植物根际芽胞杆菌菌种资源采集及其具有过水解催化活性水解酶基因的克隆

[背景]芽胞杆菌源枯草杆菌蛋白酶(subtilisin carlsberg)、乙酰基木聚糖酯酶(acetyl xylan esterase)和头孢菌素乙酰水解酶(cephalosporin acetyl hydrolase)具有较高的过水解催化活性,有商业开发价值。[目的]挖掘芽胞杆菌菌株中具有过水解酶催化活性的水解酶蛋白基因,为后续制备过水解酶及酶法合成过氧乙酸奠定基础。[方法]利用定向筛选培养基,从植物根际及纳豆产品中筛选产蛋白酶芽胞杆菌候选菌株,并利用RFLP及16S rRNA基因对其进行鉴定。从蛋白酶高产芽胞杆菌菌株中克隆枯草杆菌蛋白酶、乙酰木聚糖醋酶和头孢菌素乙酰水解酶的全长基因。[结果]从植物根际土壤及纳豆产品中共分离到85个候选菌株,RFLP及16S rRNA基因鉴定结果表明候选菌株均为芽胞杆菌,分别属于Bacillus subtilis、Bacillus cereus、Bacillus pumilus和Bacillus megaterium四个类群。从B.subtilis NSYT-3克隆的枯草杆菌蛋白酶基因编码的多肽链全长381个氨基酸,从B.pumilus OSLJ-3克隆得到的乙酰基木聚糖酯酶基因编码的多肽链全长320个氨基酸,从B.subtilis NSYT-3克隆的头孢菌素乙酰水解酶基因编码的多肽链全长318个氨基酸,3D结构模拟表明这3个酶蛋白均具有α/β水解酶折叠家族蛋白结构特点。[结论]芽胞杆菌源具过水解催化活性水解酶基因的克隆,为后续开发酶法合成过氧乙酸工艺奠定了基础。     

芽胞杆菌肽聚糖水解酶的功能研究进展

长久以来,肽聚糖水解酶都被认为是破坏性的角色,如噬菌体利用它裂解宿主以释放病毒粒子,一些细菌分泌它来溶解竞争对手等。但近几年来,随着研究的不断深入发现,在细胞生长代谢过程中,它的积极作用更为重要。综述了芽胞杆菌肽聚糖水解酶在细胞整个生命过程中的功能,包括细胞生长、分裂、能动性、芽胞形成及萌发、蛋白分泌、信息传递、先天性免疫以及致病性各个方面,为研究芽胞杆菌及其他微生物中肽聚糖水解酶和细胞代谢调节奠定了理论基础。     

芽胞形成相关基因缺失对解淀粉芽胞杆菌生物量及胞外酶表达的影响

解淀粉芽胞杆菌作为公认的安全生产宿主菌(GRAS),在高效表达异源蛋白方面具有巨大的发展潜力和应用前景。本研究以解淀粉芽胞杆菌TCCC111018为出发菌株,通过敲除芽胞形成相关基因(spo0A,sigF和sigE),构建了一系列突变菌株(BAΔspo0A,BAΔsigF,BAΔsigE),并对突变株生物量和胞外酶表达量进行分析比较。结果表明,与出发菌株BAΔupp相比,菌株BAΔspo0A的生物量及胞外酶表达量显著降低,BAΔsigE无明显变化,而菌株BAΔsigF的生产性能有显著提升,菌体生长的稳定期延长约6 h,产酶时间提前,耐酸性α-淀粉酶与碱性蛋白酶酶活分别提高了25.2%和21.3%,该菌株的成功构建为工业酶生产宿主提供了新的选择,也为异源酶的高效生产提出了一种新的策略。     

生防解淀粉芽胞杆菌的研究进展

解淀粉芽胞杆菌(Bacillus amyloliquefaciens)因能抑制植物病害和促进植物生长而被广泛研究。随着测序技术的不断发展,解淀粉芽胞杆菌菌株的全基因组序列陆续被测定,综述了其拮抗作用相关的功能基因、生防机制及植物-病原物-生防菌的相互作用,并对后续研究趋势进行了展望,为解淀粉芽胞杆菌的深入研究及其更好的应用提供理论参考     

苏云金芽胞杆菌clpP基因缺失对碱刺激敏感性的影响

[目的]比较苏云金芽胞杆菌与枯草芽胞杆菌在碱性培养条件下生长情况,明确clpp基因在碱刺激条件下的作用.[方法]采用同源重组技术敲除苏云金芽胞杆菌HD73菌株clpP基因,通过在不同pH下生长曲线的测定明确了clpP基因缺失突变体对碱性环境的敏感性,测定clpp基因的缺失对芽胞形成率、芽胞萌发效率和盐胁迫的影响.[结果]苏云金芽胞杆菌在碱刺激后,当培养基pH值为8.9-9.1时可以恢复生长,而枯草芽胞杆菌在pH值为8.2-8.4时可以恢复生长,说明苏云金芽胞杆菌对碱性环境适应能力更强,这有助于作为病原菌的Bt适应昆虫中肠的碱性环境.clpp基因缺失对芽胞形成率和萌发效率没有明显的影响.在将培养基中NaOH终浓度调节至30 mmol/L NaOH时,clpp基因缺失突变体的生长较出发菌株慢.说明ClpP在苏云金芽胞杆菌对碱性环境的适应过程中具有重要作用.     

解淀粉芽胞杆菌产酶多样性及应用研究进展

解淀粉芽胞杆菌(Bacillus amyloliquefaciens)能产生多种有研究价值的酶类,其酶具有明显的多样性,在众多领域,如轻工业、农业、种植业、养殖业、食品加工业、果蔬的采后保鲜、饲料业等行业具有重要价值。对解淀粉芽胞杆菌产生的不同类型的蛋白酶、降解多糖的重要酶类、脂类代谢相关的酶类以及其他几种重要的酶类进行了归类,并对其在各领域的应用及前景进行综述。     

解淀粉芽胞杆菌分离鉴定及培养优化的研究进展

解淀粉芽胞杆菌(Bacillus amyloliquefaciens)是芽胞杆菌属的一个种,广泛存在于自然界,具有丰富的生境多样性,其芽胞具有抗逆性,可抵抗不良环境,同时能产生多种抗菌物质,如脂肽、抗菌蛋白和多种酶类,能抗植物病原真菌和有害细菌,该菌能形成生物膜,定植于植物根系,促进植物生长,因此对该菌进行分离鉴定以及培养优化具有重要意义。截至目前,已经从各种生境中分离、鉴定了多种有应用价值的解淀粉芽胞杆菌,通过不同策略对该菌的培养基和培养条件进行优化,使该菌相关功能得以提高。本文综述了近年来对解淀粉芽胞杆菌的生境多样性、分离鉴定及培养基和培养条件优化的策略,简要归纳了国内外关于解淀粉芽胞杆菌重要的工业和商业产品,为更好地研究和应用解淀粉芽胞杆菌提供必要的参考和借鉴。     

地衣芽胞杆菌FLP/FRT基因编辑系统的构建及验证

地衣芽胞杆菌有效的基因编辑工具有限,为了拓展和丰富其基因编辑手段,在地衣芽胞杆菌中构建一个抗性标记可重复使用的FLP/FRT基因编辑系统,并通过敲除α-淀粉酶基因amyL、蛋白酶基因aprE及敲入外源透明颤菌血红蛋白基因vgb对该系统进行初步验证。首先以温敏质粒pNZT1为载体分别构建amyL和aprE基因敲除质粒pNZTT-AFKF和pNZTT-EFKF,两个敲除质粒各自包含针对目标基因的同源臂、抗性基因及同向的FRT位点;将敲除质粒转化地衣芽胞杆菌并经过两次同源交换过程实现目标基因的敲除;最后导入一个FLP重组酶表达质粒通过FLP/FRT系统的重组作用介导抗性基因的回收。为进一步验证本系统的实用性及编辑效率,构建了包含透明颤菌血红蛋白编码基因vgb表达盒及基因组丙酮酸甲酸裂解酶编码基因pflB敲除盒的重组质粒pNZTK-PFTF-vgb,并以此进行外源基因vgb在基因组上的定向敲入。结果显示,成功敲除amyL及aprE并回收了抗性标记卡那霉素基因,敲除后淀粉酶活和蛋白酶活分别减少95.3%和80.4%;vgb基因成功整合入基因组pflB位点并回收了抗性标记四环素基因,并利用荧光定量PCR技术检测到vgb的整合表达。文中首次建立了一个适用于地衣芽胞杆菌的、抗性标记可重复使用的FLP/FRT基因编辑系统,并进行了基因敲除及基因敲入验证,为地衣芽胞杆菌遗传改造提供了良好的方法参考。     

多粘类芽胞杆菌SC2基因敲除体系的建立

摘要:【目的】建立多粘类芽胞杆菌SC2 的基因敲除体系。【方法】利用电转化把温敏型自杀质粒pRN5101导入到多粘类芽胞杆菌SC2中。采用基因重组技术敲除SC2 中的多粘菌素基因E(pmxE),得到突变株SC2-E。利用抗细菌性能检测和高效液相色谱分析合成多粘菌素的能力,来证实pmxE基因是否被敲除。【结果】成功构建了多粘类芽胞杆菌SC2 的基因敲除体系。pRN5101转入SC2后能够在28℃复制,39℃自杀。突变株失去了合成多粘菌素的能力,成功敲除pmxE基因,验证了此体系的可用性。【结论】首次构建了多粘类芽胞杆菌的基因敲除体系,拓展了pRN5101的使用范围,为研究多粘类芽胞杆菌的基因功能提供了高效的遗传操作工具。     

通过失活Sec途径阻遏蛋白和胞外蛋白酶提高地衣芽孢杆菌产碱性蛋白酶的能力

为了探究Sec分泌途径对地衣芽孢杆菌(Bacillus licheniformis)碱性蛋白酶产量的影响,对地衣芽孢杆菌TCCC11470 (BL ΔuppΔepsΔpgs)中的分子伴侣阻遏蛋白基因hrcA和基因组中3个Sec途径分泌的胞外蛋白酶基因epr、bpr和vpr进行叠加敲除。通过对比分析基因缺失前后的碱性蛋白酶酶活力发现,敲除菌株TCCC11470ΔhrcA和TCCC11470ΔhrcAΔeprΔbprΔvpr在42 h的碱性蛋白酶酶活力分别达到18 521.2 U/mL和20 048.5 U/mL,分别高出对照菌株BLΔuppΔepsΔpgs(14 478.6 U/mL) 27.9%和38.5%。这一结果指出,通过改进Sec分泌通路可以显著提升碱性蛋白酶的催化效能,为构建优化的工业酶生产宿主提供了新思路和研究方向。     

Taxonomic status of the species of the green algal genusNeochloris

Komárek has recently reviewed the various species assigned to the green algal genusNeochloris Starr (Chlorococcales, Chlorococcaceae) and removed those with uninucleate vegetative cells to a new genus,Ettlia. Watanabe & Floyd, unaware ofKomárek's work, also reviewed the species ofNeochloris and distributed them among three genera—Neochloris, Chlorococcopsis gen. nov., andParietochloris gen. nov.—on the basis of details of the covering of the zoospore and the arrangement of the basal bodies of the flagellar apparatus. This paper reconciles these two treatments and makes additional recommendations at the ranks of genus, family, order, and class.     

Hectorella: A member of the betalain-suborderChenopodiineae of the orderCentrospermae

Roots ofHectorella caespitosa Hook. f. were induced to produce a red pigment which was shown to be a betalain and not an anthocyanin. These data indicate thatHectorella belongs to theChenopodiineae, the betalain suborder of theCentrospermae, and excludes alignment with the anthocyanin family theCaryophyllaceae.     

Succession in the southern part of the Canadian boreal forest

Forest succession following fire in a forest mosaic of northwestern Quebec has been studied in order to: (1) describe the successional pathways using communities of different ages and (2) evaluate convergence of successional pathways and possible effect of fire suppression on the establishment of steady-state communities. As a first step, ordination and classification techniques were used in order to remove changes in forest composition which are related to abiotic conditions. Then, ordinations based on tree diameter distributions were used to study shifts in species composition in relation to time since the last fire.Even under similar abiotic conditions, successional pathways are numerous. However, regardless of forest composition after fire, most stands show convergence toward dominance of Thuja occidentalis and Picea mariana on xeric sites and dominance of Abies balsamea and Thuja occidentalis on more mesic sites. Stable communities of >300 yr occur on xeric sites while on mesic sites directional succession still occurs after 224 yr. Nearly all species involved in succession are present in the first 50 yr following fire. Only Abies balsamea and Thuja occidentalis increase significantly in frequency during succession. Following initial establishment, successional processes can generally be explained by species longevity and shade tolerance. Early successional species may be abundant in the canopy for more than 200 yr while the rapid decrease of Picea glauca, a late successional species could be related to spruce budworm outbreaks. Considering the short fire rotation observed (about 150 yr), a steady-state forest is unlikely to occur under natural conditions, though it may be possible if fire is controlled.     

The purification of the three trimannosyl-N-acetylchitobiose pentasaccharides from the urine of swainsonine-intoxicated sheep

A simple method for the preparative resolution of three Man₃GlcNAc₂ isomers called Ia, Ib and II has been designed. It consists mainly of the use of concanavalin A-Sepharose which allowed the total purification of Man₃GlcNAc₂-Ia, and then of anion-exchange resin in borate buffer-gradient to separate the Ib and II isomers. The purity of each oligosaccharide was checked by two HPLC methods. The use of these oligosaccharides for different analytical and biosynthetic purposes is discussed, and the unexpected resistance of one of the Man₃GlcNAc₂ alditols to the action of endo--N-acetylglucosaminidase H is noted.     

Evaluation of the Pro-Lab ID ring system for the identification of medically important yeasts

We evaluated 151 coded isolates of medically important yeast species belonging to the genera Candida, Cryptococcus, Geotrichum, Rhodoturula, Saccharomyces and Torulopsis using the newly developed rapid Pro-Lab Identification Ring, PL 960 system (PLID-Ring). All isolates were concurrently identified by the API 20C and conventional procedures comprising macro- and micromorphology, assimilation and fermentation of various carbon and nitrogen compounds. The PLID-Ring system identified isolates of Candida albicans, C. kefyr, C. krusei, C. lusitaniae, C. parapsilosis, Rhodotorula rubra, and Torulopsis glabrata with 100% accuracy in 24 h. This system identified C guilliermondii and S. cerevisiae isolates with an accuracy of 90% and 86%, respectively, while those belonging to Cr. neoformans, T. candida (= C. famata), C. rugosa and C. tropicalis were identified with 38.4%, 50%, 12.5% and 50% accuracy, respectively. Three isolates of Cr. laurentii were not identified by the PLID-Ring system. The overall accuracy of the PLID-Ring system was 81.45% (123 of 151 isolates). However, the system does not include species such as Cr. laurentii in its data base. When these three Cr. laurentii isolates were excluded from the evaluation, the accuracy of the PLID-Ring system increased from 81.45% to 83.1%.     

Comparative analysis of the Kekkon molecules,related members of the LIG superfamily

Leucine-rich repeats (LRRs) and immunoglobulin (Ig) domains represent two of the most abundant sequence elements in metazoan proteomes. Despite this prevalence, comparatively few molecules containing both LRR and Ig (LIG) modules exist, and fewer still have been functionally defined. One LIG whose function has been investigated is the Drosophila protein Kekkon1 (Kek1). In vivo studies have demonstrated a role for Kek1 in Epidermal Growth Factor Receptor (EGFR) signaling and have suggested a role in neuronal pathfinding. Kek1 is the founding member of the Kek family, a group of six Drosophila transmembrane proteins that contain seven LRRs and a single Ig in their extracellular domains. While this arrangement of domains predicts a possible role as cell adhesion molecules (CAMs), to date little is known about the function or evolutionary relationship of these additional Kek molecules. Here we report that orthologs of Kek1, Kek2, Kek5, and Kek6 exist in the mosquito, Anopheles gambiae, and the honeybee, Apis mellifera, indicating that this family has been conserved for ~300 million years of evolutionary time. Comparative sequence analyses reveal remarkable identity among these orthologs, primarily in their extracellular regions. In contrast, the intracellular regions are more divergent, exhibiting only small pockets of conservation. In addition, we provide support for the general notion that these molecules may share common functions as CAMs, by demonstrating that Kek family members can form homotypic and heterotypic complexes.Edited by D. TautzChristina M. MacLaren, Timothy A. Evans and Diego Alvarado contributed equally to this work     

Evaluation of antiprotozoal and antimycobacterial activities of the resin glycosides and the other metabolites of Scrophularia cryptophila

Resin glycosides are secondary metabolites exclusive to the convolvulaceous plants. In this study, crypthophilic acids A–C (1–3), the first resin glycosides occurring in another family (Scrophulariaceae), and the other constituents of Scrophularia cryptophila were examined for in vitro antiprotozoal and antimycobacterial potentials. Except for crypthophilic acid B (2), all tested compounds exhibited growth-inhibitory effect against Trypanosoma brucei rhodesiense, with l-tryptophan (6) and buddlejasaponin III (7) being the most potent ones (IC₅₀'s 4.1 and 9.7 μg/ml). In contrast, the activity towards Trypanosoma cruzi was poor, and only crypthophilic acid C (3), 6 and 7 were trypanocidal at concentrations above 40 μg/ml. With the exception of 2 and 6, all compounds were active against Leishmania donovani. Harpagide (4) and 3 emerged as the best leishmanicidal agents (IC₅₀'s 2.0 and 5.8 μg/ml). Only compounds 3, 6 and 7 showed antimalarial activity against Plasmodium falciparum with IC₅₀ values of 4.2, 16.6 and 22.4 μg/ml. Overall the best and broadest spectrum activity was presented by compounds 3 and 7, as they inhibited all four parasitic protozoa. None of the isolates had significant activity against Mycobacterium tuberculosis (MICs >100 μg/ml) or were toxic towards mammalian (L6) cells. This is the first report of antiprotozoal activity for natural resin glycosides, as well as for harpagide (4), acetylharpagide (5), tryptophan (6) and buddlejasaponin III (7).     

Evolution of the Australian flora through the Tertiary

The evolution of the Australian flora through the Tertiary has not been reviewed by a megafossil palaeobotanist for almost a century. Based on material available and published studies, the Australian Tertiary can be considered in three units: Eocene, Oligo-Miocene, and Pliocene. Key taxa in the Eocene includeCasuarinaceae, Proteaceae, Fagaceae, Podocarpaceae, andLauraceae. Many known Eocene deposits are interpreted as warm, humid rainforests.Oligo-Miocene floras reflect a climatic deterioration, with greater occurrence of sclerophylly and xerophylly. A reduction of tropical taxa is seen with an increase ofMyrtaceae and earliest records ofMimosaceae, Chenopodiaceae, andPoaceae. These trends continue into the Pliocene, which is not well represented in Australia.Problems needing to be addressed include the discrepancy between the pollen and megafossil records ofNothofagus, the origins of Australia's wet tropical taxa, and the role of fire in the early evolution of the Australian flora.     

Progress on the genetics of the N2-fixing actinorhizal symbiont Frankia

The actinomycete Frankia is of fundamental and ecological interests for several reasons including its wide distribution, its ability to fix nitrogen, differentiate into sporangium and vesicle (specialized cell for nitrogen-fixation), and to nodulate plants from about 24 genera. Here, we present a review on the genetics performed so far on Frankia. At the end of July 2001, 293 kbp of Frankia DNA sequences were found in the databases. Thirty five percent of these sequences corresponded to full gene or gene cluster sequences. These genes could be divided according to their role into 6 key activities: gene translation (rrnA and tRNA ^pro gene), proteolysis (pcr genes), assimilation of ammonium (glnA and glnII), protection against superoxide ions (sodF), nitrogen fixation (nif cluster), and plasmid replication. We present a review of these genetic islands; their function, expression, localization and particular properties are discussed. A comparative analysis of Frankia nif genes from various strains and species is presented. An improved nomenclature for some of these genes is suggested to avoid conflicts. Frankia plasmids DNA sequences are also presented. The novel trends in Frankia genetics are described.