IMPORTANCEIn Gram-positive bacteria, the global regulator CcpA controls a large number of important physiological and metabolic processes. Although a typical consensus CcpA-binding site, cre, has been identified, it remains poorly explored for the diversity of CcpA-mediated catabolite regulation. Here, we discovered a novel flexible CcpA-binding site architecture (crevar) that is highly variable in both length and base composition but follows certain principles, providing new insights into how CcpA can differentially recognize a variety of target genes to form a complicated regulatory network. A comprehensive search further revealed the wide distribution of crevar sites in Gram-positive bacteria, indicating it may have a universal function. This finding is the first to characterize such a highly flexible transcription factor-binding site architecture, which would be valuable for deeper understanding of CcpA-mediated global catabolite regulation in bacteria.
Catabolite control protein A (CcpA) is the master regulator in Gram-positive bacteria that mediates carbon catabolite repression (CCR) and carbon catabolite activation (CCA), two fundamental regulatory mechanisms that enable competitive advantages in carbon catabolism. It is generally regarded that CcpA exerts its regulatory role by binding to a typical 14- to 16-nucleotide (nt) consensus site that is called a catabolite response element (cre) within the target regions. However, here we report a previously unknown noncanonical flexible architecture of the CcpA-binding site in solventogenic clostridia, providing new mechanistic insights into catabolite regulation. This novel CcpA-binding site, named crevar, has a unique architecture that consists of two inverted repeats and an intervening spacer, all of which are variable in nucleotide composition and length, except for a 6-bp core palindromic sequence (TGTAAA/TTTACA). It was found that the length of the intervening spacer of crevar can affect CcpA binding affinity, and moreover, the core palindromic sequence of crevar is the key structure for regulation. Such a variable architecture of crevar shows potential importance for CcpA’s diverse and fine regulation. A total of 103 potential crevar sites were discovered in solventogenic Clostridium acetobutylicum, of which 42 sites were picked out for electrophoretic mobility shift assays (EMSAs), and 30 sites were confirmed to be bound by CcpA. These 30 crevar sites are associated with 27 genes involved in many important pathways. Also of significance, the crevar sites are found to be widespread and function in a great number of taxonomically different Gram-positive bacteria, including pathogens, suggesting their global role in Gram-positive bacteria.
INTRODUCTIONCarbon catabolite repression (CCR) and carbon catabolite activation (CCA) are two of the most fundamental regulatory mechanisms in microbes (1, 2), enabling them to adapt quickly to environmental changes. In Gram-positive bacteria, the master regulator mediating CCR and CCA is catabolite control protein A (CcpA), a protein of the LacI-GalR family (3). CcpA is a pleiotropic regulator involved in many important cellular processes, including bacterial pathogenicity (4–6).It is known that CcpA executes its regulation via binding to a so-called catabolite-responsive element (cre) within the promoter or protein-coding regions of the target genes (7). The consensus sequence of cre has been determined to be TGWAANCGNTNWCA in Bacillus subtilis, a model organism of Gram-positive bacteria, in which N represents any base and W represents A or T (8). Additional cres identified later in B. subtilis also closely match this consensus sequence (7, 9). However, it has recently been found that, in some cases, CcpA employed two different binding motifs (one is a typical cre and the other one is an atypical cre) to regulate the central carbon metabolism (10). This indicates that the general understanding of CcpA activity is superficial and the mechanism by which CcpA exerts its regulation is more sophisticated than we know.Solventogenic clostridia are of great interest because they are able to produce a series of bulk chemicals (11), among which n-butanol and ethanol are important liquid fuels. Our research group previously revealed a core cre consensus sequence, WTGWAAACGWTWWCAW (where W represents A or T) that is responsible for CcpA binding in Clostridium acetobutylicum, a typical species of solventogenic clostridia (12). This sequence is highly similar to that of B. subtilis, but meanwhile, a large number of genes that exhibited greatly altered transcription after ccpA inactivation do not contain the cre sites in their promoter or protein-coding region (12), thus raising the question as to whether there exist atypical binding sites recognized by CcpA.Here, we identified a novel flexible architecture of binding sites recognized by CcpA. This binding motif was then shown to be widespread in Gram-positive bacteria, indicating its importance in CcpA-mediated regulation. Based on these results, we identify a large number of new target genes controlled by CcpA and thereby chart a more complete CcpA regulatory network in C. acetobutylicum.RESULTSDiscovery of novel transcriptional binding sites for CcpA regulation.Based on our previously performed comparative transcriptomic analysis (12), we found that, among a total of 1,394 genes that showed greatly altered transcription after ccpA inactivation, only 154 genes contain the typical cre (WTGWAAACGWTWWCAW [W represents A or T]) sites within their promoter or coding region. Thus, the presence of noncanonical CcpA-binding sites within these genes is strongly suggested. To explore this possibility, we chose sol (CAP0162-0164), a key operon responsible for acid reassimilation and solvent formation in C. acetobutylicum (13), containing no typical cre sites but showing high binding affinity to CcpA (12), for a detailed examination. In the first step, a 663-bp promoter region (−663 to −1 bp relative to the translational start point) of sol was divided into three fragments (213, 350, and 100 bp) for electrophoretic mobility shift assays (EMSAs) (Fig. 1A). A strongly shifted band was observed for the 350-bp fragment (Fig. 1B), suggesting the existence of binding sites within this region. Next, this 350-bp fragment was further divided into three 170-bp segments (Psol-170-1, Psol-170-2, and Psol-170-3), with 80 bp overlapping one by one, for EMSAs (Fig. 1C). Interestingly, a DNA bind shift was observed for all three segments (Fig. 1D), implying that there may exist more than one CcpA-binding site within the promoter region of sol.FIG 1 Functional analysis of the CcpA-binding sites in the noncoding region of the sol operon. (A) The noncoding region of the sol operon was divided into three fragments (213, 350, and 100 bp). The −10 region and −35 region are underlined. The two transcription start sites (13) are indicated by bent arrows. (B) EMSAs of His6-CcpA binding to the 213-, 350-, and 100-bp fragments labeled with Cy5. (C) The 350-bp fragment of sol was further divided into three 170-bp fragments (Psol-170-1, Psol-170-2, and Psol-170-3). (D) EMSAs of His6-CcpA binding to Psol-170-1, Psol-170-2 and Psol-170-3.To confirm this hypothesis, Psol-170-1, which had the strongest shifted signal among these three segments, was gradually truncated, and the resulting three truncated fragments, namely, Psol-170-1 minus 20, 40, and 60 bp, respectively, were examined (Fig. 2A). The results showed that the affinity of Psol-170-1 for CcpA was almost completely abolished with a 40- or 60-bp deletion (Fig. 2B), suggesting a binding site overlapping or within the deleted region. Encouragingly, visual scanning of this 60-bp region identified a 41-nucleotide (nt) palindromic sequence (AAACTGCTAAATGTAAA-TTATACG-TTTACATTTAGCAGTTT) comprising two 17-nt inverted repeats separated by 7 nt (Fig. 2C). According to the characteristics of this palindromic sequence, we further found two other similar palindromic sequences within the 350-bp fragment, which harbor 6- and 9-nt inverted repeats separated by 8 and 18 nt, respectively (Fig. 2D). A common feature of these three palindromic sequences (designated sol-41, sol-20, and sol-36, respectively) is the two repeats that contain the core palindromic sequence TGTAAA/TTTACA, as well as the intervening spacer region; the difference is the variable length of the two repeats and the intervening spacer region. Thus, this yielded the architecture NxTGTAAA-Yx-TTTACAMx (where Y represents any base, N and M also represent any base but are complementary to each other, and x represents the base number) (Fig. 2E). Compared with the known cre consensus in bacteria such as Bacillus (14), Lactobacillus (15), and Staphylococcus species (16), this binding site architecture is quite distinct, which is an inverted TA-rich sequence separated by a variable (length and nucleotide) spacer region. This architecture is different from all known CcpA-binding cre motifs, which are normally 14 or 16 bp in length, including several highly conserved nucleotides (7, 17–20). We named this novel flexible CcpA binding site architecture “crevar”.FIG 2 Identification of novel CcpA-binding sites in C. acetobutylicum. (A) Truncation of Psol-170-1. Sequences 20, 40, and 60 bp in length were cut off from Psol-170-1, yielding three truncated fragments (Psol-170-1-20, Psol-170-1-40, and Psol-170-1-60). (B) EMSAs of His6-CcpA binding to Psol-170-1 and the three truncated fragments. His6-CcpA concentrations of 0 to 1.0 µM were used. (C) Mining of a potential CcpA-binding site in Psol-170-1 via visual analysis. The putative CcpA-binding site is represented by a box. The two inverted repeats and the intervening spacer of the bind site are colored red and green. (D) Three putative CcpA-binding sites (sol-41, sol-20, and sol-36) found in the 350-bp noncoding region of sol. These three sites share two conserved inverted repeats (TGTAAA/TTTACA), which are highlighted in boldface and italics. (E) A common sequence (NxTGTAAA-Yx-TTTACAMx) was derived from these three CcpA-binding sites. N and M represent any bases complementary to each other, Y represents any base, and x represents the number of the bases included in the two inverted repeats and intervening spacer, which was variable.Wide occurrence of the novel binding site crevar in C. acetobutylicum.To explore the distribution of the crevar sites in C. acetobutylicum, we performed a genome-wide scan using the RegPredict web server (21), in which the two 6-nt repeats of crevar were fixed but the length of the intervening region was variable, covering 0 to 40 nt. The search result revealed 103 potential crevar sites that belong to 99 genes (see Table S1 in the supplemental material). Next, 42 (the associated genes showed ≥2-fold transcriptional changes after ccpA inactivation) (12) of these 103 potential crevar sites were picked out for EMSAs to examine the quality of the prediction result. Finally, 30 crevar sites were confirmed to be bound by CcpA, including the above-mentioned three crevar sites in the upstream region of sol (see Fig. 4C) and the other 27 crevar sites that are associated with 26 genes (see Fig. S1 in the supplemental material). Among these 30 crevar sites, 20 sites are located in promoter regions, whereas 10 sites are inside protein-coding regions (see Table S2 in the supplemental material). The majority of these crevar-associated genes can be grouped into certain functional subsets (Fig. 3). Several genes are involved in important bioprocesses, including substance transport and metabolism, redox balancing, sporulation, and solvent production. Besides, by comparing the distribution of cre and crevar in the genome, crevar sites were found to be predominantly associated with genes belonging to certain function categories, i.e., sporulation, solvent production, and purine and pyrimidine metabolism (Fig. 3), which suggests that crevar may play more important roles in CcpA regulation of these genes, thus enabling a more comprehensive regulatory network of CcpA.10.1128/mBio.02004-16.1TABLE S1 Putative CcpA-binding crevar sites in the chromosome of C. acetobutylicum. Download TABLE S1, DOCX file, 0.03 MB.Copyright © 2017 Yang et al.2017Yang et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.10.1128/mBio.02004-16.2FIG S1 Verification of new CcpA regulatory targets containing crevar. Shown are results from EMSAs of CcpA binding to 26 oligonucleotides containing putative crevar sites in C. acetobutylicum. The oligonucleotide derived from the promoter region of thl (CAC2873) was used as the negative control. The promoter sequence of CAC2517 contains two putative crevar sites. Download FIG S1, DOCX file, 0.4 MB.Copyright © 2017 Yang et al.2017Yang et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.10.1128/mBio.02004-16.3TABLE S2 CcpA-binding crevar sites identified in C. acetobutylicum. Download TABLE S2, DOCX file, 0.02 MB.Copyright © 2017 Yang et al.2017Yang et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.FIG 3 Overview of the genes affected by CcpA. The genes that have been confirmed to be directly regulated by CcpA (from EMSA data in Fig. S1) via crevar sites are shown in red; the cre-associated genes that were predicted to be significantly affected by CcpA (>2-fold transcriptional change after ccpA inactivation) according to our previous work (12) are shown in black; and the genes associated with both cre and crevar and also showing significant transcriptional changes (>2-fold) after ccpA inactivation are shown in blue. The genes associated with multiple cre or crevar sites are annotated with the number of sites given in parentheses. CAC, genes located in chromosome of C. acetobutylicum; CAP, genes located in the megaplasmid of C. acetobutylicum.Characterization of the binding motif crevar.Next, we attempted to assess the importance of the signature sequences of crevar for CcpA binding. The two inverted repeats and the intervening spacers of the three crevar sites (sol-41, sol-20, and sol-36) mentioned above were mutated (Fig. 4A, B, and D), and then the binding activities of CcpA with the three mutated 120-bp sequences were determined. The results showed that the mutations at two repeats completely abolished the binding of CcpA to sol-41, whereas a light binding to sol-20 and sol-36 was maintained (Fig. 4C). In contrast, mutations in the intervening region weakened, to different extent, the binding affinities of CcpA to sol-41, sol-20, and sol-36 (Fig. 4E). These findings suggest that both the two inverted repeats and intervening spacer are crucial for CcpA-crevar binding.FIG 4 Mutational analysis of the crevar sites in the noncoding region of sol. (A) The 350-bp sequence containing the three putative crevar sites was divided into three 120-bp fragments (sol-120-1, sol-120-2, and sol-120-3), each of which harbored one crevar site (sol-41, sol-20, and sol-36), which is represented by a red box. (B) Mutation of the two inverted repeats of the three crevar sites. The two 6-nt repeats of sol-41, sol-20, and sol-36 were all mutated to GGGGGG, yielding sol-41-mr, sol-20-mr, and sol-36-mr, respectively. (C) EMSAs of His6-CcpA binding to the three 120-bp fragments as well as their derived fragments mutated in the two repeats. (D) Mutation of the intervening spacer of each of the three crevar sites (sol-41-ms, sol-20-ms, and sol-36-ms). (E) EMSAs of His6-CcpA binding to the three 120-bp fragments as well as their derived fragments mutated in the intervening spacers.In addition to in vitro experiments, we also examined CcpA binding to crevar sites in vivo by using a reporter gene. As shown in Fig. S2 in the supplemental material, the LacZ activity assay revealed that single mutation of either the sol-41 or sol-20 site and mutation of both the sol-41 and sol-20 sites resulted in significantly decreased strength of promoter Psol in the wild-type strain, whereas no significant difference was observed in the 824ccpA strain (in which ccpA was disrupted). This further confirmed that sol-41 and sol-20 are the CcpA-binding sites.10.1128/mBio.02004-16.4FIG S2 Validation of CcpA-binding sites in the noncoding region of the sol operon. (A) The mutation of sol-41 and sol-20 in the promoter region of the sol operon. sol-41 and sol-20 are mutated separately or simultaneously. Psol and its mutated derivatives are cloned into the lacZ reporter plasmid. (B) β-Galactosidase assay of the plasmids constructed in panel A at 69 h in both the C. acetobutylicum wild-type strain (824) and ccpA-inactivated (824ccpA) strain. Means and standard deviations were from two independent biological replicates (***, P ≤ 0.001, **, P ≤ 0.01, and *, P ≤ 0.05, Student’s t test). Download FIG S2, DOCX file, 0.1 MB.Copyright © 2017 Yang et al.2017Yang et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.Specific to the two inverted 6-nt repeats, since they were important for CcpA-crevar binding, we attempted to examine whether each nucleotide is essential. Thus, each nucleotide in the two 6-nt repeats of sol-41 was separately mutated, yielding 12 derivative probes for EMSA analysis (Fig. 5A). The EMSA results showed that single mutation of each one of the outer five nucleotides (L1, L2, L3, L4, and L5 or R2, R3, R4, R5, and R6) thoroughly eliminated CcpA–sol-41 binding, while mutation of L6 or R1 still retained a slight binding (Fig. 5B). Next, the in vivo experiments using a lacZ reporter were performed to see the strength variations between Psol and its 12 derivatives. While no significant difference was observed in 824ccpA (the control with ccpA disruption), all 12 single mutations resulted in greatly decreased LacZ activity in the wild-type strain (Fig. 5C), which are consistent with the in vitro EMSA results. These findings demonstrate that each nucleotide in the two 6-nt arms of crevar is important for CcpA binding.FIG 5 Characterization of the novel CcpA-binding sites in C. acetobutylicum. (A) Single point mutation of the inverted repeats of sol-41. The mutation site is marked in red. (B) EMSAs of His6-CcpA binding to sol-120-1 containing sol-41 and its mutated derivatives. Concentrations of 0 to 1.0 µM of His6-CcpA were used. (C) In vivo assay of Psol and its derivatives in both the C. acetobutylicum wild-type and ccpA-inactivated strain. The data represent the average from two independent samples.The variable intervening region length of crevar sites affects CcpA binding affinity.Since the intervening spacer of crevar is variable, the question arose as to whether the spacer length affected CcpA-DNA binding. We explored this possibility by using promoter Pcac0804-15(wt), which contains a 27-nt crevar site with a 15-nt intervening spacer (Fig. 6A). Here, the major reason for choosing Pcac0804-15(wt) for investigation is that, among the 15 genes identified to contain a sole crevar site in their promoter regions (Fig. 3), the cac0804 gene was the only one showing steady and significant upregulation (over 2-fold) in transcriptional level after ccpA overexpression (data not shown), indicating a high CcpA binding affinity to the crevar site in Pcac0804-15(wt). When this 15-nt spacer was truncated to 10 nt, a significantly altered CcpA-DNA binding affinity occurred (Fig. 6B and C); further truncated to 6 nt, no binding affinity changes were found between Pcac0804-6 and Pcac0804-15(wt) (Fig. 6C). In contrast, for the ccpA-inactivated strain (used here as a control), no significant differences in LacZ expression were observed after truncation of the spacer (Fig. 6C). These results suggest that the intervening spacer length within crevar sites can influence CcpA-crevar binding affinity.FIG 6 Influence of the intervening spacer length within crevar on CcpA regulation. (A) Construction of artificial promoters harboring the crevar sites with different spacer lengths. The two inverted repeats and the intervening spacer are highlighted with red and green, respectively. Pcac0804-15(wt) is the natural promoter sequence of the cac0804 gene. Pcac0804-mu was constructed as a negative control by mutating the two inverted repeats of Pcac0804-15(wt). (B) EMSAs of His6-CcpA binding to Pcac0804-15(wt), Pcac0804-10, Pcac0804-6, and Pcac0804-mu. (C) In vivo assay of CcpA regulation of Pcac0804-15(wt) and its derivatives in both the C. acetobutylicum wild-type strain (824) and ccpA-inactivated strain (824ccpA). Means and standard deviations were from two independent biological replicates (**, P ≤ 0.01, Student’s t test).The crevar sites are widely distributed in Gram-positive bacteria.Because the crevar sites occurred frequently in the C. acetobutylicum genome, we are curious whether this cis element is also present in other bacteria. To this end, we performed genome-wide searches in the classes Clostridia and Bacilli, two large groups in Gram-positive bacteria. Surprisingly, the crevar sites were found in the genome of several members of these two classes, including pathogens, and were especially abundant in Clostridium and Bacillus species, in which over 100 crevar sites were predicted to be present in Clostridium acetobutylicum, Clostridium cellulolyticum, Clostridium difficile, and Bacillus cereus (see Table S3 in the supplemental material). Importantly, like those identified in C. acetobutylicum, the crevar sites present in these species also exhibited high diversity in the two inverted repeats and intervening spacer regions (Table S3). To our knowledge, only very few proteins have been found capable of recognizing DNA sequence separated by a variable spacer (22–25); however, crevar-like binding motifs that contain such a highly flexible spacer region have not been reported.10.1128/mBio.02004-16.5TABLE S3 Putative crevar sites in class Clostridia and class Bacilli. Download Table S3, DOCX file, 0.1 MB.Copyright © 2017 Yang et al.2017Yang et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.We chose five potential crevar sites from both B. subtilis (BSU10020, BSU14580, BSU22720, BSU27620, and BSU35080) and C. perfringens (CPF0042, CPF0484, CPF0526, CPF0580, and CPF1663) for EMSA verification. B. subtilis CcpA and C. perfringens CcpA were purified and used for functional analysis of the crevar sites in B. subtilis and C. perfringens, respectively. Encouragingly, among these candidates, a substantial DNA band shift was observed for six crevar sites (BSU22720, BSU27620, BSU35080, CPF0526, CPF0580, and CPF1663) (Fig. 7), indicating a high reliability of the predicted crevar sites in the classes Clostridia and Bacilli.FIG 7 EMSA verification of CcpA binding to the putative crevar sites in B. subtilis and C. perfringens. The sequences highlighted with red indicate the putative crevar sites. B. subtilis CcpA and C. perfringens CcpA were purified and used for functional analysis of the crevar sites in B. subtilis and C. perfringens, respectively.DISCUSSIONAs an important regulator in Gram-positive bacteria, CcpA has remained little understood with respect to its pleiotropic regulatory function. This study has expanded CcpA’s target genes to a broader range in clostridia as well as some other Gram-positive bacteria, thereby providing new insights into CcpA regulation. Importantly, identification of the novel crevar sites revealed a flexible binding site architecture used by CcpA to regulate its target genes. The variation in both the intervening spacer region and two inverted repeats of this crevar motif, as well as its widespread occurrence in Gram-positive bacteria, suggests a more complex CcpA regulation than was previously understood.To date, only very few proteins have been found capable of recognizing repeats separated by a variable spacer. As an example, the Escherichia coli cyclic AMP (cAMP) receptor protein (CRP)-binding sites contain a 6- or 8-bp spacer (22); additionally, the E. coli CytR repressor, with the assistance of the CRP, can recognize two inverted repeats separated by 10 to 13 bp or direct repeats separated by 1 bp (23). A latest example is the E. coli transcription factor HipB, which can recognize palindromic sequences with variable intervening spacer regions (24); moreover, the crystal structures of the HipB-HipA-hipBA promoter complex showed that HipBA binding to DNA with a long spacer can be achieved by DNA extrusion (25).Here, the variation in the crevar sites suggests diversity in the CcpA-binding sites for both repressed and activated target genes. The most distinct feature of crevar is its intervening spacer region, which is flexible in both length (0- to 40-nt span) and base composition. This feature makes crevar quite different from all known CcpA-binding cre motifs, which are normally 14 or 16 bp in length, including several highly conserved nucleotides (7, 17–20). For typical cre sites, the base variations may cause them to display different bend angles during CcpA binding; CcpA is also able to adjust its conformation to meet the changes in target DNA (7). However, such changes in binding angle appeared insufficient to affect the affinity of the DNA for CcpA (7). In contrast, for the atypical crevar, the spacer within its motif is variable in both length and base composition, which may cause greater changes in CcpA conformation during its binding to the targets.Given the wide variation of the crevar sites as well as the coexistence of crevar and cre, we propose that this variability may be an effective mechanism for the diverse regulation of CcpA in Gram-positive bacteria. First, the variable spacer might affect the binding affinity of CcpA for its targets, which would enable CcpA to produce diverse regulatory outputs. For example, the regulation of E. coli CytR, a regulator belonging to the LacI family, was affected by artificially altering the half-site spacing in its binding sites, and the maximum changes in CytR regulation occurred in the short spacing variants (26). Second, the coexistence of crevar and cre suggests a complementary or independent role of crevar relative to cre in CcpA regulation, which would confer more choices to CcpA during its regulation. At least in some cases, CcpA may require more than one binding site to exert sophisticated gene regulation. For example, it has been found that CcpA employed two different binding motifs (WWGAAARCGYTTTCWW and TTTTYHWDHHWWTTTY) to regulate the central carbon metabolism in Streptococcus suis (10); besides, crevar sites were predominantly found to be related to genes of certain function categories (Fig. 3), indicating a more important role of crevar, rather than cre, in CcpA regulation of these genes.Another interesting finding here is that the widespread occurrence of the crevar sites in Gram-positive bacteria, especially classes Clostridia and Bacilli. Using B. subtilis as an example, many essential genes involved in core metabolism were shown to be controlled by CcpA via crevar (Table S3), although the predicted crevar sites appeared to be fewer than the typical cre sites, which were previously estimated to number over 100 in B. subtilis (9, 27). The validation experiments in B. subtilis also supported this finding (Fig. 7). More importantly, the crevar sites were found to be associated with several essential genes in the pathogenic bacteria, such as genes responsible for the phosphotransferase (PTS) system, cell motility and division, DNA replication and mismatch repair, and sporulation (Table S3). Of note, the crevar sites were also present in the promoter or protein-coding regions of certain toxin or virulence genes, e.g., the texT gene in Clostridium tetani (28) and a possible virulence factor gene (SE0184) in Staphylococcus epidermidis (29) (Table S3). All of these findings further suggest the potential importance of crevar as a cis element.It should be noted for the two 6-nt-sequence core region that although this sequence appears to be conserved in the crevar motif, we cannot exclude the possibility that the sequence is also changeable to a certain extent without impacting CcpA recognition of the targets. In this study, we used the common sequence (TGTAAA-Yx-TTTACA) that was extracted from the three binding sites upstream of the sol genes as a template, in which the two inverted repeats were fixed. Apparently, using such a template to search for more CcpA-binding sites has limited the 6-nt core region of the repeats; thus, the yielded binding sites do not reflect all the potential variations in this region. To determine the occurrence frequency of each base at each location of this 6-bp inverted repeat, the strategy such as chromatin immunoprecipitation followed by high-throughput sequencing (chromatin immunoprecipitation sequencing [ChIP-seq]) should be useful. This study is under way.In summary, we have discovered an unrealized highly flexible architecture of CcpA-binding sites. The motif crevar, which is variable in both the two repeats and the intervening spacer region, provides new insight into the structure of CcpA recognition sites in Gram-positive bacteria. Such a variation of crevar may provide an effective means to CcpA for fine-tuning the regulatory network. Given the wide distribution of the crevar in Gram-positive bacteria, it is conceivable that this flexible motif plays an important role in CcpA-mediated regulation of cellular properties.MATERIALS AND METHODSStrains and plasmid construction.The strains and plasmids used in this work are listed in Table S4 in the supplemental material. To express the CcpA protein of C. acetobutylicum, ccpA (CAC3037) was PCR amplified and cloned into pET-28a (Novagen, Madison, WI), yielding the plasmid pET-28a-ccpAcac. Similarly, pET-28a-ccpAbsu and pET-28a-ccpAcpf were constructed to express CcpA from Bacillus subtilis and Clostridium perfringens. pET-28a-HPrK and pGEX4T1-HPr were used for HPr kinase (HPrK) and HPr expression (30). Psol and Pcac0804 and their derivatives were PCR amplified and cloned into pIMP1-lacZ (31) for β-galactosidase assays.10.1128/mBio.02004-16.6TABLE S4 Strains and plasmids used in this study. Download Table S4, DOCX file, 0.03 MB.Copyright © 2017 Yang et al.2017Yang et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.Media and cultivation conditions.Escherichia coli was grown in Luria-Bertani (LB) medium at 37°C with the addition of chloramphenicol (Chloromycetin [25 µg/ml]), kanamycin (50 µg/ml), ampicillin (100 µg/ml), and spectinomycin (50 µg/ml) when needed. C. acetobutylicum was cultivated in CGM medium (32) and P2 medium (33). Thiamphenicol (8 µg/ml) were added to the P2 medium when needed.Identification of CcpA-binding sites.The RegPredict web server (21) was used to search all potential crevar sites on the genome of C. acetobutylicum based on the architecture of the template TGTAAA-Yx-TTTACA (Yx ranged from 0 to 40 nt). The search regions cover nucleotide positions from −500 to +2000 relative to the translational start sites of all the genes. The results were further artificially analyzed to eliminate the redundant data.Protein overexpression and purification.The His6-tagged CcpA and HPrK and glutathione S-transferase (GST)-tagged HPr were expressed and purified as described previously (12, 30). The purified proteins were checked by SDS-PAGE.EMSAs.The DNA probes used in EMSAs were generated as follows. First, the unlabeled DNA fragments were amplified from the genome using specific primer pairs containing a universal sequence (5′ AGCCAGTGGCGATAAG 3′) at the 5′ terminal. Second, the DNA fragments were Cy5 labeled by PCR using the universal primer 5′ AGCCAGTGGCGATAAG 3′, with Cy5 labeled at the 5′ end. Finally, the resulting Cy5-labeled probes were recovered by agarose gel electrophoresis.The EMSAs with C. acetobutylicum CcpA were performed as described previously (30). The EMSAs with B. subtilis CcpA and C. perfringens CcpA were performed similarly, except that the phosphorylated Hpr was not used.β-Galactosidase assays.The C. acetobutylicum strains harboring the plasmids pIMP1-Psol-lacZ, pIMP1-Pcac0804-lacZ, and their derivatives (listed in Table S4) were grown in P2 medium containing 60 g/liter glucose as the sole carbon source. CaCO3 was added at 0.5% (wt/vol) to the medium to control pH. The cell pellets were harvested by centrifugation (5,000 × g, 4°C, 10 min), dissolved in B-PER reagent (Thermo Scientific Pierce), and vortexed for 1 min for cell lysis. The cell lysate was then heat treated at 60°C for 30 min to remove the heat-unstable proteins. Finally, the cell lysate was centrifuged at 12,000 × g for 30 min, and the supernatant was used for β-galactosidase assays as previously reported (34).