产S-雌马酚梭菌C1三代全基因组测序及功能基因筛选鉴定

【目的】挖掘产S-雌马酚梭菌C1转化大豆苷元产生S-雌马酚的功能基因,为梭菌C1的S-雌马酚转化机制研究提供参考,并为利用合成生物学方法生产S-雌马酚提供新基因资源。【方法】利用GridION测序平台,对梭菌C1进行第三代全基因组测序、基因组组装和功能注释等分析,从C1菌全基因组中筛选和鉴定参与S-雌马酚生物转化的功能基因。【结果】C1全基因组大小为3 035 113 bp,预测编码3 166个基因,包含53个tRNA、15个rRNA、4个ncRNA和1个基因岛。通过生物信息学分析,发现C1-07020基因编码蛋白与已报道的Lactococcus sp.20-92大豆苷元还原酶具有44.8%的氨基酸序列相似性和相同的3个功能保守结构域,体外蛋白功能验证表明,C1-07020具有大豆苷元还原酶功能。此外,C1菌中没有发现与已知产S-雌马酚菌相似的功能基因簇或大豆苷元还原酶以外的其他功能基因。【结论】在C1中鉴定到一个新的产S-雌马酚功能基因,并发现了C1可能具备特殊的产S-雌马酚机制,实验所获基础数据可为进一步挖掘产S-雌马酚新功能基因、了解S-雌马酚的生成机制及体外产S-雌马酚基因资源...     

微生物转化影响不同内源雌激素水平大鼠粪便雌马酚含量和菌群结构

雌马酚(Equol)是肠道中特定细菌转化大豆异黄酮的产物,与其前体大豆苷元(Daidzein)相比,雌马酚具有更强的生物学活性。【目的】研究口服雌马酚产生菌对大鼠转化大豆苷元能力的可能促进作用及内源雌激素对大鼠肠道菌群的可能影响。【方法】使用平均体重为211±9g的卵巢摘除和假手术雌性大鼠各30只,分别随机分为5组,并灌胃蒸馏水、雌二醇、大豆苷元、雌马酚和大豆苷元+雌马酚产生菌ZX7。【结果】从灌胃第2天开始,接受大豆苷元后大鼠粪样中始终具有较高水平的雌马酚,显著高于对照和雌二醇组(P<0.01);灌胃大豆苷元+雌马酚产生菌ZX7的大鼠和直接灌胃雌马酚的大鼠在粪样雌马酚含量上十分接近;DGGE图谱的PCA分析显示,卵巢摘除大鼠和假手术大鼠粪便菌群存在明显差异;大鼠粪便拟杆菌门细菌数量与粪样中雌马酚水平显著正相关。【结论】大鼠肠道固有菌群中可能存在能够将大豆苷元转化为雌马酚的细菌,利用外源菌株改变大鼠雌马酚产生能力具有一定的可行性,不同的内源雌激素水平可能影响大鼠肠道菌群结构,拟杆菌门细菌可能在大豆苷元的生物转化过程中起着十分重要的作用。     

鸡源奇异变形杆菌分离鉴定及生物学特性

【背景】奇异变形杆菌(Proteusmirabilis,PM)是人畜共患病原菌,在自然界分布广泛。近年来,随着畜禽奇异变形杆菌病发病率上升和耐药性增强,亟需开展对该菌的防控研究。【目的】分离鉴定鸡源奇异变形杆菌,鉴定其耐药性、致病性、生物被膜形成能力等生物学特性。【方法】采用聚合酶链式反应(Polymerase Chain Reaction,PCR)方法鉴定了从2019-2020年病鸡中分离的52株临床奇异变形杆菌。分别采用药敏试验、PCR、结晶紫染色法对临床菌株的耐药性、毒力基因及生物膜的形成进行研究。【结果】选择14株代表性菌株进行16SrRNA基因测序,结果表明所测分离菌株均为奇异变形杆菌。所有分离菌株均对克林霉素、阿奇霉素、红霉素、四环素和利福平耐药,对氯霉素和头孢曲松外的抗生素耐药性均高于50%。对分离的奇异变形杆菌的13个毒力基因检测表明,所有菌株均能检测到hpmA、hpmB、rpoA、mrpA、fliL、zapA、ureC、atfC、atfA、pmfA,而ucaA和rsbA检出率分别为19.23%(10/52)和48.08%(25/52),hlyA未检出。对分离株生物被膜形成能力检测结果表明,所有菌株均能形成生物被膜,19.23%(10/52)的分离菌株形成生物被膜能力强,而且25℃条件下成膜能力比37℃更强。【结论】鸡源奇异变形杆菌携带多种毒力基因,具有较强生物被膜形成能力,耐药模式多样且日趋严重,应进一步加强对奇异变形杆菌在动物致病性和耐药性方面的监控,以降低其感染风险。     

一株将甜菊苷转化为甜茶甙的细菌鉴定及转化特性

摘要：【目的】筛选一株对甜菊苷具有特异转化性能的细菌,并对该菌及转化产物进行鉴定,探讨转化酶及酶对甜菊苷的转化特性。【方法】通过16S rDNA序列分析,构建该菌系统进化树,结合菌体形态及菌落特征,确立该菌系统发育学地位。通过高效液相色谱（HPLC）及液质联用（LC－MS）法检测并鉴定转化产物。用菌液直接对甜菊苷进行转化以研究菌的转化能力。用静息细胞、胞外液和胞内液对甜菊糖分别转化法,确定转化酶与菌体的关系,并用该酶液进行转化特性研究。【结果】该菌株与黄杆菌属的16S rDNA序列相似性为99%,结合菌体     

兼性肠球菌Enterococcus hirae AUH-HM195对黄豆苷原的开环转化

摘要：【目的】从褐马鸡粪样中分离对大豆异黄酮黄豆苷原具有转化作用的功能微生物菌株。【方法】在厌氧工作站内对褐马鸡新鲜粪样进行梯度稀释后涂板,从板上挑取单菌落与底物黄豆苷原厌氧混合培养,用高效液相色谱检测底物被转化情况。【结果】分离出一株对黄豆苷原具开环转化作用的革兰氏阳性兼性好氧菌株AUH-HM195（EU919863）,经BLAST比对,该菌株的16S rDNA基因全序与肠球菌属菌株Enterococcus hirae (DSM20160) 的相似性为100%。根据保留时间、代谢产物最大紫外吸图谱以及核     

雌马酚产生细菌及其雌马酚合成代谢机制

大豆食品中通常富含染料木素和大豆苷元等异黄酮素,人和动物肠道中的某些细菌具有将异黄酮素代谢转化为S-雌马酚的能力。到目前为止,S-雌马酚被认为是一种具有潜在健康调节作用的化合物。啮齿类动物均具备产雌马酚的能力,但不同人群之间存在差异,产雌马酚细菌是否存在可能是造成这种差异的重要原因;不同产雌马酚细菌的代谢机制可能不同,并影响机体最终产雌马酚的能力。本文对已知的各种产雌马酚细菌及其细菌的雌马酚合成机制进行综述,以期为进一步了解雌马酚产生个体差异、雌马酚代谢转化效率、体外雌马酚的发酵生产,以及临床产雌马酚细菌的应用等提供理论参考。     

重组酿酒酵母催化二氢大豆苷元生产雌马酚

雌马酚是大豆异黄酮的代谢产物,是一种天然的选择性雌激素受体调节剂,稳定性和生物学活性高。为实现雌马酚的微生物合成,采用模块途径工程策略,构建编码雌马酚合成关键酶基因 orf-1、orf-2和orf-3 的表达载体,成功用于转化酿酒酵母BY4741,得到工程菌株。结果表明,工程菌株有效表达了外源基因,并可将大豆异黄酮代谢中间体二氢大豆苷元转化为雌马酚。为构建从头合成雌马酚的微生物细胞工厂提供了重要科学参考。     

人肠道产柚苷酶菌株分离、鉴定及其对柚皮苷的转化特性

【目的】从健康人肠道微生物菌群中分离能将柚皮苷高效转化为柚皮素的特定细菌菌株,对分离得到的菌株进行菌种鉴定并研究菌株对柚皮苷转化特性,目的是为柚皮素的高效生物合成提供新细菌资源。【方法】取健康人新鲜粪样,在厌氧工作站内培养24 h后进行梯度稀释并涂板,从板上挑取单菌落与底物柚皮苷进行厌氧培养,用高效液相色谱检测底物被转化情况。根据16S rDNA序列及相关生理生化特性分析,对所分离的柚皮苷转化菌进行菌种鉴定,并测定转化菌株对底物柚皮苷的转化动态和转化能力。【结果】从人肠道菌群中分离得到4株能将柚皮苷转化为柚皮素的细菌菌株,分别命名为AUH-JLD3、AUH-JLD7、AUHJLD104和AUH-JLD109。根据16S rDNA序列分析,结合形态学及相关生理生化特性等,将其分别鉴定为布劳特氏菌(Blautia sp.AUH-JLD3)、肠球菌(Enterococcus sp.AUH-JLD7)、拟杆菌(Bacteroides sp.AUHJLD104)和巴氏链球菌(Streptococcus pasteurianus subsp.AUH-JLD109)。转化动态研究结果表明,所分离的4株细菌菌株均能在12 h内将0.2 mmol/L底物柚皮苷转化为柚皮素;转化能力测定结果显示,菌株AUHJLD3、AUH-JLD7、AUH-JLD104及AUH-JLD109能够高效转化底物柚皮苷的最大浓度分别为0.2 mmol/L(平均转化率66.67%)、0.8 mmol/L(平均转化率86.49%)、0.2 mmol/L(平均转化率73.68%)以及1.6 mmol/L(平均转化率93.20%)。【结论】本研究首次从人肠道菌群中分离得到4株能将柚皮苷转化为柚皮素的细菌菌株,其中巴氏链球菌AUH-JLD109对底物柚皮苷转化能力最强。     

鹅源奇异变形杆菌的分离鉴定及毒力基因检测

【背景】奇异变形杆菌(Proteusmirabilis)是一种人兽共患机会致病菌,革兰氏阴性,兼性厌氧,呈多形性。【目的】探明内蒙古自治区通辽市某养殖场雏鹅发生死亡的病因,研究致病菌的分类地位和毒力基因携带情况。【方法】通过病原菌的筛查和感染试验确定发病原因,对其16S rRNA基因测序,建立病原菌系统进化树,结合BD PhoenixTM 100全自动微生物鉴定仪对病原菌进行分析鉴定,综合判定致病菌的种类及其分类地位;根据已报道的基因序列合成奇异变形杆菌的ure C、zapA、mrp A、ucaA、rsbA、pmfA、atfA、atfC8个毒力基因引物,通过PCR扩增研究病原菌毒力基因携带情况。【结果】致病茵的16Sr RNA基因与已报道的奇异变形杆菌(P.mirabilis)相似性在99%以上,通过构建系统进化树和BDPhoenixTM100全自动微生物鉴定仪鉴定,确定该菌株为P.mirabilis并编号为AYQ-1;8种毒力基因在该致病菌中均被检测到;雏鹅腹腔注射感染可导致雏鹅死亡,半致死浓度(LD_(50))为1.51×10~7 CFU/mL;药敏试验结果显示,该菌对链霉素、氨曲南和氧氟沙星等19种药物敏感,对万古霉素、青霉素和四环素等15种药物耐药。【结论】AYQ-1经鉴定为鹅奇异变形杆菌,毒力基因型为ureC(+)、zapA(+)、mrpA(+)、ucaA(+)、rsbA(+)、pmfA(+)、atfA(+)、atfC(+)型。     

一株养殖水体中亚硝酸盐去除菌的鉴定及其去除条件

【目的】从养殖污泥中分离筛选优良亚硝酸盐去除菌,并对其去除条件进行研究。【方法】从养殖污泥中分离亚硝酸盐去除菌,进一步通过测定比较分离菌株对亚硝酸盐的去除率,筛选优良的亚硝酸盐去除菌,通过API ID32GN细菌鉴定系统以及16S rDNA序列分析法对其进行鉴定,并采用单因子法研究其去除亚硝酸盐的条件。【结果】从养殖污泥中分离筛选了一株优良的亚硝酸盐去除菌AQ-3,其对50 mg/L亚硝酸盐的去除率高达99.47%。菌株AQ-3被鉴定为鲍曼氏不动杆菌(Acinetobacter baumannii)(GenBank登录号:JF751054.1),其16S rDNA序列与基因库中不动杆菌属菌株的16S rDNA序列有99%?100%的同源性,而且与鲍曼氏不动杆菌KF714株(GenBank登录号:AB109775)的亲缘关系最近。菌株AQ-3去除亚硝酸盐的最适初始pH范围为7?9,最佳碳源为乙酸钠和丁二酸钠,而且随着初始菌浓度的不断增大,菌株AQ-3对亚硝酸盐的去除率显著升高;随着亚硝酸盐浓度的不断增大,菌株AQ-3对亚硝酸盐的去除率逐渐降低。【结论】在丰富亚硝酸盐去除菌种质资源的同时,为该菌在养殖水体中的实际应用提供了理论基础。     

Enantioselective synthesis of S-equol from dihydrodaidzein by a newly isolated anaerobic human intestinal bacterium

A newly isolated rod-shaped, gram-negative anaerobic bacterium from human feces, named Julong 732, was found to be capable of metabolizing the isoflavone dihydrodaidzein to S-equol under anaerobic conditions. The metabolite, equol, was identified by using electron impact ionization mass spectrometry, (1)H and (13)C nuclear magnetic resonance spectroscopy, and UV spectral analyses. However, strain Julong 732 was not able to produce equol from daidzein, and tetrahydrodaidzein and dehydroequol, which are most likely intermediates in the anaerobic metabolism of dihydrodaidzein, were not detected in bacterial culture medium containing dihydrodaidzein. Chiral stationary-phase high-performance liquid chromatography eluted only one metabolite, S-equol, which was produced from a bacterial culture containing a racemic mixture of dihydrodaidzein. Strain Julong 732 did not show racemase activity to transform R-equol to S-equol and vice versa. Its full 16S rRNA gene sequence (1,429 bp) had 92.8% similarity to that of Eggerthella hongkongenis HKU10. This is the first report of a single bacterium capable of converting a racemic mixture of dihydrodaidzein to enantiomeric pure S-equol.     

兔肠道大豆异黄酮还原菌株的分离鉴定及其转化特性

【目的】从兔新鲜粪样中分离对大豆异黄酮黄豆苷原和染料木素具有转化作用的特定细菌菌株。【方法】在厌氧工作站内对獭兔新鲜粪样进行梯度稀释后涂板,挑取单菌落与底物黄豆苷原和染料木素分别厌氧混合培养,用高效液相色谱检测底物被转化情况。【结果】分离得到一株对大豆异黄酮黄豆苷原和染料木素均具有转化作用的革兰氏阳性严格厌氧细菌菌株AUH-JLR41(KJ188150)。根据产物的高效液相保留时间、紫外吸收图谱和质谱分析结果,将菌株AUH-JLR41代谢底物黄豆苷原和染料木素生成的产物分别鉴定为二氢黄豆苷原和二氢染料木素。经手性高效液相系统检测,产物二氢黄豆苷原和二氢染料木素均呈现两个等面积物质峰,表明这两个产物的对映体过量率均为0。通过转化动态研究发现,菌株AUH-JLR41分别在底物黄豆苷原和染料木素加入48 h和72 h后将底物全部转化为产物,该菌株能转化底物黄豆苷原和染料木素的最大浓度均为0.6 mmol/L。经BLAST比对,菌株AUH-JLR41的16S r RNA基因序列与斯奈克氏菌属菌株Slackia equolifaciens DZE(EU377663)的相似性高达99.6%。【结论】兔肠道分离的斯奈克氏菌属菌株Slackia sp.AUH-JLR41在厌氧条件下能将大豆异黄酮黄豆苷原和染料木素分别还原为二氢黄豆苷原和二氢染料木素。     

Isolation of an anaerobic intestinal bacterium capable of cleaving the C-ring of the isoflavonoid daidzein

Colonic bacteria were screened for bacteria involved in the conversion of phytoestrogens. A gram-positive anaerobic bacterium, strain HGH 136, capable of conversion of the isoflavonoid daidzein, was isolated and identified as a Clostridium sp. The bacterium cleaved the C-ring of daidzein to produce O-demethylangolensin ( O-Dma). This compound was identified by comparison of the HPLC retention time and UV spectrum of the metabolite with chemically synthesized O-Dma. The identity of the metabolite was confirmed by liquid chromatography-mass spectrometry and NMR using synthetic O-Dma as a standard. The bacterium incubated with synthetic dihydrodaidzein also produced O-Dma. After 3 days of incubation, 28% of added daidzein and 12% of added dihydrodaidzein were converted to O-Dma. This is the first study in which an anaerobic bacterium involved in the ring cleavage of daidzein to produce O-Dma has been identified.     

Enhanced biosynthesis of dihydrodaidzein and dihydrogenistein by a newly isolated bovine rumen anaerobic bacterium

A rod-shaped and Gram-positive anaerobic bacterium, named Niu-O16, which was isolated from bovine rumen contents, was found to be capable of anaerobically converting isoflavones daidzein and genistein to dihydrodaidzein (DHD) and dihydrogenistein (DHG), respectively. The metabolites DHD and DHG were identified using EI-MS and NMR spectrometric analyses. Stereoisomeric metabolites, which were separated on chiral stationary phase HPLC, were formed in equal amounts by the strain Niu-O16. Tautomerization reaction occurred on the B-ring of DHD and DHG seems to be attributed to the equal production of stereoisomeric metabolites. For the synthesis of DHD, the strain Niu-O16 showed an optimal pH range from 6.0 to 7.0 and completely reduced up to 800 microM of daidzein to DHD with the initial OD600nm=1.0 and pH 7.0 for 3 days incubation. The strain Niu-O16, showed relatively faster reduction activity toward daidzein to produce DHD than the previously isolated human intestinal bacterium Clostridium sp. HGH6.     

Screening and identification of antimicrobial compounds from Streptomyces bottropensis suppressing rice bacterial blight

Xanthomonas oryzae pv. oryzae (Xoo) is the most devastating pathogen to Oryza sativa and has been shown to cause bacterial blight. Two bioactive compounds showing antimicrobial activities against Xoo strain KACC 10331 were isolated from a Streptomyces bottropensis strain. The ethyl acetate extract was fractionated on a Sephadex LH-20 column, and then purified by preparative HPLC. The purified compounds were identified as bottromycin A2 and dunaimycin D3S by HR/MS and 1H NMR analyses. The MIC value against Xoo and the lowest concentration still capable of suppressing rice bacterial blight were 2 microgram/ml and 16 microgram/ml for bottromycin A2, and 64 microgram/ml and 0.06 microgram/ml for dunaimycin D3S, respectively. These two compounds were shown to exert different bioactivities in vitro and in rice leaf explants.     

Characterization of the fungicidal activity of Calothrix elenkinii using chemical methods and microscopy

An investigation was directed towards biochemical characterization of cyanobacterium Calothrix elenkinii and analysis of the chemical nature and mode of action of its fungicidal metabolite(s) against oomycete Pythium debaryanum. Biochemical characterization of the culture in terms of carbohydrate utilization revealed the facultative nature of C. elenkinii. Unique antibiotic markers were also found for this strain. 16S rDNA sequencing of the strain revealed 98% similarity with Calothrix sp. PCC7101. The fungicidal activity was tested by disc diffusion assay of different fractions of the culture filtrate. A minimum inhibitory concentration of 10 microl was recorded for ethyl acetate fraction of the 7-weeks old culture filtrates. HPLC, followed by NMR spectral analysis demonstrated the presence of a substituted benzoic acid in the ethyl acetate fraction. Microscopic examination revealed distinct granulation, followed by disintegration of the hyphae of Pythium sp., indicating the presence of an active metabolite in the culture filtrates of Calothrix sp. The fungicidal activity of C. elenkinii can be attributed to the presence of 3-acetyl-2-hydroxy-6-methoxy-4-methyl benzoic acid. This is the first report of a benzoic acid derivative having fungicidal activity in cyanobacteria.     

Enantioselective Synthesis of S-Equol from Dihydrodaidzein by a Newly Isolated Anaerobic Human Intestinal Bacterium

A newly isolated rod-shaped, gram-negative anaerobic bacterium from human feces, named Julong 732, was found to be capable of metabolizing the isoflavone dihydrodaidzein to S-equol under anaerobic conditions. The metabolite, equol, was identified by using electron impact ionization mass spectrometry, ¹H and ¹³C nuclear magnetic resonance spectroscopy, and UV spectral analyses. However, strain Julong 732 was not able to produce equol from daidzein, and tetrahydrodaidzein and dehydroequol, which are most likely intermediates in the anaerobic metabolism of dihydrodaidzein, were not detected in bacterial culture medium containing dihydrodaidzein. Chiral stationary-phase high-performance liquid chromatography eluted only one metabolite, S-equol, which was produced from a bacterial culture containing a racemic mixture of dihydrodaidzein. Strain Julong 732 did not show racemase activity to transform R-equol to S-equol and vice versa. Its full 16S rRNA gene sequence (1,429 bp) had 92.8% similarity to that of Eggerthella hongkongenis HKU10. This is the first report of a single bacterium capable of converting a racemic mixture of dihydrodaidzein to enantiomeric pure S-equol.     

Complete genomic sequence of the equol-producing bacterium Eggerthella sp. strain YY7918, isolated from adult human intestine

Eggerthella sp. strain YY7918 was isolated from the intestinal flora of a healthy human. It metabolizes daidzein (a soybean isoflavonoid) and produces S-equol, which has stronger estrogenic activities than daidzein. Here, we report the finished and annotated genomic sequence of this organism.     

Identification and biocontrol efficacy of Streptomyces miharaensis producing filipin III against Fusarium wilt

A number of bacterial strains were isolated from the internal tissue of Trapa japonica. Of these, strain KPE62302H, which had a 16S rDNA sequence identical to that of Streptomyces miharaensis showed antifungal activity against several plant pathogens. Treatment of seeds with strain KPE62302H induced a significant reduction in the incidence of Fusarium wilt in tomato plants compared with untreated controls. An antifungal substance (FP-1) was purified from the culture extract of strain KPE62302H using C18 flash and Sephadex LH-20 column chromatography and reverse phase HPLC. Extensive spectrometric analysis using MS and NMR identified this as filipin III. FP-1 inhibited the mycelial growth of plant pathogenic fungi such as Alternaria mali, Aspergillus niger, Colletotrichum gloeosporioides, C. orbiculare, Cylindrocarpon destructans, Diaporthe citiri, Fusarium oxysporum at 1-10 μg ml(-1) and also markedly inhibited the development of Fusarium wilt caused by F. oxysporum f.sp. lycopersici in tomato plants by treatment with 10 μg ml(-1) under greenhouse conditions. The efficacy of FP-1 against Fusarium wilt was comparable to that of the synthetic fungicide benomyl. An egfp -tagged strain of KPE62302H confirmed its ability to colonize tomato plants.