层出镰刀菌乌头酸酶家族的功能研究

【目的】层出镰刀菌是引起苜蓿根腐病的主要病原菌之一,探究层出镰刀菌中乌头酸酶家族蛋白的功能特性,为深入认识层出镰刀菌基础生理代谢的分子机制提供依据。【方法】利用hmmsearch工具,对真菌中含有乌头酸酶结构域的蛋白进行检索,并进行系统进化分析;通过实时荧光定量PCR及SWISS MODEL建模技术分别分析FpACO基因的表达模式与蛋白结构;利用同源重组双交换方法构建层出镰刀菌乌头酸酶基因敲除突变体;分析ΔFpACO3、ΔFpACO4-1、ΔFpACO4-2敲除突变体的生长、产孢、孢子形态、环境胁迫响应及致病力等表型变化;进一步测定敲除突变体中线粒体代谢相关生理生化指标的变化情况。【结果】FpACO4-1与FpACO4-2在产孢及孢子形态发生中发挥作用;FpACO3、FpACO4-1、FpACO4-2参与调控层出镰刀菌对细胞壁胁迫及金属离子胁迫的敏感性;FpACO3、FpACO4-1、FpACO4-2影响线粒体代谢,包括总乌头酸酶活性、ATP含量、过氧化氢含量及三羧酸循环关键基因表达等。【结论】乌头酸酶家族参与调控层出镰刀菌产孢、孢子形态分化、细胞壁胁迫及金属离子胁迫响应和线粒体代谢等过程。     

双组分系统EnvZ/OmpR促进副溶血弧菌抵抗碱胁迫的作用机制

【目的】探究双组分系统(two-component system,TCS)EnvZ/OmpR对副溶血弧菌(Vibrio parahaemolyticus,VP)抵抗碱胁迫的作用机制。【方法】用SMART在线工具(https://smart.embl.de/)鉴定出副溶血弧菌基因组中的双组分系统EnvZ/OmpR,再利用同源重组技术将envZ和ompR基因分别进行缺失,构建相应回补株,比较各菌株的生长曲线来检测相应基因对细菌适应高渗透胁迫和碱胁迫的作用,并结合qRT-PCR及荧光检测系统,筛选参与EnvZ/OmpR抵抗碱胁迫的下游靶基因,鉴定该双组分系统对下游基因的调控机制。【结果】在副溶血弧菌基因组中鉴定出vp0155/vp0154编码EnvZ/OmpR双组分系统同源蛋白。△ompR菌株在高渗透胁迫和碱胁迫中的生长能力明显弱于野生株,而回补株C△ompR、△envZ和C△envZ菌株生长能力与野生株类似。在△ompR菌株中,孔道蛋白基因vp1218、vp0493、vpa1745、vpa0085与vpa1308的转录水平均明显低于野生株,并且发现这些孔道蛋白基因缺失株(△vpa1308除外)在碱性环境中生长能力均明显弱于野生株。OmpR蛋白可直接抑制调控因子AphB基因转录,而△aphB菌株在碱胁迫中的生长能力明显强于野生株。此外,AphB蛋白可直接抑制孔道蛋白基因vp0493和vpa0085转录。【结论】双组分系统EnvZ/OmpR促进副溶血弧菌抵抗碱胁迫,其中OmpR蛋白可通过抑制调控因子AphB的表达,以促进部分孔道蛋白的表达,从而增强副溶血弧菌抵抗碱胁迫的能力。     

海产品嗜冷菌波罗的海希瓦氏菌中3个冷激蛋白功能分析

【目的】波罗的海希瓦氏菌是冷藏海产品中常见的腐败菌,而该菌中关于冷激蛋白的功能研究尚未见报道。本研究从分子生物学角度分析波罗的海希瓦氏菌中3个冷激蛋白各自的功能。【方法】采用BEAST软件分析γ-变形菌纲中部分食源性微生物的冷激蛋白进化时间,接着利用实时荧光定量PCR方法检测波罗的海希瓦氏菌3个冷激蛋白基因的表达规律,进而构建3个冷激蛋白的基因敲除株,分析敲除株在不同温度和不同环境胁迫条件下的生长状况、群体感应现象以及致腐能力,最后构建3个冷激蛋白的异源表达菌株并分析它们在不同温度和不同环境胁迫条件下的生长状况。【结果】波罗的海希瓦氏菌中鉴定到3个冷激蛋白,分别为cspC、cspD、cspG。所有γ-变形菌纲的cspD基因单独聚成一支,并于1 109.6百万年前与其他csp基因相分离,波罗的海希瓦氏菌的cspC和cspG在858.8百万年前互相分开。cspG基因是波罗的海希瓦氏菌低温生存的必需基因,且广泛响应环境胁迫条件;cspC基因对cspG基因功能的实施起辅助作用;cspD不响应冷激,但却会随生长阶段的变化而发生变化。此外,cspC基因和cspG基因在低温条件下与细菌的致腐能力相关。【结论】波罗的海希瓦氏菌3个冷激蛋白基因各有不同,且cspC基因和cspG基因与该菌致腐能力有关,这为今后研究腐败菌的冷适应和致腐机制提供了新思路。     

单增李斯特菌LPXTG蛋白Lmo0880在感染致病中的作用

【目的】通过构建单核细胞增生李斯特菌(单增李斯特菌) LPXTG蛋白Lmo0880的基因缺失菌株和回补菌株,探究Lmo0880在细菌生长、细胞感染和宿主感染等方面发挥的作用。【方法】利用同源重组原理构建lmo0880的基因缺失株及回补株,比较野生株、缺失株和回补株在生长能力、细胞黏附与侵袭和胞内增殖能力等方面的差异,从而鉴定Lmo0880在单增李斯特菌感染宿主中的作用。【结果】缺失lmo0880基因后,单增李斯特菌在生长能力上无明显变化;对细胞的黏附能力无显著差异,但对细胞侵袭能力、胞内增殖能力、小鼠致病力和小鼠组织定殖能力显著降低。【结论】本研究阐明了单增李斯特菌LPXTG蛋白Lmo0880在细胞侵袭、胞内增殖和组织定殖等方面发挥的重要作用。     

低温下阻遏蛋白MogR对单增李斯特菌鞭毛形成的影响

【目的】单核细胞增生李斯特菌(简称单增李斯特菌) (Listeria monocytogenes, Lm)是食源性病原菌,可以引发李斯特菌病(listeriosis)。Lm能在低温下生长,对冷藏食品的安全构成严重威胁,并对人类健康造成潜在的危害。Lm能低温生长与抑制鞭毛基因表达以减少鞭毛的合成有关。MogR是Lm鞭毛基因转录的阻遏蛋白,在机体里或37 ℃环境下具有阻遏作用,Lm不产生鞭毛;然而,当Lm处于20-0 ℃时MogR无阻遏作用,Lm产生鞭毛。我们研究发现在4 ℃生长条件下Lm鞭毛合成减少,但具体的分子机制尚未明确。本文探究在4 ℃下Lm鞭毛合成少与MogR起阻遏作用的关系。【方法】以Lm ATCC 19115为亲本株,分别构建了MogR和鞭毛丝蛋白FlaA的缺失株ΔmogR和ΔflaA (作为无鞭毛对照株)及其回补株cΔmogR和cΔflaA,测定了菌株在4、28、37 ℃时的运动性、鞭毛产生情况和鞭毛基因表达量,并对所构建的菌株进行了4、28、37 ℃时生长曲线的测定。【结果】在4 ℃时,mogR缺失后,菌株运动性显著强于亲本株(P<0.01),鞭毛合成量显著多于亲本株(P<0.001),鞭毛基因转录水平显著高于亲本株(P<0.001);缺失株ΔmogR的生长能力显著弱于亲本株(P<0.05)。回补株cΔmogR的运动能力、鞭毛合成量和鞭毛基因转录水平与亲本株相比,无显著性差异。【结论】在4 ℃时,Lm鞭毛产量少与MogR对鞭毛基因起转录抑制作用有关,低温条件下Lm能生长繁殖与MogR对鞭毛基因表达的抑制作用有关。本研究结果为揭示Lm低温生长机制提供了新的信息。     

wbnH2基因对北京欧文氏菌生物学特性及致病力的影响

【背景】北京欧文氏菌(Erwinia beijingensis)引起的刺芹侧耳细菌性软腐病(bacterial soft rot)给企业带来了严重的经济损失。wbnH2糖基转移酶基因在北京欧文氏菌中的生物学功能尚不明确。【目的】构建wbnH2基因的缺失株Δ-wbnH2和回补株C-wbnH2,探究wbnH2基因对北京欧文氏菌致病性的影响。【方法】采用同源重组方法构建北京欧文氏菌LMG 27579^TwbnH2基因缺失突变株Δ-wbnH2,并对基因缺失菌株的致病性、生长速度、运动能力、生物膜形成能力、黏附能力等生物学特性与野生型菌株进行比较分析;采用广宿主质粒pBBR1MCS2构建回补株C-wbnH2,排除了极性效应引起的突变株表型变化。【结果】与野生型菌株相比,基因缺失株Δ-wbnH2的生长速度无明显差别。但是wbnH2基因的缺失导致多糖分泌、生物膜形成能力、黏附能力、致病能力明显下降。【结论】wbnH2基因影响北京欧文氏菌多糖分泌、生物膜的形成能力、黏附能力及致病力,表明该基因在北京欧文氏菌致病过程中起重要作用,本研究为软腐病的防控提供了理论基础。     

拟态弧菌金属蛋白酶基因PrtV缺失株的构建及生物学特性分析

【背景】研究发现PrtV基因编码含多囊肾病(polycystic kidney disease)结构域的金属蛋白酶,其在多种细菌的致病过程中具有重要作用。拟态弧菌是一种感染多种水生动物的重要病原菌,PrtV基因在拟态弧菌致病中的作用尚不清楚。【目的】探究PrtV基因对拟态弧菌致病相关生物学特性的影响。【方法】采用自然转化的方法构建拟态弧菌PrtV基因缺失株(ΔPrtV),同时通过基因与质粒重组后电转化导入缺失株构建回补株(ΔPrtV/pPrtV),对突变株的生长特性、生化特征、生物被膜形成、自聚集能力、胞外产物卵磷脂酶和蛋白酶活性,以及致病性和细胞毒性等进行分析。【结果】与野生株相比,缺失株的生长特性、生物被膜形成、自聚集能力和卵磷脂酶活性无变化,但分解尿素、甘氨酸、香豆酸盐、鸟氨酸和赖氨酸的理化特性改变;胞外产物蛋白酶活性显著降低(P<0.05),细胞毒性显著下降(P<0.05),对杂交鲇的致病力下降10倍。【结论】PrtV基因与拟态弧菌的细胞毒性及致病性等多种生物学特性有关。该结果为进一步解析拟态弧菌PrtV基因功能及其致病机制提供了依据。     

基因CfATG6和CfATG14参与调控果生刺盘孢细胞自噬和致病力

【目的】炭疽病是油茶的主要病害,由刺盘孢属的多种真菌引起,其中果生刺盘孢分布范围最广、分离率最高,是油茶炭疽病的主要致病菌。研究自噬相关蛋白CfAtg6和CfAtg14的生物学功能,为进一步揭示果生刺盘孢通过细胞自噬调控致病的分子机制,并为油茶炭疽病的防治提供理论基础。【方法】根据同源重组原理,通过聚乙二醇(polyethylene glycol, PEG)介导的方法,在果生刺盘孢中敲除基因CfATG6和CfATG14,并进一步获得回补菌株ΔCfatg6-C和ΔCfatg14-C。【结果】酵母双杂交试验结果显示,果生刺盘孢蛋白CfAtg6和CfAtg14可能存在互作关系。生物学表型测定结果表明,相较于野生型和回补菌株,突变体ΔCfatg6和ΔCfatg14均表现出营养生长速率显著减慢,附着胞形成率分别只有野生型的5%和18%;突变体ΔCfatg6和ΔCfatg14致病力均极显著减弱,造成的油茶叶片病斑面积少于野生型和回补菌株的1/3;CfATG6和CfATG14基因缺失突变体均丧失转运和降解CfAtg8蛋白的能力,并对细胞壁胁迫更敏感。突变体ΔCfatg6的分生孢子产量显著降低,仅为野生型的20%左右;氧化胁迫试验结果表明,相较于野生型和回补菌株,过氧化氢对突变体的生长抑制率升高10%左右。内质网压力胁迫试验表明,ΔCfatg14对二硫苏糖醇抑制率升高5%以上。【结论】自噬相关基因CfATG6和CfATG14参与调控了果生刺盘孢生长发育、细胞自噬和致病力。     

猪链球菌2型硫氧还蛋白还原酶介导细菌应激与致病作用

【背景】硫氧还蛋白还原酶（thioredoxin reductase,TRR）是硫氧还蛋白系统关键组成部分,对病原菌应对体内外氧化应激、调节细菌稳态和介导致病过程具有重要作用。【目的】探究硫氧还蛋白还原酶TRR在人畜共患猪链球菌2型感染过程中参与的生物学效应。【方法】同源重组法构建猪链球菌2型硫氧还蛋白还原酶trr基因缺失株（Δtrr）及回补株（cΔtrr）,通过细菌染色、点板计数、体外细胞和动物感染模型等试验比较分析trr基因对细菌形态、抗应激反应及致病过程的影响。【结果】缺失trr对猪链球菌2型形态与生长特性的影响不大,但可增强细菌抗热应激、氧化应激和酸应激能力,缺失株对上皮细胞黏附力下降,侵袭进入脑血管内皮细胞作用显著降低,易于被吞噬细胞吞噬清除,对小鼠模型致病效应显著减弱。【结论】猪链球菌2型TRR因子参与细菌应激反应,介导细菌黏附、侵袭等致病过程,是猪链球菌2型新的潜在毒力因子。     

铜绿假单胞菌fur基因缺失突变株的构建及其表型分析

铁摄取调节蛋白(ferric uptake regulator, Fur)是控制铜绿假单胞菌铁代谢和毒力的关键调节因子。许多课题组尝试构建铜绿假单胞菌fur的缺失突变株均失败,因此铜绿假单胞菌的fur一直被认为是必需基因,这导致其生物学功能一直未得到全面的解析。【目的】构建铜绿假单胞菌fur的缺失突变株,并对该突变株的表型进行分析。【方法】以铜绿假单胞菌PAO1为亲本菌株,通过同源重组的方法构建fur缺失突变株,研究该基因对铜绿假单胞菌生长、铁载体生物合成、抗氧胁迫能力、鞭毛形成、生物被膜形成和毒力等的影响。同时,通过遗传分析对fur缺失突变株生长缺陷表型的原因进行探究。【结果】本研究成功构建了铜绿假单胞菌fur基因的缺失突变株,发现缺失突变fur极大地限制了铜绿假单胞菌的生长能力,并降低了该菌对限铁环境的生长适应性,但不影响该菌对高铁环境的生长适应性。铜绿假单胞菌Δfur的这种生长缺陷表型是细胞生长增殖变慢造成的,而不是诱导细胞死亡引起的。然而,其他异源的fur基因能完全互补Δfur的这种生长缺陷表型,暗示铜绿假单胞菌的Fur蛋白在功能上不存在独特性。尽管Fur与毒素-抗毒素系统PacTA存在功能关联性,但是铜绿假单胞菌Δfur的这种生长缺陷表型却与PacT毒素无关。除了影响铜绿假单胞菌的生长表型,缺失突变fur还使铜绿假单胞菌丧失了对铁载体生物合成的抑制作用,导致该菌对H₂O₂更敏感并丧失了鞭毛的形成能力,同时降低了该菌对大蜡螟幼虫的毒力。此外,缺失突变fur还显著提升了铜绿假单胞菌的胞内环二鸟苷酸(cyclic diguanylate, c-di-GMP)水平,从而诱导pelF和pslA基因的表达,进而促进铜绿假单胞菌生物被膜的形成。【结论】fur是可以缺失的非必需基因,在铜绿假单胞菌的正常生长、铁载体生物合成、抗氧胁迫能力、鞭毛形成、生物被膜形成和毒力等方面都发挥着十分重要的作用,这为针对铜绿假单胞菌的疫苗和抗菌药物开发奠定了基础。     

Removal of wastewater pathogen indicators in a constructed wetland in Leon, Spain

This paper studies the seasonal presence and removal of the pathogenous micro-organisms Escherichia coli, total coliforms (TC), Clostridium perfringens (Cp), faecal streptococci (FS), Giardia cysts, Cryptosporidium oocysts and helminth eggs, in a constructed wetland treatment system. The removal efficiency of this system with respect to the indicator micro-organisms achieved maximum values in spring and autumn at 99.9% for E. coli and TC, respectively, in winter at 97.0% for FS, in summer at 100% for Clostridium and throughout the year, also at 100%, in the case of Giardia cysts, Cryptosporidium oocysts and helminth eggs. In general, very low protozoan and helminth egg counts were found, and the system demonstrated efficient reduction of the wastewater indicator pathogens.     

Vermicomposting in the management of pig-waste in Hong Kong

The treatment and disposal of pig-waste in Hong Kong has received much attention in recent years but, following any of the presently used treatment processes, solids remain to be further stabilized. Vermicomposting is a waste stabilization technique which converts waste into potentially recyclable materials such as worm protein and worm casts. The earthworm, Pheretima asiatica, can stabilize most of the solids arising from the treatment of pig-waste, including raw pig manure, suggesting that vermicomposting has a high potential as a unit process in the management of pig-waste in Hong Kong.S.H. Wong is with the Environmental Protection Department, Hong Kong; and D.A. Griffiths is with the Department of Botany, University of Hong Kong, Hong Kong.     

Mycoflora of Argentinian corn harvested in the main production area in 1990

Corn (Zea mays) is the main cereal produced in and exported from Argentina. The risk of contamination by mycotoxins is related to the mycoflora associated with the corn kernels. This paper reports on the identification of internal and external mycoflora of corn kernels harvested in the main production area in Argentina in 1990. A mycological survey was carried out on 178 corn samples, from five locations in that area and the isolation frequency and relative density of the prevalent fungal genera compared. GenusFusarium was the most prevalent component of the internal seedborne mycoflora in the five locations.Penicillium was prevalent in all locations, taking into account the frequency. However, this genus was predominant only in two locations, when the relative density was considered. The predominantFusarium wasF. moniliforme and the most frequently isolated species ofAlternaria, Aspergillus andPenicillium wereA. alternata, A. flavus andP. decumbens, respectively.Diplodia species were not isolated from any of the samples.     

Inbreeding in a french peasant population

Genealogical pedigrees were collected from 976 farming couples who lived in a canton of central France in the XIXth and XXth century. Several methods (genealogical, isonymic and probability of origin of genes) were used to appraise the evolution of the biological structure of this population, which has gone through two phases. The first phase from 1800 to the World War I, shows gene flows between the farming population of the canton and those of neighbouring rural districts which are linked to an increase in consanguineous marriages. The second phase, from 1920 onwards, shows important gene flow from distant areas. This trend implies a decrease in consanguineous marriages.     

Lactic acid bacteria in fermented foods in Thailand

Traditional fermented foods (fish, meat and vegetable products), produced by many different processes, are eaten in many parts of Thailand. Lactic acid bacteria are responsible for the souring and ripening of these foods. Homofermentative strains of Lactobacillus pentosus, L. plantarum and Pediococcus pentosaceus are dominant in foods with low salt concentrations whereas P. halophilus strains are present in foods containing high salt. Strains of Lactobacillus sake, other Lactobacillus spp., P. acidilactici and P. urinaeequi are frequently found. Heterofermentative strains of L. brevis, L. confusus, L. fermentum, L. vaccinostercus, other Lactobacillus spp., and of Leuconostoc spp. are distributed as minor bacteria and strains of Staphylococcus, Enterococcus and Halobacterium are occasionally isolated.S. Tanasupawat is with the Department of Microbiology, Faculty of Pharmaceutical Sciences. Chulalongkorn University, Bangkok 10330, Thailand; K. Komagata is with the Department of Agricultural Chemistry, Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagaya-ku, Tokyo 156, Japan.     

Isolation of spore-forming bacilli from mosquitoes in natural breeding habitats in Iraq

New isolates of spore-forming bacilli from larvae and pupae of 3 species of mosquitoes are recorded in central Iraq.Bacillus sphaericus Meyer and Neide was isolated fromCulex pipiens (L.) larva.Bacillus carotarum Koch andBacillus cereus Frankland & Frankland were isolated fromTheobaldia longiareolata (Macquart) pupae.Bacillus laterosporus Laubach andBacillus thuringiensis (H 18) were isolated fromAedes caspius (Pallas) larvae. In addition, unidentifiedBacillus spp. were isolated fromCx. pipiens, T. longiareolata andAe. caspius larvae. Examination of soil samples collected from mosquito natural breeding habitats revealed isolates ofB. cereus, Bacillus thuringiensis H4a 4b; H 12 and H 16 and an unidentifiedBacillus sp.

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Résumé Des souches bactériennes sporogènes sont isolées de moustiques qui se trouvent dans la région centrale de l'Irak. Les résultats obtenus sont les suivants:Bacillus sphaericus Meyer & Neide [Culex pipiens (L.), larve],Bacillus carotarum Koch etBacillus cereus Frankland & Frankland [Theobaldia longiareolata (Macquart), nymphe],Bacillus laterosporus Laubach etBacillus thuringiensis (H 18) [Aedes caspius (Pallas), larvel]. L'examen des larves deCulex pipiens. T. longiareolata etAe. caspius, ainsi que l'analyse des échantillons du sol prélevés dans la région montrent la présence deBacillus cereus, Bacillus thuringiensis H4a 4b; H12 plus H16 et d'autresBacillus non identifiés.

     

The role of natural enemies in controllingIcerya purchasi in South Australia

The obscure occurrence of the cottony-cushion scaleIcerya purchasi Maskell in its native country Australia is generally attributed to its natural enemies. Twelve natural-enemy-exclusion experiments were conducted at monthly intervals to confirm the role of natural enemies. Each experiment had uncaged, open-caged, and caged treatments. The natural enemies were active throughout the year. The percentage of scales surviving to adults in the cages was significantly higher than in the open-caged treatments, except the April (first) cohort. In both open-caged and uncaged treatments, the percentage of scales surviving to adults was similar and matched the changes in the numbers of natural enemies, thus confirming their importance in controllingI. purchasi in South Australia.      

Phylogenetic relationships in theCampanulales based onrbcL sequences

Phylogenetic relationships within the angiosperm orderCampanulales were investigated by comparative sequencing of the chloroplast generbcL. CompleterbcL sequences were obtained for ten species in six families within the order. These data were analyzed along with previously publishedrbcL sequences from other taxa (for a total of 117 species) within the subclassAsteridae and outgroups, producing 32 equally parsimonious trees. A subset consisting of 44 of these taxa was then chosen and more rigorous analyses performed, resulting in four equally parsimonious trees. Results indicate that two major clades roughly corresponding to traditionally circumscribedAsterales andCampanulales exist as sister taxa. In particular, therbcL trees indicate thatSphenoclea is not a member ofCampanulales orAsterales, thatPentaphragma is more closely allied toAsterales thanCampanulales, that theCyphiaceae are not monophyletic, thatCampanulaceae andLobeliaceae are not sister taxa, and thatStylidiaceae are correctly placed withinCampanulales.     

New species and new combinations in Peruvian Orchidaceae

seventeen new species and combinations are proposed in the generaChondrorhyncha, Cischweinfia, Cochlioda, Eloyella, Encyclia, Kefersteinia, Koellensteinia, Macroclinium, Rodriguezia, Solenidiopsis, andStenia. All new species are illustrated. A key is provided for 2-flowered species ofMacroclinium, PeruvianSigmatostalix, and PeruvianStenia. Solenidium (Solenidiopsis) peruvianum Schltr. is lectotypified.