cellular processes, such as glucose repression bacteria, which was first characterized as a transcrip- on D-xylose metabolism in C. acetobutylicum by our la- tional regulator responsible for glucose repression on α- boratory [11]; also efficient sporulation and enterotoxin amylase synthesis in Bacillus subtilis, a model firmicute gene regulation [12], biofilm formation [13] and glid- bacterium [1,2]. The later studies have demonstrated ing in pathogen C. perfringens [14]; and repression that B. subtilis CcpA is a master repressor or activator of toxin gene expression in C. difficile [15]. to many cellular processes, such as sugar transport [3,4], Gram-positive C. acetobutylicum has been used to pro- glycolysis [5,6], ammonium assimilation [7,8] and duce organic solvents through a so-called ABE (acetone- D-xylose and L-arabinose metabolism [9,10]. In butanol-ethanol) fermentation process [16,17]. Among several solvents that ABE fermentation process produces, * Correspondence: [email protected] butanol has attracted significant attention in recent years †Equal contributors as a potential next-generation liquid fuel [18,19]. Com- 1Key Laboratory of Synthetic Biology, Institute of Plant Physiology and pared to the other existing biofuels, such as ethanol, bu- Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China tanol offers advantages as a gasoline substitute because Full list of author information is available at the end of the article of its higher energy content and hydrophobicity [20]. © 2012 Ren et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ren et al. BMC Genomics 2012, 13:349 Page 2 of 20 http://www.biomedcentral.com/1471-2164/13/349 C. acetobutylicum genome contains one ccpA gene repression on non-preferred carbon sources utilization; [21,22], the product of which shares 42% amino acid iii) regulation of acidogenesis and solventogenesis path- identity with that of B. subtilis. However, currently very ways; and iv) necessary for efficient sporulation. The little is known about the regulatory roles of CcpA in C. study revealed novel aspects of CcpA regulatory func- acetobutylicum. Our previous analysis showed that C. tions in C. acetobutylicum, offering new targets for fur- acetobutylicum ccpA mutant established significant ther engineering this solventogenic clostridia. phenotypic changes (e.g., deficiency in acids re-assimila- tion) compared to its parental strain [11], suggesting that Results and discussion in addition to regulation of D-xylose metabolism, CcpA Global changes of transcriptome of C. acetobutylicum may be involved in regulation of other cellular processes caused by ccpA inactivation in C. acetobutylicum. Since the regulatory roles of C. acetobutylicum CcpA in Since CcpA likely exerts pleiotropic regulation in fermenting D-glucose and D-xylose mixture (simulating C. acetobutylicum, it thus raises an intriguing question, lignocellulosic hydrolysates components) are of great namely whether C. acetobutylicum CcpA is involved in interest, these two mono sugars were used as the carbon regulating specific cellular processes that are closely sources in our fermentation experiments, from which related to its industry applications, such as acids samples were taken for microarray assay. We have previ- re-assimilation, solvents forming, sporulation and cap- ously found that inactivation of the ccpA gene caused ability of utilizing non-preferable sugars [19,23-26]. Posi- acids accumulation in C. acetobutylicum, resulting in a tive modification of regulation of these cellular processes deficient growth [11]. Therefore, in order to create a will result in significant improvement in terms of eco- normal growth profile for the ccpA-inactivated strain nomic feasibility of industrial-scale butanol production. (824ccpA), pH was controlled (≥5.0) during the fermen- In this study, we employed a comparative transcrip- tation process for the 824ccpA as well as the wild type tome analysis in combination with genetic and biochem- strain (824WT) (Figure 1). Microarray analysis was per- ical validation to determine the possible regulatory formed to investigate global control of gene expression functions of CcpA in C. acetobutylicum. The results by CcpA during growth on D-glucose and D-xylose at showed CcpA is an important pleiotropic regulator in four time points, at which A600 values of the 824WT and C. acetobutylicum: i) activating genes in D-glucose-spe- 824ccpA were similar (Figure 1A). The time point M cific phosphotransferase system (PTS), enhancing D-glu- (middle exponential phase) and L (late exponential cose utilization; ii) mediating carbon catabolite phase) were in acidogenic phase, while the time point T Figure 1 Comparison of the growth, sugar consumption and production of solvents (butanol and acetone) and butyrate between the 824ccpA (Square) and 824WT (triangle) strains when fermenting D-glucose and D-xylose with pH control. pH value was kept over 5.0 by using 9% (w/v) aqueous ammonia. Samples (indicated by arrows) for microarray analysis were collected in four time points: middle (M) and late (L)-exponential growth phase; transition phase (T) and stationary phase (S). Ren et al. BMC Genomics 2012, 13:349 Page 3 of 20 http://www.biomedcentral.com/1471-2164/13/349 (transition phase) and S (stationary phase) were in shift the motif comparison utility TOMTOM [3], indicating phase (shifting from acidogenesis to solventogenesis) that these CRE sequences are reliable. In the contrary, and solventogenic phase, respectively. In addition, in despite that some down-regulated genes were found to order to confirm whether the genes involved in carbohy- harbor putative CRE sites (Addition file 3), no such a drates uptake and metabolism, which were found to be conserved motif can be retrieved from the promoter repressed by CcpA, are also subject to “glucose repres- sequences of these genes, thus indicating that the CcpA- sion” (also called Carbon Catabolite Repression, CCR), binding sites of most of the down-regulated genes are the transcriptional levels of these genes in 824WT were atypical or they might be indirectly regulated by CcpA. also investigated using microarray in the presence Next, we adopted HMM (Hidden Markov Model) [31], (time point S) and absence (time point S2 and S3) of which was built based on the above CRE sequences, to D-glucose (Additional file 1A: Figure S1). search all potential CcpA-binding CRE sites in C. aceto- butylicum. The bit score assigned by HmmSearch pro- (a) Overview of DNA microarray analysis gram reflects the similarity of a sequence match Comparative transcriptomic analysis of the 824ccpA and to a profile Hidden Markov Model [32]. The result 824WT showed that 636, 596, 608 and 936 genes were (Additional file 3: Table S2) showed that 148 and 224 differentially expressed (change fold ≥2.0) in the phase putative CRE sites were detected upstream of the Open M, L, T and S, respectively (Additional file 2: Table S1). Reading Frame (ORF) and within ORF region, respect-