肠道内产丁酸细菌及其产物丁酸生理功能的研究进展

产丁酸细菌是利用糖类发酵产生丁酸的一类细菌,代表种是丁酸梭菌。在动物及人类肠道内存在的产丁酸细菌主要是梭菌属、柔嫩梭菌属、罗斯式菌属、真菌属及丁酸弧菌属。本文一方面介绍部分肠道内产丁酸细菌的种类、特点及膳食纤维的摄入和肠道益生菌对产丁酸细菌的影响,另一方面对其主要代谢产物丁酸在体内的生理功能进行探讨,以期为产丁酸细菌的应用及产品开发提供理论依据。     

Tandem use of selenocysteine: adaptation of a selenoprotein glutaredoxin for reduction of selenoprotein methionine sulfoxide reductase

Several engineered selenocysteine (Sec)-containing glutaredoxins (Grxs) and their enzymatic properties have been reported, but natural selenoprotein Grxs have not been previously characterized. We expressed a bacterial selenoprotein Grx from Clostridium sp. (also known as Alkaliphilus oremlandii) OhILAs in Escherichia coli and characterized this selenoenzyme and its natural Cys homologues in Clostridium and E. coli. The selenoprotein Grx had a 200-fold higher activity than its Sec-to-Cys mutant form, suggesting that Sec is essential for catalysis by this thiol-disulfide oxidoreductase. Kinetic analysis also showed that the selenoprotein Grx had a 10-fold lower K(m) than Cys homologues. Interestingly, this selenoenzyme efficiently reduced a Clostridium selenoprotein methionine sulfoxide reductase A (MsrA), suggesting that it is the natural reductant for the protein that is not reducible by thioredoxin, a common reductant for Cys-containing MsrAs. We also found that the selenoprotein Grx could not efficiently reduce a Cys version of Clostridium MsrA, whereas natural Clostridium and E. coli Cys-containing Grxs, which efficiently reduce Cys-containing MsrAs, poorly acted on the selenoprotein MsrA. This specificity for MsrA reduction could explain why Sec is utilized in Clostridium Grx and more generally provides a novel example of the use of Sec in biological systems.     

Clostridium saccharogumia sp. nov. and Lactonifactor longoviformis gen. nov., sp. nov., two novel human faecal bacteria involved in the conversion of the dietary phytoestrogen secoisolariciresinol diglucoside

Two anaerobic bacteria involved in the conversion of the plant lignan secoisolariciresinol diglucoside were isolated from faeces of a healthy male adult. The first isolate, strain SDG-Mt85-3Db, was a mesophilic strictly anaerobic Gram-positive helically coiled rod. Based on 16S r RNA gene sequence analysis, its nearest relatives were Clostridium cocleatum (96.7% similarity) and Clostridium ramosum (96.6%). In contrast to these species, the isolate was devoid of alpha-galactosidase and -glucosidase and did not grow on maltose, melibiose, raffinose, rhamnose and trehalose. The hypothesis that strain SDG-Mt85-3Db represents a new bacterial species of the Clostridium cluster XVIII was confirmed by DNA-DNA hybridisation experiments. The G+C content of DNA of strain SDG-Mt85-3Db (30.7+/-0.8 mol%) was comparable with that of Clostridium butyricum, the type species of the genus Clostridium. The name Clostridium saccharogumia is proposed for strain SDG-Mt85-3Db (=DSM 17460T=CCUG 51486T). The second isolate, strain ED-Mt61/PYG-s6, was a mesophilic strictly anaerobic Gram-positive regular rod. Based on 16S rRNA gene sequence analysis, its nearest relatives were Clostridium amygdalinum (93.3%), Clostridium saccharolyticum (93.1%) and Ruminococcus productus (93.0%). The isolate differed from these species in its ability to dehydrogenate enterodiol. It also possessed alpha-arabinosidase and -galactosidase and had a higher G+C content of DNA (48.0 mol%). According to these findings, it is proposed to create a novel genus, Lactonifactor, and a novel species, Lactonifactor longoviformis, to accommodate strain ED-Mt61/PYG-s6. The type strain is DSM 17459T (=CCUG 51487T).     

丙酮丁醇梭菌的基因编辑工具及代谢工程改造

丙酮丁醇梭菌作为极具潜力的新型生物燃料丁醇的生产菌,受到各国研究学者的广泛关注。通过丙酮丁醇梭菌(ABE)发酵生产丁醇,由于生产成本高,限制了其工业化应用。随着基因组学和分子生物学的快速发展,适用于丙酮丁醇的基因编辑工具不断发展并应用于提高菌株的发酵性能。本文对丙酮丁醇梭菌基因编辑工具和代谢工程改造取得的进展进行综述。     

艰难梭菌的研究进展

艰难梭菌为革兰阳性厌氧芽胞杆菌,可引起艰难梭菌相关性腹泻,导致一系列肠道感染症状和相关临床表现。近年来由于高致病株的出现、菌株耐药性的增加,艰难梭菌感染在全球呈蔓延趋势。本文就艰难梭菌的耐药机制、检测技术、防治及国内感染现状等作一简要综述。     

Mechanisms of the cytopathic action of actin-ADP-ribosylating toxins

Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, and Clostridium spiroforme toxin ADP-ribosylate actin monomers. Toxin-induced ADP-ribosylation disturbs the cellular equilibrium between monomeric and polymeric actin and traps monomeric actin in its unpolymerized form, thereby depolymerizing actin filaments and destroying the microfilament network. Furthermore, the toxins ADP-ribosylate gelsolin actin complexes. These modifications may contribute to the cytopathic action of the toxins.     

Specificity of bile salt sulfatase activity from Clostridium sp. strains S1.

Clostridium sp. strain S1, an unnamed bile acid-desulfating strain from rat intestinal microflora (S.M. Huijghebaert, J. A. Mertens, and H. J. Eyssen, Appl. Environ. Microbiol. 43:185-192, 1982), was examined for its ability to desulfate different bile acid sulfates and steroid sulfates in growing cultures. Clostridium sp. strain S1 desulfated the 3 alpha-monosulfates of chenodeoxycholic, deoxycholic, and cholic acid, but not their 7 alpha- or 12 alpha-monosulfates. Among the 3-sulfates of the 5 alpha- and 5 beta-bile acids, only bile acid-3-sulfates with an equatorial sulfate group were desulfated. Hence, Clostridium sp. strain S1 desulfated the 3-sulfates of bile acids with a 3 alpha, 5 beta-, a 3 beta, 5 alpha- or a 3 beta, delta 5-structure. In contrast, the bile acid-3-sulfates with a 3 beta, 5 beta- or a 3 alpha, 5 alpha-structure were not desulfated. In addition, Clostridium sp. strain S1 did not hydrolyze the equatorial 3-sulfate esters of C19 and C21 steroids and cholesterol or the phenolic 3-sulfate esters of estrone and estradiol. 23-Nordeoxycholic acid with a C-23 carboxyl group was also not desulfated, in contrast to the 5 beta-bile acid 3 alpha-sulfates with a C-24 or C-26 carboxyl group. Therefore, the specificity of the sulfatase of Clostridium sp. strain S1 is related to the location of the sulfate group on the bile acid molecule, the equatorial orientation of the sulfate group, and the structure of the C-17 side chain, its carboxyl group, and chain length.     

粪菌移植治疗艰难梭菌感染的研究进展

艰难梭菌感染(Clostridium difficile infection,CDI)是院内抗生素相关性腹泻的最重要因素之一,其主要临床表现包括血便、腹泻、中毒性巨结肠、伪膜性肠炎等。近年来,CDI发病率、复发率、死亡率和治疗费用均明显增加,但其各种治疗方法均有局限性,尤其是抗生素治疗复发性艰难梭菌感染已面临许多棘手问题。目前证实肠道菌群失调和CDI感染关系密切,肠道菌群紊乱后导致艰难梭菌过度繁殖并释放毒素,可导致艰难梭菌感染。粪菌移植作为重建肠道菌群的重要方式,已成为复发性、难治性艰难梭菌感染最有效的治疗方式之一,不良反应极少。本文就国内外粪菌移植治疗艰难梭菌感染的研究进展作一综述。     

艰难梭菌荚膜致病作用的初步研究

本文在我室先前研究的基础上,以3株艰难梭菌的荚膜多糖提取物、全菌悬液以及全菌免疫血清分别在小鼠中进行半数致死量和保护试验等动物实验,以探讨艰难梭菌荚膜的致病作用。结果测得其中一株艰难梭菌的荚膜多糖对小鼠有致死毒性,其LD50为2.83mg,特异性抗血清保护试验证实艰难梭菌免疫血清对动物有保护作用。实验结果可能为进一步研究艰难梭菌荚膜的致病作用提供了参考资料。     

丙酮丁醇梭菌(Clostridium acetobutylicum)原生质体融合育种研究

为了提高丙酮丁醇梭菌(Clostridium acetobutylicum)的丁醇耐受能力和培养基总糖产丁醇的转化率,通过原生质体融合的方法,研究了溶菌酶浓度及其作用时间、再生培养基种类、55℃条件下菌体致死时间、不同PEG分子量以及作用时间、Ca^2+和Mg^2+不同的添加量对丙酮丁醇梭菌原生质体制备、融合、再生的影响,得到了一套比较系统的丙酮丁醇梭菌的原生质体融合条件,同时通过气相色谱检测了融合菌的产溶剂能力并计算总糖转化率。结果显示,最终得到的215I菌株的总糖转化率比原始菌株提高了34.7%,产丁醇能力比原始菌株提高了32.2%,并且发现1株融合菌能产生新物质。原生质体融合方法在丙酸丁醇梭菌育种方面有广泛的应用潜力,通过融合得到的菌株为丁醇生产奠定了基础。     

Clostridium barkeri sp. n

Clostridium barkeri sp. n. has been described, and its relationship to other clostridia is discussed.     

Biofilm-growing intestinal anaerobic bacteria

Sessile growth of anaerobic bacteria from the human intestinal tract has been poorly investigated, so far. We recently reported data on the close association existing between biliary stent clogging and polymicrobial biofilm development in its lumen. By exploiting the explanted stents as a rich source of anaerobic bacterial strains belonging to the genera Bacteroides, Clostridium, Fusobacterium, Finegoldia, Prevotella, and Veillonella, the present study focused on their ability to adhere, to grow in sessile mode and to form in vitro mono- or dual-species biofilms. Experiments on dual-species biofilm formation were planned on the basis of the anaerobic strains isolated from each clogged biliary stent, by selecting those in which a couple of anaerobic strains belonging to different species contributed to the polymicrobial biofilm development. Then, strains were investigated by field emission scanning electron microscopy and confocal laser scanning microscopy to reveal if they are able to grow as mono- and/or dual-species biofilms. As far as we know, this is the first report on the ability to adhere and form mono/dual-species biofilms exhibited by strains belonging to the species Bacteroides oralis, Clostridium difficile, Clostridium baratii, Clostridium fallax, Clostridium bifermentans, Finegoldia magna, and Fusobacterium necrophorum.     

兼性厌氧复合菌群H纤维素降解和产乙醇能力及生态组成初探

通过限制性培养条件和连续继代培养,筛选获得了一组具有高效稳定降解纤维素能力的复合菌群H。该菌群在传代30代以上仍能保持各项性状稳定,其工作pH为6～9,3 d可以完全降解置于100 mL PCS缓冲液培养基中的滤纸,发酵液中能够检出1.54 g/L乙醇。通过16S rDNA扩增和DGGE的方法,对菌群在不同阶段的微生物组成进行了研究,确定了琥珀酸嗜热梭菌Clostridium thermo succinogene、产气荚膜梭菌Clostridium straminisolvens和紫色板蓝根梭菌Clostridium isatidis等多种可直接实现纤维素到乙醇转化的菌株。菌群通过菌种之间的协同作用,共同维持了体系的稳定及降解能力的稳定。明确菌系的组成,对于进一步研究菌群降解机理、优化菌群和提高乙醇产率意义重大。     

Clostridium absonum from gas gangrene

A Clostridium perfringens-like strain was isolated from a case of gas gangrene. The morphological properties and the lecithinase reaction of the isolate were very similar to those of C. perfringens; however, the lecithinase reaction was only slightly suppressed by C. perfringens alpha-antitoxin serum and the organism was identified as Clostridium absonum from its biochemical properties.     

羊源丁酸梭菌HDRyYB1发酵工艺的优化

【目的】目前,国内外鲜有关于羊源丁酸梭菌的报道。本课题选用羊源丁酸梭菌HDRy YB1为研究对象,对其发酵工艺进行优化,为该菌株作为饲料添加剂应用于畜牧业生产奠定基础。【方法】采用Plackett-Burman(PB)试验设计法和响应面法分析并优化显著影响HDRy YB1菌株发酵液中芽胞数的培养基成分。【结果】发酵培养基中的面粉浓度、鱼粉浓度和米粉浓度显著影响发酵液中的芽胞数,优化后的发酵培养基组分(质量体积比)为:面粉3.72%、鱼粉0.90%、米粉3.96%、酵母粉0.60%、Na Cl 0.19%、Mg SO4·7H2O 0.19%、KH2PO4 0.01%、Na HCO3 0.01%、Ca CO3 0.48%;培养参数为:37°C,初始p H为7.2-7.4,瓶装量100/250,接种量3%。在此条件下,HDRy YB1菌株发酵完全(18 h)的芽胞数为1.478×108 CFU/m L,是优化前的2.7倍。【结论】HDRy YB1菌株发酵培养基得到了优化,优化后的培养基可用于后期的扩大发酵试验,验证其在实践生产中的应用价值。     

Different substrate recognition requirements for cleavage of synaptobrevin-2 by Clostridium baratii and Clostridium botulinum type F neurotoxins

Botulinum neurotoxins (BoNTs) cause botulism, which can be fatal if it is untreated. BoNTs cleave proteins necessary for nerve transmission, resulting in paralysis. The in vivo protein target has been reported for all seven serotypes of BoNT, i.e., serotypes A to G. Knowledge of the cleavage sites has led to the development of several assays to detect BoNT based on its ability to cleave a peptide substrate derived from its in vivo protein target. Most serotypes of BoNT can be subdivided into subtypes, and previously, we demonstrated that three of the currently known subtypes of BoNT/F cleave a peptide substrate, a shortened version of synaptobrevin-2, between Q58 and K59. However, our research indicated that Clostridium baratii type F toxin did not cleave this peptide. In this study, we detail experiments demonstrating that Clostridium baratii type F toxin cleaves recombinant synaptobrevin-2 in the same location as that cleaved by proteolytic F toxin. In addition, we demonstrate that Clostridium baratii type F toxin can cleave a peptide substrate based on the sequence of synaptobrevin-2. This peptide substrate is an N-terminal extension of the original peptide substrate used for detection of other BoNT/F toxins and can be used to detect four of the currently known BoNT/F subtypes by mass spectrometry.     

Clostridiolysin S,a Post-translationally Modified Biotoxin from Clostridium botulinum

Through elaboration of its botulinum toxins, Clostridium botulinum produces clinical syndromes of infant botulism, wound botulism, and other invasive infections. Using comparative genomic analysis, an orphan nine-gene cluster was identified in C. botulinum and the related foodborne pathogen Clostridium sporogenes that resembled the biosynthetic machinery for streptolysin S, a key virulence factor from group A Streptococcus responsible for its hallmark β-hemolytic phenotype. Genetic complementation, in vitro reconstitution, mass spectral analysis, and plasmid intergrational mutagenesis demonstrate that the streptolysin S-like gene cluster from Clostridium sp. is responsible for the biogenesis of a novel post-translationally modified hemolytic toxin, clostridiolysin S.     

Antibacterial Effect of Cysteine-Nitrosothiol and Possible Percursors Thereof

The postulated intermediate of nitrite-myoglobin reaction, cysteine-nitrosothiol, was prepared and its antibacterial effect was tested on Salmonella strains, Streptococcus faecium, and spores and vegetative cells of Clostridium sporogenes. Cysteine-nitrosothiol showed a higher inhibitory effect than nitrite. Preliminary results on the effect of simultaneous use of nitrite and cysteine on Clostridium sporogenes spores were also presented.     

Detection, cloning, and sequence analysis of an indigenous plasmid from cellulolytic clostridial strain MCF1

Nucleotide sequence analysis of a 2451-bp plasmid (pMCF1) from a cellulolytic Clostridium revealed that the protein specified by the largest open reading frame (ORF1) was homologous to RepB of Clostridium butyricum plasmid pCB101. The data suggest that pMCF1 belongs to the pC194 family of rolling-circle replicating plasmids and the ORF1 protein functions as its replication protein.