Phosphoproteomic investigation of a solvent producing bacterium Clostridium acetobutylicum

In this study, we employed TiO? enrichment and high accuracy liquid chromatography-mass spectrometry-mass spectrometry to identify the phosphoproteome of Clostridium acetobutyicum ATCC824 in acidogenesis and solventogenesis. As many as 82 phosphopeptides in 61 proteins, with 107 phosphorylated sites on serine, threonine, or tyrosine, were identified with high confidence. We detected 52 phosphopeptides from 44 proteins in acidogenesis and 70 phosphopeptides from 51 proteins in solventogenesis, respectively. Bioinformatic analysis revealed most of the phosphoproteins located in cytoplasm and participated in carbon metabolism. Based on comparison between the two stages, we found 27 stage-specific phosphorylated proteins (10 in acidogenesis and 17 in solventogenesis), some of which were solvent production-related enzymes and metabolic regulators, showed significantly different phosphorylated status. Further analysis indicated that protein phosphorylation could be involved in the shift of stages or in solvent production pathway directly. Comparison against several other organisms revealed the evolutionary diversity among them on phosphorylation level in spite of their high homology on protein sequence level.     

Identification and characterization of two functionally unknown genes involved in butanol tolerance of Clostridium acetobutylicum

Solvents toxicity is a major limiting factor hampering the cost-effective biotechnological production of chemicals. In Clostridium acetobutylicum, a functionally unknown protein (encoded by SMB_G1518) with a hypothetical alcohol interacting domain was identified. Disruption of SMB_G1518 and/or its downstream gene SMB_G1519 resulted in increased butanol tolerance, while overexpression of SMB_G1518-1519 decreased butanol tolerance. In addition, SMB_G1518-1519 also influences the production of pyruvate:ferredoxin oxidoreductase (PFOR) and flagellar protein hag, the maintenance of cell motility. We conclude that the system of SMB_G1518-1519 protein plays a role in the butanol sensitivity/tolerance phenotype of C. acetobutylicum, and can be considered as potential targets for engineering alcohol tolerance.     

细胞分裂蛋白编码基因的敲除对丙酮丁醇梭菌溶剂合成与细胞形态的影响

丙酮丁醇梭菌是生物丁醇合成的重要菌株,近年来,研究者们利用基因编辑等技术对其进行菌株改造。通过对丙酮丁醇梭菌中3个细胞分裂蛋白(RodA、DivIVA、DivIB)编码基因(cac1251、cac2118、cac2125)进行敲除,发现cac2118敲除菌株的细胞在产溶剂期为球状形态,细胞变小,ABE发酵的丁醇得率为0.19 g/g,与野生型相比提高了5.6%。cac1251敲除菌株的葡萄糖消耗量和丁醇产量与野生型相比降低了33.9%和56.3%,分别为47.3 g/L和5.6 g/L。cac1251和cac2125的敲除对细胞生长有显著影响,菌体浓度最大值与野生型相比分别降低了40.4%和38.3%。研究表明细胞分裂蛋白DivIVA对细胞的形态和大小调控起重要作用;细胞分裂蛋白RodA和DivIB调控细胞分裂进程,进而影响细胞生长和溶剂合成进程。     

Nitrogen-fixation genes and nitrogenase activity in Clostridium acetobutylicum and Clostridium beijerinckii

Several solvent-producing clostridia, including Clostridium acetobutylicum and C. beijerinckii, were previously shown to be nitrogen-fixing organisms based on the incorporation of ¹⁵N₂ into cellular material. The key nitrogen-fixation (nif) genes, including nifH, nifD, and nifK for nitrogenase component proteins as well as nifE, nifN, nifB and nifV for synthesis of the iron–molybdenum cofactor (FeMoco) of nitrogenase, have now been identified in C. acetobutylicum or C. beijerinckii or both. The organization of these genes is similar to the distinctive pattern that was first observed in Clostridium pasteurianum, with the nifN and nifB genes fused into the nifN-B gene and with the nifV gene split into the nifVω and nifVα genes. The corresponding nif genes of these three clostridial species are highly related to each other. However, in the two solvent-producing clostridia, the nifH and nifD genes are interspersed by two glnB-like genes, which are absent in the corresponding region in C. pasteurianum. However, the nifN-B and nifVω genes of C. pasteurianum are interspersed by the putative modA and modB genes (for molybdate transport), which are absent in the corresponding region in C. acetobutylicum. C. acetobutylicum and C. beijerinckii grew well under nitrogen-fixing conditions, and the acetylene-reducing activity of nitrogenase was measured in the two species. Acetone, butanol, and isopropanol production occurred in nitrogen-fixing cultures, but the peak of nitrogen-fixing activity preceded the active solventogenic phase. Journal of Industrial Microbiology & Biotechnology (2001) 27, 281–286. Received 02 September 2000/ Accepted in revised form 22 November 2000     

Culture conditions for growth and solvent biosynthesis by a modified Clostridium acetobutylicum

Summary A modified strain of Clostridium acetobutylicum and the fermentation medium conditions for good growth of the culture and normal production of solvents are described. The pretreatment of the culture with butyric-acid-enriched medium increased the final solvent yield on sugar and lowered the residual butyric acid accumulation. In a complex medium, relatively high concentrations of yeast extract (7.5 g/l) and ammonium sulphate (3 g/l to 6 g/l) were required for normal solvent synthesis. The nitrogen requirements for cellular growth and solvent production were distinctively different. Production of solvents and growth of the culture were dependent on the concentration of para-aminobenzoic acid and relatively independent of the variations of the initial pH of the medium in the range of 4.6 to 6.3. Solvent production was obtained with initial glucose concentrations of 20.5 g/l to 70 g/l, resulting in a maximum solvent concentration of 22 g/l and a maximum yield on glucose of 32.7%.     

Stability of solvent formation in Clostridium acetobutylicum during repeated subculturing

Summary Clostridium acetobutylicum ATCC 824 was submitted to repeated subculturing at 24-hour intervals for 218 days. The organism retained its ability to form solvents, although the fermentation slowly became increasingly acidogenic during the first 200 days. Except for the initial spore inoculum, the cultures were not subjected to heat shocking between the serial transfers. When the inoculum volume was doubled from 3.3% to 6.7% after 200 days of subculturing, the product formation pattern quickly shifted back from acids to primarily butanol. Acetone production also resumed after being undetectable for more than 50 days. The relative formation of acetate and ethanol remained nearly constant throughout the experiments, while the formation of butyrate mirrored that of butanol.     

Metronidazole activation and isolation of Clostridium acetobutylicum electron transport genes

An Escherichia coli F19 recA, nitrate reductase-deficient mutant was constructed by transposon mutagenesis and shown to be resistant to metronidazole. This mutant was a most suitable host for the isolation of Clostridium acetobutylicum genes on recombinant plasmids, which activated metronidazole and rendered the E. coli F19 strain sensitive to metronidazole. Twenty-five E. coli F19 clones containing different recombinant plasmids were isolated and classified into five groups on the basis of their sensitivity to metronidazole. The clones were tested for nitrate reductase, pyruvate-ferredoxin oxidoreductase, and hydrogenase activities. DNA hybridization and restriction endonuclease mapping revealed that four of the C. acetobutylicum insert DNA fragments on recombinant plasmids were linked in an 11.1-kb chromosomal fragment. DNA sequencing and amino acid homology studies indicated that this DNA fragment contained a flavodoxin gene which encoded a protein of 160 amino acids that activated metronidazole and made the E. coli F19 mutant very sensitive to metronidazole. The flavodoxin and hydrogenase genes which are involved in electron transfer systems were linked on the 11.1-kb DNA fragment from C. acetobutylicum.     

Tn916-induced mutants of Clostridium acetobutylicum defective in regulation of solvent formation

Sixteen Tn916-induced mutants of Clostridium acetobutylicum were selected that were defective in the production of acetone and butanol. Formation of ethanol, however, was only partially affected. The strains differed with respect to the degree of solvent formation ability and could be assigned to three different groups. Type I mutants (2 strains) were completely defective in acetone and butanol production and contained one or three copies of Tn916 in the chromosome. Analysis of the mutants for enzymes responsible for solvent production revealed the presence of a formerly unknown, specific acetaldehyde dehydrogenase. The data obtained also strongly indicate that the NADP⁺-dependent alcohol dehydrogenase is in vivo reponsible for ethanol formation, whereas the NAD⁺-dependent alcohol dehydrogenase is probably involved in butanol production. No activity of this enzyme together with all other enzymes in the acetone and butanol pathway could be found in type I strains. All tetracycline-resistant mutants obtained did no longer sporulate.Non-standard abbreviations AADC acetoacetate decarboxylase - AcaDH acetaldehyde dehydrogenase - BuaDH butyraldehyde dehydrogenase - CoA-TF acetoacetyl coenzyme A: acetate/butyrate: coenzyme A transferase - NAD-ADH, NAD⁺ dependent alcohol dehydrogenase - NADP-ADH, NADP⁺ dependent alcohol dehydrogenase     

Studies on the stability of solvent production by Clostridium acetobutylicum in continuous culture

Various methods of continuous flow culture of Clostridium acetobutylicum NCIB 8052 were investigated, with the aim of obtaining prolonged production of acetone and butanol. In ammonia-limited chemostat culture, maximal concentrations of solvents were obtained at pH 5–5 at a relatively high biomass concentration of 1.3–2.0 g/1 dry weight maintained at a dilution rate of 0.06/h. Similar dependence of solvent production on the sustenance of a relatively high cell density was observed in magnesium- or phosphate-limited chemostat cultures. Solvent production was always transient, however, with a shift to production of only acetic and butyric acids being observed after 4–16 volume changes. Longer term solvent production was obtainable under conditions of glucose limitation but the solvent yield was low. Cultivation in a pH-auxostat permitted solvent production in reasonably high yield over at least 70 volume changes with no signs of culture degeneration. Although none of the continuous flow cultures achieved a true steady state, we conclude that turbidostat or pH-auxostat culture are the methods of choice for continuous solvent production by Cl. acetobutylicum NCIB 8052.     

Stability of solvent production by Clostridium acetobutylicum in continuous culture: strain differences

W oolley , R.C. & M orris , J.G. 1990. Stability of solvent production by Clostridium acetobutylicum in continuous culture: strain differences. Journal of Applied Bacteriology 69 , 718–728.
Several strains of Clostridium acetobutylicum , including strains ATCC 824 and DSM 1731, continue to produce solvents during prolonged periods of chemostat culture. In such cultures, dominance is established by asporogenous mutant(s) that retain the ability to produce solvents. Strain NCIB 8052 (which is not identical with ATCC 824) behaved differently in that its chemostat cultures invariably became acidogenic due to ultimate selection of asporogenous mutant(s) unable to produce solvents, incapable of synthesizing granulose, and demonstrating enhanced sensitivity to environmental stresses of various types. These mutants spontaneously reverted, at a low but measurable frequency, to the parental phenotype, indicating thai their multiple loss of capacities was the pleiotropic consequence of a lesion in some global regulatory gene. Their resemblance to previously described cls mutants of strain P262 and the possible nature of the affected regulatory gene are discussed. A simple tetrazolium blue plate assay procedure is described which allows visual discrimination between solvent-producing and non-solventogenic colonies of Cl. ocetobutylicum .     

Mutagenesis of Clostridium acetobutylicum

Mutagenesis of the obligate anaerobe Clostridium acetobutylicum was best accomplished using agents (e.g. ethyl methane sulphonate or N -methyl- N '-nitro- N -nitrosoguanidine) which are believed to act by a direct mutagenic mechanism. Other agents (e.g. u.v. radiation) whose effectiveness relies on misrepair of damaged DNA via an error-prone pathway, were poor mutagens of this organism. Procedures are described which readily yielded a variety of auxotrophic and other useful mutant strains of Cl. acetobutylicum and related saccharolytic clostridia.     

Mutagenesis of Clostridium acetobutylicum

Mutagenesis of the obligate anaerobe Clostridium acetobutylicum was best accomplished using agents (e.g. ethyl methane sulphonate or N-methyl-N'-nitro-N-nitrosoguanidine) which are believed to act by a direct mutagenic mechanism. Other agents (e.g. u.v. radiation) whose effectiveness relies on misrepair of damaged DNA via an error-prone pathway, were poor mutagens of this organism. Procedures are described which readily yielded a variety of auxotrophic and other useful mutant strains of Cl. acetobutylicum and related saccharolytic clostridia.     

Comparative investigations of growth and solvent formation in ‘Clostridium saccharoperbutylacetonicum’ DSM 2152 and Clostridium acetobutylicum DSM 792

The butanol and acetone-producing strain DSM 2152, invalidly described as ‘Clostridium saccharoperbutylacetonicum’ is compared with the type strain C. acetobutylicum, DSM 792, with respect to solvent and acid formation at varying pH values and growth rates. Batch cultures, product-limited chemostat and pH-auxostat cultures were used for characterization. Under all conditions strain DSM 2152 produced much lower amounts of butyric and acetic acids than the type strain. The pH optimum for solvent formation was higher, ie 5.5 instead of 4.5. Solvent formation occurred at higher dilution rates, but below 0.1 h⁻¹ a lower solvent concentration was obtained, indicating that acid production was too low to provide a sufficient amount for acetone formation. The results are discussed in the light of recent publications on the taxonomy of butanol-acetone producing clostridia using 16S rRNA sequence analysis and other nucleic acid data. The presently suggested ‘phylogenetic’ classification of the collective species, C. acetobutylicum, is also reflected in the fermentation characteristics. Received 21 December 1998/ Accepted in revised form 22 January 1999