Genetic disruption of the bacterial raiA motif noncoding RNA causes defects in sporulation and aggregation.

Several structured noncoding RNAs in bacteria are essential contributors to fundamental cellular processes. Thus, discoveries of additional ncRNA classes provide opportunities to uncover and explore biochemical mechanisms relevant to other major and potentially ancient processes. A candidate structured ncRNA named the "raiA motif" has been found via bioinformatic analyses in over 2,500 bacterial species. The gene coding for the RNA typically resides between the raiA and comFC genes of many species of Bacillota and Actinomycetota. Structural probing of the raiA motif RNA from the Gram-positive anaerobe Clostridium acetobutylicum confirms key features of its sophisticated secondary structure model. Expression analysis of raiA motif RNA reveals that the RNA is constitutively produced but reaches peak abundance during the transition from exponential growth to stationary phase. The raiA motif RNA becomes the fourth most abundant RNA in C. acetobutylicum, excluding ribosomal RNAs and transfer RNAs. Genetic disruption of the raiA motif RNA causes cells to exhibit substantially decreased spore formation and diminished ability to aggregate. Restoration of normal cellular function in this knock-out strain is achieved by expression of a raiA motif gene from a plasmid. These results demonstrate that raiA motif RNAs normally participate in major cell differentiation processes by operating as a trans-acting factor.

Gene essentiality in the solventogenic Clostridium acetobutylicum DSM 792.

Clostridium acetobutylicum is a solventogenic, anaerobic, gram-positive bacterium that is commonly considered the model organism for studying acetone-butanol-ethanol fermentation. The need to produce these chemicals sustainably and with a minimal impact on the environment has revived the interest in research on this bacterium. The recent development of efficient genetic tools allows to better understand the physiology of this micro-organism, aiming at improving its fermentation capacities. Knowledge about gene essentiality would guide the future genetic editing strategies and support the understanding of crucial cellular functions in this bacterium. In this work, we applied a transposon insertion site sequencing method to generate large mutant libraries containing millions of independent mutants that allowed us to identify a core group of 418 essential genes needed for in vitro development. Future research on this significant biocatalyst will be guided by the data provided in this work, which will serve as a valuable resource for the community. IMPORTANCE: Clostridium acetobutylicum is a leading candidate to synthesize valuable compounds like three and four carbons alcohols. Its ability to convert carbohydrates into a mixture of acetone, butanol, and ethanol as well as other chemicals of interest upon genetic engineering makes it an advantageous organism for the valorization of lignocellulose-derived sugar mixtures. Since, genetic optimization depends on the fundamental insights supplied by accurate gene function assignment, gene essentiality analysis is of great interest as it can shed light on the function of many genes whose functions are still to be confirmed. The data obtained in this study will be of great value for the research community aiming to develop C. acetobutylicum as a platform organism for the production of chemicals of interest.

Efforts to install a heterologous Wood-Ljungdahl pathway in Clostridium acetobutylicum enable the identification of the native tetrahydrofolate (THF) cycle and result in early induction of solvents.

Here, we report the construction of a Clostridium acetobutylicum strain ATCC 824 (pCD07239) by heterologous expression of carbonyl branch genes (CD630_0723∼CD630_0729) from Clostridium difficile, aimed at installing a heterologous Wood-Ljungdahl pathway (WLP). As part of this effort, in order to validate the methyl branch of the WLP in the C. acetobutylicum, we performed 13C-tracing analysis on knockdown mutants of four genes responsible for the formation of 5-methyl-tetrahydrofolate (5-methyl-THF) from formate: CA_C3201, CA_C2310, CA_C2083, and CA_C0291. While C. acetobutylicum 824 (pCD07239) could not grow autotrophically, in heterotrophic fermentation, it began producing butanol at the early growth phase (OD600 of 0.80; 0.162 g/L butanol). In contrast, solvent production in the parent strain did not begin until the early stationary phase (OD600 of 7.40). This study offers valuable insights for future research on biobutanol production during the early growth phase.

Alleviation of Carbon Catabolite Repression through araR and xylR Inactivation in Clostridium acetobutylicum DSM 792.

Efficient bioconversion processes of lignocellulose-derived carbohydrates into chemicals have received increasing interest in the last decades since they represent a promising alternative to petro-based processes. Despite efforts to adapt microorganisms to the use of such substrates, one of their major limitations remains their inability to consume multiple sugars simultaneously. In particular, the solventogenic model organism Clostridium acetobutylicum struggles to efficiently use second generation (2G) substrates because of carbon catabolite repression mechanisms that prevent the assimilation of xylose and arabinose in the presence of glucose. In this study, we addressed this issue by inactivating genes encoding transcriptional repressors involved in such mechanisms in the C. acetobutylicum strain DSM 792. Our results showed that the deletion of the two putative copies of xylR (CA_C2613 and CA_C3673) had little or no effect on the ability of the strain to consume xylose. Unlikely, the deletion of araR (CA_C1340) led to a 2.5-fold growth rate increase on xylose. The deletion of both araR and xylR genes resulted in the coassimilation of arabinose together with glucose, while xylose consumption remained inefficient. Transcriptional analyses of the wild-type strain and mutants grown on glucose, arabinose, xylose, and combinations of them provided a crucial, global overview of regulations triggered by the products of both araR and xylR in C. acetobutylicum. As suggested by these data, overexpression of xylA and xylB led to further improvement of pentose assimilation. Those results represent a step forward in the development of genetically modified strains of C. acetobutylicum able to coassimilate lignocellulosic-derived sugars. IMPORTANCE C. acetobutylicum is a strong candidate to produce chemicals of interest such as C3 and C4 alcohols. Used for more than a century for its capacity to produce a mixture of acetone, butanol, and ethanol from first generation (1G) substrates, its natural ability to assimilate a wide variety of monoosides also predisposes it as an auspicious organism for the valorization of lignocellulose-derived sugar mixtures. To achieve this purpose, a better understanding of carbon catabolite repression mechanisms is essential. The work done here provides critical knowledge on how these mechanisms occur during growth on glucose, arabinose, and xylose mixtures, as well as strategies to tackle them.

A division of labor between two biotin protein ligase homologs.

Group I biotin protein ligases (BPLs) catalyze the covalent attachment of biotin to its cognate acceptor proteins. In contrast, Group II BPLs have an additional N-terminal DNA-binding domain and function not only in biotinylation but also in transcriptional regulation of genes of biotin biosynthesis and transport. Most bacteria contain only a single biotin protein ligase, whereas Clostridium acetobutylicum contains two biotin protein ligase homologs: BplA and BirA'. Sequence alignments showed that BplA is a typical group I BPL, whereas BirA' lacked the C-terminal domain conserved throughout extant BPL proteins. This raised the questions of why two BPL homologs are needed and why the apparently defective BirA' has been retained. We have used in vivo and in vitro assays to show that BplA is a functional BPL whereas BirA' acts as a biotin sensor involved in transcriptional regulation of biotin transport. We also successfully converted BirA' into a functional biotin protein ligase with regulatory activity by fusing it to the C-terminal domain from BplA. Finally, we provide evidence that BplA and BirA' interact in vivo.

Effects of orphan histidine kinases on clostridial sporulation progression and metabolism.

Solventogenesis and sporulation of clostridia are the main responsive adaptations to the acidic environment during acetone-butanol-ethanol (ABE) fermentation. It was hypothesized that five orphan histidine kinases (HKs) including Cac3319, Cac0323, Cac0903, Cac2730, and Cac0437 determined the cell fates between sporulation and solventogenesis. In this study, the comparative genomic analysis revealed that a mutation in cac0437 appeared to contribute to the nonsporulating feature of ATCC 55025. Hence, the individual and interactive roles of five HKs in regulating cell growth, metabolism, and sporulation were investigated. The fermentation results of mutants with different HK expression levels suggested that cac3319 and cac0437 played critical roles in regulating sporulation and acids and butanol biosynthesis. Morphological analysis revealed that cac3319 knockout abolished sporulation (Stage 0) whereas cac3319 overexpression promoted spore development (Stage VII), and cac0437 knockout initiated but blocked sporulation before Stage II, indicating the progression of sporulation was altered through engineering HKs. By combinatorial HKs knockout, the interactive effects between two different HKs were investigated. This study elucidated the regulatory roles of HKs in clostridial differentiation and demonstrated that HK engineering can be effectively used to control sporulation and enhance butanol biosynthesis.

High-rate continuous n-butanol production by Clostridium acetobutylicum from glucose and butyric acid in a single-pass fibrous-bed bioreactor.

Biobutanol produced in acetone-butanol-ethanol (ABE) fermentation at batch mode cannot compete with chemically derived butanol because of the low reactor productivity. Continuous fermentation can dramatically enhance productivity and lower capital and operating costs, but are rarely used in industrial fermentation because of increased risks of culture degeneration, cell washout, and contamination. In this study, cells of the asporogenous Clostridium acetobutylicum ATCC55025 were immobilized in a single-pass fibrous-bed bioreactor (FBB) for continuous production of butanol from glucose and butyrate at various dilution rates. Butyric acid in the feed medium helped maintaining cells in the solventogenic phase for stable continuous butanol production. At a dilution rate of 1.88 h-1 , butanol was produced at 9.55 g/L, with a yield of 0.24 g/g and productivity of 16.8 g/L/h, which was the highest productivity ever achieved for biobutanol fermentation and an 80-fold improvement over the conventional ABE fermentation. The extremely high productivity was attributed to the high density of viable cells (~100 g/L at >70% viability) immobilized in the fibrous matrix, which also enabled the cells to better tolerate butanol and butyric acid. The FBB was stable for continuous operation for an extended period of over 1 month.

Oxidoreduction potential controlling for increasing the fermentability of enzymatically hydrolyzed steam-exploded corn stover for butanol production.

BACKGROUND: Lignocellulosic biomass is recognized as an effective potential substrate for biobutanol production. Though many pretreatment and detoxification methods have been set up, the fermentability of detoxicated lignocellulosic substrate is still far lower than that of starchy feedstocks. On the other hand, the number of recent efforts on rational metabolic engineering approaches to increase butanol production in Clostridium strains is also quite limited, demonstrating the physiological complexity of solventogenic clostridia. In fact, the strain performance is greatly impacted by process control. developing efficient process control strategies could be a feasible solution to this problem. RESULTS: In this study, oxidoreduction potential (ORP) controlling was applied to increase the fermentability of enzymatically hydrolyzed steam-exploded corn stover (SECS) for butanol production. When ORP of detoxicated SECS was controlled at - 350 mV, the period of fermentation was shortened by 6 h with an increase of 27.5% in the total solvent (to 18.1 g/L) and 34.2% in butanol (to 10.2 g/L) respectively. Silico modeling revealed that the fluxes of NADPH, NADH and ATP strongly differed between the different scenarios. Quantitative analysis showed that intracellular concentrations of ATP, NADPH/NADP+, and NADH/NAD+ were increased by 25.1%, 81.8%, and 62.5%. ORP controlling also resulted in a 2.1-fold increase in butyraldehyde dehydrogenase, a 1.2-fold increase in butanol dehydrogenase and 29% increase in the cell integrity. CONCLUSION: ORP control strategy effectively changed the intracellular metabolic spectrum and significantly improved Clostridium cell growth and butanol production. The working mechanism can be summarized into three aspects: First, Glycolysis and TCA circulation pathways were strengthened through key nodes such as pyruvate carboxylase [EC: 6.4.1.1], which provided sufficient NADH and NADPH for the cell. Second, sufficient ATP was provided to avoid "acid crash". Third, the key enzymes activities regulating butanol biosynthesis and cell membrane integrity were improved.

A high-efficient strategy for combinatorial engineering paralogous gene family: A case study on histidine kinases in Clostridium.

Microorganisms harbor bulks of functionally similar or undefined genes, which belong to paralogous gene family. There is a necessity of exploring combinatorial or interactive functions of these genes, but conventional loss-of-function strategy with one-by-one rounds suffers extremely low efficiency for generating mutant libraries with all gene permutations. Here, taking histidine kinases (HKs) in Clostridium acetobutylicum as a proof-of-concept, we developed a multi-plasmid cotransformation strategy for generating all theoretical HKs combinations in one round. For five HKs with 31 theoretical combinations, the library containing 22 mutants within all the possible HKs-inactivated combinations was constructed with 11 days compared to 242 days by conventional strategy, while the other 9 combinations cannot survive. Six mutants with the enhanced butanol production and tolerance were obtained with changes of cell development during fermentation, one of which could produce 54.2% more butanol (56.4% more solvents), while the butanol production of other mutants was unchanged or decreased. The cotransformation strategy demonstrated potentials for fast exploring pleiotropic function of paralogous family genes in cell survival, cell development, and target product metabolism.

Enforcing ATP hydrolysis enhanced anaerobic glycolysis and promoted solvent production in Clostridium acetobutylicum.

BACKGROUND: The intracellular ATP level is an indicator of cellular energy state and plays a critical role in regulating cellular metabolism. Depletion of intracellular ATP in (facultative) aerobes can enhance glycolysis, thereby promoting end product formation. In the present study, we examined this s trategy in anaerobic ABE (acetone-butanol-ethanol) fermentation using Clostridium acetobutylicum DSM 1731. RESULTS: Following overexpression of atpAGD encoding the subunits of water-soluble, ATP-hydrolyzing F1-ATPase, the intracellular ATP level of 1731(pITF1) was significantly reduced compared to control 1731(pIMP1) over the entire batch fermentation. The glucose uptake was markedly enhanced, achieving a 78.8% increase of volumetric glucose utilization rate during the first 18 h. In addition, an early onset of acid re-assimilation and solventogenesis in concomitant with the decreased intracellular ATP level was evident. Consequently, the total solvent production was significantly improved with remarkable increases in yield (14.5%), titer (9.9%) and productivity (5.3%). Further genome-scale metabolic modeling revealed that many metabolic fluxes in 1731(pITF1) were significantly elevated compared to 1731(pIMP1) in acidogenic phase, including those from glycolysis, tricarboxylic cycle, and pyruvate metabolism; this indicates significant metabolic changes in response to intracellular ATP depletion. CONCLUSIONS: In C. acetobutylicum DSM 1731, depletion of intracellular ATP significantly increased glycolytic rate, enhanced solvent production, and resulted in a wide range of metabolic changes. Our findings provide a novel strategy for engineering solvent-producing C. acetobutylicum, and many other anaerobic microbial cell factories.

Response characteristics of the membrane integrity and physiological activities of the mutant strain Y217 under exogenous butanol stress.

Butanol inhibits bacterial activity by destroying the cell membrane of Clostridium acetobutylicum strains and altering functionality. Butanol toxicity also results in destruction of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS), thereby preventing glucose transport and phosphorylation and inhibiting transmembrane transport and assimilation of sugars, amino acids, and other nutrients. In this study, based on the addition of exogenous butanol, the tangible macro indicators of changes in the carbon ion beam irradiation-mutant Y217 morphology were observed using scanning electron microscopy (SEM). The mutant has lower microbial adhesion to hydrocarbon (MATH) value than C. acetobutylicum ATCC 824 strain. FDA fluorescence intensity and conductivity studies demonstrated the intrinsically low membrane permeability of the mutant membrane, with membrane potential remaining relatively stable. Monounsaturated FAs (MUFAs) accounted for 35.17% of the mutant membrane, and the saturated fatty acids (SFA)/unsaturated fatty acids (UFA) ratio in the mutant cell membrane was 1.65. In addition, we conducted DNA-level analysis of the mutant strain Y217. Expectedly, through screening, we found gene mutant sites encoding membrane-related functions in the mutant, including ATP-binding cassette (ABC) transporter-related genes, predicted membrane proteins, and the PTS transport system. It is noteworthy that an unreported predicted membrane protein (CAC 3309) may be related to changes in mutant cell membrane properties. KEY POINTS: • Mutant Y217 exhibited better membrane integrity and permeability. • Mutant Y217 was more resistant to butanol toxicity. • Some membrane-related genes of mutant Y217 were mutated.

Improved CRISPR/Cas9 Tools for the Rapid Metabolic Engineering of Clostridium acetobutylicum.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated proteins)9 tools have revolutionized biology-several highly efficient tools have been constructed that have resulted in the ability to quickly engineer model bacteria, for example, Escherichia coli. However, the use of CRISPR/Cas9 tools has lagged behind in non-model bacteria, hampering engineering efforts. Here, we developed improved CRISPR/Cas9 tools to enable efficient rapid metabolic engineering of the industrially relevant bacterium Clostridium acetobutylicum. Previous efforts to implement a CRISPR/Cas9 system in C. acetobutylicum have been hampered by the lack of tightly controlled inducible systems along with large plasmids resulting in low transformation efficiencies. We successfully integrated the cas9 gene from Streptococcuspyogenes into the genome under control of the xylose inducible system from Clostridium difficile, which we then showed resulted in a tightly controlled system. We then optimized the length of the editing cassette, resulting in a small editing plasmid, which also contained the upp gene in order to rapidly lose the plasmid using the upp/5-fluorouracil counter-selection system. We used this system to perform individual and sequential deletions of ldhA and the ptb-buk operon.

RRNPP-type quorum sensing affects solvent formation and sporulation in Clostridium acetobutylicum.

The strictly anaerobic bacterium Clostridium acetobutylicum is well known for its ability to convert sugars into organic acids and solvents, most notably the potential biofuel butanol. However, the regulation of its fermentation metabolism, in particular the shift from acid to solvent production, remains poorly understood. The aim of this study was to investigate whether cell-cell communication plays a role in controlling the timing of this shift or the extent of solvent formation. Analysis of the available C. acetobutylicum genome sequences revealed the presence of eight putative RRNPP-type quorum-sensing systems, here designated qssA to qssH, each consisting of an RRNPP-type regulator gene followed by a small open reading frame encoding a putative signalling peptide precursor. The identified regulator and signal peptide precursor genes were designated qsrA to qsrH and qspA to qspH, respectively. Triplicate regulator mutants were generated in strain ATCC 824 for each of the eight systems and screened for phenotypic changes. The qsrB mutants showed increased solvent formation during early solventogenesis and hence the QssB system was selected for further characterization. Overexpression of qsrB severely reduced solvent and endospore formation and this effect could be overcome by adding short synthetic peptides to the culture medium representing a specific region of the QspB signalling peptide precursor. In addition, overexpression of qspB increased the production of acetone and butanol and the initial (48 h) titre of heat-resistant endospores. Together, these findings establish a role for QssB quorum sensing in the regulation of early solventogenesis and sporulation in C. acetobutylicum.

A CRISPR/Anti-CRISPR Genome Editing Approach Underlines the Synergy of Butanol Dehydrogenases in Clostridium acetobutylicum DSM 792.

Although Clostridium acetobutylicum is the model organism for the study of acetone-butanol-ethanol (ABE) fermentation, its characterization has long been impeded by the lack of efficient genome editing tools. In particular, the contribution of alcohol dehydrogenases to solventogenesis in this bacterium has mostly been studied with the generation of single-gene deletion strains. In this study, the three butanol dehydrogenase-encoding genes located on the chromosome of the DSM 792 reference strain were deleted iteratively by using a recently developed CRISPR-Cas9 tool improved by using an anti-CRISPR protein-encoding gene, acrIIA4 Although the literature has previously shown that inactivation of either bdhA, bdhB, or bdhC had only moderate effects on the strain, this study shows that clean deletion of both bdhA and bdhB strongly impaired solvent production and that a triple mutant ΔbdhA ΔbdhB ΔbdhC was even more affected. Complementation experiments confirmed the key role of these enzymes and the capacity of each bdh copy to fully restore efficient ABE fermentation in the triple deletion strain.IMPORTANCE An efficient CRISPR-Cas9 editing tool based on a previous two-plasmid system was developed for Clostridium acetobutylicum and used to investigate the contribution of chromosomal butanol dehydrogenase genes during solventogenesis. Thanks to the control of cas9 expression by inducible promoters and of Cas9-guide RNA (gRNA) complex activity by an anti-CRISPR protein, this genetic tool allows relatively fast, precise, markerless, and iterative modifications in the genome of this bacterium and potentially of other bacterial species. As an example, scarless mutants in which up to three genes coding for alcohol dehydrogenases are inactivated were then constructed and characterized through fermentation assays. The results obtained show that in C. acetobutylicum, other enzymes than the well-known AdhE1 are crucial for the synthesis of alcohol and, more globally, to perform efficient solventogenesis.

Development of Strong Anaerobic Fluorescent Reporters for Clostridium acetobutylicum and Clostridium ljungdahlii Using HaloTag and SNAP-tag Proteins.

One of the biggest limitations in the study and engineering of anaerobic Clostridium organisms is the lack of strong fluorescent reporters capable of strong and real-time fluorescence. Recently, we developed a strong fluorescent reporter system for Clostridium organisms based on the FAST protein. Here, we report the development of two new strong fluorescent reporter systems for Clostridium organisms based on the HaloTag and SNAP-tag proteins, which produce strong fluorescent signals when covalently bound to fluorogenic ligands. These new fluorescent reporters are orthogonal to the FAST ligands and to each other, allowing for simultaneous labeling and visualization. We used HaloTag and SNAP-tag to label the strictly anaerobic organisms Clostridium acetobutylicum and Clostridium ljungdahlii We have also identified a new strong promoter for protein expression in C. acetobutylicum, based on the phosphotransacetylase gene (pta) from C. ljungdahlii Furthermore, the HaloTag and the SNAP-tag, in combination with the previously described FAST system, were successfully used to measure cell populations in bacterial mixed cultures and showed the simultaneous orthogonal labeling of HaloTag and SNAP-tag together with the FAST protein reporter. Finally, we show the expression of recombinant fusion protein of FAST and the ZapA division protein (from C. acetobutylicum) in C. ljungdahlii. The availability of multiple strong fluorescent reporters is a major addition to the genetic toolkit of Clostridium and other anaerobes that will lead to better understanding of these unique organisms.IMPORTANCE Up to this point, assays and methods involving fluorescent reporter proteins were unavailable or limited in Clostridium organisms and other strict anaerobes. Green fluorescent protein (GFP), mCherry, and flavin-binding proteins (and their derivatives) have been used only in a few clostridia with limited success and yielded low fluorescence compared to aerobic microbial systems. Recently, we reported a new strong fluorescent reporter system based on the FAST protein as a first step in expanding the fluorescence-based reporters for Clostridium and other anaerobic microbial platforms. Additional strong orthogonal fluorescent proteins, with distinct emission spectra are needed to allow for (i) multispecies tracking within the growing field of microbial cocultures and microbiomes, (ii) protein localization and tracking in anaerobes, and (iii) identification and development of natural and synthetic promoters, ribosome-binding sites (RBS), and terminators for optimal protein expression in anaerobes. Here, we present two new strong fluorescent reporter systems based on the HaloTag and SNAP-tag proteins.

Efficient butanol production under aerobic conditions by coculture of Clostridium acetobutylicum and Nesterenkonia sp. strain F.

Clostridium acetobutylicum is widely used for the microbial production of butanol in a process known as acetone-butanol-ethanol (ABE) fermentation. However, this process suffers from several disadvantages including high oxygen sensitivity of the bacterium which makes the process complicated and necessitate oxygen elimination in the culture medium. Nesterenkonia sp. strain F has attracted interests as the only known non-Clostridia microorganism with inherent capability of butanol production even in the presence of oxygen. This bacterium is not delimited by oxygen sensitivity, a challenge in butanol biosynthesis, but the butanol titer was far below Clostridia. In this study, Nesterenkonia sp. strain F was cocultivated with C. acetobutylicum to form a powerful "coculture" for butanol production thereby eliminating the need for oxygen removal before fermentation. The response surface method was used for obtaining optimal inoculation amount/time and media formulation. The highest yield, 0.31 g/g ABE (13.6 g/L butanol), was obtained by a coculture initiated with 1.5 mg/L Nesterenkonia sp. strain F and inoculated with 15 mg/L C. acetobutylicum after 1.5 hr in a medium containing 67 g/L glucose, 2.2 g/L yeast extract, 4 g/L peptone, and 1.4% (vol/vol) P2 solution. After butanol toxicity assessment, where Nesterenkonia sp. strain F showed no butanol toxicity, the coculture was implemented in a 2 L fermenter with continual aeration leading to 20 g/L ABE.

Genetic sensor-regulators functional in Clostridia.

This study addressed the functionality of genetic circuits carrying natural regulatory elements of Clostridium acetobutylicum ATCC 824 in the presence of the respective inducer molecules. Specifically, promoters and their regulators involved in diverse carbon source utilization were characterized using mCherryOpt or beta-galactosidase as a reporter. Consequently, most of the genetic circuits tested in this study were functional in Clostridium acetobutylicum ATCC 824 in the presence of an inducer, leading to the expression of reporter proteins. These genetic sensor-regulators were found to be transferable to another Clostridium species, such as Clostridium beijerinckii NCIMB 8052. The gradual expression of reporter protein was observed as a function of the carbohydrates of interest. A xylose-inducible promoter allows a titratable and robust expression of a reporter protein with stringency and efficacy. This xylose-inducible circuit was seen to enable induction of the expression of reporter proteins in the presence of actual sugar mixtures incorporated in woody hydrolysate wherein glucose and xylose are present as predominant carbon sources.

Biochemical characterization of an esterase from Clostridium acetobutylicum with novel GYSMG pentapeptide motif at the catalytic domain.

Gene CA_C0816 codes for a serine hydrolase protein from Clostridium acetobutylicum (ATCC 824) a member of hormone-sensitive lipase of lipolytic family IV. This gene was overexpressed in E. coli strain BL21and purified using Ni2+-NTA affinity chromatography. Size exclusion chromatography revealed that the protein is a dimer in solution. Optimum pH and temperature for recombinant Clostridium acetobutylicum esterase (Ca-Est) were found to be 7.0 and 60 °C, respectively. This enzyme exhibited high preference for p-nitrophenyl butyrate. KM and kcat/KM of the enzyme were 24.90 µM and 25.13 s-1 µM-1, respectively. Sequence analysis of Ca-Est predicts the presence of catalytic amino acids Ser 89, His 224, and Glu 196, presence of novel GYSMG conserved sequence (instead of GDSAG and GTSAG motif), and undescribed variation of HGSG motif. Site-directed mutagenesis confirmed that Ser 89 and His 224 play a major role in catalysis. This study reports that Ca-Est is hormone-sensitive lipase with novel GYSMG pentapeptide motif at a catalytic domain.

Effects of Spo0A on Clostridium acetobutylicum with an emphasis on biofilm formation.

Clostridium acetobutylicum is a well-known strain for biofuel production. In previous work, it was found that this strain formed biofilm readily during fermentation processes. Biofilm formation could protect cells and enhance productivities under environmental stresses in our previous work. To explore the molecular mechanism of biofilm formation, Spo0A of C. acetobutylicum was selected to investigate its influences on biofilm formation and other physiological performances. When spo0A gene was disrupted, the spo0A mutant could hardly form biofilm. The aggregation and adhesion abilities of the spo0A mutant as well as its swarming motility were dramatically reduced compared to those of wild type strain. Sporulation was also negatively influenced by spo0A disruption, and solvent production was almost undetectable in the spo0A mutant fermentation. Furthermore, proteomic differences between wild type strain and the spo0A mutant were consistent with physiological performances. This is the first study confirming a genetic clue to C. acetobutylicum biofilm and will be valuable for biofilm optimization through genetic engineering in the future.

A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST).

Visualizing protein localization and characterizing gene expression activity in live Clostridium cells is limited for lack of a real-time, highly fluorescent, oxygen-independent reporter system. Enzymatic reporter systems have been used successfully for many years with Clostridium spp.; however, these assays do not allow for real-time analysis of gene expression activity with flow cytometry or for visualizing protein localization through fusion proteins. Commonly used fluorescent reporter proteins require oxygen for chromophore maturation and cannot be used for most strictly anaerobic Clostridium organisms. Here we show that the fluorescence-activating and absorption-shifting tag protein (FAST), when associated with the fluorogenic ligand 4-hydroxy-3-methylbenzylidene-rhodanine (HMBR; now commercially available) and other commercially available ligands, is highly fluorescent in Clostridium acetobutylicum under anaerobic conditions. Using flow cytometry and a fluorescence microplate reader, we demonstrated FAST as a reporter system by employing the promoters of the C. acetobutylicum thiolase (thl), acetoacetate decarboxylase (adc), and phosphotransbutyrylase (ptb) metabolic genes, as well as a mutant Pthl and modified ribosome binding site (RBS) versions of Padc and Pptb Flow cytometry-based sorting was efficient and fast in sorting FAST-expressing cells, and positively and negatively sorted cells could be effectively recultured. FAST was also used to tag and examine protein localization of the predicted cell division FtsZ partner protein, ZapA, to visualize the divisome localization in live C. acetobutylicum cells. Our findings suggest that FAST can be used to further investigate Clostridium divisomes and more broadly the localization and expression levels of other proteins in Clostridium organisms, thus enabling cell biology studies with these organisms.IMPORTANCE FAST in association with the fluorogenic ligand HMBR is characterized as a successful, highly fluorescent reporter system in C. acetobutylicum FAST can be used to distinguish between promoters in live cells using flow cytometry or a fluorescence microplate reader and can be used to tag and examine protein localization in live, anaerobically grown cells. Given that FAST is highly fluorescent under anaerobic conditions, it can be used in several applications of this and likely many Clostridium organisms and other strict anaerobes, including studies involving cell sorting, sporulation dynamics, and population characterization in pure as well as mixed cultures, such as those in various native or synthetic microbiomes and syntrophic cultures.