益生芽胞杆菌肠道微生态学研究进展

芽胞杆菌作为微生态制剂的一种重要来源细菌,在维护人和动物肠道健康、促进微生态平衡方面起着重要的作用。但是对于益生芽胞杆菌的作用机制却知之甚少。近年来,人们对芽胞杆菌在肠道微生物调控方面做了大量的基础研究,随着芽胞杆菌肠道分子微生态学研究的不断深入,人们发现芽胞杆菌的芽孢能够在体内迅速萌发增殖,从而起到免疫促进、改善肠道菌群以及相关的代谢酶活性等生理作用。本文从芽胞杆菌在胃肠道中的分布与定植、芽胞杆菌在胃肠道中的增殖以及芽胞杆菌与肠道菌群之间的关系等三个方面综述了益生芽胞杆菌对肠道微生态的调控作用的最新研究进展,为指导芽胞杆菌作为微生态制剂机制研究和应用提供一定的理论参考。     

感受态还是芽胞？细胞命运决定的遗传调控网络

枯草芽胞杆菌作为一般认为安全(GRAS,Generally recognized as safe)菌株,被广泛应用于饲料、食品、生物防治等领域,同时,枯草芽胞杆菌作为表达宿主在工业酶的应用中扮演重要角色。然而,低效的芽胞形成率与感受态效率极大限制了枯草芽胞杆菌的应用潜力。尽管已有大量关于芽胞形成与感受态形成的分子遗传机制的研究报道,但是通过遗传改造提高枯草芽胞杆菌芽胞形成率与感受态效率的研究报道并不多。可能的原因是芽胞形成与感受态形成作为枯草芽胞杆菌生长后期两个主要的发育事件,受胞内复杂的遗传调控机制操纵,且两个遗传通路之间存在相互调控关系,对遗传改造工作形成挑战。随着基因工程与代谢工程研究的不断发展,积累了大量关于细胞生长、代谢与发育等方面的遗传信息,通过综合这些遗传信息构建细胞遗传调控网络,用于指导生产实践,已经成为当前研究的热点之一。基于此,本文简要概述了枯草芽胞杆菌芽胞形成和感受态形成的遗传通路,初步探讨了芽胞形成与感受态形成之间的遗传调控网络,及细胞在生长后期的遗传决定机制,并讨论了该遗传调控网络对枯草芽孢杆菌及其近缘种应用研究的指导作用。     

芽胞杆菌发酵产2,3-丁二醇的研究进展及展望

综述了近年来利用芽胞杆菌生产2,3-丁二醇(2,3-BD)的研究进展,包括生产菌株的筛选、影响芽胞杆菌发酵2,3-丁二醇的因素、芽胞杆菌2,3-丁二醇代谢途径及调控等方面,并对其研究方向进行了展望。     

凝结芽胞杆菌生物学特性及其在家禽生产中的应用

作为一种新兴的微生物饲料添加剂,凝结芽胞杆菌既表现出类乳酸菌的益生特性,又具有芽胞杆菌抗逆性好、易储存的优点,有利于产业化及市场推广。凝结芽胞杆菌能够产生有机酸及多种酶类、修复受损的肠黏膜、抑制病原菌繁殖及促进营养物质的消化吸收,在家禽生产中受到广泛关注和应用。目前关于凝结芽胞杆菌的研究不够深入,其生物学特性及作用机制还在进一步研究中,还需加深其在畜牧业中的应用研究。本文主要对凝结芽胞杆菌近几年的研究进展及其在家禽生产中的应用进行综述,为今后凝结芽胞杆菌的研发及应用提供参考依据。     

枯草芽胞杆菌芽胞表面展示研究进展

枯草芽胞杆菌芽胞表面展示技术是把枯草芽胞杆菌作为芽胞表面展示的宿主来展示目的蛋白的一种技术。该技术不仅具备芽胞表面展示技术可展示分子量较大的目的蛋白、目的蛋白无需跨膜及芽胞的极强抗逆性等特点外,同时由于该技术的宿主菌--枯草芽胞杆菌的分子生物学信息研究得比较清楚、安全性高而被广泛应用。介绍了枯草芽胞杆菌表面展示近10年在生产疫苗和固定化酶方面的进展,并对如何提高表面展示目的蛋白的产量做了简要概述。     

枯草芽胞杆菌基因组删减对异源酶表达影响的研究进展

枯草芽胞杆菌作为一种遗传背景清晰、基因编辑成熟的革兰氏阳性菌,是多种重要工业酶的生产宿主。随着转录组、蛋白质组、代谢组等多组学测序和分析技术的发展,通过合理设计简化枯草芽胞杆菌基因组,减少细胞内冗余的调控和代谢网络,使得细胞更精简且便于控制,展现出了枯草芽胞杆菌作为异源酶表达宿主细胞的应用潜力。本文简要综述了枯草芽胞杆菌基因组删减的研究进展,归纳了必需基因的确定方法,重点介绍了枯草芽胞杆菌通过删减基因组提升异源酶表达的研究进展及删减策略,充分展示了枯草芽胞杆菌基因组删减在构建异源酶表达底盘细胞中的重要作用。     

PlcR在炭疽芽胞杆菌A16R中的功能研究

炭疽芽胞杆菌(Bacillus anthracis)、蜡样芽胞杆菌(B. cereus)和苏云金芽胞杆菌(B. thuringiensis)均属于蜡样芽胞杆菌群,在遗传学上有很高的相似性。PlcR (Phospholipase C regulator)在蜡样芽胞杆菌中是十分重要的调控因子,但plcR基因在炭疽芽胞杆菌中发生一个无义突变导致在炭疽芽胞杆菌中产生一个截短PlcR蛋白。为了研究plcR基因对炭疽芽胞杆菌功能的影响,文章以蜡样芽胞杆菌CMCC6330基因组为模板,构建重组表达质粒pBE2A-plcR后导入炭疽芽胞杆菌疫苗株A16R中获得重组菌株,对其进行表型分析。结果显示,炭疽芽胞杆菌重组菌株的溶血活性基本没有恢复,但恢复了部分神经鞘磷脂酶活性,表明将蜡样芽胞杆菌的plcR基因导入炭疽芽胞杆菌后,可以直接激活神经鞘磷脂酶活性。     

死亡谷芽胞杆菌研究进展

死亡谷芽胞杆菌(Bacillus vallismortis)是一种好氧、产胞的革兰氏阳性细菌,归类于枯草芽胞杆菌(Bacillus subtilis)群,对环境抗逆性强,且具有优良的生物活性,可以被广泛地应用到农业、医药及环境治理等领域。本文从死亡谷芽胞杆菌的亲缘性、抑菌活性、降解活性、生物吸附活性及产酶情况等方面做了总结,为死亡谷芽胞杆菌的进一步研究与应用提供理论依据。     

凝结芽胞杆菌的研究与应用进展

凝结芽胞杆菌是近年来快速走红的一种益生菌,他以其独特的优越性迅速地崛起于益生菌研究领域。在国外对其的研究已经进行了很多年,且生产了许多凝结芽胞杆菌产品,并得到了人们的普遍认可。在中国有关于凝结芽胞杆菌的研究还处于起步阶段。本文主要阐述了凝结芽胞杆菌的发现、发展、作用及其研究与应用。     

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。     

Na(+)-dependent, active nucleoside transport in S49 mouse lymphoma cells and loss in AE-1 mutant deficient in facilitated nucleoside transport

S49 murine lymphoma cells were examined for expression of various nucleoside transport systems using a non-metabolized nucleoside, formycin B, as substrate. Nitrobenzylthioinosine (NBTI)-sensitive, facilitated transport was the primary nucleoside transport system of the cells. The cells also expressed very low levels of NBTI-resistant, facilitated nucleoside transport as well as of Na(+)-dependent, concentrative formycin B transport. Concentrative transport was specific for uridine and purine nucleosides, just as the concentrative nucleoside transporters of other mouse and rat cells. A nucleoside transport mutant of S49 cells, AE-1, lacked both the NBTI-sensitive, facilitated and Na(+)-dependent, concentrative formycin B transport activity, but Na(+)-dependent, concentrative transport of alpha-aminoisobutyrate was not affected.     

Effect of Nucleotides and Other Inhibitors on the Inactivation of Glutamate Decarboxylase

Abstract: The effects of 17 nucleotides and nucleotide analogs and 11 other compounds on the glutamate-promoted inactivation of brain glutamate decarboxylase were examined. Among the nucleotides, the major determinant of potency was the polyphosphate chain, Glutamate-promoted inactivation was strongly enhanced by low concentrations (<100 μM) of adenosine tetraphosphate and all eight nucleoside triphosphates tested. Nucleoside diphosphates enhanced inactivation, but were much less effective than the nucleoside triphosphates; nucleoside monophosphates were not effective. Modification of the polyphosphate chain of the nucleoside triphosphates also affected potency; adenylylimidodiphosphate and α,β-methylene ATP were about as effective as nucleoside diphosphates, but α,β-methylene ATP was nearly as effective as ATP. The nucleoside base had only a small effect on potency; purine nucleotides were more potent than pyrimidine nucleotides, and one nucleotide with a tricyclic base, 1, N⁶-etheno ATP, was as effective as the purine nucleoside triphosphates. The 2'-hydroxyl group of ribose was unimportant, since deoxy ATP was as effective as ATP. Three nonnucleotide polyanions were strong promoters of inactivation; inositol hexasulfate and 5-phosphorylribose 1-pyrophosphate were at least as effective as ATP; inositol hexaphosphate (phytate) was as effective as the nucleoside diphosphates. These results suggest that a major determinant of potency was a strong negative charge on the molecule. Negative charge was not sufficient, however, since fructose 1,6-bisphosphate did not promote inactivation. Inactivation by all of these compounds was slow, requiring more than 20 min for full effect. Two competitive inhibitors, chloride and glutarate, acted immediately and also reduced rather than enhanced glutamate-promoted inactivation.     

Structure of the fluorescent nucleoside of yeast phenylalanine transfer ribonucleic acid

The structure of enzymatically isolated Y nucleoside of yeast phenylalanine tRNA was established by comparing its absorption, fluorescence, and mass spectra to that of the free base. The site of ribosylation was tentatively deduced by comparing the behavior under acid conditions of the natural nucleoside to that of synthetic Y nucleoside analogs. Our results indicate that the aglycone of the enzymatically isolated nucleoside has the same structure as the free base excised by acid treatment of phenylalanine tRNA, and that the ribose is probably attached to the N-3 position of the tricyclic nucleus.     

In vitro packaging of bacteriophage T7 DNA requires ATP.

Removal of nucleoside triphosphates from extracts prepared from bacteriophage T7-infected Escherichia coli results in a stringent requirement for added ATP to form infective phage particles by in vitro packaging of bacteriophage T7 DNA. Optimal packaging efficiency was achieved at a concentration of about 1.25 mM. Other nucleoside triphosphates could be substituted for ATP, but none of the common nucleoside triphosphates was as effective as ATP in promoting in vitro encapsulation.     

Biosynthesis of nucleoside analogues via thermostable nucleoside phosphorylase

Biocatalyzed synthesis of nucleoside analogues was carried out using two thermostable nucleoside phosphorylases from the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix K1. The synthesis of the 2,6-diaminopurine nucleoside and 5-methyluridine was used as a reaction model to test the process. Both the purine nucleoside phosphorylase (apPNP) and uridine phosphorylase (apUP) were functionally expressed in Escherichia coli. The recombinant enzymes were characterized after purification, and both enzymes showed high thermostability and broad substrate specificity. Both enzymes retained 100 % of their activity after 60 min at high temperature, and the optimum temperature for the enzymes was 90–100 °C. The nucleoside phosphorylases obtained from A. pernix are valuable industrial biocatalysts for high-temperature reactions that produce nucleoside drugs in high yields.     

Adenosine triphosphatase activity in the carotid body of the cat

Summary Three kinds of nucleoside phosphatases were demonstrated histochemically in the cat carotid body with nucleoside triphosphate, nucleoside disphosphate and nucleoside monophosphate as substrates. Each of these enzyme activities exhibited the substrate specificity respectively. The nucleoside triphosphatase activity showed specific localization in association with the parenchymal cells of the carotid body.The electronmicroscopy revealed that the reaction product was located on and between the two apposing plasma membranes of type I and type II cells, of a type II cell and its wrapping axons and of the intricate basal infolding of a type II cell itself.Some possible functions of the adenosine triphosphatase in the carotid body are discussed.     

Synthesis and Anti-HIV Activity of Novel 4′-Trifluoromethylated 5′-Deoxycarbocyclic Nucleoside Phosphonic Acids

Efficient synthetic route to novel 4′-trifluoromethylated 5′-deoxycarbocyclic nucleoside phosphonic acids was described from α-trifluoromethyl-α,β-unsaturated ester. Coupling of purine nucleosidic bases with cyclopentanol using a Mitsunobu reaction gave the nucleoside intermediates which were further phosphonated and hydrolyzed to reach desired nucleoside analogs. Synthesized nucleoside analogs were tested for anti-HIV activity as well as cytotoxicity. Adenine analog 22 shows significant anti-HIV activity (EC₅₀ = 8.3 μM) up to 100 μM.     

Differential recognition of Toxoplasma gondii recombinant nucleoside triphosphate hydrolase isoforms by naturally infected human sera

Toxoplasma gondii possesses a highly active nucleoside triphosphate hydrolase, which has been shown to be an immunodominant antigen in mice and humans. Two isoforms (I and II) which exhibit different activities with respect to hydrolysis of ATP exist. Past studies suggest that all strains of T. gondii contain the less active nucleoside triphosphate hydrolase II, whilst only virulent strains contain the nucleoside triphosphate hydrolase I isoform. In order to further investigate the correlation between nucleoside triphosphate hydrolase isoform and biological significance, we cloned and expressed as glutathione S-transferase fusion proteins the full-length nucleoside triphosphate hydrolase I and II isoforms and two truncations of the nucleoside triphosphate hydrolase I isoform in Escherichia coli. We then used ELISAs with the full-length recombinant nucleoside triphosphate hydrolases as antigens to examine 188 naturally infected T. gondii-positive sera and 83 T. gondii-negative sera for antibody reactivity. All positive sera reacted to T. gondii whole tachyzoite lysate antigen, 31 sera reacted to both nucleoside triphosphate hydrolase isoforms, three sera reacted specifically to nucleoside triphosphate hydrolase I and two sera reacted to only nucleoside triphosphate hydrolase II. Immunoblot analysis of the five sera reacting to either nucleoside triphosphate hydrolase I or II revealed both quantitative and qualitative differences in reactivity to the two isoforms. Comparative immunoblot analysis using the truncations of the nucleoside triphosphate hydrolase I isoform, and one of these positive sera identified a presumptive differential epitope between the nucleoside triphosphate hydrolase I and II isoforms within an 81-aa region (aa 445–526) at the C-terminus of the nucleoside triphosphate hydrolase I isoform. This differential reactivity was further localised to the 12-residue region of greatest variability between the two isoforms (residues 488–499) using synthetic peptides. This is the first report where naturally infected human sera have been used to identify a differential epitope. Because this region is essential for substrate binding, an antibody response to this region may play some role in inhibition of this highly active enzyme.     

Reverse and forward reactions of carbamyl phosphokinase from Streptococcus faecalis R. Participation of nucleotides and reaction mechanisms

The participation of Mg complex of nucleoside diphosphates and nucleoside triphosphates in the reverse and forward reactions catalyzed by purified carbamyl phosphokinase (ATP : carbamate phosphotransferase, EC 2.7.2.2) of Streptococcus faecalis R, ATCC-8043 were studied. The results of initial velocity studies of approx. 1 mM free Mg2+ concentration have indicated that in the reverse reaction MgdADP was as effective a substrate as MgADP. The phosphoryl group transfer from carbamyl phosphate to MgGDP, MgCDP and MgUDP was also observed at relatively higher concentrations of the enzyme and respective magnesium nucleoside diphosphate. In the forward direction MgdATP was found to be as efficient a phosphate donor as MgATP. On the other hand, Mg complexes of GTP, CTP and UTP were ineffective even at higher concentrations of the enzyme and respective magnesium nucleoside triphosphate. Product inhibition studies carried out at non-inhibitory level of approx. 1 mM free Mg2+ concentration have revealed that the enzyme has two distinct sites, one for nucleoside diphosphate or nucleoside triphosphate and the other for carbamyl phosphate or carbamate, and its reaction with the substrates is of the random type. Further tests of numerical values for kinetic constants have indicated that they are partially consistent with the Haldane relationship which is characteristic of rapid equilibrium and random mechanism.