Production of 1,3-Propanediol by Clostridium butyricum VPI 3266 in continuous cultures with high yield and productivity

The effects of dilution rate and substrate feed concentration on continuous glycerol fermentation by Clostridium butyricum VPI 3266, a natural 1,3-propanediol producer, were evaluated in this work. A high and constant 1,3-propanediol yield (around 0.65 mol/mol), close to the theoretical value, was obtained irrespective of substrate feed concentration or dilution rate. Improvement of 1,3-propanediol volumetric productivity was achieved by increasing the dilution rate, at a fixed feed substrate concentration of 30, 60 or 70 g l⁻¹. Higher 1,3-propanediol final concentrations and volumetric productivities were also obtained when glycerol feed concentration was increased from 30 to 60 g l⁻¹, at D=0.05–0.3 h⁻¹, and from 60–70 g l⁻¹, at D=0.05 and 0.1 h⁻¹·30 g l⁻¹ of 1,3-propanediol and the highest reported value of productivity, 10.3 g l⁻¹ h⁻¹, was achieved at D=0.30 h⁻¹ and 60 g l⁻¹ of feed glycerol. A switch to an acetate/butyrate ratio higher than one was observed for 60 g l⁻¹ of feed glycerol and a dilution rate higher than 0.10 h⁻¹; moreover, at D=0.30 h⁻¹ 3-hydroxypropionaldehyde accumulation was observed for the first time in the fermentation broth of C. butyricum.     

Fibronectin‐binding proteins of Clostridium perfringens recognize the III1‐C fragment of fibronectin

The Clostridium perfringens strain 13 genome contains two genes (fbpA, fbpB) that encode putative Fbp. Both rFbpA and rFbpB were purified and their reactivity with human serum Fn was analyzed. To determine the region of the Fn molecule recognized by rFbp, a plate binding assay using N‐terminal 70‐kDa peptide, III₁‐C peptide, and 110‐kDa peptide containing III_2–10 of Fn was performed. Both rFbp bound to the III₁‐C peptide of Fn but not to the other peptides. However, the III₁‐C fragment of Fn is known to be cryptic in serum Fn. Then, rFbp‐BP from Fn were purified by rFbp‐affinity chromatography. The yield of purified proteins was approximately 1% of the applied Fn on a protein basis. Western blotting analysis of the rFbp‐BP, using four different anti‐Fn monoclonal antibodies, revealed that the rFbp‐BP carried partial Fn antigenicity. Bindings of rFbp to rFbp‐BP were inhibited by the presence of the III₁‐C peptide, suggesting that rFbp‐BP express the III₁‐C fragment. The binding of Fn to III₁‐C was inhibited by the presence of either rFbpA or rFbpB. This result that suggests C. perfringens Fbps may inhibit the formation of Fn‐matrix in vivo.     

酪酸梭菌活菌散在治疗手足口病中的应用

目的观察和评价酪酸梭菌活菌散治疗手足口病的临床疗效。方法将34例手足口病患儿随机分为治疗组和对照组,治疗组18例,对照组16例,2组均给予对症支持治疗。其中治疗组在治疗的同时加用酪酸梭菌活菌散,1周岁以下,0．5g／次,1周岁以上,1g／次,3次／d,温水送服。对照组不用酪酸梭菌活菌散治疗。对两组疱疹、食欲、体温变化情况及腹泻等并发症进行统计分析。结果治疗组起效时间、疱疹消失时间、食欲正常时间和体温恢复正常时间显著短于对照组,差异具有统计学意义（P〈0．05或0．01）。结论酪酸梭菌活菌散治疗手足口病疗效显著,具有积极的临床意义。     

Variations in lethal toxin and cholesterol-dependent cytolysin production correspond to differences in cytotoxicity among strains of Clostridium sordellii

Clostridium sordellii is an emerging human pathogen and frequent contaminant of cadaver-derived tissue transplant material. Herein, we provide data suggesting the potential for severe C. sordellii-associated disease may be dictated by whether the specific strain produces lethal toxin (TcsL) or sordellilysin (SDL), a cholesterol-dependent cytolysin. The virulence factor profiles of 14 C. sordellii isolates were determined, and culture supernatant from six of the isolates was found to be cytotoxic to mammalian cells; yet, only one of these strains conferred cytotoxicity via production of TcsL. Cytotoxicity of TcsL- strains correlated with the production of sordellilysin, which was also recognized by an antiperfringolysin O antibody. However, supernatant from TcsL+, SDL- strains demonstrated a lower LD50 relative to TcsL-, SDL+ strains, suggesting the potential for severe C. sordellii-associated disease may be determined by the particular strain colonizing the host.     

The use of clostridial spores for cancer treatment

Hypoxic/necrotic regions, absent in normal tissues, can be exploited to target tumours in cancer therapy using nonpathogenic strains of the bacterial genus Clostridium. Following administration of Clostridium spores to tumour-bearing organisms, these spores can only germinate within the hypoxic/necrotic regions of solid tumours, proving their exquisite selectivity. Low oxygen tension is a common feature of solid tumours, which may arise from the unique physiological environment, generated to a large extent by the abnormal tumour vasculature, and provides as such a niche for anaerobic bacteria. Some clostridia tested clearly showed innate oncolytic activity, but they could not completely eradicate the tumour. Recombinant clostridia producing prodrug-converting enzymes or cytokines resulted in the production of such proteins solely within the tumour, and where applicable, could convert the prodrug in a toxic compound. Moreover, in some cases, tumour eradication or tumour control could be observed. This review brings an overview of the relative successes and failures of the Clostridium-directed tumour therapy with both wild-type strains and strains producing proteins useful in antitumour therapy.     

Evaluation of an alternative method for the enumeration and confirmation of Clostridium perfringens from treated and untreated sewages

AIMS: Clostridium perfringens is recommended as a suitable indicator bacterium for human enteric viruses, Giardia cysts and Cryptosporidium oocysts in finished water and in the assessment and evaluation of water treatment. Several agars and confirmation procedures were evaluated in parallel with the Australian/New Zealand Standard (AS/NZ) Method for the enumeration of Cl. perfringens from treated and untreated sewage samples. METHODS AND RESULTS: The current AS/NZ method utilizes tryptose sulfite cycloserine agar (TSC), lactose gelatin medium (LG) and nitrate motility medium (NM) at an incubation temperature of 37 degrees C. Sixty treated and untreated sewage samples were used to evaluate TSC agar, membrane Cl. perfringens agar (mCP), Perfringens agar (OPSP) and Perfringens agar with 4-methylumbelliferyl phosphate (OPSP-MUP) for enumeration of Clostridium. An incubation temperature of 44 degrees C for 24 h was used for comparison. Confirmation procedures were also evaluated using 103 isolates and included LG and NM, ortho-nitrophenyl-beta-D-galactopyranoside (ONPG) with MUP (ONPG-MUP) and phosphatase reagent (PR). OPSP compared favourably with TSC agar. One false negative result was obtained from each of the LG/NM and ONPG-MUP procedures. No false results were obtained using the PR confirmation procedure. CONCLUSIONS: OPSP agar and PR were determined as suitable replacements for the AS/NZ Standard procedure with no interference from spreading organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a simple and rapid method for isolating and enumerating Cl. perfringens from sewage samples and confirmed results can be reported more quickly due to shorter analytical turnaround times.     

Evidence for a synergistic salt-protein interaction -- complex patterns of activation vs. inhibition of nitrogenase by salt

The molybdenum nitrogenase enzyme system, comprised of the MoFe protein and the Fe protein, catalyzes the reduction of atmospheric N(2) to NH(3). Interactions between these two proteins and between Fe protein and nucleotides (MgADP and MgATP) are crucial to catalysis. It is well established that salts are inhibitors of nitrogenase catalysis that target these interactions. However, the implications of salt effects are often overlooked. We have reexamined salt effects in light of a comprehensive framework for nitrogenase interactions to offer an in-depth analysis of the sources of salt inhibition and underlying apparent cooperativity. More importantly, we have identified patterns of salt activation of nitrogenase that correspond to at least two mechanisms. One of these mechanisms is that charge screening of MoFe protein-Fe protein interactions in the nitrogenase complex accelerates the rate of nitrogenase complex dissociation, which is the rate-limiting step of catalysis. This kind of salt activation operates under conditions of high catalytic activity and low salt concentrations that may resemble those found in vivo. While simple kinetic arguments are strong evidence for this kind of salt activation, further confirmation was sought by demonstrating that tight complexes that have previously displayed little or no activity due to the inability of Fe protein to dissociate from the complex are activated by the presence of salt. This occurs for the combination Azotobacter vinelandii MoFe protein with: (a) the L127Delta Fe protein; and (b) Clostridium pasteurianum Fe protein. The curvature of activation vs. salt implies a synergistic salt-protein interaction.     

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.     

Cellular stability of Rho-GTPases glucosylated by Clostridium difficile toxin B

Mono-glucosylation of Rho, Rac, and Cdc42 by Clostridium difficile toxin B (TcdB) induces changes of actin dynamics and apoptosis. When fibroblasts were treated with TcdB, an apparent decrease of the cellular Rac1 level was observed when applying anti-Rac1(Mab 102). This decrease was not based on degradation as inhibition of the proteasome by lactacystin did not stabilise cellular Rac1 levels. The application of anti-Rac1 (Mab 23A8) showed that the cellular Rac1 level slightly increased in TcdB-treated fibroblasts; thus, the apparent loss of cellular Rac1 was not due to degradation but due to impaired recognition of glucosylated Rac1 by anti-Rac1 (Mab 102). In contrast, recognition of RhoA by anti-RhoA (Mab 26C4) and Cdc42 by anti-Cdc42 (Mab 44) was not altered by glucosylation; a transient decrease of cellular RhoA and Cdc42 in TcdB-treated fibroblasts was indeed due to proteasomal degradation, as inhibition of the proteasome by lactacystin stabilised both cellular RhoA and Cdc42 levels. The finding that the apparent decrease of Rac1 reflects Rac1 glucosylation offers a valuable tool to determine Rac1 glucosylation.