Genetic manipulation of butyrate formation pathways in Clostridium butyricum

Clostridium butyricum is one of the commonly used species for fermentative hydrogen production. While producing H₂, it can produce acids (lactic, acetic and butyric acids) and CO₂, as well as a small amount of ethanol. It has been proposed that elimination of competing pathways, such as the butyrate formation pathway, should increase H₂ yields in Clostridium species. However, the application of this strategy has been hindered by the unavailability of genetic tools for these organisms. In this study, we successfully transferred a plasmid (pMTL007) to C. butyricum by inter-specific conjugation with Escherichia coli and disrupted hbd, the gene encoding β-hydroxybutyryl-CoA dehydrogenase in C. butyricum. Fermentation data showed that inactivation of hbd in C. butyricum eliminated the butyrate formation pathway, resulting in a significant increase in ethanol production and an obvious decrease in H₂ yield compared with the wild type strain. However, under low partial pressure of H₂, the hbd-deficient strain showed increased H₂ production with the simultaneous decrease of ethanol production, indicating that H₂ production by C. butyricum may compete for NADH with the ethanol formation pathway. Together with the discovery of a potential bifurcating hydrogenase, this study extends our understanding of the mechanism of H₂ production by C. butyricum.     

p-Cresol formation by cell-free extracts of Clostridium difficile

Cell-free extracts of Clostridium difficile were shown to form p-cresol by decarboxylation of p-hydroxyphenylacetic acid. This activity required both high and low molecular weight fractions. The active component of the low molecular weight fraction had properties of an amino acid and could be replaced by serine, threonine or the corresponding alpha keto acids. Pyruvate was shown to function catalytically. Since the high molecular weight fraction was O₂-sensitive and since dithionite was as effective as pyruvate with some high molecular weight fractions, the alpha keto acids probably serve as low potential reducing agents in this system. Because of instability, the p-cresolforming enzyme could not be purified.     

Binary toxin producing Clostridium difficile strains

Clostridium difficile produces three toxins, TcdA, TcdB and CDT. TcdA and TcdB are single-stranded molecules acting as glucosyltransferases specific for small GTPases. CDT is an actin specific ADP-ribosylating binary toxin characteristically composed of two independent components, enzymatic CDTa (48 kDa) and binding CDTb (99 kDa). The cdtA and cdtB genes were sequenced in two CDT-positive strains of C. difficile (CD 196 and 8864) and at least two CDT-negative strains with truncated form of binary toxin genes are known (VPI 10463 and C. difficile genome strain 630). The prevalence of binary toxin producing strains is estimated to be from 1.6% to 5.5%, although a much higher proportion has been reported in some studies. The role of the binary toxin as an additional virulence factor is discussed.     

Production of Cellulase by Clostridium Papyrosolvens CFR-703

Clostridium papyrosolvens producing filter paperase, carboxymethyl cellulase and cellobiase under anaerobic cultivation conditions at 35 °C is described. Higher activities of filter paperase and carboxymethylcellulases were assayed in 48 h culture filtrate, while maximum cellobiase accumulated in the culture broth at 72 h. Filter paperase, carboxymethylcellulase and cellobiase activities were optimum at 35 °C and pH values of 7.0, 6.5 and 7.5 respectively. Cultivation of the strain in 1000 ml Hungate bottles with 1% cellulose at pH 6.5 and 35 °C produced carboxymethyl cellulase, filter paperase and cellobiase activities of 45, 35 and 20 IU/ml respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Anaerobic degradation of 1,3-propanediol by sulfate-reducing and by fermenting bacteria

Three strains of strictly anaerobic Gram-negative, non-sporeforming, motile bacteria were enriched and isolated from freshwater sediments with 1,3-propanediol as sole energy and carbon source. Strain OttPdl was a sulfate-reducing bacterium which grew also with lactate, ethanol, propanol, butanol, 1,4-butanediol, formate or hydrogen plus CO₂, the latter only in the presence of acetate. In the absence of sulfate, most of these substrates were fermented to the respective fatty acids in syntrophic cooperation with Methanospirillum hungatei. Sulfur, thiosulfate, or sulfite were reduced, nitrate not. The other two isolates degraded propanediol only in coculture with Methanospirillum hungatei. Strain OttGlycl grew in pure culture with acetoin and with glycerol in the presence of acetate. Strain WoAcl grew in pure culture only with acetoin. Both strains did not grow with other substrates, and did not reduce nitrate, sulfate, sulfur, thiosulfate or sulfite. The isolates were affiliated with the genera Desulfovibrio and Pelobacter. The pathways of propanediol degradation and the ecological importance of this process are discussed.     

Effect of growth supplements and whey pretreatment on butyric acid production by Clostridium butyricum

Improved production of butyrate (up to 19 g/l) from whey by Clostridium butyricum was achieved by adding either yeast extract (5 g/l) or biotin (50 g/l). Hydrolysed lactose and proteolysed whey were less effective even with added biotin.The authors are with the Department of Biochemical Technology, Faculty of Chemical Technology, Slovak Technical University, Radlinského 9, Bratislava 812 37, Slovakia     

Optimization of medium composition for butyric acid production by Clostridium thermobutyricum using response surface methodology

The optimal medium for butyric acid production by Clostridium thermobutyricum in a shake flask culture was studied using statistical experimental design and analysis. The optimal composition of the fermentation medium for maximum butyric acid yield, as determined on the basis of a three-level four-factor Box-Behnken design (BBD), was obtained by response surface methodology (RSM). The high correlation between the predicted and observed values indicated the validity of the model. A maximum butyric acid yield of 12.05 g/l was obtained at K₂HPO₄ 7.2 g/l, 34.9 g/l glucose, 20 g/l yeast extract, and 15 g/l acetate, which compared well to the predicated production of 12.13 g/l.     

华南吊灯花属(夹竹桃科)植物小志

通过野外调查、标本查阅及文献考证,订正了华南吊灯花属(Ceropegia Linn.)植物,将狭瓣吊灯花(C. angustilimba Merr.)从吊灯花(C. trichantha Hemsl.)的异名中予以恢复,提供了二者的形态描述,确认华南地区记录的柳叶吊灯花(C. salicifolia H. Huber)实为剑叶吊灯花(C. dolichophylla Schltr.)的错误鉴定,并提供了后者的形态描述。     

Spermine stimulation of phospholipase C fromCatharanthus roseus transformed roots

Polyamines (Pas) are aliphatic amines that are ubiquitous in all living organisms and regulate a broad spectrum of physiological processes. It has been suggested that they can act through a signal transduction pathway. Using Catharanthus roseus hairy roots as a model we determined the levels of Pas throughout a culture cycle. We found that there is a peak in the intracellular concentration of Pas during the first six days of culture. The effect of Pas on phospholipase C (PLC) activity was also investigated. Putrescine, spermidine and spermine were added in vitro to the PLC assay. Putrescine did not modify PLC activity; spermidine inhibited the enzyme but at very high, non-physiological concentrations; and spermine increased the PLC activity four-fold at physiological concentrations. Our results suggest that spermine could regulate root growth by regulating the PLC signal transduction mechanism.     

FTIR-spectroscopic studies of the fine structure of nitrocellulose treated by Desulfovibrio desulfuricans

Most studies have concluded that nitrocellulose (NC) with high degree of nitrogen content is resistant to biodegradation. Our results demonstrated that NC (>11%N) does undergo biotransformation in the presence of sulfate-reducing bacteria Desulfovibrio desulfuricans 1388. FTIR analyses indicated that the substitution of nitro groups for OH(-) groups took place. The spectrum of precipitate obtained after acetone extraction of NC resembled mainly the spectrum of native cellulose. Thus the synthetic unbiodegradable polymer was transformed to the natural compound accessible for microorganisms.     

Seeing green bacteria in a new light: genomics-enabled studies of the photosynthetic apparatus in green sulfur bacteria and filamentous anoxygenic phototrophic bacteria

Based upon their photosynthetic nature and the presence of a unique light-harvesting antenna structure, the chlorosome, the photosynthetic green bacteria are defined as a distinctive group in the Bacteria. However, members of the two taxa that comprise this group, the green sulfur bacteria (Chlorobi) and the filamentous anoxygenic phototrophic bacteria (Chloroflexales), are otherwise quite different, both physiologically and phylogenetically. This review summarizes how genome sequence information facilitated studies of the biosynthesis and function of the photosynthetic apparatus and the oxidation of inorganic sulfur compounds in two model organisms that represent these taxa, Chlorobium tepidum and Chloroflexus aurantiacus. The genes involved in bacteriochlorophyll (BChl) c and carotenoid biosynthesis in these two organisms were identified by sequence homology with known BChl a and carotenoid biosynthesis enzymes, gene cluster analysis in Cfx. aurantiacus, and gene inactivation studies in Chl. tepidum. Based on these results, BChl a and BChl c biosynthesis is similar in the two organisms, whereas carotenoid biosynthesis differs significantly. In agreement with its facultative anaerobic nature, Cfx. aurantiacus in some cases apparently produces structurally different enzymes for heme and BChl biosynthesis, in which one enzyme functions under anoxic conditions and the other performs the same reaction under oxic conditions. The Chl. tepidum mutants produced with modified BChl c and carotenoid species also allow the functions of these pigments to be studied in vivo.     

Cytokinesis in brown algae: studies of asymmetric division in fucoid zygotes

Summary. The mechanism of cytokinesis was investigated during the first asymmetric division in fucoid zygotes. A plate of actin assembled midway between daughter nuclei where microtubules interdigitated and defined the cytokinetic plane. A membrane was then deposited in islands throughout the cytokinetic plane; the islands eventually fused into a continuous partition membrane and cell plate material was deposited in the intermembrane space. All of these structures matured from the center of the cell outward (centrifugal maturation). Pharmacological agents were used to investigate the roles of microtubules, actin, and secretion in cytokinesis. The findings indicate a mechanism of cytokinesis that may be unique to the brown algae.Correspondence and reprints: Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112-0840, U.S.A.     

Metabolism of lactose by Clostridium thermolacticum growing in continuous culture

The objective of the present study was to characterize the metabolism of Clostridium thermolacticum, a thermophilic anaerobic bacterium, growing continuously on lactose (10 g l⁻¹) and to determine the enzymes involved in the pathways leading to the formation of the fermentation products. Biomass and metabolites concentration were measured at steady-state for different dilution rates, from 0.013 to 0.19 h⁻¹. Acetate, ethanol, hydrogen and carbon dioxide were produced at all dilution rates, whereas lactate was detected only for dilution rates below 0.06 h⁻¹. The presence of several key enzymes involved in lactose metabolism, including beta-galactosidase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase, acetate kinase, ethanol dehydrogenase and lactate dehydrogenase, was demonstrated. Finally, the intracellular level of NADH, NAD⁺, ATP and ADP was also measured for different dilution rates. The production of ethanol and lactate appeared to be linked with the re-oxidation of NADH produced during glycolysis, whereas hydrogen produced should come from reduced ferredoxin generated during pyruvate decarboxylation. To produce more hydrogen or more acetate from lactose, it thus appears that an efficient H₂ removal system should be used, based on a physical (membrane) or a biological approach, respectively, by cultivating C. thermolacticum with efficient H₂ scavenging and acetate producing microorganisms.     

Regulation of isocitrate lyase in a mutant of Rhodopseudomonas capsulata adapted to growth on acetate

Studies on acetate utilization by Rhodopseudomonas capsulata strain St. Louis indicated that the wild type grew poorly on acetate and made little if any of the glyoxylate cycle enzyme isocitrate lyase. A spontaneous mutant, Ac-l, capable of vigorous and immediate growth on acetate and exhibiting high levels of isocitrate lyase activity, was isolated in the course of those studies.Isocitrate lyase was not formed when the mutant was grown on malate. Addition of malate to cultures of Ac-l growing on acetate resulted in loss of the enzyme by dilution through growth.Starvation of acetate-grown Ac-l for acetate resulted in a rapid and complete loss of isocitrate lyase activity which was shown to be energy dependent. Readdition of acetate to a starved culture previously grown on acetate resulted in a rapid recovery of enzyme activity. The recovery required energy and was sensitive to chloramphenicol inhibition at any time during the recovery phase.     

Heterologous expression of the<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> alcohol acetyltransferase genes in<Emphasis Type="Italic"> Clostridium acetobutylicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis> for the production of isoamyl acetate

Esters are formed by the condensation of acids with alcohols. The esters isoamyl acetate and butyl butyrate are used for food and beverage flavorings. Alcohol acetyltransferase is one enzyme responsible for the production of esters from acetyl-CoA and different alcohol substrates. The genes ATF1 and ATF2, encoding alcohol acetyltransferases from the yeast Saccharomyces cerevisiae have been sequenced and characterized. The production of acids and alcohols in mass quantities by the industrially important Clostridium acetobutylicum makes it a potential organism for exploitation of alcohol acetyltransferase activity. This report focuses on the heterologous expression of the alcohol acetyltransferases in Escherichia coli and C. acetobutylicum. ATF1 and ATF2 were cloned and expressed in E. coli and ATF2 was expressed in C. acetobutylicum. Isoamyl acetate production from the substrate isoamyl alcohol in E. coli and C. acetobutylicum cultures was determined by head-space gas analysis. Alcohol acetyltransferase I produced more than twice as much isoamyl acetate as alcohol acetyltransferase II when expressed from a high-copy expression vector. The effect of substrate levels on ester production was explored in the two bacterial hosts to demonstrate the efficacy of utilizing ATF1and ATF2 in bacteria for ester production.     

Organic matter fractions by SP-MAS C NMR and microbial communities involved in the suppression of Fusarium wilt in organic growth media

Fusarium wilt is an economically important disease in carnation and tomato plants. The use of suppressive plant growth media has become an alternative method for plant disease control due to the lack of effective chemical control measures. Plant disease suppressiveness is sustained only in plant growth media with an adequate organic matter (OM) composition. Carbohydrate polymers are the most important sources of carbon nutrient for microbial community in these media, mainly consisting of cellulose and hemicellulose. This determines microbial activity, biomass and selects microbial communities in plant growth media, which are reported factors associated with Fusarium wilt suppressiveness.This work determined OM carbon functional groups using Single Pulse Magic Angle Spinning ¹³C-Nuclear Magnetic Resonance (SP-MAS ¹³C-NMR) in three plant growth media with different suppressiveness levels to Fusarium wilt in two crops, carnation and tomato. We propose that the critical role of OM to sustain naturally occurring suppressiveness in those media is not related with cellulose reserve. This could be explained because cellulose protected by lignin encrustation is not available to microbial degradation, meaning that cellulose availability is critical to sustenance of microorganism-mediated biological control. However, the hemicellulose relative abundance (peak 175 ppm) was associated to Fusarium wilt suppression level in plant growth media studied.Carbon source availability in OM was related to microbial biomass and econutritional group population densities involved in biocontrol. For these composts, Bacillus spp., oligotrophic and cellulolytic actinomycetes, and oligotrophic actinomycetes/oligotrophic bacteria and cellulolytic actinomycetes/cellulolytic bacteria ratios were indicated as microbial populations potentially involved in suppression.     

Acetyl coenzyme A and coenzyme A contents of growing Clostridium kluyveri as determined by isotope assays

CoASH and some of its acyl derivatives, especially acetyl-SCoA, occupy a central position in the energy metabolism of the anaerobic Clostridium kluyveri, both as intermediates and as regulatory effectors. The steady state concentrations of these compounds were determined in growing cultures of this organism using an anaerobic and fast deproteinization technique and radio isotope assays. Acetyl-SCoA was determined as [1-¹⁴C]citrate formed in the presence of [4-¹⁴C]oxaloacetate and citrate synthase; 0.49 mol/g cell wet wt. were found CoASH, CoAS-SCoA after borohydride reduction, and total acyl derivatives of coenzyme A after hydrolysis of the thiol esters were converted to thioethers with [2,3-¹⁴C]N-ethylmaleimide and brought to radiochemical purity by chromatographic methods. While disulfides of coenzyme A were undetectable, 0.13 mol CoASH and 1.17 mol of total acyl-SCoA per g wet wt. were found. These data are consistent with the regulatory scheme of the energy metabolism of C. kluyveri previously proposed.Abbreviations DTE dithioerythritol - NEM N-ethylmaleimide - NES N-ethylsuccinimide Enzymes (EC 2.7.2.1) Acetate kinase, ATP: acetate phosphotransferase - (EC 3.1.3.1) Alkaline phosphatase, orthophosphoric monoester phosphohydrolase - (GOT) Aspartate aminotransferase - (EC 2.6.1.1) L-aspartate:2-oxoglutarate aminotransferase - (CS) Citrate synthase - (EC 4.1.3.7) citrate oxaloacetate-lyase (pro 3S-CH₂COOacetyl-CoA) - (EC 2.8.3.8) CoA-transferase, acyl-CoA:acetate CoA-transferase - (EC 1.1.1.37) Malate dehydrogenase, L-malate:NAD⁺ oxidoreductase - (EC 1.18.1.3) NADH:ferredoxin reductase, ferredoxin:NAD⁺ oxidoreductase - (EC 3.1.4.1) Phosphodiesterase (snake venom), orthophosphoric diester phosphohydrolase - (EC 2.3.1.8) Phosphotransacetylase, acetyl-CoA:orthophosphate acetyltransferase - (EC 2.3.1.9) Thiolase, acetyl-CoA:acetyl-CoA C-acetyltransferase A preliminary account of this work has been given (Decker et al. 1976)