解纤维梭菌脱水四环素诱导的ClosTron基因打靶系统构建

【背景】解纤维梭菌是发酵木质纤维素生产乙醇的中温模式菌株,构建可控诱导的基因打靶技术是研究解纤维梭菌遗传调控的重要手段。【目的】在解纤维梭菌中构建脱水四环素诱导的ClosTron基因打靶系统。【方法】首先,分析解纤维梭菌对脱水四环素的敏感性,筛选合适的诱导剂浓度,并以β-葡萄糖苷酶为报告基因,检测其在解纤维梭菌中的诱导效果。然后,将脱水四环素诱导型操纵子与II型内含子元件组合,构建脱水四环素诱导的ClosTron质粒。最后,以解纤维梭菌mspI、ldh和ack为例,检测其在解纤维梭菌中的打靶效率。【结果】脱水四环素诱导系统在解纤维梭菌中能够诱导ClosTron元件表达,在mspI、ldh和ack这3个基因位点的打靶效率分别为29.48%±15.51%、23.61%±7.08%和28.09%±6.97%。【结论】在解纤维梭菌中成功构建脱水四环素诱导的ClosTron基因打靶系统,为梭菌基因工程改造奠定了基础。     

异化铁还原细菌LQ25还原重金属Cr (VI)的特性研究

[背景] 异化铁还原细菌能够在还原Fe （III）的同时将毒性较大的Cr （VI）还原成毒性较小的Cr （III）,解决铬污染的问题。[目的] 基于丁酸梭菌（Clostridium butyricum） LQ25异化铁还原过程制备生物磁铁矿,开展异化铁还原细菌还原Cr （VI）的特性研究。[方法] 构建以氢氧化铁为电子受体和葡萄糖为电子供体的异化铁培养体系。菌株LQ25培养结束时制备生物磁铁矿。设置不同初始Cr （VI）浓度（5、10、15、25和30 mg/L）,分别测定菌株LQ25对Cr （VI）还原效率以及生物磁铁矿对Cr （VI）的还原效率。[结果] 菌株LQ25在设置的Cr （VI）浓度范围内都能良好生长。当Cr （VI）浓度为15 mg/L时,在异化铁培养条件下,菌株LQ25对Cr （VI）的还原率为63.45%±5.13%,生物磁铁矿对Cr （VI）的还原率为87.73%±9.12%,相比菌株还原Cr （VI）的效率提高38%。pH变化能影响生物磁铁矿对Cr （VI）的还原率,当pH 2.0时,生物磁铁矿对Cr （VI）的还原率最高,几乎达到100%。电子显微镜观察发现生物磁铁矿表面有许多孔隙,X-射线衍射图谱显示生物磁铁矿中Fe （II）的存在形式是Fe （OH）₂。[结论] 基于异化铁还原细菌制备生物磁铁矿可用于还原Cr （VI）,这是一种有效去除Cr （VI）的途径。     

The Synthesis and Biology of Fluorinated Prostacyclins

Abstract

Clostridium thermocellum produces a highly active cellulase system that consists of a high-M_r multienzyme complex termed cellulosome. Hydrolytic components of the cellulosome are organized around a large, noncatalytic glycoprotein termed CipA that acts both as a scaffolding component and a cellulose-binding factor. Catalytic subunits of the cellulosome bear conserved, noncatalytic subdomains, termed dockerin domains, which bind to receptor domains of CipA, termed cohesin domains. CipA includes nine cohesin domains, a cellulose-binding domain, and a specialized dockerin domain. Proteins of the cell envelope carrying cohesin domains that specifically bind the dockerin domain of CipA have been identified. These proteins may mediate anchoring of the cellulosomes to the cell surface. Cellulase complexes similar to the cellulosome of C. thermocellum are produced by several cellulolytic clostridia. High-Mr multienzyme complexes have also been identified in anaerobic rumen fungi. The architecture of the fungal complexes also seems to rely on the interaction of conserved, noncatalytic docking domains with a scaffolding component. However, the sequence of the fungal docking domains bears no resemblance to the clostridial dockerin domains, suggesting that the fungal and clostridial complexes arose independently.     

Computer-Intensive Statistical Procedures

The overall goal of this review is to summarize the current body of knowledge about the structure and function of major proteins of Bacillus anthracis and/or similar spore-forming organisms. B. anthracis is a key spore-forming biological threat agent, as well as human and animal Gram-positive bacterial pathogen. The structural information described here is limited to approximately the last 5 years. This information is then related to the role of the selected proteins in pathogenesis and in the possible development of novel vaccine and/or other antimicrobial agents against spore-forming organisms, including anthrax, a disease caused by B. anthracis.

Among spore-forming bacteria, Bacillus and Clostridium species are the predominant spore-forming bacilli that cause serious diseases. The biochemical properties and mechanism of catalysis of the novel spore germination protease that degrades small, acid-soluble proteins protecting DNA against damage, a cofactor independent phosphoglycerate mutase, NAD⁺ synthetase, and the three know B. anthracis toxins, protective antigen, lethal factor, and edema factor are described. The studies described in this work review and unify selected information critical for the prevention of microbial diseases such as anthrax. A strategy for the structure-guided development of new prophylactic and therapeutic agents is discussed.     

EFFECT OF FERMENTATION PARAMETERS ON BIO-ALCOHOLS PRODUCTION FROM GLYCEROL USING IMMOBILIZED Clostridium pasteurianum: AN OPTIMIZATION STUDY

This article addresses the issue of effect of fermentation parameters for conversion of glycerol (in both pure and crude form) into three value-added products, namely, ethanol, butanol, and 1,3-propanediol (1,3-PDO), by immobilized Clostridium pasteurianum and thereby addresses the statistical optimization of this process. The analysis of effect of different process parameters such as agitation rate, fermentation temperature, medium pH, and initial glycerol concentration indicated that medium pH was the most critical factor for total alcohols production in case of pure glycerol as fermentation substrate. On the other hand, initial glycerol concentration was the most significant factor for fermentation with crude glycerol. An interesting observation was that the optimized set of fermentation parameters was found to be independent of the type of glycerol (either pure or crude) used. At optimum conditions of agitation rate (200 rpm), initial glycerol concentration (25 g/L), fermentation temperature (30°C), and medium pH (7.0), the total alcohols production was almost equal in anaerobic shake flasks and 2-L bioreactor. This essentially means that at optimum process parameters, the scale of operation does not affect the output of the process. The immobilized cells could be reused for multiple cycles for both pure and crude glycerol fermentation.     

OPTIMIZATION OF AFFINITY DIGESTION FOR THE ISOLATION OF CELLULOSOMES FROM Clostridium thermocellum

The affinity digestion process for cellulase purification consisting of binding to amorphous cellulose, and amorphous cellulose hydrolysis in the presence of dialysis (Morag et al., 1991), was optimized to obtain high activity recoveries and consistent protein recoveries in the isolation of Clostridium thermocellum cellulase. Experiments were conducted using crude supernatant prepared from C. thermocellum grown on either Avicel or cellobiose. While no difference was observed between Avicel-grown or cellobiose-grown cellulase in the adsorption step, differences were observed during the hydrolysis step. The optimal amorphous cellulose loading was found to be 3 mg amorphous cellulose per milligram supernatant protein. At this loading, 90–100% of activity in the crude supernatant was adsorbed. Twenty-four-hour incubation with the amorphous cellulose during the adsorption stage was found to result in maximal and stable adsorption of activity to the substrate. By fitting the adsorption data to the Langmuir model, an adsorption constant of 410 L/g and a binding capacity of 0.249 g cellulase/g cellulose were obtained. The optimal length of time for hydrolysis was found to be 3 hr for cellulase purified from Avicel cultures and 4 hr for cellulase purified from cellobiose cultures. These loadings and incubation times allowed for more than 85% activity recovery.     

Host response to intravenous injection of epsilon toxin in mouse model: A proteomic view

Epsilon toxin (ETX) is an extremely potent pore‐forming toxin and a category B biological agent. ETX is a major virulence determinant of Clostridium perfringens toxinotypes B and D, and is implicated in pathogenesis of rapidly fatal economically important pulpy kidney disease in lambs caused by toxinotype D. Despite being a toxin, ETX can be utilized as a tool to target glutamatergic neurons and for drug delivery into the CNS. 2DE‐MS approach was employed to elucidate the host response to ETX following intravenous injection in mouse model. In total, 136 proteins were identified either differentially expressed in brain (18) and kidney (33); showing specific interaction with ETX from lysates of brain (4), kidney (21), or from plasma (42); and urine markers (18) of intoxication. Differentially expressed proteins in kidney included those involved in calcium homeostasis and cytoskeletal organization. Proteins involved in ER and oxidative stress and energy metabolism also showed differential levels in the target tissue after ETX treatment. The known functions of the proteins differentially expressed and those interacting with ETX indicate involvement of interlinked pathways. This study provides first proteomic account of host response to ETX exposure providing clues to mechanism of toxicity and potential therapeutic targets.     

Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies

Clostridium perfringens epsilon toxin (Etx) is a pore‐forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx‐H149A), previously reported to have reduced, but not abolished, toxicity. The three‐dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx‐H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx‐H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx‐H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx‐H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx‐H149A identified a glycan (β‐octyl‐glucoside) binding site in domain III of Etx‐H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity.     

Inhibitory Activity of (9R,10S,12Z)-9,10-Dihydroxy-8-oxo-octadecenoic Acid,Its C-9 Epimer and Their Derivatives toward the Growth of Tea Pollen Tubes

The inhibitory activity of (9R,10S,12Z)-9,10-dihydroxy-8-oxooctadecenoic acid and its diacetate, acetonide and methyl ester toward tea pollen tube growth were different, the inhibition by the diacetate being the strongest. Each compound of the fatty acid and its derivatives exhibited more inhibition than its C-9 epimer. The fatty acid and its C-9 epimer showed the same toxicity against HeLa cells.     

Enhanced butanol production by eukaryotic Saccharomyces cerevisiae engineered to contain an improved pathway

Compared with ethanol, butanol has more advantageous physical properties as a fuel, and biobutanol is thus considered a promising biofuel material. Biobutanol has often been produced by Clostridium species; however, because they are strictly anaerobic microorganisms, these species are challenging to work with. We attempted to introduce the butanol production pathway into yeast Saccharomyces cerevisiae, which is a well-known microorganism that is tolerant to organic solvents. 1-Butanol was found to be produced at very low levels when the butanol production pathway of Clostridium acetobutylicum was simply introduced into S. cerevisiae. The elimination of glycerol production pathway in the yeast contributed to the enhancement of 1-butanol production. In addition, by the use of trans-enoyl-CoA reductase in the engineered pathway, 1-butanol production was markedly enhanced to yield 14.1 mg/L after 48 h of cultivation.