The transcriptional program underlying the physiology of clostridial sporulation
© Jones et al.; licensee BioMed Central Ltd. 2008
Received: 5 March 2008
Accepted: 16 July 2008
Published: 16 July 2008
Clostridia are ancient soil organisms of major importance to human and animal health and physiology, cellulose degradation, and the production of biofuels from renewable resources. Elucidation of their sporulation program is critical for understanding important clostridial programs pertaining to their physiology and their industrial or environmental applications.
Using a sensitive DNA-microarray platform and 25 sampling timepoints, we reveal the genome-scale transcriptional basis of the Clostridium acetobutylicum sporulation program carried deep into stationary phase. A significant fraction of the genes displayed temporal expression in six distinct clusters of expression, which were analyzed with assistance from ontological classifications in order to illuminate all known physiological observations and differentiation stages of this industrial organism. The dynamic orchestration of all known sporulation sigma factors was investigated, whereby in addition to their transcriptional profiles, both in terms of intensity and differential expression, their activity was assessed by the average transcriptional patterns of putative canonical genes of their regulon. All sigma factors of unknown function were investigated by combining transcriptional data with predicted promoter binding motifs and antisense-RNA downregulation to provide a preliminary assessment of their roles in sporulation. Downregulation of two of these sigma factors, CAC1766 and CAP0167, affected the developmental process of sporulation and are apparently novel sporulation-related sigma factors.
This is the first detailed roadmap of clostridial sporulation, the most detailed transcriptional study ever reported for a strict anaerobe and endospore former, and the first reported holistic effort to illuminate cellular physiology and differentiation of a lesser known organism.
Clostridia are of major importance to human and animal health and physiology, cellulose degradation, bioremediation, and for the production of biofuels and chemicals from renewable resources . These obligate anaerobic, Gram-positive, endospore-forming firmicutes include several major human and animal pathogens, such as C. botulinum, C. perfringens, C. difficile, and C. tetani, the cellulolytic C. thermocellum and C. phytofermentans, several ethanologenic , and many solventogenic (butanol, acetone and ethanol) species . Their sporulation/differentiation program is critical for understanding important cellular functions or programs, yet it remains largely unknown. We have recently examined the similarity of the clostridia and bacilli sporulation programs using information from sequenced clostridial genomes . We concluded that, based on genomic information alone, the two programs are substantially different, reflecting the different evolutionary age and roles of these two genera. We have also argued that C. acetobutylicum is a good model organism for all clostridia . Transcriptional or functional genomic information is, however, necessary for detailing these differences and for understanding clostridial differentiation and physiology. Key issues awaiting resolution include: the identification of the mid to late sigma and sporulation factors and their regulons; the orchestration and timing of their action; the set of genes employed by the cells in the mid and late stages of spore maturation; identification of candidate histidine kinases that might be capable of phosphorylating the master regulator (Spo0A) of sporulation; and some functional assessment of the roles of several sigma factors of unknown function encoded by the C. acetobutylicum genome. Furthermore, an understanding of the transcriptional basis of the complex physiology of this organism will go a long way to improve our ability to metabolically engineer, for practical applications, its complex sporulation and metabolic programs. Such information generates tremendous new opportunities for further exploration of this complex anaerobe and its clostridial relatives, and constitutes a firm basis for future detailed genetic and functional studies.
Using a limited in scope and resolution transcriptional study, we have previously shown that it is possible to use DNA-microarray-based transcriptional analysis to generate valuable functional information related to stress response [4, 5], initiation of sporulation  and the early sporulation program of C. acetobutylicum . In order to be able to accurately study the transcriptional orchestration underlying the complete sporulation program of the cells, it was necessary to develop a more sensitive and accurate microarray platform, a better mRNA isolation protocol (in order to isolate RNA from the mid and late stationary phases), as well as to use a much higher frequency of observation and sampling. We also aimed to employ more sophisticated bioinformatic tools in order to globally interrogate any desirable cellular program and relate it to the characteristic phenotypic metabolism and sporulation of this organism. The results of this extensive study are presented here as a single, undivided story, which offers unprecedented insights and a tremendous wealth of information for further explorations. Furthermore, it serves as a paradigm of what can be effectively accomplished with the now highly accurate DNA-microarray analysis in generating a robust transcriptional roadmap and in illuminating the physiology of a lesser understood organism.
Results and discussion
Metabolism and differentiation of C. acetobutylicum: identification of a new cell type?
The transcriptional program of clostridial differentiation
Six distinct clusters of temporal expression patterns were selected (Figure 1c,d) by K-means to achieve a balance between inter- and intra-cluster variability. To examine transcriptional changes in larger functional groups (for example, transcription, motility, translation), each cluster was analyzed according to the Cluster of Orthologous Groups of proteins (COG) classification  and the functional genome annotation . To determine if a COG functional group was overrepresented in any of the K-means clusters, first the percentage of each group in the genome was determined, and then the percentage of each group was determined in each of the K-means clusters. By comparing the percentage in the K-means clusters to the genome percentage, we could identify overrepresented groups (Additional data file 2).
Exponential phase: motility, chemotaxis, nucleotide and primary metabolism
The first cluster contains 134 genes highly expressed during exponential growth (hours 6 to 10; see Additional data file 2 for a list of the genes). This cluster characterizes highly motile vegetative cells (Figure 1b, I) and, given the minimal amount of knowledge on the genes responsible for motility and chemotaxis in clostridia, our analysis offers the possibility of identifying these genes at the genome scale . This cluster includes the flagella structural components flagellin and flbD, the main chemotaxis response regulator, cheY (CAC0122; responsible for flagellar rotation in B. subtilis ), as well as several methyl-accepting chemotaxis receptor genes (CAC0432, CAC0443, CAC0542, CAC1600, CAP0048). COG analysis showed that genes related to cell motility (COG class N) and nucleotide transport and metabolism (COG class F) were overrepresented in this cluster (Additional data file 2). In order to investigate cell motility further, all genes that fell within this COG class were hierarchically clustered according to their expression profiles (see Additional data file 3 for Figure S2 and discussion). Interestingly, the two main cell motility gene clusters, the first including most of the flagellar assembly and motor proteins and the second containing most of the known chemotaxis proteins, clustered together and displayed a bimodal expression pattern (Figure S2). The genes were not only expressed during exponential phase but also during late stationary phase, around hour 38, which is consistent with the observation that a motile cell population was again observed in late stationary phase. Included in the category of nucleotide transport and metabolism are several purine and pyrimidine biosynthesis genes: a set of five consecutive genes, purECFMN, the bi-functional purQ/L gene, purA, pyrPR, pyrD, and pyrI. Two other purine synthesis genes (purH, purD) showed very similar profiles but were not classified within this cluster by the clustering algorithm. Vegetative cells, which correspond to this cluster, produce ATP through acidogenesis, whereby the cells uptake glucose and convert it to acetic and butyric acid. Because glucose is the main energy source, multiple genes for glucose transport were included within this cluster, including the glucose-specific phosphotransferase gene, ptsG, the glucose kinase glcK and CAP0131, the gene most similar to B. subtilis glucose permease glcP. The genes required for the metabolism of glucose to pyruvate did not show temporal regulation, suggesting that expression of these genes is constitutive-like (see Additional data file 3 for Figure S3 and discussion). Acetic acid production genes pta and ack were not temporally expressed, but butyrate production genes ptb and buk were. Though expressed throughout exponential phase, the expression of both ptb and buk slightly peaked during late exponential phase, as previously seen , and thus fall in the transitional (second) cluster. Analysis of the expression patterns of all the genes involved in acidogenesis, not just the differentially expressed genes discussed here, is included in Figure S3 in Additional data file 3. Finally, the expression patterns of the two classes of hydrogenases (iron only and nickel-iron) were investigated (Figure S3 in Additional data file 3). hydA, the iron only hydrogenase that catalyzes the production of molecular hydrogen, was expressed only during exponential phase, whereas the iron-nickel hydrogenase, mbhS and mbhL, was expressed throughout stationary phase.
Initiation of sporulation: abrB, sinR, lipid and iron metabolism
The transitional phase is captured by 139 genes in the second cluster (Figure 1c,d; Additional data file 2). It is made up of genes that show elevated expression between hours 10 and 18 and is when solvent formation was initiated. This cluster characterizes the shift from vegetative cells to cells committing to sporulation and thus includes two important regulators of sporulation, abrB (CAC0310) and sinR (CAC0549), which are discussed in more detail below. Also characteristic of this shift from vegetative growth to sporulation was the overrepresentation of genes related to energy production and conversion (COG class C), since sporulation is an energy intensive process. Solvent production began in the transitional phase, though the genes responsible for solvent production fall in the next (third) cluster; the third cluster partially overlaps with this second cluster but is distinguished by a sustained expression pattern. In response to these solvents, C. acetobutylicum undergoes a change in its membrane composition and fluidity, generally decreasing the ratio between unsaturated to saturated fatty acids [16–18]. Consistent with this change, genes related to lipid metabolism (COG class I) were overrepresented in this cluster. To further investigate this COG class, all genes identified as COG class I were hierarchically clustered (see Additional data file 3 for Figure S4 and discussion). Seven genes that were upregulated just before the onset of sporulation fall within the same operon and are related to fatty acid synthesis. In contrast, many of the most characterized genes involved in fatty acid synthesis (accBC, fabDFZ, and acp) maintain a fairly flat profile throughout the timecourse (Figure S4 in Additional data file 3). Also within this cluster is the gene responsible for cyclopropane fatty acid synthesis (cfa), though classified in COG class M (cell envelope biogenesis) and not COG class I. Importantly, the ratio of cyclopropane fatty acids in the outer membrane has been shown to increase as cells enter stationary phase [18, 19], but the overexpression of this gene alone was unable to produce a solvent tolerant strain . Though not overrepresented in this cluster, all the genes within COG class M were also hierarchically clustered (see Additional data file 3 for Figure S5 and discussion). The transitional cluster also included several genes related to iron transport and regulation like the fur family iron uptake regulator CAC2634, the iron permease CAC0788, feoA, feoB, fhuC, and two iron-regulated transporters (CAC3288, CAC3290), which is consistent with the earlier, more limited data . Significantly, iron-limitation has been found to promote solventogenesis .
Solventogenesis, clostridial form, stress proteins, and early sigma factors
The third cluster (Figure 1c,d; Additional data file 2) of 175 upregulated genes represents the solventogenic/stationary phase as it contains all key solventogenic genes. This cluster characterizes the transcriptional pattern of clostridial cells, the unique developmental stage in clostridia and first recognizable cell type of the sporulation cascade, and exhibited a longer upregulation of gene expression than the previous two clusters. Indeed, its range overlapped the previous (second) and the next two (fourth and fifth) clusters. The clostridial form is generally recognized to be the form responsible for solvent production [8, 21] and is distinguished morphologically as swollen cell forms with phase bright granulose within the cell . This cluster captures both of these characteristics with the inclusion of the solventogenic genes and several granulose formation genes. The solventogenic genes adhE1-ctfA-ctfB, adc, and bdhB were initially induced during transitional phase, the second cluster, but were expressed throughout stationary phase and were thus placed within this cluster. Two granulose formation genes, glgC (CAC2237) and CAC2240, and a granulose degradation gene, glgP (CAC1664), were included within this cluster. The other two granulose formation genes, glgD (CAC2238) and glgA (CAC2239), though not included in this cluster, displayed a similar expression profile to glgC and CAC2240. The concomitant requirement of NADH during butanol production drove the expression of three genes involved in NAD formation: nadABC. Expression of the stress-response gene hsp18, a heat-shock related chaperone, and the ctsR-yacH-yacI-clpC operon, containing the molecular chaperone clpC and the stress-gene repressor ctsR, also fell in this cluster and paralleled the expression of the solventogenic genes (see Additional data file 3 for Figure S6). Other important stress-response genes, groEL-groES (CAC2703-04) and hrcA-grpE-dnaK-dnaJ (CAC1280-83), mirrored this expression pattern, though were not differentially expressed according to the strict criteria employed for selecting the genes of Figure 2c,d (Figure S6 in Additional data file 3). Although genes encoded on the pSOL1 megaplasmid  represent less than 5% of the genome, they constitute 15% of genes in this cluster. pSOL1 harbors all essential solvent-formation genes and, importantly, some unknown gene(s) essential for sporulation . Besides the genes listed in this cluster, the vast majority of the genes located on pSOL1 were expressed throughout stationary phase, with most being upregulated at the onset of solventogenesis (see Additional data file 3 for Figure S7). Several key sporulation-specific sigma factors (σF, σE, σG) and the σF-associated anti-sigma factors in the form of the tricistronic spoIIA operon (CAC2308-06) belong to this cluster along with one of the two paralogs of spoVS (CAC1750) and one of three spoVD paralogs (CAP0150). The second spoVS paralog (CAC1817) did not meet the threshold of expression in 12 of the 25 timepoints; the other two paralogs of spoVD (CAC0329, CAC2130) were above the expression cutoff but did not show significant temporal regulation. Of unknown significance was the expression of a large cluster of genes involved in the biosynthesis of the branched-chain amino acids valine, leucine and isoleucine (CAC3169-74) coinciding with the onset of solventogenesis, as shown before [7, 23], as well as the upregulation of several glycosyltranferases (see Additional data file 3 for Figure S8). The upregulation of valine, leucine, and isoleucine synthesis genes could be indicative of a membrane fluidity adaptation . In B. subtilis, these branched-chain amino acids can be converted into branched-chain fatty acids and change the membrane fluidity , and under cold shock stress, B. subtilis downregulates a number of genes related to valine, leucine, and isoleucine synthesis . Therefore, this upregulation may be another mechanism to change membrane fluidity, though the ratio of unbranched and branched fatty acids has not been reported in studies investigating membrane composition [16–18, 26].
Stationary phase carbohydrate (beyond glucose) and amino acid metabolism
The fourth cluster (Figure 1c,d; Additional data file 2) of 84 genes represents a sharp induction of expression between 18 and 24 hours (early stationary phase). This cluster falls within the stationary (third) cluster described above. This is a compact group, with 70% belonging to one of three COG categories: carbohydrate transport and metabolism, transport and metabolism of amino acids, and inorganic ion transport and metabolism. A number of different carbohydrate substrate pathways, from monosaccharides (fructose, galactose, mannose, and xylose) to disaccharides (lactose, maltose, and sucrose) to complex carbohydrates (cellulose, glycogen, starch, and xylan), were investigated, and many exhibited upregulation during stationary phase, though only a few are highly expressed (see Additional data file 3 for Figure S9). The significance of this upregulation of non-glucose pathways is unknown, because sufficient glucose remains in the media (approximately 200 mM or about 44% of the initial glucose level). Of particular interest was the upregulation of several genes related to starch and xylan degradation (Figure S9 in Additional data file 3). The two annotated α-amylases (CAP0098 and CAP0168) along with the less characterized glucosidases and glucoamylase were all upregulated throughout stationary phase and a number were highly expressed, like CAC2810 and CAP0098. Also upregulated were the predicted xylanases CAC2383, CAP0054, and CAC1037, with CAP0054 and CAC1037 being highly expressed during stationary phase. Mirroring this pattern were CAC1086, a xylose associated transcriptional regulator, and the highly expressed CAC2612, a xylulose kinase. The genes related to glycogen metabolism are believed to be involved in granulose formation, as discussed earlier. Several genes for arginine biosynthesis (argF, argGH, argDB, argCJ, carB) were induced during this time, probably as a result of its depletion in the culture medium.
Genes underlying the activation of the sporulation machinery and the genes for tryptophan and histidine biosynthesis
The fifth cluster (Figure 1c,d; Additional data file 2), representing the middle stationary phase, contains 120 genes mainly expressed between hours 24 and 36, and again falls within the stationary (third) cluster described above. Most of the genes in this cluster activate the sporulation-related sigma factors (σF, σE, σG) or are putatively regulated by them. These include spoIIE, the phosphatase that dephosphorylates SpoIIAA and results in the activation of σF, and the σE-dependent operons spoVR (involved in cortex synthesis), spoIIIAA-AH (required for the activation of σG), and spoIVA (involved in cortex formation and spore coat assembly). The σG-dependent spoVT gene has two paralogs in C. acetobutylicum (CAC3214, CAC3649); the transcriptional pattern suggests that CAC3214, included in this cluster, is the real spoVT. Sporulation-related genes included in this cluster are three cotF genes, one cotJ gene, one cotS gene, the spore maturation protein B, a small acid soluble protein (CAC2365), and two spore lytic enzymes (CAC0686, CAC3244). Though several sporulation-related genes are included in the next (sixth) cluster as well, most, beyond those listed here, are upregulated in mid-stationary phase (see Additional data file 3 for Figure S10 and discussion). Seven genes of the putative operon (CAC3157-63) encoding genes for tryptophan synthesis from chorismate and ten genes for histidine synthesis (CAC0935-43, CAC3031) were also included here.
Spore maturation and late-stationary phase vegetative cells
The sixth cluster, representative of the late stationary phase, includes 162 genes mainly expressed after hour 36 (Figure 1c,d; Additional data file 2). This cluster captured the expression profiles of the forespore and endospore forms, free spores, and late-stage vegetative-like cells. The endospore form represents the last stage before mature spores are released, and therefore fewer sporulation-related genes are within this cluster than previous ones. The sporulation-related genes included in this cluster are two small acid-soluble proteins (CAC1522 and CAC2372), a spore germination protein (CAC3302), a spore coat biosynthesis protein (CAC2190) and a spore protease (CAC1275). Also within this cluster are the two phosphotransferase genes, CAC2958 (a galactitol-specific transporter) and CAC2965 (a lactose-specific transporter), another annotated cheY (CAC2218), various enzymes related to different sugar pathways (CAC2180, CAC2250, CAC2954), and two glycosyltransferases (CAC2172, CAC3049). Expression of these genes may be reflective of the late-stage vegetative-like cells observed during microscopy and demonstrate they have a different genetic profile compared to the early vegetative cells. Interestingly, this cluster is enriched in defense mechanism genes (COG class V) like a phospholipase (CAC3026) and multidrug transporters that may play a role in resistance to a variety of environmental toxins.
General processes: cell division and ribosomal proteins
Two additional gene classes (cell division and ribosomal proteins), though not overrepresented in any of the six clusters described above, were investigated because of their importance in cellular processes and interesting expression patterns. COG class D (cell division and chromosome partitioning), besides important genes for vegetative symmetric division, includes ftsAZ, important for both symmetric and asymmetric cell division, and soj (a regulator of spo0J) and spoIIIE, important for proper chromosomal partitioning between the mother cell and prespore. These genes, along with several uncharacterized genes, were upregulated at the beginning of sporulation (see Additional data file 3 for Figure S11). Almost all the ribosomal proteins were downregulated as the culture entered stationary phase, and interestingly, about half of those downregulated genes were again upregulated in mid-stationary phase and remained upregulated until late-stationary phase (see Additional data file 3 for Figure S12). This upregulation is likely related to the late-stage vegetative-like cells seen.
Expression and activity patterns of sporulation-related sigma factors and related genes
Expression of sporulation transcription factors
Deduced activity profiles of sporulation factors
Can we deduce the activation and processing of σF, σE, and σGfrom transcriptional data?
In B. subtilis, the sigma factors downstream of Spo0A (σF, σE, and σG) are all regulated by a complex network of interactions . We desired to examine if our transcriptional data could be used to do a first test to determine whether the mechanisms employed in the B. subtilis model are valid for C. acetobutylicum. In B. subtilis, σF is held inactive in the pre-divisional cell by the anti-σF factor SpoIIAB. σF is released when the anti-anti-σF factor SpoIIAA is dephosphorylated by SpoIIE, resulting in SpoIIAA binding to SpoIIAB, which then releases σF. In C. acetobutylicum, spoIIAB (CAC2307) and spoIIAA (CAC2308) are transcribed on the same operon as sigF (Figure 3e), but spoIIE (CAC3205) is transcribed separately. The initial increase in σF activity during the transitional phase was not accompanied by an increase in spoIIE expression, but the peak in σF activity did occur after spoIIE upregulation (Figure 4c). Despite the sustained level of σF activity, sigF and spoIIE decreased in expression, though spoIIE expression did increase slightly again after 48 hours (Figure 4c). In B. subtilis, the pro-σE translated from the sigE gene undergoes processing from SpoIIGA, which must interact with SpoIIR in order to accomplish the σE activation. In C. acetobutylicum, SpoIIGA (CAC1694) is transcribed on the same operon as sigE (Figure 3f), and SpoIIR is coded by CAC2898. σE activity increased with the induction of spoIIR (Figure 4d), suggesting a similar mechanism as in B. subtilis. Finally, σG activation in B. subtilis is dependent upon the eight genes within the spoIIIA operon. Here, the second and larger increase in σG activity followed peak expression of the spoIIIA operon, but the early increase in σG activity was not characterized by a large induction of spoIIIA expression (Figure 4e). We tentatively conclude that the B. subtilis processing and activation model does generally hold true in C. acetobutylicum, but further investigation is needed to determine the exact timing and interaction of the various factors and their activators.
Is there a functional sigK?
In B. subtilis, σK is formed by splicing together two genes (spoIVCB and spoIIIC), both under the control of σE and SpoIIID , separated by a skin element . In contrast, a single gene encoding σK has been annotated in C. acetobutylicum . The gene was initially identified using a PCR-approach  and was later detected by primer extension in a phosphate-limited, continuous culture of C. acetobutylicum DSM 1731 . spoIIID, which controls sigK expression with σE in B. subtilis, reached peak expression at hour 30, which is consistent with it being under σE control (Figure 3d) . However, at no timepoint in this study did sigK exceed the cutoff expression criterion. Q-RT-PCR also showed a significantly lower sigK induction compared to the other sigma factors and suggests the transcript, if expressed, is at much lower levels than any other gene analyzed (Figure 2). The putative main σK processing enzyme, SpoIVFB (CAC1253), also did not exceed the cutoff criterion. To help determine if there is an active σK, we investigated two genes controlled by σK in B. subtilis. yabG (CAC2905), which encodes a protein involved in spore coat assembly, was upregulated mid-stationary phase and peaked at hour 30 (Figure 3d), and spsF (CAC2190), involved in spore coat synthesis, was not upregulated until late stationary phase, at hour 38 (Figure 3d). From these two genes, it is difficult to determine whether a functional sigK gene exists or not. Clearly they are both transcribed, but based on its expression pattern, yabG could fall under the control of σE instead of σK. spsF upregulation is late enough to possibly indicate σK regulation though. Ideally, more genes need to be investigated to draw firmer conclusions, but because few σK regulon homologs exist in C. acetobutylicum, we cannot currently determine if there is σK activity or not.
Distinct profiles of sensory histidine kinases: which for Spo0A?
Revisiting the orphan kinases
As discussed, phosphorylated Spo0A is responsible for initiating sporulation in both bacilli and clostridia along with solvent formation in C. acetobutylicum. In bacilli, Spo0A is phosphorylated via a multi-component phosphorelay , initiated by five orphan histidine kinases, KinA-E (kinases that lack an adjacent response regulator); this phosphorelay system is absent in all sequenced clostridia . Alternatively, Spo0A in clostridia may be directly phosphorylated by a histidine kinase, orphan or not, as was hypothesized in [1, 7]. This alternative was demonstrated in C. botulinum, where the orphan kinase CBO1120 was able to phosphorylate Spo0A . In C. acetobutylicum, five true orphan kinases have been identified with a sixth orphan, CAC2220, identified as CheA, which has a known response regulator .
Non-orphan kinase expression
Though primarily interested in orphan kinases because of the similarity to the B. subtilis model, a two-component response system could also be responsible for the phosphorylation of Spo0A. The remaining 30 annotated histidine kinases were also investigated to determine if any displayed a peak in expression before the initial induction of the sol operon (Additional data file 5). Six kinases (Figure 5d,e) were found to have a peak in expression at 8 hours. CAC0290 and CAC3430 subsequently decreased in expression while CAC0225 and CAC0863 maintained expression at initial levels. Despite a dip in expression at hour 9, CAC1582 maintained an increased expression level from 8 hours on. CAC2434 peaked at hour 8, dropped back to initial levels, but then steadily increased with the second induction of the sol operon.
Sigma factors of unknown function: a first assessment of their functional roles
Transcriptional analysis of the sigma factors of unknown function
Loss of pSOL1 impairs sporulation at the level of spo0A expression [7, 48], thus generating increased interest for sigma factors located on the pSOL1 plasmid as these may play a role in the regulation of sporulation. Two sigma factors, CAP0157 and CAP0167, are located on pSOL1 and are annotated as 'special sigma factor (σF/σE/σG family)' and 'specialized sigma factor (σF/σE family)', respectively. It was predicted that CAP0167 is putatively co-transcribed with CAP0166 from a promoter of the σF/σG family  and it displayed an expression pattern similar to that of spo0A, consistent with the computational prediction of an 0A box  and two reverse 0A boxes in its promoter region (Figure 6a). CAP0157 was expressed from an unidentified promoter late in the timecourse (40+ hours) and thus may be involved in late-stage sporulation, despite its low level of expression at hour 20 (Figure 6a). CAC3267, putatively the fourth gene in an operon starting with CAC3270 and ending with CAC3264 , was mainly expressed during early exponential growth (Figure 6a), then decreased, and peaked again around 14 hours, after which expression decreased again. This pattern of expression suggests that it plays a role in vegetative growth and possibly early sporulation. CAC0550, putatively transcribed from a σA promoter as a single cistron , was mainly transcribed early with its expression ending after 20-24 hours (Figure 6b), suggesting that it is not involved in sporulation. CAC1766, expressed from an unknown promoter, displayed a unique pattern with a progressive buildup starting around hours 8-12 and a distinct peak around hour 22 (Figure 6b). CAC2052 is annotated as 'DNA-dependent RNA polymerase σ-subunit' and was putatively expressed together with CAC2053, a hypothetical protein, from a σA and/or a σF/σG promoter . Our data suggest that it is unlikely to be transcribed from a σF/σG promoter without any other effectors, as their transcription peaked at hour 16, when there was very little (if any) σF or σG activity (Figure 6b).
Phylogenetic tree comparison
To help determine a possible function for these sigma factors, a phylogenetic tree was constructed of σ70 sigma factors from ten species, including B. subtilis and all sequenced clostridial species. The resulting tree (Additional data file 6) contains eleven major branches, and of these, seven can be definitively classified based on known sigma factors within the branch. These categories are extracytoplasmic function (ECF), sporulation factors (sigF, sigE, and sigG), sigH, sigA (a basal sigma factor), sigD (regulates chemotaxis and motility), and sigB (a general response sigma factor). Two factors, CAC3267 and CAC1766, fell within ECF branches. CAC3267 fell within an ECF branch close to the B. subtilis σV, a sigma factor of unknown function, and σM, a sigma factor essential for growth and survival in high salt concentrations. CAC1766 fell within a different ECF branch close to B. subtilis σZ, a sigma factor of unknown function, and CAC1509, a sigma factor expressed for less than eight consecutive timepoints. The remaining four factors fell within clusters with other clostridial sigma factors of unknown function, though several could have possible ECF function.
Antisense RNA knock-down of four sigma factors: 'fat' clostridial forms and enhanced glucose metabolism
Of the six expressed sigma factors of unknown function, CAP0157, CAP0167, CAC2052, and CAC1766 were chosen for further study because the timing and shape of their expression patterns suggested potential involvement in sporulation and/or solventogenesis. Since the two processes are coupled, phenotypic changes in differentiation may affect solvent production, as has been previously observed [4, 6, 29, 33, 49]. Antisense RNA (asRNA) knock-down was chosen over knocking out the genes, because knockouts are still extremely difficult to produce in this and all other clostridia. Indeed, to date, only a handful of knockouts have been created [29, 50–53], and these have only been achieved after screening thousands of transformants [51–53]. Recently, a group II intron system has been developed for clostridia , but this system was not yet available when these experiments were carried out. In contrast, asRNA is relatively quick, has been shown to reduce gene expression by up to 90% [33, 55, 56] and has been used to knock-down a large number of genes with a high level of specificity [33, 49, 55–59]. asRNA constructs (see Additional data file 7 for specific sequences used) were designed against CAP0157, CAP0167, CAC2052, and CAC1766 along with CAC2053 and CAP0166, the first genes in the operons predicted to contain CAC2052 and CAP0167, respectively . Cultures of these strains were examined and compared against the wild type (WT) and plasmid control strain 824(pSOS95del) for cell morphology differences and metabolic changes.
Microscopy results from the asRNA-strain cultures revealed both novel morphologies and apparently altered differentiation (Figure 6d). Most notable were changes in strains asCAP0166, asCAP0167 and asCAC1766. Typical WT cultures display a predominately vegetative, symmetrically dividing population through 72 hours as evidenced by the thin, rod-shaped, phase dark cells (Figure 6d, I). By 72 hours, WT cultures exhibited only a small percentage of swollen, cigar-shaped clostridial forms and then a proportional population of free spores by 96 hours.
pSOS95del cultures exhibited clostridial forms by 48 hours, suggesting an accelerated differentiation compared to WT, as has been seen before in our laboratory (Figure 6d, II). Moreover, a greater percentage of clostridial forms and free spores compared to WT were observed at 72 and 96 hours, respectively. asCAP0166 cultures generated a large percentage of clostridial forms and endospores/free spores by hours 48 and 72, respectively (Figure 6d, III). This differentiation is accelerated in comparison to pSOS95del. By hour 96, asCAP0166 cultures exhibited predominately vegetative cells apparently derived from germinated spores (data not shown). asCAP0167 cultures also exhibited accelerated differentiation and displayed a novel (to our knowledge) form of cellular morphology that was most profoundly observable at 72 hours (Figure 6d, IV). This novel morphology has qualities of an excessively swollen clostridial cigar-form (which makes them look much shorter than normal clostridial forms), with what appears to be endospore formation occurring, but without the associated phase bright characteristics seen in the 72 hour asCAP0166 cultures. The asCAP0166 culture displayed cells in this novel morphological state as well, but to a lesser extent, although it is possible that because of its faster sporulation, such cell forms appeared prior to 72 hours. The asCAC1766 cultures also exhibited altered differentiation; most importantly, at 72 hours the majority of the cells exhibited a very swollen clostridial-form morphology similar to that in the asCAP0167 cultures at 72 hours, but slightly more elongated (Figure 6d, V).
Concentrations of glucose, acetone, and butanol for asRNA strains
This detailed and previously unrevealed transcriptional roadmap has allowed for the first time a complete investigation of the genetic events associated with clostridial differentiation. We were able to link distinct and striking global transcriptional changes to previously known important morphological and physiological changes. To date, this is the most complete genetic analysis of the different morphological forms: vegetative, clostridial, and forespore/endospore. Importantly, this analysis was performed on a mixed culture, which may either dilute or produce noise in the data, but investigation of the clusters identified revealed that these clusters do capture important known processes. We were also able to identify a cell population late in the timecourse similar to vegetative cells. Visually, these late cells looked and acted like vegetative cells, and transcriptionally, they were also fairly similar. The major cell motility and chemotaxis genes were upregulated both early and late in the timecourse (Figure S2 in Additional data file 3), as were the ribosomal proteins (Figure S12 in Additional data file 3). Also, the cell division associated genes rodA, ftsE, and ftsX follow the same transcriptional pattern of both early and late expression (Figure S11 in Additional data file 3). Although, these cells stain differently from the early vegetative cells, probably due to changes in membrane structure in response to the presence of solvents and do not produce detectable levels of acids or solvents, we believe these cells are germinated cells from spores produced early in the timecourse. While the triggers for both sporulation and germination are not known , the culture late in the timecourse is less acidic because of the acid reassimilation, and pH has been shown to be a trigger for sporulation .
This study has also allowed the first full comparison to the widely studied B. subtilis sporulation program. We have confidently identified the temporal orchestration of all known sporulation-related transcription factors and conclude the Bacillus model generally holds true with the cascade progressing in the following manner: σH, Spo0A, σF, σE, and σG (Figure 4f). In addition, we can conclude that the major activating/processing proteins involved in sigma factor activation in B. subtilis play a similar role in C. acetobutylicum, though additional investigation is needed to clarify their role. Of significance is the lack of sigK signal. The genes responsible for transcribing sigK in B. subtilis, sigE and spoIIID, were expressed, but the putative processing enzyme spoIVFB was not. Two genes under the control of σK in B. subtilis were expressed, but their expression patterns are not consistent with each other. Based on the expression pattern of yabG, it could be controlled by σE, while the late expression of spsF could be an indication of σK activity.
Materials and methods
Two cultures of C. acetobutylicum ATCC 824 were grown in pH controlled (pH >5) bioreactors (Bioflow II and 110, New Brunswick Scientific, Edison, NJ, USA) . Cell density, substrate and product concentrations were analyzed as described .
RNA isolation and cDNA labeling
Samples were collected by centrifuging 3-10 ml of culture at 5,000×g for 10 minutes, 4°C and storing the cell pellets at -85°C. Prior to RNA isolation, cells were washed in 1 ml SET buffer (25% sucrose, 50 mM EDTA [pH 8.0], and 50 mM Tris-HCl [pH 8.0]) and centrifuged at 5,000×g for 10 minutes, 4°C. Pellets were processed similarly to  but with the noted modifications. Cells were lysed by resuspending in 220 μl SET buffer with 20 mg/ml lysozyme (Sigma, St. Louis, MO, USA) and 4.55 U/ml proteinase K (Roche, Indianapolis, IN, USA) and incubated at room temperature for 6 minutes. Following incubation, 40 mg of acid-washed glass beads (≤106 μm; Sigma) were added to the solution, and the mixture was continuously vortexed for 4 minutes at room temperature. Immediately afterwards, 1 ml of ice cold TRIzol (Invitrogen, Carlsbad, CA, USA) was added; 500 μl of sample was diluted with an equal volume of ice cold TRIzol and purified. Following dilution, 200 μl of ice cold chloroform was added to each sample, mixed vigorously for 15 s, and incubated at room temperature for 3 minutes. Samples were then centrifuged at 12,000 rpm in a tabletop microcentrifuge for 15 minutes at 4°C. The upper phase was saved and diluted by adding 500 μl of 70% ethanol. Samples were then applied to the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), following the manufacturer's instructions. To minimize genomic DNA contamination, samples were incubated with the RW1 buffer at room temperature for 4 minutes. The method disrupted all cell types equally, as evidenced by microscopy (data not shown). cDNA was generated and labeled as described . The reference RNA pool contained 25 μg of RNA from samples taken from the same culture at 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 44, 48, 54, 58, and 66 h.
Agilent technology 22k arrays, (GEO accession number GPL4412) as described in , were hybridized, washed, and scanned per Agilent's recommendations. Spot quantification employed Agilent's eXtended Dynamic Range technique with gains of 100% and 10% (Agilent's Feature Extraction software (v. 9.1)). Normalization and slide averaging was carried out as described [7, 63]. A minimum intensity of 50 intensity units was used as described . Microarray data have been deposited in the Gene Expression Omnibus database under accession number GSE6094. To gain a qualitative measure of the abundance of an mRNA transcript, the averaged normalized log mean intensity values were ranked on a scale of 1 (lowest intensity value) to 100 (highest intensity value). Genes were clustered using TIGR's MEV program .
Q-RT-PCR was performed as described . Specific primer sequences are included in Additional data file 9; CAC3571 was used as the housekeeping gene.
For light microscopy, samples were stored at -85°C after 15% glycerol was added to the sampled culture. Samples were then pelleted, washed twice with 1% w/v NaCl and fixed using 50 μl of 0.05% HCl/0.5% NaCl solution to a final count of 106 cells/μl. Slides were imaged using a Leica widefield microscope with either phase contrast or Syto-9 and PI dyes (Invitrogen LIVE/DEAD BacLight Kit) to distinguish cell morphology.
For electron microscopy, samples were fixed by addition of 16% paraformaldehyde and 8% glutaraldehyde to the culture medium for a final concentration of 2% paraformaldehyde and 2% glutaraldehyde. For cultures grown on plates, colonies were scraped from the agar and suspended in 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Cultures were fixed for 1 h at room temperature, pelleted and resuspended in buffer.
For transmission electron microscopy, bacteria were pelleted, embedded in 4% agar and cut into 1 mm × 1 mm cubes. The samples were washed three times for 15 minutes in 0.1 M sodium cacodylate buffer (pH 7.4), fixed in 1% osmium tetroxide in buffer for 2 h, and then washed extensively with buffer and double de-ionized water. Following dehydration in an ascending series of ethanol (25, 50, 75, 95, 100, 100%; 15 minutes each), the samples were infiltrated with Embed-812 resin in 100% ethanol (1:3, 1:2, 1:1, 2:1, 3:1; 1 h each) and then several changes in 100% resin. After an overnight infiltration in 100% resin, the samples were embedded in BEEM capsules and polymerized at 65°C for 48 h. Blocks were sectioned on a Reichert-Jung UltracutE ultramicrotome and ultrathin sections were collected onto formvar-carbon coated copper grids. Sections were stained with methanolic uranyl acetate and Reynolds' lead citrate  and viewed on a Zeiss CEM 902 transmission electron microscope at 80 kV. Images were recorded with an Olympus Soft Imaging System GmbH Megaview II digital camera. Brightness levels were adjusted in the images so that the background between images appeared similar.
For scanning electron microscopy, fixed samples were incubated on poly-L-lysine coated silica wafers for 1 h and then rinsed three times for 15 minutes in 0.1 M sodium cacodylate buffer (pH 7.4). The samples were fixed with 1% osmium tetroxide in buffer for 2 h, washed in buffer and double de-ionized water, and then dehydrated in ethanol (25, 50, 75, 95, 100, 100%; 15 minutes each). The wafers were critical point dried in an Autosamdri 815B critical point drier and mounted onto aluminum stubs with silver paint. The samples were coated with Au/Pd with a Denton Bench Top Turbo III sputter-coater and viewed with a Hitachi 4700 FESEM at 3.0 kV.
Phylogenetic tree generation
Based on the genome annotations available at NCBI, we considered any sigma factor that was annotated as σ70 or unannotated. A second filter was applied by requiring that all the sequences should contain a Region 2, the most conserved region of the σ70 protein. All members of this class of sigma factor contain Region 2, and it was modeled with the HMM pfam04542. This criterion removed CAC0550, CAC1766 and CAP0157, but they were added to the list again despite their lack of a Region 2. The alignment was made using ClustalW 1.83 using the default settings and visualized as a radial tree as created by Phylodraw v. 0.8 from Pusan National University.
Generation and characterization of antisense strains
Oligonucleotides were designed to produce asRNA complementary to the upstream 20 bp and first 30-40 bp of the targeted genes' transcripts (Additional data file 7). The constructs were cloned into pSOS95del under the control of a thiolase (thl) promoter and confirmed by restriction digest. Plasmids were then methylated and transformed into C. acetobutylicum ATCC 824, as previously described [33, 55, 56]. Strains were grown in 10 ml cultures and characterized using microscopy and HPLC to analyze final product concentrations .
Additional data files
The following additional data are available. Additional data file 1 is a figure comparing the present microarray study to an earlier microarray study that examined the early sporulation of C. acetobutylicum followed by a brief discussion. Additional data file 2 contains tables detailing the COG analysis for each cluster and all the genes placed in each cluster. Additional data file 3 contains figures of the transcriptional profiles, in terms of both intensity and differential expression, of specific gene clusters with brief discussions following several figures. Additional data file 4 is a composite figure showing the individual expression profiles of the genes that were standardized and averaged and is followed by a brief discussion on how the genes used to construct the deduced activity plots were chosen. Additional data file 5 is a figure showing the differential expression and intensity of all annotated histidine kinases and response regulators. Additional data file 6 is a figure showing the phylogenetic tree resulting from the alignment of the σ70-related and unannotated sigma factors from ten bacterial species. Additional data file 7 is a table listing the sequences for each asRNA construct. Additional data file 8 contains figures showing additional TEM images of the plasmid control strain, asCAP0167, and asCAC1766. Additional data file 9 is a table listing the primer sequences used in the Q-RT-PCR experiments.
Cluster of Orthologous Groups
quantitative reverse transcription PCR
transmission electron microscopy
We acknowledge the use of the Northwestern University Keck Biophysics Facility, the Northwestern University Biological Imaging Facility for the light microscopy, and Shannon Modla in the Delaware Biotechnology Institute Bio-Imaging Facility for the electron microscopy. Supported by NSF grant (BES-0418157) and an NIH/NIGMS Biotechnology Training grant (T32-GM08449) fellowship for Bryan Tracy.
- Paredes CJ, Alsaker KV, Papoutsakis ET: A comparative genomic view of clostridial sporulation and physiology. Nat Rev Microbiol. 2005, 3: 969-978. 10.1038/nrmicro1288.PubMedView ArticleGoogle Scholar
- Demain AL, Newcomb M, Wu JH: Cellulase, clostridia, and ethanol. Microbiol Mol Biol Rev. 2005, 69: 124-154. 10.1128/MMBR.69.1.124-154.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Woods DR: The genetic engineering of microbial solvent production. Trends Biotechnol. 1995, 13: 259-264. 10.1016/S0167-7799(00)88960-X.PubMedView ArticleGoogle Scholar
- Alsaker KV, Spitzer TR, Papoutsakis ET: Transcriptional analysis of spo0A overexpression in Clostridium acetobutylicum and its effect on the cell's response to butanol stress. J Bacteriol. 2004, 186: 1959-1971. 10.1128/JB.186.7.1959-1971.2004.PubMedPubMed CentralView ArticleGoogle Scholar
- Tomas CA, Beamish J, Papoutsakis ET: Transcriptional analysis of butanol stress and tolerance in Clostridium acetobutylicum. J Bacteriol. 2004, 186: 2006-2018. 10.1128/JB.186.7.2006-2018.2004.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhao Y, Tomas CA, Rudolph FB, Papoutsakis ET, Bennett GN: Intracellular butyryl phosphate and acetyl phosphate concentrations in Clostridium acetobutylicum and their implications for solvent formation. Appl Environ Microbiol. 2005, 71: 530-537. 10.1128/AEM.71.1.530-537.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Alsaker KV, Papoutsakis ET: Transcriptional program of early sporulation and stationary-phase events in Clostridium acetobutylicum. J Bacteriol. 2005, 187: 7103-7118. 10.1128/JB.187.20.7103-7118.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Jones DT, Westhuizen van der A, Long S, Allcock ER, Reid SJ, Woods DR: Solvent production and morphological changes in Clostridium acetobutylicum. Appl Environ Microbiol. 1982, 43: 1434-1439.PubMedPubMed CentralGoogle Scholar
- Long S, Jones DT, Woods DR: Sporulation of Clostridium acetobutylicum P262 in a defined medium. Appl Environ Microbiol. 1983, 45: 1389-1393.PubMedPubMed CentralGoogle Scholar
- Comas-Riu J, Vives-Rego J: Cytometric monitoring of growth, sporogenesis and spore cell sorting in Paenibacillus polymyxa (formerly Bacillus polymyxa). J Appl Microbiol. 2002, 92: 475-481. 10.1046/j.1365-2672.2002.01549.x.PubMedView ArticleGoogle Scholar
- Yang H, Haddad H, Tomas C, Alsaker K, Papoutsakis ET: A segmental nearest neighbor normalization and gene identification method gives superior results for DNA-array analysis. Proc Natl Acad Sci USA. 2003, 100: 1122-1127. 10.1073/pnas.0237337100.PubMedPubMed CentralView ArticleGoogle Scholar
- Tatusov RL, Galperin MY, Natale DA, Koonin EV: The COG database: a tool for genome-scale analysis of protein functions and evolution. Nucleic Acids Res. 2000, 28: 33-36. 10.1093/nar/28.1.33.PubMedPubMed CentralView ArticleGoogle Scholar
- Nölling J, Breton G, Omelchenko MV, Makarova KS, Zeng Q, Gibson R, Lee HM, Dubois J, Qiu D, Hitti J, GTC Sequencing Center Production, Finishing, and Bioinformatics Teams, Wolf YI, Tatusov RL, Sabathe F, Doucette-Stamm L, Soucaille P, Daly MJ, Bennett GN, Koonin EV, Smith DR: Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J Bacteriol. 2001, 183: 4823-4838. 10.1128/JB.183.16.4823-4838.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- Lyristis M, Boynton ZL, Petersen D, Kan Z, Bennett GN, Rudolph FB: Cloning, sequencing, and characterization of the gene encoding flagellin, flaC, and the post-translational modification of flagellin, FlaC, from Clostridium acetobutylicum ATCC824. Anaerobe. 2000, 6: 69-79. 10.1006/anae.1999.0311.View ArticleGoogle Scholar
- Welch M, Oosawa K, Aizawa SI, Eisenbach M: Effects of phosphorylation, Mg2+, and conformation of the chemotaxis protein CheY on its binding to the flagellar switch protein FliM. Biochemistry. 1994, 33: 10470-10476. 10.1021/bi00200a031.PubMedView ArticleGoogle Scholar
- Baer SH, Blaschek HP, Smith TL: Effect of butanol challenge and temperature on lipid composition and membrane fluidity of butanol-tolerant Clostridium acetobutylicum. Appl Environ Microbiol. 1987, 53: 2854-2861.PubMedPubMed CentralGoogle Scholar
- Lepage C, Fayolle F, Hermann M, Vandecasteele J-P: Changes in membrane lipid composition of Clostridium acetobutylicum during acetone-butanol fermentation: effects of solvents, growth temperature and pH. J Gen Microbiol. 1987, 133: 103-110.Google Scholar
- Vollherbst-Schneck K, Sands JA, Montenecourt BS: Effect of butanol on lipid composition and fluidity of Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol. 1984, 47: 193-194.PubMedPubMed CentralGoogle Scholar
- Zhao Y, Hindorff LA, Chuang A, Monroe-Augustus M, Lyristis M, Harrison ML, Rudolph FB, Bennett GN: Expression of a cloned cyclopropane fatty acid synthase gene reduces solvent formation in Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol. 2003, 69: 2831-2841. 10.1128/AEM.69.5.2831-2841.2003.PubMedPubMed CentralView ArticleGoogle Scholar
- Peguin S, Soucaille P: Modulation of carbon and electron flow in Clostridium acetobutylicum by iron limitation and methyl viologen addition. Appl Environ Microbiol. 1995, 61: 403-405.PubMedPubMed CentralGoogle Scholar
- Jones DT, Woods DR: Acetone-butanol fermentation revisited. Microbiol Rev. 1986, 50: 484-524.PubMedPubMed CentralGoogle Scholar
- Cornillot E, Nair RV, Papoutsakis ET, Soucaille P: The genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 reside on a large plasmid whose loss leads to degeneration of the strain. J Bacteriol. 1997, 179: 5442-5447.PubMedPubMed CentralGoogle Scholar
- Schaffer S, Isci N, Zickner B, Dürre P: Changes in protein synthesis and identification of proteins specifically induced during solventogenesis in Clostridium acetobutylicum. Electrophoresis. 2002, 23: 110-121. 10.1002/1522-2683(200201)23:1<110::AID-ELPS110>3.0.CO;2-G.PubMedView ArticleGoogle Scholar
- Mansilla MC, Cybulski LE, Albanesi D, de Mendoza D: Control of membrane lipid fluidity by molecular thermosensors. J Bacteriol. 2004, 186: 6681-6688. 10.1128/JB.186.20.6681-6688.2004.PubMedPubMed CentralView ArticleGoogle Scholar
- Kaan T, Homuth G, Mader U, Bandow J, Schweder T: Genome-wide transcriptional profiling of the Bacillus subtilis cold-shock response. Microbiology. 2002, 148: 3441-3455.PubMedView ArticleGoogle Scholar
- Johnston NC, Goldfine H: Lipid composition in the classification of the butyric acid-producing clostridia. J Gen Microbiol. 1983, 129: 1075-1081.PubMedGoogle Scholar
- Burbulys D, Trach KA, Hoch JA: Initiation of sporulation in B. subtilis is controlled by a multicomponent phosphorelay. Cell. 1991, 64: 545-552. 10.1016/0092-8674(91)90238-T.PubMedView ArticleGoogle Scholar
- Stragier P, Losick R: Molecular genetics of sporulation in Bacillus subtilis. Annu Rev Genet. 1996, 30: 297-241. 10.1146/annurev.genet.30.1.297.PubMedView ArticleGoogle Scholar
- Harris LM, Welker NE, Papoutsakis ET: Northern, morphological, and fermentation analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824. J Bacteriol. 2002, 184: 3586-3597. 10.1128/JB.184.13.3586-3597.2002.PubMedPubMed CentralView ArticleGoogle Scholar
- Molle V, Fujita M, Jensen ST, Eichenberger P, Gonzalez-Pastor JE, Liu JS, Losick R: The Spo0A regulon of Bacillus subtilis. Mol Microbiol. 2003, 50: 1683-1701. 10.1046/j.1365-2958.2003.03818.x.PubMedView ArticleGoogle Scholar
- Cervin MA, Lewis RJ, Brannigan JA, Spiegelman GB: The Bacillus subtilis regulator SinR inhibits spoIIG promoter transcription in vitro without displacing RNA polymerase. Nucleic Acids Res. 1998, 26: 3806-3812. 10.1093/nar/26.16.3806.PubMedPubMed CentralView ArticleGoogle Scholar
- Mandic-Mulec I, Doukhan L, Smith I: The Bacillus subtilis SinR protein is a repressor of the key sporulation gene spo0A. J Bacteriol. 1995, 177: 4619-4627.PubMedPubMed CentralGoogle Scholar
- Scotcher MC, Rudolph FB, Bennett GN: Expression of abrB310 and sinR, and effects of decreased abrB310 expression on the transition from acidogenesis to solventogenesis, in Clostridium acetobutylicum ATCC 824. Appl Environ Microbiol. 2005, 71: 1987-1995. 10.1128/AEM.71.4.1987-1995.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Perego M, Spiegelman GB, Hoch JA: Structure of the gene for the transition state regulator, abrB: regulator synthesis is controlled by the spo0A sporulation gene in Bacillus subtilis. Mol Microbiol. 1988, 2: 689-699. 10.1111/j.1365-2958.1988.tb00079.x.PubMedView ArticleGoogle Scholar
- Chary VK, Meloni M, Hilbert DW, Piggot PJ: Control of the expression and compartmentalization of (sigma)G activity during sporulation of Bacillus subtilis by regulators of (sigma)F and (sigma)E. J Bacteriol. 2005, 187: 6832-6840. 10.1128/JB.187.19.6832-6840.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Sun DX, Cabrera-Martinez RM, Setlow P: Control of transcription of the Bacillus subtilis spoIIIG gene, which codes for the forespore-specific transcription factor sigma G. J Bacteriol. 1991, 173: 2977-2984.PubMedPubMed CentralGoogle Scholar
- Paredes CJ, Rigoutsos I, Papoutsakis ET: Transcriptional organization of the Clostridium acetobutylicum genome. Nucleic Acids Res. 2004, 32: 1973-1981. 10.1093/nar/gkh509.PubMedPubMed CentralView ArticleGoogle Scholar
- Kroos L, Kunkel B, Losick R: Switch protein alters specificity of RNA polymerase containing a compartment-specific sigma factor. Science. 1989, 243: 526-529. 10.1126/science.2492118.PubMedView ArticleGoogle Scholar
- Stragier P, Kunkel B, Kroos L, Losick R: Chromosomal rearrangement generating a composite gene for a developmental transcription factor. Science. 1989, 243: 507-512. 10.1126/science.2536191.PubMedView ArticleGoogle Scholar
- Sauer U, Treuner A, Buchholz M, Santangelo JD, Dürre P: Sporulation and primary sigma factor homologous genes in Clostridium acetobutylicum. J Bacteriol. 1994, 176: 6572-6582.PubMedPubMed CentralGoogle Scholar
- Santangelo JD, Kuhn A, Treuner-Lange A, Dürre P: Sporulation and time course expression of sigma-factor homologous genes in Clostridium acetobutylicum. FEMS Microbiol Lett. 1998, 161: 157-164. 10.1111/j.1574-6968.1998.tb12943.x.PubMedView ArticleGoogle Scholar
- Tatti KM, Jones CH, Moran CP: Genetic evidence for interaction of sigma E with the spoIIID promoter in Bacillus subtilis. J Bacteriol. 1991, 173: 7828-7833.PubMedPubMed CentralGoogle Scholar
- Piggot PJ, Hilbert DW: Sporulation of Bacillus subtilis. Curr Opin Microbiol. 2004, 7: 579-586. 10.1016/j.mib.2004.10.001.PubMedView ArticleGoogle Scholar
- Wörner K, Szurmant H, Chiang C, Hoch JA: Phosphorylation and functional analysis of the sporulation initiation factor Spo0A from Clostridium botulinum. Mol Microbiol. 2006, 59: 1000-1012. 10.1111/j.1365-2958.2005.04988.x.PubMedView ArticleGoogle Scholar
- Jiang M, Shao W, Perego M, Hoch JA: Multiple histidine kinases regulate entry into stationary phase and sporulation in Bacillus subtilis. Mol Microbiol. 2000, 38: 535-542. 10.1046/j.1365-2958.2000.02148.x.PubMedView ArticleGoogle Scholar
- Dartois V, Djavakhishvili T, Hoch JA: Identification of a membrane protein involved in activation of the KinB pathway to sporulation in Bacillus subtilis. J Bacteriol. 1996, 178: 1178-1186.PubMedPubMed CentralGoogle Scholar
- LeDeaux JR, Grossman AD: Isolation and characterization of kinC, a gene that encodes a sensor kinase homologous to the sporulation sensor kinases KinA and KinB in Bacillus subtilis. J Bacteriol. 1995, 177: 166-175.PubMedPubMed CentralGoogle Scholar
- Alsaker KV, Paredes CJ, Papoutsakis ET: Design, optimization and validation of genomic DNA microarrays for examining the Clostridium acetobutylicum transcriptome. Biotechnol Bioprocess Eng. 2005, 10: 432-443.View ArticleGoogle Scholar
- Scotcher MC, Bennett GN: SpoIIE regulates sporulation but does not directly affect solventogenesis in Clostridium acetobutylicum ATCC 824. J Bacteriol. 2005, 187: 1930-1936. 10.1128/JB.187.6.1930-1936.2005.PubMedPubMed CentralView ArticleGoogle Scholar
- Green EM, Boynton ZL, Harris LM, Rudolph FB, Papoutsakis ET, Bennett GN: Genetic manipulation of acid formation pathways by gene inactivation in Clostridium acetobutylicum ATCC 824. Microbiology. 1996, 142: 2079-2086.PubMedView ArticleGoogle Scholar
- Huang IH, Waters M, Grau RR, Sarker MR: Disruption of the gene (spo0A) encoding sporulation transcription factor blocks endospore formation and enterotoxin production in enterotoxigenic Clostridium perfringens type A. FEMS Microbiol Lett. 2004, 233: 233-240. 10.1111/j.1574-6968.2004.tb09487.x.PubMedView ArticleGoogle Scholar
- Raju D, Waters M, Setlow P, Sarker MR: Investigating the role of small, acid-soluble spore proteins (SASPs) in the resistance of Clostridium perfringens spores to heat. BMC Microbiol. 2006, 6: 50-10.1186/1471-2180-6-50.PubMedPubMed CentralView ArticleGoogle Scholar
- Sarker MR, Carman RJ, McClane BA: Inactivation of the gene (cpe) encoding Clostridium perfringens enterotoxin eliminates the ability of two cpe-positive C. perfringens type A human gastrointestinal disease isolates to affect rabbit ileal loops. Mol Microbiol. 1999, 33: 946-958. 10.1046/j.1365-2958.1999.01534.x.PubMedView ArticleGoogle Scholar
- Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP: The ClosTron: a universal gene knock-out system for the genus Clostridium. J Microbiol Methods. 2007, 70: 452-464. 10.1016/j.mimet.2007.05.021.PubMedView ArticleGoogle Scholar
- Desai RP, Papoutsakis ET: Antisense RNA strategies for metabolic engineering of Clostridium acetobutylicum. Appl Environ Microbiol. 1999, 65: 936-945.PubMedPubMed CentralGoogle Scholar
- Tummala SB, Welker NE, Papoutsakis ET: Design of antisense RNA constructs for downregulation of the acetone formation pathway of Clostridium acetobutylicum. J Bacteriol. 2003, 185: 1923-1934. 10.1128/JB.185.6.1923-1934.2003.PubMedPubMed CentralView ArticleGoogle Scholar
- Perret S, Maamar H, Belaich JP, Tardif C: Use of antisense RNA to modify the composition of cellulosomes produced by Clostridium cellulolyticum. Mol Microbiol. 2004, 51: 599-607. 10.1046/j.1365-2958.2003.03860.x.PubMedView ArticleGoogle Scholar
- Raju D, Setlow P, Sarker MR: Antisense-RNA-mediated decreased synthesis of small, acid-soluble spore proteins leads to decreased resistance of Clostridium perfringens spores to moist heat and UV radiation. Appl Environ Microbiol. 2007, 73: 2048-2053. 10.1128/AEM.02500-06.PubMedPubMed CentralView ArticleGoogle Scholar
- Tummala SB, Junne SG, Papoutsakis ET: Antisense RNA downregulation of coenzyme A transferase combined with alcohol-aldehyde dehydrogenase overexpression leads to predominantly alcohologenic Clostridium acetobutylicum fermentations. J Bacteriol. 2003, 185: 3644-3653. 10.1128/JB.185.12.3644-3653.2003.PubMedPubMed CentralView ArticleGoogle Scholar
- Tomas CA, Welker NE, Papoutsakis ET: Overexpression of groESL in Clostridium acetobutylicum results in increased solvent production and tolerance, prolonged metabolism, and changes in the cell's transcriptional program. Appl Environ Microbiol. 2003, 69: 4951-4965. 10.1128/AEM.69.8.4951-4965.2003.PubMedPubMed CentralView ArticleGoogle Scholar
- Asai K, Ishiwata K, Matsuzaki K, Sadaie Y: A viable Bacillus subtilis strain without functional extracytoplasmic function sigma genes. J Bacteriol. 2008, 190: 2633-2636. 10.1128/JB.01859-07.PubMedPubMed CentralView ArticleGoogle Scholar
- Mascher T, Hachmann AB, Helmann JD: Regulatory overlap and functional redundancy among Bacillus subtilis extracytoplasmic function sigma factors. J Bacteriol. 2007, 189: 6919-6927. 10.1128/JB.00904-07.PubMedPubMed CentralView ArticleGoogle Scholar
- Paredes CJ, Senger RS, Spath IS, Borden JR, Sillers R, Papoutsakis ET: A general framework for designing and validating oligomer-based DNA microarrays and its application to Clostridium acetobutylicum. Appl Environ Microbiol. 2007, 73: 4631-4638. 10.1128/AEM.00144-07.PubMedPubMed CentralView ArticleGoogle Scholar
- Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, Klapa M, Currier T, Thiagarajan M, Sturn A, Snuffin M, Rezantsev A, Popov D, Ryltsov A, Kostukovich E, Borisovsky I, Liu Z, Vinsavich A, Trush V, Quackenbush J: TM4: a free, open-source system for microarray data management and analysis. Biotechniques. 2003, 34: 374-378.PubMedGoogle Scholar
- Reynolds ES: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963, 17: 208-212. 10.1083/jcb.17.1.208.PubMedPubMed CentralView ArticleGoogle Scholar
- Frey M: Hydrogenases: hydrogen-activating enzymes. Chembiochem. 2002, 3: 153-160. 10.1002/1439-7633(20020301)3:2/3<153::AID-CBIC153>3.0.CO;2-B.PubMedView ArticleGoogle Scholar
- Gorwa MF, Croux C, Soucaille P: Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824. J Bacteriol. 1996, 178: 2668-2675.PubMedPubMed CentralGoogle Scholar
- Moir A, Corfe BM, Behravan J: Spore germination. Cell Mol Life Sci. 2002, 59: 403-409. 10.1007/s00018-002-8432-8.PubMedView ArticleGoogle Scholar
- Igarashi T, Setlow P: Transcription of the Bacillus subtilis gerK operon, which encodes a spore germinant receptor, and comparison with that of operons encoding other germinant receptors. J Bacteriol. 2006, 188: 4131-4136. 10.1128/JB.00265-06.PubMedPubMed CentralView ArticleGoogle Scholar
- Dürre P, Hollergschwandner C: Initiation of endospore formation in Clostridium acetobutylicum. Anaerobe. 2004, 10: 69-74. 10.1016/j.anaerobe.2003.11.001.PubMedView ArticleGoogle Scholar
- Makino S, Moriyama R: Hydrolysis of cortex peptidoglycan during bacterial spore germination. Med Sci Monit. 2002, 8: RA119-127.PubMedGoogle Scholar
- Ishikawa S, Yamane K, Sekiguchi J: Regulation and characterization of a newly deduced cell wall hydrolase gene (cwlJ) which affects germination of Bacillus subtilis spores. J Bacteriol. 1998, 180: 1375-1380.PubMedPubMed CentralGoogle Scholar
- Kodama T, Takamatsu H, Asai K, Kobayashi K, Ogasawara N, Watabe K: The Bacillus subtilis yaaH gene is transcribed by SigE RNA polymerase during sporulation, and its product is involved in germination of spores. J Bacteriol. 1999, 181: 4584-4591.PubMedPubMed CentralGoogle Scholar
- Moriyama R, Fukuoka H, Miyata S, Kudoh S, Hattori A, Kozuka S, Yasuda Y, Tochikubo K, Makino S: Expression of a germination-specific amidase, SleB, of bacilli in the forespore compartment of sporulating cells and its localization on the exterior side of the cortex in dormant spores. J Bacteriol. 1999, 181: 2373-2378.PubMedPubMed CentralGoogle Scholar
- Setlow P: Mechanisms which contribute to the long-term survival of spores of Bacillus species. Soc Appl Bacteriol Symp Ser. 1994, 23: 49S-60S.PubMedGoogle Scholar
- Bourne N, FitzJames PC, Aronson AI: Structural and germination defects of Bacillus subtilis spores with altered contents of a spore coat protein. J Bacteriol. 1991, 173: 6618-6625.PubMedPubMed CentralGoogle Scholar
- Roels S, Driks A, Losick R: Characterization of spoIVA, a sporulation gene involved in coat morphogenesis in Bacillus subtilis. J Bacteriol. 1992, 174: 575-585.PubMedPubMed CentralGoogle Scholar
- Takamatsu H, Imamura A, Kodama T, Asai K, Ogasawara N, Watabe K: The yabG gene of Bacillus subtilis encodes a sporulation specific protease which is involved in the processing of several spore coat proteins. FEMS Microbiol Lett. 2000, 192: 33-38. 10.1111/j.1574-6968.2000.tb09355.x.PubMedView ArticleGoogle Scholar
- Takamatsu H, Watabe K: Assembly and genetics of spore protective structures. Cell Mol Life Sci. 2002, 59: 434-444. 10.1007/s00018-002-8436-4.PubMedView ArticleGoogle Scholar
- Driks A: Proteins of the spore core and coat. Bacillus subtilis and its Closest Relatives: From Genes to Cells. Edited by: Sonenshein AL, Hoch JA, Losick R. 2002, Washington, DC: American Society for Microbiology, 527-535.View ArticleGoogle Scholar
- Britton RA, Eichenberger P, Gonzalez-Pastor JE, Fawcett P, Monson R, Losick R, Grossman AD: Genome-wide analysis of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis. J Bacteriol. 2002, 184: 4881-4890. 10.1128/JB.184.17.4881-4890.2002.PubMedPubMed CentralView ArticleGoogle Scholar
- Wang ST, Setlow B, Conlon EM, Lyon JL, Imamura D, Sato T, Setlow P, Losick R, Eichenberger P: The forespore line of gene expression in Bacillus subtilis. J Mol Biol. 2006, 358: 16-37. 10.1016/j.jmb.2006.01.059.PubMedView ArticleGoogle Scholar
- Eichenberger P, Fujita M, Jensen ST, Conlon EM, Rudner DZ, Wang ST, Ferguson C, Haga K, Sato T, Liu JS, Losick R: The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis. PLoS Biol. 2004, 2: e328-10.1371/journal.pbio.0020328.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to 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.