Comparison of Metagenomic DNA Extraction Methods for Soil Sediments of High Elevation Puga Hot Spring in Ladakh,India to Explore Bacterial Diversity

Extraction of good-quality metagenomic DNA from extreme environments is quite challenging, particularly from high elevation hot spring sediments. Low microbial load, high humic acid content and other contaminants complicate the process of extraction of metagenomic DNA from hot spring sediments. In the present study, efficacy of five manual DNA extraction protocols with modifications has been evaluated for metagenomic DNA extraction from boron–sulfur rich high elevation Puga hot spring sediments. Best suited protocol was identified based on the cell lysis efficiency, DNA yield, humic acid content, PCR reproducibility and representation of bacterial diversity. Quantity as well as quality of crude metagenomic DNA differed remarkably between various protocols used and were not pure enough to give PCR amplification using 16S rRNA bacterial and archaeal primers. Crude metagenomic DNA extracted using five different DNA extraction protocols was purified using spin column based purification method. Even after purification, only three protocols C, D and E yielded metagenomic DNA that could be amplified using both archaeal and bacterial primers. To evaluate the degree of microbial diversity represented by protocols C, D and E, phylogenetic genes amplified were subjected to amplified ribosomal DNA restriction analysis (ARDRA) and denaturing gradient gel electrophoresis analysis (DGGE) analysis. ARDRA banding pattern of amplicons generated for all the three extraction protocols, i.e., C, D and E were found to be similar. DGGE of protocol E derived amplicons resulted in the similar number of dominant bands but a greater number of non-dominant bands, i.e., the highest microbial diversity in comparison to protocols C and D, respectively. In the present study, protocol E developed from Yeates et al. protocol has been found to be best in terms of DNA yield, DNA purity and bacterial diversity depiction associated with boron–sulfur rich sediment of high elevation hot springs.     

Intratracheal immunization with pili protein protects against mortality associated with Pseudomonas aeruginosa pneumonia in mice

We examined the protective effect of intratracheal immunization with Pseudomonas aeruginosa pili protein against respiratory infection caused by P. aeruginosa. Mice were immunized intratracheally or subcutaneously with purified pili protein or bovine serum albumin as a control. Intratracheally but not subcutaneously pili protein-immunized mice showed significant improvement of survival after intratracheal challenge with the PAO1 strain. Furthermore, bacterial cell counts in pili protein-immunized murine lungs were significantly decreased compared to controls at 18 h after the challenge. Antipili protein antibody titers in bronchoalveolar lavage fluid of intratracheally pili protein-immunized mice were higher than in bovine serum albumin immunized mice. However, antipili antibody titers were not increased in bronchoalveolar lavage fluid of subcutaneously pili protein-immunized mice, despite the high serum antipili antibody titers. Inoculation of P. aeruginosa induced immediate increases in interleukin-12 and interferon-gamma in bronchoalveolar lavage fluid of pili protein-immunized mice, reflecting an adequate and rapid immune response against P. aeruginosa respiratory tract infection. Our findings suggest that intratracheal pili protein immunization is effective against respiratory tract infection caused by P. aeruginosa in mice.     

Expression, purification, and crystallization of endopolygalacturonase from a pathogenic fungus, Stereum purpureum, in Escherichia coli

Endopolygalacturonases (EC 3.2.1.15) catalyze random hydrolysis of the alpha-1,4 glycosidic linkages in polygalacturonic acid, a component of pectin. Previously, we reported crystal structures of endogenously produced Stereum purprureum endopolygalacturonase I (endoPG I), both in its native form and complexed with its product, galacturonate. However, the substrate-binding mechanism of endoPG I is still unclear, because crystals have not yet been obtained with a substrate analog, or with mutant enzymes that can bind substrates. We describe here an expression system using Escherichia coli and a purification method to prepare functionally active endoPG I for such mutation and crystallographic studies. Expression in E. coli strain Origami (DE3) provided a soluble and active enzyme with proper disulfide bond formation, whereas the enzyme expressed in BL21 (DE3) was localized in inclusion bodies. A sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography. The specific activity of recombinant endoPG I was equivalent to that of the enzyme produced by S. purpureum. Recombinant endoPG I was crystallized under the same conditions as those used for the native enzyme produced by S. purpureum. The crystals diffracted beyond 1.0 A resolution with synchrotron radiation.     

山西阳泉老窑水硫酸盐还原菌的分离鉴定及其还原功能驯化

【背景】含硫煤矿开采后,地表水/地下水回流至采空区形成酸性老窑水,含有高浓度重金属离子和硫酸盐,严重危害生态系统健康。利用微生物自身生长处理老窑水具有成本低、环境友好等特点,具有良好的应用前景。目前利用的硫酸盐还原菌大多只在适宜温度和中性pH条件下具有较高活性,在北方低温和酸性条件下难以发挥作用。【目的】本研究旨在从山西阳泉山底河流域的老窑水环境中分离硫酸盐还原菌,并调节温度和pH进行驯化,从而得到高效耐低温耐酸菌株,为北方老窑水微生物治理提供可用菌种资源。【方法】对山底河流域典型老窑水样品中的微生物进行富集培养,并筛选硫酸盐还原菌。通过革兰氏染色、扫描电镜对菌株形貌特性进行表征,利用16SrRNA基因序列比对进行菌种鉴定,探究其生长特性和硫酸盐还原性能。在此基础上降低温度和pH,对高效还原硫酸盐菌株进行驯化,探讨其在北方老窑水污染治理中的应用潜力。【结果】本研究筛选得到2株硫酸盐还原菌,命名为YQ-1和YQ-2,分别属于革兰氏阴性瘤胃解蛋白质菌属(Proteiniclasticum)和脱硫弧菌属(Desulfovibrio)。在30°C、pH 7.5条件下,YQ-1和YQ-2对1 1...     

Effect of bacterial lypopolysaccharide on penicillin-induced seizure activity in rats

In experiments on Wistar rats, we found that, within an early period (2 to 4 h) after injection of bacterial lypopolysaccharide, LPS (0.1 mg/kg, i.p.), the latency of generalized seizures induced by injection of benzylpenicillin (sodium salt; 3.0 million IU/kg, i.p.) became significantly shorter, while the severity of seizure manifestations was higher than in the control group. Within this period, the power of oscillations of the delta and alpha-frequency ranges increased in the frontal cortex and hippocampal structures; fast ECoG components (beta and gamma rhythms) were suppressed, and the power of the theta activity decreased. In the hippocampal structures, these changes were more clearly pronounced, as compared with the neocortex. Within a later period of the action of LPS (12 to 18 h from the moment of injection), the latency of penicillin-induced seizures significantly exceeded the control value, and the severity of such seizures was lower. Under such conditions, we observed a smaller power of the synchronized activity of delta and alpha frequencies combined with intensification of the theta activity (most clearly pronounced in the hippocampal structures), and also an increase in the power of “desynchronized” rhythms (beta and gamma oscillations) in the cortex and hippocampus. Neirofiziologiya/Neurophysiology, Vol. 40, No. 3, pp. 236–241, May–June, 2008.     

干热处理对甜瓜种子活力和细菌性果斑病抑制能力的影响

以厚皮甜瓜品种‘哈密绿’种子为材料,在70℃、75℃和80℃温度下分别处理24h、48h和72h,研究不同干热处理对甜瓜种子活力萌发和生理生化指标变化及细菌性果斑病的防治效果。结果显示:(1)随着处理温度的升高和时间的延长,干热处理甜瓜种子发芽指标和成苗率显著下降,而70℃处理24h和48h对种子发芽指标(活力指数除外)和成苗率无显著影响。(2)与对照相比,干热处理种子胚芽中的SOD活性、可溶性糖和脯氨酸含量升高,在同一温度处理下,SOD活性和可溶性糖含量随着处理时间的延长基本呈增长趋势,脯氨酸含量则随着处理时间的延长呈下降趋势,POD和APX活性以及可溶性蛋白含量变化随着温度的升高和处理时间的延长呈下降趋势;与对照相比,胚芽中CAT活性在70℃处理下降低,而在75℃和80℃处理下升高,但其随着处理时间的延长呈下降趋势;胚芽中O-·2产生速率在70℃和75℃处理下与对照接近,而在80℃处理下随着处理时间的延长呈上升趋势,且均显著高于对照。(3)随着处理温度的升高和时间的延长,甜瓜接菌种子幼苗细菌性果斑病发病率较对照显著降低。研究表明,适宜干热处理温度和时间诱导甜瓜种子中抗氧化酶活性增强,渗透调节物质含量增加,超氧阴离子产生速率降低,种子活力和出苗率有效提高,‘哈密绿’种子有效、安全的干热处理组合为70℃、48h。     

外源基因表达造成的群体感应细菌生存压力的建模分析

外源基因的表达及其对细菌种群的影响对于群体感应系统和合成生物学产业的研究具有重要意义。然而,人们对于表达外源蛋白的细菌本身的行为模式仍然知之甚少。为了研究菌落生长和外源基因表达的过程究竟受到哪些因素的影响,文中测量了受Lux类受体调控的外源基因在N-酰基高丝氨酸内酯 (N-acyl homoserine lactone,N-AHL) 信号分子诱导下的表达,并模拟了其对细菌种群动态的影响。文中建立了一个假设性的数学模型,对信号分子诱导表达下细菌种群生长受影响的现象进行了分析。先前的研究通常将细菌种群生长受群体感应系统影响的现象归咎于合成群体感应信号分子的消耗与N-AHL信号分子的毒性,文中提供了对于这种生存压力的另一种可能的解释。     

新疆玛纳斯热气泉免培养土壤细菌多样性分析

【目的】了解新疆玛纳斯热气泉土壤免培养细菌群落组成及多样性。【方法】采用免培养法直接从土壤样品中提取总DNA,利用细菌通用引物对土壤总DNA进行16S rDNA扩增,构建细菌16SrDNA文库。使用HaeⅢ限制性内切酶对阳性克隆进行限制性片段长度多态性分析(restriction fragment length polymorphism,RFLP),挑选具有不同酶切图谱的克隆进行测序、比对并构建16SrDNA系统发育树。【结果】从土壤细菌16S rDNA文库中随机挑选了170个阳性克隆,共得到29个不同的分类单元(operational taxonomic unit,OTU)。系统发育分析归为6个门:酸杆菌门(Acidobacteria)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)和浮霉菌门(Planctomycetes)。其中厚壁菌门(Firmicutes)为绝对优势类群,占整个细菌文库的71%。29个OTUs中有14条序列与GenBank中相关序列的相似性低于97%(序列长度约1.5kb),占序列总数的48%。【结论】热气泉土壤细菌种群多样性较低,但存在大量潜在细菌新种。     

Eicosanoids in insect immunity: bacterial infection stimulates hemocytic phospholipase A2 activity in tobacco hornworms

Intracellular phospholipase A(2) (PLA(2)) is responsible for releasing arachidonic acid from cellular phospholipids, and is thought to be the first step in eicosanoid biosynthesis. Intracellular PLA(2)s have been characterized in fat body and hemocytes from tobacco hornworms, Manduca sexta. Here we show that bacterial challenge stimulated increased PLA(2) activity in isolated hemocyte preparations, relative to control hemocyte preparations that were challenged with water. The increased activity was detected as early as 15 s post-challenge and lasted for at least 1 h. The increased activity depended on a minimum bacterial challenge dose, and was inhibited in reactions conducted in the presence of oleyoxyethylphosphorylcholine, a site-specific PLA(2) inhibitor. In independent experiments with serum prepared from whole hemolymph, we found no PLA(2) activity was secreted into serum during the first 24 h following bacterial infection. We infer that a hemocytic intracellular PLA(2) activity is increased immediately an infection is detected. The significance of this enzyme lies in its role in launching the biosynthesis of eicosanoids, which mediate cellular immune reactions to bacterial infection.     

MicroRNAs and their role in viral infection

Recently, a class of about 22 nucleotides (nt) small RNA has been discovered in many eukaryotes, termed microRNAs (miRNAs), which have a variety of functions. Many recent findings have demonstrated that viruses can also encode their own miRNAs. Meanwhile, other findings reveal a relationship between host miRNA and viral infection. These findings suggest a tight relationship between host and viral infection via miRNA pathway. This article introduces the miRNAs encoded by viruses and reviews the advances of the interaction of the mammalian host miRNAs and viral infection.