宏基因组学技术及其在微生物学研究中的应用

自然环境中超过99%的微生物不能用传统的方法进行纯培养,这大大限制了人类认识微生物世界的视野。宏基因组学是20世纪90年代发展起来的,它是通过直接提取环境中全部微生物的总基因组DNA并克隆到合适的可培养微生物宿主中来筛选目的基因的新方法。它已广泛应用于农业、林业、土壤、海洋、人体医学等各个领域,从发酵工艺到生物能源再到环境治理等与人们生活息息相关的各个方面都起到突出的作用。本文重点介绍了宏基因组学在微生物学研究中的应用,旨在说明宏基因组技术可以获得难以人工培养的微生物基因组信息,这使我们在很大程度上提高了对环境中微生物资源多样性的认识,并使其极大的为人类有效开发利用。     

基于微生物基因组的新型天然产物发现策略

微生物天然产物的生物合成是一个从基因到化合物的过程。微生物基因组内未被发掘的孤儿或沉默途径所编码的新型次级代谢产物的合成潜力远远超过现已发现的代谢产物数量。微生物基因组大规模测序的广泛开展,为天然产物的发现和研究提供了新的研究领域和契机。本文主要介绍基于微生物基因组序列的新型天然产物的发现策略及其研究进展。可以预计,随着实验技术的不断发展,采用基于基因的药物发现模式可以充分发掘微生物产生天然产物的潜力,微生物基因组内的孤儿或沉默途径编码的未知代谢产物将成为新药开发的重要源泉。     

美科学家在土壤中发现了新型抗生素

据 BBC 1月20日报道,美国科学家正在呼吁公众加入一项土壤样本全球征集行动,以便其利用土壤中某种特殊成分研制新药。洛克菲勒大学研究团队最近在土壤样本中发现一种潜在的新型抗生素,科学家们大受鼓舞,并打算从世界各地征集土壤样本进行检测。该研究小组说,大自然中存在许多未经培养的细菌,可以提供大量的新型有机分子,其中许多可被拿来研制新药。基因编码小分子实验室的负责人布雷迪博士说：“我们希望通过努力,了解自然界微生物多样性以及化学多样性的分布,借以发现新的药物并改良现有的药物。”“我们希望得到帮助,能够收集到来自世界各地的土壤样本。”研究小组说道。     

乌头根际土壤细菌群落多样性的PCR-DGGE分析

目的:研究不同生长期乌头根际土壤微生物细菌群落结构的差异。方法:采用PCR-DGGE技术,研究不同产地不同健康状况的乌头根际土壤细菌群落结构的变化。结果:健康与患病乌头根际土壤细菌群落结构存在差异性,且患病植株具有能引起植物患病的病原种。结论:患病植株改变了乌头根际土壤的微生物细菌群落多样性,致病菌的存在导致了乌头植株的患病。根际土壤的健康状况对土壤微生物群落结构有显著影响,根际微生物群落结构的不同将导致植物对病原菌的抗性及品种差异。     

乌头根际土壤真菌群落多样性的PCR-DGGE分析

目的:研究不同生长期乌头根际土壤微生物真菌群落结构的差异。方法:采用PCR-DGGE技术,研究不同产地不同健康状况的乌头根际土壤真菌群落结构的变化。结果:健康与患病乌头根际土壤真菌群落结构存在差异性,且致病菌与正常菌株之间存在着拮抗关系。结论:患病植株改变了乌头根际土壤的微生物真菌群落多样性,致病菌的存在抑制了正常植株真菌群落的繁殖。根际土壤的健康状况对土壤微生物群落结构有显著影响,根际微生物群落结构的不同将导致植物对病原菌的抗性及品种差异。     

中医药院校药类《微生物学》病原微生物教学内容改革

结合当前药学教学改革背景和药学类《微生物学》教学现状,分析了药学类专业《微生物学》中病原微生物教学内容改革的必要性,并根据药学专业特性对病原微生物的教学内容作了相应的优化设计,建议将细菌、病毒和真菌各论进行合并,单独列出"药物微生物多样性"一章,淡化纯病原的讲解,加强药效及药品质量检测相关微生物的内容;淡化病原的诊疗技术,强化微生物的检测与应用。     

丹参栽培对土壤微生物区系组成特征的影响

目的 研究栽培丹参对土壤微生物区系组成的影响.方法 采集中国丹参主产地的丹参根际土和对照土,采用人工培养法培养土壤微生物,数据采用SPSS软件分析.结果 栽培丹参可使根际土的微生物数量增加;氨化细菌、好气性固氮细菌、好氧纤维素分解菌、芳香族化合物分解菌、有机磷分解菌、无机磷分解菌、铁细菌和硅酸盐分解菌减少,亚硝化细菌、硝化细菌、反硝化细菌、嫌气性固氮菌、厌氧纤维素分解菌、硫化细菌和反硫化细菌增加;增加了根际土的细菌多样性,影响其分布;抑制硅酸盐分解菌繁殖,促进硝化细菌繁殖;栽培丹参与野生丹参的根际微生物有较大差异.结论 栽培丹参可筛选根际土壤中的微生物,破坏了原有的种群结构;硅酸盐分解菌是丹参栽培的有益微生物菌群.     

红树林土壤微生物与其代谢产物研究进展

分布于海岸潮间带的红树林独特土壤生境中蕴藏着丰富的微生物资源,然而,由于认识水平和技术手段的限制,至今人们对红树林土壤微生物的系统了解仍较为有限。近年来,随着科学技术的发展,红树林土壤微生物及其相关活性代谢产物的研究引起了较多的关注。文中主要阐述了红树林土壤微生物物种多样性,代谢产物及其活性物质,生物修复作用等相关方面的研究进展,并展望了该领域研究的重要性和发展趋势。     

乌头不同生长期根际土壤微生物动态变化特征的研究

目的:研究不同生长期患病与健康乌头植株根际土壤微生物动态变化特征。方法:采用纯培养法测定土壤微生物的数量。结果:不同月份及不同生长状况的乌头根际土壤细菌数量具有显著性差异、且患病植株随着生长期不同呈逐渐下降趋势、健康植株则呈先降低后增强的趋势;而放线菌和真菌数量变化不显著。结论:健康与患病植株细菌数量差异较大,研究结果为乌头病害研究提供了科学参考依据。     

微生物药物外排泵及其抑制剂研究

细菌自身的不断进化及抗生素的不合理使用导致了耐药性细菌的出现和蔓延，微生物的主动外排是导致细菌产生多重耐药的重要原因。微生物外排泵的研究是目前的热点之一，也被认为是开发新抗生素的理想靶标。本文从基因组和比较基因组水平对外排泵的类型、作用机制、表达调控及研究方法等方面概述，并结合正在开展的分枝杆菌的药物外排泵进行了比较基因组分析。这些工作有利于开发新的抗生素或抗生素增效剂。     

Metagenomic sequencing-driven multidisciplinary approaches to shed light on the untapped microbial natural products

The advantage of metagenomics over the culture-based natural product (NP) discovery pipeline is the ability to access the biosynthetic potential of uncultivable microbes. Advances in DNA sequencing are revolutionizing conventional metagenomics approaches for microbial NP discovery. The genomes of (in)cultivable bugs can be resolved straightforwardly from environmental samples, enabling in situ prediction of biosynthetic gene clusters (BGCs). The predicted chemical diversities could be realized not only by heterologous expression of gene clusters originating from DNA synthesis or direct cloning, but also potentially by bioinformatic-directed organic synthesis or chemoenzymatic total synthesis. In this review, we suggest that metagenomic sequencing in tandem with multidisciplinary approaches will form a versatile platform to shed light on a plethora of microbial ‘dark matter’.     

Studies on screening and isolation of esterase inhibitors from soil microorganisms(I). Identification of strain DMC-498 producing esterase inhibitors

□To find microorganisms producing esterase inhibitors, microbes were isolated from soil samples that were collected at different locations in Korea and screened for inhibitory activities. One of the inhibitor-producing strains was named strain DMC-498. This strain was found to be a new species of the genusStreptomyces by comparison with the characteristics of morphology and metabolism of the other species of the genus.     

Soil surface colonization by phototrophic indigenous organisms,in two contrasted soils treated by formulated maize herbicide mixtures

Soil phototrophic microorganisms, contributors to soil health and food webs, share their particular metabolism with plants. Current agricultural practices employ mixtures of pesticides to ensure the crops yields and can potentially impair these non-target organisms. However despite this environmental reality, studies dealing the susceptibility of phototrophic microorganisms to pesticide mixtures are scarce. We designed a 3 months microcosm study to assess the ecotoxicity of realistic herbicide mixtures of formulated S-metolachlor (Dual Gold Safeneur^®), mesotrione (Callisto^®) and nicosulfuron (Milagro^®) on phototrophic communities of two soils (Limagne vertisol and Versailles luvisol). The soils presented different colonizing communities, with diatoms and chlorophyceae dominating communities in Limagne soil and cyanobacteria and bryophyta communities in Versailles soil. The results highlighted the strong impairment of Dual Gold Safeneur^® treated microcosms on the biomass and the composition of both soil phototrophic communities, with no resilience after a delay of 3 months. This study also excluded any significant mixture effect on these organisms for Callisto^® and Milagro^® herbicides. We strongly recommend carrying on extensive soil studies on S-metolachlor and its commercial formulations, in order to reconsider its use from an ecotoxicological point of view.     

Effects of tetracycline on the relationship between the microbial community and oxidative stress in earthworms based on canonical correlation analysis

In this study, Eisenia fetida was taken as the test organism and tetracycline was taken as the stress compound. The artificial soil test was conducted to study the utilization intensity of different carbon sources (the Biolog-microplate supplied) by microorganisms under different stress times and stress concentrations. The changes in the in vivo key enzymes activities of earthworms and oxidative stress indicators, such as malondialdehyde (MDA), were explored. The canonical correlation analysis method was the first used to establish a analysis-model to explore the relationship between the functional diversity of microbial community and the oxidative stress in earthworms in vivo under different stress times and concentrations. Research shows: 1) after tetracycline stress, in the earthworm, the CAT, POD, SOD, GPX were related to the microbes that use carbohydrate carbon sources; the GST and AChE were related to the microbes that use polymer carbon sources; the MDA was related to the microbes that use carbon sources: amino acid, carboxylic acid and phenolic acid. 2) Under low concentrations of tetracycline stress, there was no significant relationship between the functional diversity of the microbial communities and the effects of oxidative stress at this concentration. The high concentration of tetracycline can be utilized to screen probiotics that alleviate the effects of oxidative stress. 3) The utilization of carbon sources by microbial community in the earthworm after stress can be used as biomarker of ecotoxicology. It provides a basic theoretical for adding beneficial carbon sources to combat oxidative damage in vivo.     

Effect of PGPR Serratia marcescens BC-3 and AMF Glomus intraradices on phytoremediation of petroleum contaminated soil

Soil contamination caused by petroleum hydrocarbons has become a worldwide environmental problem. Microorganism combined with phytoremediation appears to be more effective for removal and/or degradation of petroleum hydrocarbons from impacted soils. The current study investigated the effect of inoculated with PGPR Serratia marcescens BC-3 alone or in combination with AMF Glomus intraradices on the phytoremediation of petroleum-contaminated soil. Pot experiments were conducted to analyze the effect on plant and soil for 90 days in greenhouse. The inoculation treatments showed higher plant biomass and antioxidant enzyme activities than the non inoculation control. Inoculation treatments also improved rhizosphere microbial populations in petroleum contaminated soil. The degradation rate of total petroleum hydrocarbons with PGPR and AMP co-inoculation treatment was up to 72.24 %. The results indicated that plant combined with microorganisms for remediation of petroleum hydrocarbons would be a feasible method.     

Structure-toxicity relationship of chloroacetanilide herbicides: Relative impact on soil microorganisms

The research was carried out to ascertain the effect of three chloroacetanilide herbicides, alachlor, butachlor and pretilachlor on soil microbial biomass growth and activity. Laboratory experiments were performed in a silty clay loam soil to relate changes of soil enzymatic activity to the herbicide persistence under laboratory condition up to 42 days at three application rates. The results showed that all the three herbicides caused enhancement of dehydrogenase activity. Higher concentrations of herbicide resulted in enhancement of the enzymatic activity. In addition, a similar trend was observed in β-glucosidase and acid phosphatase activity, although urease activity decreased upon incubation for 42 days as compared with initial soil incubation values. Based on the extent of impact for dehydrogenase activity in soil, the order was pretilachlor>alachlor>butachlor; whereas in case of urease activity, the order changed to pretilachlor>butachlor>alachlor. The soil half-lives of alachlor, butachlor and pretilachlor respectively, were 9.3, 12.7 and 7.3 days, which could be accounted for in terms of their respective chemical structures, as well as variable adsorption, degradation, differential effects of the agents on soil microbes. Soil management practices and the differing physicochemical properties of the herbicides may contribute to their rates of decay in soil.     

Toll-like receptors: role of inflammation and commensal bacteria

TLR ligands are present on both commensal and pathogenic microbes. Intestinal epithelial cells (IECs) have been observed to be largely unresponsive to TLR ligands. This observation has partly been explained by the fact that TLR expression on IECs is sparse. The discovery of the Toll-like receptors finally identified the innate immune receptors that were responsible for many of the innate immune functions that had been studied for many years. Interestingly, TLRs seem only to be involved in the cytokine production and cellular activation in response to microbes, and do not play a significant role in the adhesion and phagocytosis of microorganisms. One member of this group, interleukin-1β (IL-1β), together with tumour-necrosis factor (TNF), is defined as an alarm cytokine. It is secreted by macrophages and initiates inflammation on activation of TLRs.     

Soil respiration,ATP content,and Photobacterium toxicity test as indicators of metal pollution in soil

The effects of heavy metal deposition from four different metalworks on soil respiration, ATP content, and toxicity to Photobacterium phosphoreum were examined. Soil samples were collected along a transect through the pollution gradients. Vegetation and soil types were homogeneous throughout the transects. Soil respiration was measured as CO₂ production in closed bottles using an IR-gas analyzer. The ATP content was measured with a bioluminometric assay after trichloracetic acid extraction of soil. The toxicities of distilled water extracts of soil samples were evaluated by the standard P. phosphoreum toxicity test and the metal content of these water extracts was measured as the bioavailable fraction of the heavy metal pollution in the soil. Soil respiration and ATP content were strongly affected by the heavy metal content in the soil. The correlation between the methods was high. The P. phosphoreum procedure can be used to estimate the toxicity of soils heavily polluted by metals. However, soil respiration and ATP content appear to be more sensitive indicators of soil pollution. © 1994 by John Wiley & Sons, Inc..