σK of Clostridium acetobutylicum is the first known sporulation-specific sigma factor with two developmentally separated roles, one early and one late in sporulation.

Sporulation in the model endospore-forming organism Bacillus subtilis proceeds via the sequential and stage-specific activation of the sporulation-specific sigma factors, σ(H) (early), σ(F), σ(E), σ(G), and σ(K) (late). Here we show that the Clostridium acetobutylicum σ(K) acts both early, prior to Spo0A expression, and late, past σ(G) activation, thus departing from the B. subtilis model. The C. acetobutylicum sigK deletion (ΔsigK) mutant was unable to sporulate, and solventogenesis, the characteristic stationary-phase phenomenon for this organism, was severely diminished. Transmission electron microscopy demonstrated that the ΔsigK mutant does not develop an asymmetric septum and produces no granulose. Complementation of sigK restored sporulation and solventogenesis to wild-type levels. Spo0A and σ(G) proteins were not detectable by Western analysis, while σ(F) protein levels were significantly reduced in the ΔsigK mutant. spo0A, sigF, sigE, sigG, spoIIE, and adhE1 transcript levels were all downregulated in the ΔsigK mutant, while those of the sigH transcript were unaffected during the exponential and transitional phases of culture. These data show that σ(K) is necessary for sporulation prior to spo0A expression. Plasmid-based expression of spo0A in the ΔsigK mutant from a nonnative promoter restored solventogenesis and the production of Spo0A, σ(F), σ(E), and σ(G), but not sporulation, which was blocked past the σ(G) stage of development, thus demonstrating that σ(K) is also necessary in late sporulation. sigK is expressed very early at low levels in exponential phase but is strongly upregulated during the middle to late stationary phase. This is the first sporulation-specific sigma factor shown to have two developmentally separated roles.

Parallel labeling experiments validate Clostridium acetobutylicum metabolic network model for (13)C metabolic flux analysis.

In this work, we provide new insights into the metabolism of Clostridium acetobutylicum ATCC 824 obtained using a systematic approach for quantifying fluxes based on parallel labeling experiments and (13)C-metabolic flux analysis ((13)C-MFA). Here, cells were grown in parallel cultures with [1-(13)C]glucose and [U-(13)C]glucose as tracers and (13)C-MFA was used to quantify intracellular metabolic fluxes. Several metabolic network models were compared: an initial model based on current knowledge, and extended network models that included additional reactions that improved the fits of experimental data. While the initial network model did not produce a statistically acceptable fit of (13)C-labeling data, an extended network model with five additional reactions was able to fit all data with 292 redundant measurements. The model was subsequently trimmed to produce a minimal network model of C. acetobutylicum for (13)C-MFA, which could still reproduce all of the experimental data. The flux results provided valuable new insights into the metabolism of C. acetobutylicum. First, we found that TCA cycle was effectively incomplete, as there was no measurable flux between α-ketoglutarate and succinyl-CoA, succinate and fumarate, and malate and oxaloacetate. Second, an active pathway was identified from pyruvate to fumarate via aspartate. Third, we found that isoleucine was produced exclusively through the citramalate synthase pathway in C. acetobutylicum and that CAC3174 was likely responsible for citramalate synthase activity. These model predictions were confirmed in several follow-up tracer experiments. The validated metabolic network model established in this study can be used in future investigations for unbiased (13)C-flux measurements in C. acetobutylicum.

Workflow for quantitative proteomic analysis of Clostridium acetobutylicum ATCC 824 using iTRAQ tags.

Clostridium acetobutylicum (Cac) is an anaerobic, endospore-forming, Gram-positive bacterium with tremendous promise for use as a biocatalyst for the production of fuels and solvents. Cac proteomic sample preparation for shotgun analysis typically involves a multitude of reagents for harsh lysis conditions and to maintain protein solubility. We describe a protein extraction and preparation method for Cac that is compatible with proteomic shotgun analysis using isobaric labeling approaches. The method is applied to the analysis of Cac grown under butanol stress and labeled using iTRAQ 4-plex reagents. This method relies on the use of calcium carbonate to facilitate lysis by sonication and a commercially available kit to remove detergents prior to labeling. This workflow resulted in the identification and quantitation of 566 unique proteins using ProteinPilot software with a false discovery rate of 0.01% for peptide matches and 0.70% for protein matches. Ninety-five proteins were found to have statistically higher expression levels in butanol-stressed Cac as compared to non-stressed Cac. Sixty-one proteins were found to have statistically lower expression levels in stressed versus non-stressed cells. This method may be applicable to other Gram-positive organisms.

The Clostridium small RNome that responds to stress: the paradigm and importance of toxic metabolite stress in C. acetobutylicum.

BACKGROUND: Small non-coding RNAs (sRNA) are emerging as major components of the cell's regulatory network, several possessing their own regulons. A few sRNAs have been reported as being involved in general or toxic-metabolite stress, mostly in Gram- prokaryotes, but hardly any in Gram+ prokaryotes. Significantly, the role of sRNAs in the stress response remains poorly understood at the genome-scale level. It was previously shown that toxic-metabolite stress is one of the most comprehensive and encompassing stress responses in the cell, engaging both the general stress (or heat-shock protein, HSP) response as well as specialized metabolic programs. RESULTS: Using RNA deep sequencing (RNA-seq) we examined the sRNome of C. acetobutylicum in response to the native but toxic metabolites, butanol and butyrate. 7.5% of the RNA-seq reads mapped to genome outside annotated ORFs, thus demonstrating the richness and importance of the small RNome. We used comparative expression analysis of 113 sRNAs we had previously computationally predicted, and of annotated mRNAs to set metrics for reliably identifying sRNAs from RNA-seq data, thus discovering 46 additional sRNAs. Under metabolite stress, these 159 sRNAs displayed distinct expression patterns, a select number of which was verified by Northern analysis. We identified stress-related expression of sRNAs affecting transcriptional (6S, S-box &solB) and translational (tmRNA & SRP-RNA) processes, and 65 likely targets of the RNA chaperone Hfq. CONCLUSIONS: Our results support an important role for sRNAs for understanding the complexity of the regulatory network that underlies the stress response in Clostridium organisms, whether related to normophysiology, pathogenesis or biotechnological applications.

Inactivation of σE and σG in Clostridium acetobutylicum illuminates their roles in clostridial-cell-form biogenesis, granulose synthesis, solventogenesis, and spore morphogenesis.

Central to all clostridia is the orchestration of endospore formation (i.e., sporulation) and, specifically, the roles of differentiation-associated sigma factors. Moreover, there is considerable applied interest in understanding the roles of these sigma factors in other stationary-phase phenomena, such as solvent production (i.e., solventogenesis). Here we separately inactivated by gene disruption the major sporulation-specific sigma factors, σ(E) and σ(G), and performed an initial analysis to elucidate their roles in sporulation-related morphogenesis and solventogenesis in Clostridium acetobutylicum. The terminal differentiation phenotype for the sigE inactivation mutant stalled in sporulation prior to asymmetric septum formation, appeared vegetative-like often with an accumulation of DNA at both poles, frequently exhibited two longitudinal internal membranes, and did not synthesize granulose. The sigE inactivation mutant did produce the characteristic solvents (i.e., butanol and acetone), but the extent of solventogenesis was dependent on the physiological state of the inoculum. The sigG inactivation mutant stalled in sporulation during endospore maturation, exhibiting engulfment and partial cortex and spore coat formation. Lastly, the sigG inactivation mutant did produce granulose and exhibited wild-type-like solventogenesis.

SpoIIE is necessary for asymmetric division, sporulation, and expression of sigmaF, sigmaE, and sigmaG but does not control solvent production in Clostridium acetobutylicum ATCC 824.

In order to better characterize the initial stages of sporulation past Spo0A activation and the associated solventogenesis in the important industrial and model organism Clostridium acetobutylicum, the spoIIE gene was successfully disrupted and its expression was silenced. By silencing spoIIE, sporulation was blocked prior to asymmetric division, and no mature spores or any distinguishable morphogenetic changes developed. Upon plasmid-based complementation of spoIIE, sporulation was restored, although the number of spores formed was below that of the plasmid control strain. To investigate the impact of silencing spoIIE on the regulation of sporulation, transcript levels of sigF, sigE, and sigG were examined by semiquantitative reverse transcription-PCR, and the corresponding σF, σE, and σG protein levels were determined by Western analysis. Expression of sigF was significantly reduced in the inactivation strain, and this resulted in very low σF protein levels. Expression of sigE was barely detected, and no sigG transcript was detected at all; consequently, no σE or σG proteins were detected. These data suggest an autostimulatory role for σF in C. acetobutylicum, in contrast to the model organism for endospore formation, Bacillus subtilis, and confirm that high-level expression of σF is required for expression of σE and σG. Unlike the σF and σE inactivation strains, the SpoIIE inactivation strain did not exhibit inoculum-dependent solvent formation and produced good levels of solvents from both exponential- and stationary-phase inocula. Thus, we concluded that SpoIIE does not control solvent formation.

Inactivation of σF in Clostridium acetobutylicum ATCC 824 blocks sporulation prior to asymmetric division and abolishes σE and σG protein expression but does not block solvent formation.

Clostridium acetobutylicum is both a model organism for the understanding of sporulation in solventogenic clostridia and its relationship to solvent formation and an industrial organism for anaerobic acetone-butanol-ethanol (ABE) fermentation. How solvent production is coupled to endospore formation--both stationary-phase events--remains incompletely understood at the molecular level. Specifically, it is unclear how sporulation-specific sigma factors affect solvent formation. Here the sigF gene in C. acetobutylicum was successfully disrupted and silenced. Not only σ(F) but also the sigma factors σ(E) and σ(G) were not detected in the sigF mutant (FKO1), and differentiation was stopped prior to asymmetric division. Since plasmid expression of the spoIIA operon (spoIIAA-spoIIAB-sigF) failed to complement FKO1, the operon was integrated into the FKO1 chromosome to generate strain FKO1-C. In FKO1-C, σ(F) expression was restored along with sporulation and σ(E) and σ(G) protein expression. Quantitative reverse transcription-PCR (RT-PCR) analysis of a select set of genes (csfB, gpr, spoIIP, sigG, lonB, and spoIIR) that could be controlled by σ(F), based on the Bacillus subtilis model, indicated that sigG may be under the control of σ(F), but spoIIR, an important activator of σ(E) in B. subtilis, is not, and neither are the rest of the genes investigated. FKO1 produced solvents at a level similar to that of the parent strain, but solvent levels were dependent on the physiological state of the inoculum. Finally, the complementation strain FKO1-C is the first reported instance of purposeful integration of multiple functional genes into a clostridial chromosome--here, the C. acetobutylicum chromosome--with the aim of altering cell metabolism and differentiation.

A genomic-library based discovery of a novel, possibly synthetic, acid-tolerance mechanism in Clostridium acetobutylicum involving non-coding RNAs and ribosomal RNA processing.

We generated a genomic library from sheared Clostridium acetobutylicum ATCC 824 DNA, whereby inserts can be expressed in both directions from the thiolase promoter, P(thl). Serial transfer of library-bearing C. acetobutylicum cultures exposed to increasing butyrate concentrations enriched for inserts containing fragments of rRNA genetic loci. The selected library inserts were placed so that antisense (to the rRNAs) non-coding RNAs (ncRNAs) would be transcribed from P(thl). Different enriched inserts imparted similar butyrate-tolerance characteristics. A minimal tolerance fragment (RDNA7) was identified as the 16S-rRNA promoter region. Expressed on plasmid pRD7 off P(thl), RDNA7 can produce putative ncRNAs termed ncRNA(RD7). C. acetobutylicum 824(pRD7) showed superior resistance to butyrate and other carboxylic acids. Transcriptional analysis of butyrate stress identified 120 differentially expressed genes between 824(pRD7) and 824(pSOS95del). The few upregulated genes included the ffh gene of the putative signal recognition particle (SRP) system. Northern analysis of ncRNA(RD7) and corresponding antisense RNAs demonstrated multiple ncRNA(RD7) molecules in 824(pRD7). Several corresponding antisense RNA molecules were identified both in 824(pRD7) and 824(pSOS95del), but at much higher levels in 824(pRD7). Northern analysis of 16S rRNA expression suggested complex RDNA7-dependent rRNA processing. Our data suggest that by hybridizing against unprocessed rRNA precursors, ncRNA(RD7) alters rRNA processing, and these alterations result in acid tolerance, possibly through a mechanism involving the Ffh protein.

Recent advances in single cell protein use as a feed ingredient in aquaculture.

The global demand for high-quality, protein-rich foods will continue to increase as the global population grows, along with income levels. Aquaculture is poised to help fulfill some of this demand, and is thus the fastest growing animal protein industry. A key challenge for it, though, is sourcing a sustainable, renewable protein ingredient. Single cell protein (SCP) products, protein meals based on microbial or algal biomass, have the potential to fulfill this need. Here, we review potential sources of SCP strains and their respective production processes, highlight recent advances on identification of new SCP strains and feedstocks, and, finally, review new feeding trial data on important aquaculture species, specifically Atlantic salmon, rainbow trout, and whiteleg shrimp.

Fixation of CO2 and CO on a diverse range of carbohydrates using anaerobic, non-photosynthetic mixotrophy.

Biological CO2 fixation is an important technology that can assist in combating climate change. Here, we show an approach called anaerobic, non-photosynthetic mixotrophy can result in net CO2 fixation when using a reduced feedstock. This approach uses microbes called acetogens that are capable of concurrent utilization of both organic and inorganic substrates. In this study, we investigated the substrate utilization of 17 different acetogens, both mesophilic and thermophilic, on a variety of different carbohydrates and gases. Compared to most model acetogen strains, several non-model mesophilic strains displayed greater substrate flexibility, including the ability to utilize disaccharides, glycerol and an oligosaccharide, and growth rates. Three of these non-model strains (Blautia producta, Clostridium scatologenes and Thermoanaerobacter kivui) were chosen for further characterization, under a variety of conditions including H2- or syngas-fed sugar fermentations and a CO2-fed glycerol fermentation. In all cases, CO2 was fixed and carbon yields approached 100%. Finally, the model acetogen C. ljungdahlii was engineered to utilize glucose, a non-preferred sugar, while maintaining mixotrophic behavior. This work demonstrates the flexibility and robustness of anaerobic, non-photosynthetic mixotrophy as a technology to help reduce CO2 emissions.

CO2 fixation by anaerobic non-photosynthetic mixotrophy for improved carbon conversion.

Maximizing the conversion of biogenic carbon feedstocks into chemicals and fuels is essential for fermentation processes as feedstock costs and processing is commonly the greatest operating expense. Unfortunately, for most fermentations, over one-third of sugar carbon is lost to CO2 due to the decarboxylation of pyruvate to acetyl-CoA and limitations in the reducing power of the bio-feedstock. Here we show that anaerobic, non-photosynthetic mixotrophy, defined as the concurrent utilization of organic (for example, sugars) and inorganic (for example, CO2) substrates in a single organism, can overcome these constraints to increase product yields and reduce overall CO2 emissions. As a proof-of-concept, Clostridium ljungdahlii was engineered to produce acetone and achieved a mass yield 138% of the previous theoretical maximum using a high cell density continuous fermentation process. In addition, when enough reductant (that is, H2) is provided, the fermentation emits no CO2. Finally, we show that mixotrophy is a general trait among acetogens.

The Clostridium sporulation programs: diversity and preservation of endospore differentiation.

SUMMARY: Bacillus and Clostridium organisms initiate the sporulation process when unfavorable conditions are detected. The sporulation process is a carefully orchestrated cascade of events at both the transcriptional and posttranslational levels involving a multitude of sigma factors, transcription factors, proteases, and phosphatases. Like Bacillus genomes, sequenced Clostridium genomes contain genes for all major sporulation-specific transcription and sigma factors (spo0A, sigH, sigF, sigE, sigG, and sigK) that orchestrate the sporulation program. However, recent studies have shown that there are substantial differences in the sporulation programs between the two genera as well as among different Clostridium species. First, in the absence of a Bacillus-like phosphorelay system, activation of Spo0A in Clostridium organisms is carried out by a number of orphan histidine kinases. Second, downstream of Spo0A, the transcriptional and posttranslational regulation of the canonical set of four sporulation-specific sigma factors (σ(F), σ(E), σ(G), and σ(K)) display different patterns, not only compared to Bacillus but also among Clostridium organisms. Finally, recent studies demonstrated that σ(K), the last sigma factor to be activated according to the Bacillus subtilis model, is involved in the very early stages of sporulation in Clostridium acetobutylicum, C. perfringens, and C. botulinum as well as in the very late stages of spore maturation in C. acetobutylicum. Despite profound differences in initiation, propagation, and orchestration of expression of spore morphogenetic components, these findings demonstrate not only the robustness of the endospore sporulation program but also the plasticity of the program to generate different complex phenotypes, some apparently regulated at the epigenetic level.

Acetogenic mixotrophy: novel options for yield improvement in biofuels and biochemicals production.

Mass yields of biofuels and chemicals from sugar fermentations are limited by the decarboxylation reactions involved in Embden-Meyerhof-Parnas (EMP) glycolysis. This paper reviews one route to recapture evolved CO2 using the Wood-Ljungdahl carbon fixation pathway (WLP) in a process called anaerobic, non-photosynthetic (ANP) mixotrophic fermentation. In ANP mixotrophic fermentation, the two molecules of CO2 and eight electrons produced from glycolysis are used by the WLP to generate three molecules of acetyl-CoA from glucose, rather than the two molecules that are produced by typical fermentation processes. In this review, we define the bounds of ANP mixotrophy, calculate the potential metabolic advantages, and discuss the viability in a number of host organisms. Additionally, we highlight recent accomplishments in the field, including the recent discovery of electron bifurcation in acetogens, and close with recommendations to realize mixotrophic biofuel and biochemical production.

Complex and extensive post-transcriptional regulation revealed by integrative proteomic and transcriptomic analysis of metabolite stress response in Clostridium acetobutylicum.

BACKGROUND: Clostridium acetobutylicum is a model organism for both clostridial biology and solvent production. The organism is exposed to its own toxic metabolites butyrate and butanol, which trigger an adaptive stress response. Integrative analysis of proteomic and RNAseq data may provide novel insights into post-transcriptional regulation. RESULTS: The identified iTRAQ-based quantitative stress proteome is made up of 616 proteins with a 15 % genome coverage. The differentially expressed proteome correlated poorly with the corresponding differential RNAseq transcriptome. Up to 31 % of the differentially expressed proteins under stress displayed patterns opposite to those of the transcriptome, thus suggesting significant post-transcriptional regulation. The differential proteome of the translation machinery suggests that cells employ a different subset of ribosomal proteins under stress. Several highly upregulated proteins but with low mRNA levels possessed mRNAs with long 5'UTRs and strong RBS scores, thus supporting the argument that regulatory elements on the long 5'UTRs control their translation. For example, the oxidative stress response rubrerythrin was upregulated only at the protein level up to 40-fold without significant mRNA changes. We also identified many leaderless transcripts, several displaying different transcriptional start sites, thus suggesting mRNA-trimming mechanisms under stress. Downregulation of Rho and partner proteins pointed to changes in transcriptional elongation and termination under stress. CONCLUSIONS: The integrative proteomic-transcriptomic analysis demonstrated complex expression patterns of a large fraction of the proteome. Such patterns could not have been detected with one or the other omic analyses. Our analysis proposes the involvement of specific molecular mechanisms of post-transcriptional regulation to explain the observed complex stress response.

Clostridia: the importance of their exceptional substrate and metabolite diversity for biofuel and biorefinery applications.

Clostridia are anaerobic Firmicutes producing a large array of metabolites by utilizing simple and complex carbohydrates, such as cellulose, as well as CO2/H2 or CO. Their exceptional substrate diversity is enhanced by their ability to produce a broad spectrum of chemicals that can be used as precursors to or directly as biofuels and industrial chemicals. Genetic and genomic tools are under intense development, and recent efforts to metabolically engineer clostridia demonstrate their potential for biofuel and biorefinery applications. Pathway engineering to combine established substrate-utilization programs, such as for cellulose, CO2/H2 or CO, with desirable metabolic programs could lead to modular design of strains suitable for many applications. Engineering complex phenotypes--aerotolerance, abolished sporulation, and tolerance to toxic chemicals--could lead to superior bioprocessing strains.

Small RNAs in the genus Clostridium.

The genus Clostridium includes major human pathogens and species important to cellulose degradation, the carbon cycle, and biotechnology. Small RNAs (sRNAs) are emerging as crucial regulatory molecules in all organisms, but they have not been investigated in clostridia. Research on sRNAs in clostridia is hindered by the absence of a systematic method to identify sRNA candidates, thus delegating clostridial sRNA research to a hit-and-miss process. Thus, we wanted to develop a method to identify potential sRNAs in the Clostridium genus to open up the field of sRNA research in clostridia. Using comparative genomics analyses combined with predictions of rho-independent terminators and promoters, we predicted sRNAs in 21 clostridial genomes: Clostridium acetobutylicum, C. beijerinckii, C. botulinum (eight strains), C. cellulolyticum, C. difficile, C. kluyveri (two strains), C. novyi, C. perfringens (three strains), C. phytofermentans, C. tetani, and C. thermocellum. Although more than one-third of predicted sRNAs have Shine-Dalgarno (SD) sequences, only one-sixth have a start codon downstream of SD sequences; thus, most of the predicted sRNAs are noncoding RNAs. Quantitative reverse transcription-PCR (Q-RT-PCR) and Northern analysis were employed to test the presence of a randomly chosen set of sRNAs in C. acetobutylicum and several C. botulinum strains, leading to the confirmation of a large fraction of the tested sRNAs. We identified a conserved, novel sRNA which, together with the downstream gene coding for an ATP-binding cassette (ABC) transporter gene, responds to the antibiotic clindamycin. The number of predicted sRNAs correlated with the physiological function of the species (high for pathogens, low for cellulolytic, and intermediate for solventogenic), but not with 16S rRNA-based phylogeny.

The transcriptional program underlying the physiology of clostridial sporulation.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.