具有分泌蛋白能力的短芽孢杆菌的筛选及鉴定

短芽孢杆菌（Bacillus　brevis）具有分泌蛋白能力强和胞外蛋白酶活性低的特性,是分泌表达外源蛋白较理想的宿主。为获得分泌表达系统较理想的宿主菌,建立了短芽孢杆菌高效筛选模型,从800余株细菌中筛得8株具有高蛋白分泌能力且没有胞外蛋白酶活性的候选菌。经多相分类学初步鉴定其中5株为短芽孢杆菌。     

短短小芽孢杆菌大肠杆菌穿梭分泌表达载体的构建

应用PCR技术从具有分泌蛋白能力强且没有胞外蛋白酶活性的短短小芽孢杆菌50中分离出细胞壁蛋白基因的多启动子和信号肽编码序列,利用它与质粒pUB110和pKF3-起构建成穿梭分泌表达载体pBKE50,将α0淀粉酶基因引入该载体转化短短小芽孢杆菌50后,发现α－淀粉酶可以活性形式分泌表达,此工作为下一步建立短短小芽孢杆菌高效分泌表达系统奠定了基础。     

芽胞杆菌高效分泌表达异源蛋白的研究进展

外泌蛋白易于分离纯化,有利于以较低的生产成本获得大量的目标蛋白,因此分泌表达是一种理想的外源基因的表达方式。芽胞杆菌由于其具有较好的分泌能力,被认为是一种理想的表达外源基因的宿主。到目前为止已有大量来源于不同生物的外源基因在芽胞杆菌中实现了高效分泌表达。但是芽胞杆菌的分泌表达系统仍然存在很多问题,如对某些目的蛋白的分泌量低等,限制了其作为“细胞工厂”生产目标蛋白的应用。综述了芽胞杆菌的外泌系统,分析了芽胞杆菌分泌蛋白过程中的限制因素,并总结了相关的解决方案。     

重组枯草芽抱杆菌的构建及对街体的Cl , 2 位脱氢

本文将简单节杆茵3-甾酮-1-脱氢酶基因克隆到分泌表达载体pWB980上,并转入枯草芽孢杆菌WB600中,得到重组芽孢杆菌菌株。重组芽孢杆菌表达出的目的蛋白的分子量为55KDa,分光光度法检测到胞内和胞外可溶性部分的酶活分别为110±0．5mU和15±0．6mU每毫克蛋白,对甾体底物4-AD的转化率为45．3%,比出发茵简单节杆菌提高了将近10倍。     

短短芽胞杆菌功能基因的研究及其应用

短短芽胞杆菌分布广泛,并且在不同生长阶段,菌落形态也有所不同。目前已经完成2株短短芽胞杆菌的全基因组测序,但只对短短芽胞杆菌部分基因的功能进行了研究,如编码二硫化物氧化还原酶的基因bdb、α-乙酰乳酸脱羧酶的基因aldB、耐碱性木聚糖酶xylB基因和细胞壁的合成基因等,特别是具有抗菌活性的短杆菌酪肽的合成过程及所需酶的基因均有研究。短短芽胞杆菌在生物防治、作为分泌表达外源蛋白的宿主及环境治理等领域有广泛应用前景。     

芽胞杆菌中重组蛋白分泌表达的优化策略及研究进展

芽胞杆菌属具有良好的蛋白表达和分泌能力,在工业酶的生产中被广泛应用,是理想的工业宿主菌,但实现蛋白分泌表达的普遍高效性还存在许多瓶颈。本文综述了芽胞杆菌的蛋白分泌表达策略,从启动子、信号肽、分泌途径、宿主和培养条件这5个方面总结了提高芽胞杆菌中分泌表达重组蛋白的方法,对芽胞杆菌高效生产工业酶有一定的参考价值,最后展望了优化芽胞杆菌分泌表达的研究方向,各种新型生物技术的发展必将推进芽胞杆菌在分泌表达领域有更深入的应用。     

巨大芽孢杆菌B．m2980产孢对氧化葡萄糖酸杆菌产酸的影响

为确定维生素C二步发酵中巨大芽孢杆菌（伴生菌）芽孢形成对氧化葡萄酸杆菌（产酸菌）产酸的影响,本研究通过对巨大芽孢杆菌生长特性分析,选取培养12h（未形成芽孢）和36h（芽孢大量形成）巨大芽孢杆菌B．m2980,检测其胞外液、胞内液以及混合液对产酸菌生成2-酮基-L-古龙酸的影响。结果表明,在未开始形成芽孢时,伴生菌胞外液、胞内液及混合液对产酸菌的生长和产酸有较低的促进作用,其中胞内液的促进能力大于胞外液;在芽孢生成后,胞外液以及混合液对产酸菌生长和产酸的促进能力显著提高。     

产单核细胞李斯特菌p60蛋白N端肽聚糖结合基序在枯草芽孢杆菌中的表达

目的:构建产单核细胞李斯特菌胞外蛋白p60的N端肽聚糖结合基序(p60-N)表达载体,实现其在枯草芽孢杆菌中的分泌表达。方法:将目标基因克隆到表达载体pHT43中获得重组载体pHT43-p60-N,转化枯草芽孢杆菌WB800N获得重组工程菌株WB800N/pHT43-p60-N,在此基础上考察IPTG浓度、培养基、表达时间和温度等条件对目标蛋白表达的影响。结果:p60-N蛋白可在枯草芽孢杆菌中分泌表达,与鼠李糖乳杆菌形成的革兰阳性增强基(GEM)颗粒特异性结合,具有与天然蛋白类似的生物学活性。用GB培养基,在37℃下,加入终浓度为0.2 mmol/L的IPTG诱导12~20 h,表达量提高到28 mg/L。结论:实现了p60蛋白N端基序在枯草芽孢杆菌中的分泌表达,为进一步研究以该蛋白为基础的细菌样颗粒疫苗奠定了基础。     

3-甾酮-1-脱氢酶基因的表达及甾体转化分析

本文利用带有P43启动子的表达分泌载体pWB980,实现了简单节杆菌3-甾酮-1-脱氢酶在枯草芽孢杆菌中的表达,表达出的目的蛋白的分子量为55KDa。利用分光光度法检测得到胞内和胞外可溶性部分的酶活分别为110±0.5mU和15±0.6mU每毫克蛋白, 比出发菌株简单节杆菌提高了将近30倍。重组芽孢杆菌对甾体底物4-AD的转化率为45.3％,比出发菌株简单节杆菌提高了近10倍。利用枯草芽孢杆菌对甾体底物进行脱氢为甾体药物的生产开辟了一个新的途径。     

巨大芽孢杆菌表达系统的特点及其研究进展

巨大芽孢杆菌表达系统是口前比较成功的异源蛋白原核分泌达系统,可能成为大肠杆菌表达系统的替代品,该文对该表达系统的特点及研究进展作一简要综述.该系统质具有胞外蛋白酶活性低,质粒稳定,可以直接将表达的蛋白高效分泌到培养基中,且无内毒素,诱导剂廉价和易培养等优点.至今已有多个基因在巨大芽孢杆菌中成功获得表达,这个系统被使用得越来越多.     

Rapid identification of Lactobacillus brevis using the polymerase chain reaction

AIMS: Species-specific PCR was applied to identify Lactobacillus brevis and the sensitivity and the specificity of the protocol were determined. METHODS AND RESULTS: Strains of Lact. brevis obtained from foods, particularly dairy products, and various strain collections, were identified by PCR using primers which amplified a 1340 bp fragment within the 16S rRNA gene. The PCR product was obtained after amplification of all the Lact. brevis strains tested; the size of the amplicon was as expected. No PCR products were observed after amplification from DNA of several lactic acid bacteria (LAB) species. CONCLUSIONS: A PCR method was optimized to identify Lact. brevis. The protocol was highly efficient and sensitive. SIGNIFICANCE AND IMPACT OF THE STUDY: Conventional phenotypic methods often lead to ambiguous identification of LAB species belonging to Lact. brevis. The proposed protocol is sensitive, specific, and can be applied to total DNA extracted by use of chelating matrix with loss of neither sensitivity nor specificity.     

Ultrastructure and ribosomal RNA phylogeny of the free-living heterotrophic flagellate Dysnectes brevis n. gen., n. sp., a new member of the Fornicata

Dysnectes brevis n. gen., n. sp., a free-living heterotrophic flagellate that grows under microaerophilic conditions possesses two flagella. The posterior one lies in a ventral feeding groove, suggesting that this flagellate is an excavate. Our detailed electron microscopic observations revealed that D. brevis possesses all the key ultrastructural characters considered typical of Excavata. Among the 10 excavate groups previously recognized, D. brevis displays an evolutionary affinity to members of the Fornicata (i.e. Carpediemonas, retortamonads, and diplomonads). Firstly, a strong D. brevis-Fornicata affinity was recovered in the phylogenetic analyses of small subunit ribosomal RNA (SSU rRNA) sequences, albeit the internal branching pattern of the D. brevis+Fornicata clade was not resolved with confidence. Corresponding to the SSU rRNA phylogeny, D. brevis and the Fornicata shared the following components of the flagellar apparatus: the arched B fiber bridging the right root; a posterior basal body; and a left root. Combining both morphological and molecular phylogenetic analyses, D. brevis is classified as a new free-living excavate in the Fornicata incertae sedis.     

Cloning, characterization, and inactivation of the Bacillus brevis lon gene.

A gene of Bacillus brevis HPD31 analogous to the Escherichia coli lon gene has been cloned and characterized. The cloned gene (B. brevis lon gene) encodes a polypeptide of 779 amino acids with a molecular weight of 87,400 which resembles E. coli protease La, the lon gene product. Fifty-two percent of the amino acid residues of the two polypeptides were identical. The ATP-binding sequences found in E. coli protease La were highly conserved. The promoter of the B. brevis lon gene resembled that recognized by the major RNA polymerase of Bacillus subtilis and did not contain sequences homologous to the E. coli heat shock promoters. The B. brevis lon gene was inactivated by insertion of the neomycin resistance gene. A mutant B. brevis carrying the inactivated lon gene showed diminished ability for the degradation of abnormal polypeptides synthesized in the presence of puromycin.     

Selective medium based on tyrosine metabolism for the isolation and enumeration of Brevibacillus brevis (Bacillus brevis)

AIMS: To develop a selective medium for the enumeration of Brevibacillus brevis Nagano spores from soil and plant material. METHODS AND RESULTS: Tyrosine agar was developed as a selective medium and compared with nutrient agar for the enumeration of B. brevis Nagano spores from sterile and non-sterile plant and soil extracts. Brevibacillus brevis Nagano colonies could be easily identified only on tyrosine agar due to their clear halo and distinct colony morphology. Identification was confirmed by thin layer chromatography of the antibiotic, gramicidin S, produced by this strain. CONCLUSIONS: Tyrosine agar was shown to be a suitable selective medium for the enumeration of B. brevis Nagano. SIGNIFICANCE AND IMPACT OF THE STUDY: The medium developed, tyrosine agar, can be used to monitor the population of the biological control agent, B. brevis Nagano, and will allow detailed studies within the crop environment.     

Structural study of the basal bodies of the flagella of Bacillus brevis var. G.-B. P+

The structural organisation of the flagellum basal body was studied in Bacillus brevis var. G.-B. P+ by electron microscopy. It was compared with that of Escherichia coli MS 1350. The basal body of a B. brevis flagellum contains, in addition to two pairs of rings on a rod, another ring-like structure (d = 13.6 nm, h = 4.3 nm) which we referred to as a "collar". The collar makes the basal body of B. brevis different from that of B. subtilis, another Gram-positive bacterium. The collar seems to fasten the flagellum of B. brevis to the cell wall. We have concluded that the basal body can differ not merely among bacterial systematic groups, but also among bacteria belonging to one and the same genus. The role of individual elements in the structure of the basal body of bacterial flagella is discussed.     

Comparative study on the cell wall structure of protein-producing Bacillus brevis

Abstract Among eight strains of protein-producing Bacillus brevis , three morphological groups have been identified according to the structure of the cell walls.

• (I)

  Cell wall consisting of a peptidoglycanlayer
• (II)

  Two-layered cell wall consisting of a peptidoglycan-layer and an S-layer
• (III)

  Three-layered cell wall consisting of a peptidoglycan-layer and two S-layers

Group I and group II cell walls have not been described yet for protein-producing bacteria. The S-layers observed in this study all had hexagonal symmetry and lattice constants of approximately 18 nm. The immunological relation between the S-layer proteins of the newly isolated B. brevis strains and those of B. brevis 47 has been examined using antisera against both S-layer-proteins of B. brevis 47. S-layers from protein-producing B. brevis strains, which were adjacent to the peptidoglycan-layer, were similar to each other, whether they were the outermost cell wall layer (group II) or not (group III). However, no similarity was found between these layers and the outermost S-layer of B. brevis 47 (group III).     

Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis

Karenia brevis is a toxic marine dinoflagellate endemic to the Gulf of Mexico. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). Previously, PKS encoding genes were amplified from K. brevis culture and their similarity to a PKS gene from the closely related protist, Cryptosporidium parvum, suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. Herein we report the localization of PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate.     

Sensitivity of some Lactobacillus species of the subgenus Betabacterium to the action of bacteriocins from different species of Lactobacillus]

Sensitivity of 84 cultures of L. fermenti, 43 cultures of L. brevis, 13 cultures of L. buchneri and 2 cultures of L. cellobiosus to the effect of 39 types of bacteriocins produced by various species of lactobacilli was studied with the method of delayed antagonism. All the cultures of L. brevis, L. buchneri and L. cellobiosus and 65.5 per cent of the cultures of L. fermenti were sensitive to one or more such bacteriocins. The cultures of L. Fermenti, L. brevis, L. buchneri and L. cellobiosus were sensitive respectively to 37, 19, 16 and 9 types of the bacteriocins out of 39 types tested.     

Analysis of the structure of Bacillus brevis neutral proteinase and its biosynthesis in Bacillus subtilis cells

Amino acid sequence of neutral metalloprotease from Bac. brevis has been compared with that of Bac. amyloloquefaciens, Bac. cereus, Bac. subtilis, Bac. stearothermophilis, Bac. thermoproteolyticus (thermolysine). A sequence region from N-40 to N-1 with a significant degree of homology allowed to predict the processing site of the propart of Bac. brevis enzyme. The sequence comparison allows to put Bac. brevis enzyme within the evolutionary branch of enzymes, which includes thermolysin and proteases of Bac. cereus and Bac. stearothermophilus. Using automated Edman degradation the N-terminal sequence of Bac. brevis protease has been determined. It does not differ from the sequence predicted from the nucleotide sequence of the gene. It was shown that, when Bac. brevis gene coding for thermostable protease is expressed on a plasmid vector in Bac. subtilis cells at 37 degrees C, enzyme forms possessing low activity are secreted. The enzyme may be significantly activated without an additional cleavage or processing and the activation includes numerous conformation transition states of the protein molecule.