未/难培养微生物可培养策略研究：机遇与挑战

微生物分布广泛、种类众多、功能多样,虽体积微小但功能强大,关乎人类的安全健康和生态的稳定发展,在整个地球生命系统中起着举足轻重的作用。17世纪以来,研究者们一直努力获得、了解和利用这些微生物,然而目前分离方法的局限性使得环境中绝大部分微生物仍不能被纯培养,严重阻碍了我们对微生物生命活动规律的认知。因此,如何分离获得这些仍未被培养出来的“暗物质”是微生物研究面临的严峻挑战和重大机遇。本文分析了环境中制约微生物分离培养的因素,综述未/难培养微生物可培养研究的最新进展,着重论述优化的传统培养方法及网络导向培养、膜扩散培养、微流控分选培养和细胞分选培养等新型技术的应用,并对未来研究进行展望,探索多技术联合使用策略,为未/难微生物资源的挖掘及开发利用提供借鉴。     

Limited diffusive fluxes of substrate facilitate coexistence of two competing bacterial strains

Soils are known to support a great bacterial diversity down to the millimeter scale, but the mechanisms by which such a large diversity is sustained are largely unknown. A feature of unsaturated soils is that water usually forms thin, poorly-connected films, which limit solute diffusive fluxes. It has been proposed, but never unambiguously experimentally tested, that a low substrate diffusive flux would impact bacterial diversity, by promoting the coexistence between slow-growing bacteria and their potentially faster-growing competitors. We used a simple experimental system, based on a Petri dish and a perforated Teflon membrane to control diffusive fluxes of substrate (benzoate) whilst permitting direct observation of bacterial colonies. The system was inoculated with prescribed strains of Pseudomonas, whose growth was quantified by microscopic monitoring of the fluorescent proteins they produced. We observed that substrate diffusion limitation reduced the growth rate of the otherwise fast-growing Pseudomonas putida KT2440 strain. This strain out-competed Pseudomonas fluorescens F113 in liquid culture, but its competitive advantage was less marked on solid media, and even disappeared under conditions of low substrate diffusion. Low diffusive fluxes of substrate, characteristic of many unsaturated media (e.g. soils, food products), can thus promote bacterial coexistence in a competitive situation between two strains. This mechanism might therefore contribute to maintaining the noncompetitive diversity pattern observed in unsaturated soils.     

Comparison of plate count agar and R2A medium for enumeration of heterotrophic bacteria in natural mineral water

In order to recover as many viable bacteria as possible from natural mineral water, in this study we have compared the counts obtained with the standard method (pour plate procedure with Plate Count Agar (PCA)) and counts with alternative test methods (PCA/spread plates, R2A medium/pour plates and R2A medium/spread plates). The results showed that counts with R2A medium/spread plates at 22°C and after a 7-day incubation period were more than 343% higher than those obtained with PCA/pour plate method. At 37°C and after a 3-day incubation period, the R2A pour plate technique gave counts about 368% greater than for the standard method. Moreover, while Pseudomonas, Comamonas and Acinetobacter species were isolated both from PCA and R2A medium, Flavobacterium spp. and Arthrobacter spp. were isolated only from R2A medium. For its higher productivity, R2A medium should be recommended for heterotrophic plate counts in natural mineral water.     

肠道微生物培养的研究进展及应用

肠道中栖居着组成复杂、功能多样的微生物群,这些微生物群在宿主免疫、营养吸收、代谢调节等方面发挥着重要作用。随着测序技术的快速发展,肠道微生物研究通过16S rRNA基因测序和宏基因组测序产生了大量的数据,其中许多未组装的序列成为微生物“暗物质”。近年来,不少研究利用多种不同微生物分离培养方法,结合高通量鉴定技术,从人、小鼠、猪肠道中分离了大量的微生物,丰富了菌株资源,为解析微生物“暗物质”以及后续肠道微生物功能和应用研究提供了基础和保障。尽管微生物的可培养性受到多种因素的影响,大部分微生物尚处于“未培养”的状态,但无论是病因研究还是生理和遗传特征的解析都离不开微生物实体资源的获取。肠道微生物的分离培养对微生物研究从关联分析向菌群功能验证、因果机制解析和功能菌株开发的深入研究具有重要意义。本文旨在探讨和综述影响微生物可培养性的因素,总结回顾肠道微生物的培养方法并阐述肠道微生物培养研究的进展,以期为肠道微生物培养研究提供新的视角。     

粘细菌产生的水解酶类研究进展

粘细菌隶属于δ变形菌纲(Deltaproteobacteria),是重要的药源微生物类群,但是其分离培养困难,严重限制了粘细菌资源的发掘和开发利用。粘细菌是微生物捕食者,通过产生丰富多样的胞外水解酶,如淀粉酶、蛋白酶、几丁质酶、纤维素酶、磷酸酶、蛋白酶等来裂解其他微生物或分解纤维素等作为营养物质来源。目前,粘细菌分离纯化技术主要是利用被捕食菌或纤维素诱导法。可以说,粘细菌胞外水解酶是研究其分离培养方法的物质基础。然而,目前研究者们对粘细菌产生的水解酶类关注较少。本文主要对粘细菌产生的水解酶种类、性质及其功能进行归纳总结,为今后粘细菌分离培养技术和开发利用等相关研究提供参考。     

Characterization of a gene encoding cellulase from uncultured soil bacteria

To detect cellulases encoded by uncultured microorganisms, we constructed metagenomic libraries from Korean soil DNAs. Screenings of the libraries revealed a clone pCM2 that uses carboxymethyl cellulose (CMC) as a sole carbon source. Further analysis of the insert showed two consecutive ORFs (celM2 and xynM2) encoding proteins of 226 and 662 amino acids, respectively. A multiple sequence analysis with the deduced amino acid sequences of celM2 showed 36% sequence identity with cellulase from the Synechococcus sp., while xynM2 had 59% identity to endo-1,4-beta-xylanase A from Cellulomonas pachnodae. The highest enzymatic CMC hydrolysis was observable at pH 4.0 and 45 degrees C with recombinant CelM2 protein. Although the enzyme CelM2 additionally hydrolyzed avicel and xylan, no substrate hydrolysis was observed on oligosaccharides such as cellobiose, pNP-beta-cellobioside, pNP-beta-glucoside, and pNP-beta-xyloside. These results showed that CelM2 is a novel endo-type cellulase.     

Growth ofThiobacillus ferrooxidans on solid media containing heterotrophic bacteria

Summary the study of the chemolithotrophic bacteriumThiobacillus ferrooxidans, an organism important in mining and metallurgical applications, has always been complicated by the inability to cultivate and enumerate many wild strains on solid agar. Here, we have described a plate-count procedure that uses an acidophilic heterotrophic bacterium to scavenge organic material from normal agar, permitting colony formation of the autotroph without visible evidence of growth of the heterotroph. This technique could enable anyT. ferroxidans isolate, including those that are agar-sensitive, to grow and develop colonies on solid medium.     

Changes of α-Glycerophosphate Dehydrogenase Activity in Fatty Liver of Rats by Amino Acid Imbalance

An aminopeptidase was purified from an aqueous extract of mullet roe in the presence of 2-mercaptoethanol by fractionation with ammonium sulfate and column chromatography on DEAE-cellulose and Sephadex G-200. The molecular weight of the enzyme was 184,000 by gel filtration, and the enzyme appeared to consist of two homogenous subunits. The optimal pH and optimal temperature for activity were 7.4 and 45°C, respectively. Puromycin, p-chloromercuribenzoic acid, and o-phenanthroline inhibited the enzyme n on-competitively (their Ki = 1.34 μm, 0.113mm and 0.145 mm, respectively), while 2-mercaptoethylamine was competitive (Ki = 0.056 mm). The enzyme was also inhibited by l-amino acids, in particular glutamic acid. The enzyme could hydrolyze a variety of α-aminoacyl β-naphthylamides and was most active on l-alanyl-β-naphthylamide. Judging from these properties, the mullet roe aminopeptidase resembles soluble alanyl amino-peptidase [EC 3.4.11.14].     

环凯食(饮)具大肠菌群检验纸片检测能力测试研究

研究以营养琼脂培养基的计数结果为基准,采用幅度卫生部食检所监制的两种不同厂家生产的食（饮）具大肠菌群检验纸片作为对照,对环凯食（饮）具大肠菌群检验纸片进行了检测能力测试研究,在测试中采用10cells/mL,50cells/mL和100cells/mL3个不同菌液浓度,环凯纸片的检测结果和营养琼脂平板计数结果基本一致。与其它两种纸片的检测结果无明显差异。完全可以用于食（饮）具大肠菌群的监督检验。     

基于微生物互作的共培养分离技术研究进展

自然条件下,微生物以一种复杂的群落形式生活,细胞周围充斥着由相邻细胞产生的各类代谢物,使各细胞间存在多样的互作形式,影响彼此的生长。不同种类的菌株共培养时,营养缺陷型菌株可以利用其他菌株产生的代谢产物进行生长;共培养还可以改变微环境、刺激菌株沉默基因的表达及改变菌株的生存状态。近年来,基于模拟菌株间的互作关系而发展起来的共培养技术逐步应用于未培养微生物的分离工作中,并被认为能有效提高未培养微生物的分离效率。结合已发表的相关文献资料,综合分析潜在共培养的类群多样性以及共培养分离技术的先进性与应用现状等,以期为微生物分离技术的发展及微生物资源的发掘提供参考。     

浙江晚稻稻种非致病细菌多样性初步研究

为了解特定生态环境中晚稻稻种的非致病细菌多样性以更有效地治理水稻主要病害,于1996－2001年间对采自浙江平原、丘陵及山区的606份稻种样本进行了非致病细菌种类、频率及对水稻纹枯病和恶苗病的抑菌作用研究。从中分离出9605个菌株,经致病性测定、菌落形态及部分细菌学特征（革兰氏染色、KMB培养基上的荧光色素和扩散性非荧光色素、YDC培养基上黄色菌落的产生、好气性、鞭毛及芽孢的染色镜检等）测定后,选出代表菌株622个,连同80个对照菌株用Biolog及脂肪酸分析法（FAME）进行测试。鉴定出Pseudomonas属11个种或型及其他14属的23种非致病细菌,并发现Pseudomonas属中55％的种及其他属中49％的种存在对水稻纹枯病或恶苗病的拮抗菌株,但不同种间的抑菌率存在较大差异。3类稻区均以Bacillus spp.、Acinetobacter spp.和P.putida等3个种群为主导并同时存在许多其他相似种,其中7个常见种的分离频率在不同稻区有显著差异。     

厌氧培养皿及厌氧培养管的临床应用——对传统的厌氧菌分离培养及鉴定方法的简化

利用我们研制的厌氧培养皿及管,共检验了39份牙髓炎及牙周炎标本,分离出厌氧菌59株,标本中厌氧菌分离的阳性率为100％。其中类杆菌28株,消化链球菌9株,消化球菌7株,韦荣氏球菌5株,真杆菌2株,梭形杆菌8株。在类杆菌中产黑素类杆菌21株,占类杆菌总数的75％。结果表明使用厌氧培养皿及管研究厌氧菌简便、有效、经济,适于基层单位应用。     

Isolation and characterization of long-chain alkane–degrading Acinetobacter sp. BT1A from oil-contaminated soil in Diyarbakır,in the Southeast of Turkey

A strain of long-chain alkane–degrading bacteria, BT1A, was isolated from oil-contaminated soil in Diyarbak?r, in the southeast of Turkey. Morphological, biochemical, and physiological characterization and 16S rRNA gene sequence analysis showed that the strain BT1A was a member of Acinetobacter genus, and it was found to be closely related to Acinetobacter baumannii. The strain BT1A was able to utilize crude petroleum as carbon and energy sources in order to grow. Among the aliphatic hydrocarbons, growth was observed only in the medium containing long-chain alkanes (tridecane, pentadecane, and hexadecane) and squalene. Hexadecane was the most preferred hydrocarbon among the long-chain alkanes. Gas chromatography–mass spectrometry (GC-MS) analysis showed that BT1A degraded 83% of n-alkanes of 1% crude oil in 7 days. The present study indicates that the isolated strain can well be used for biodegradation of hydrocarbons in oil-contaminated sites.     

以固氮菌和解磷菌筛选拮抗作物病害的细菌组合

用选择性培养基从土壤分离筛选了22 种具有固N或解P能力的细菌,不进行纯化,分组混合接种于15种液体培养基,进行继代培养,每代培养48 h.第12代时,各细菌组合的菌类数目及比例达到稳定状态,共获得15个细菌组合,各组合均含有2～3种细菌.对茄黄萎病病原菌(Verticillium dahliae)、棉花立枯病病原菌(Rhizoctonia solani)和黄瓜角斑病病原菌(Pseudomonas syringae)等9种植物病原菌进行平皿抑菌试验,筛选出7个具有抑菌作用的细菌组合,进一步从植株盆栽试验中筛选出6个有防病作用的组合,其中C₇和C₁₅表现出最明显的抑菌防病效果.C₁₅保持了固N能力,C₄、C₅、C₇、C₉、C₁₄和C₁₅保持了解P能力.结果表明,这是一条可获得具有协同作用的、稳定的多功能生防菌剂的途径.     

Effect of media composition,including gelling agents,on isolation of previously uncultured rumen bacteria

The aim of this study was to develop novel anaerobic media using gellan gum for the isolation of previously uncultured rumen bacteria. Four anaerobic media, a basal liquid medium (BM) with agar (A‐BM), a modified BM (MBM) with agar (A‐MBM), an MBM with phytagel (P‐MBM) and an MBM with gelrite (G‐MBM) were used for the isolation of rumen bacteria and evaluated for the growth of previously uncultured rumen bacteria. Of the 214 isolates composed of 144 OTUs, 103 isolates (83 OTUs) were previously uncultured rumen bacteria. Most of the previously uncultured strains were obtained from A‐MBM, G‐MBM and P‐MBM, but the predominant cultural members, isolated from each medium, differed. A‐MBM and G‐MBM showed significantly higher numbers of different OTUs derived from isolates than A‐BM (P < 0·05). The Shannon index indicated that the isolates of A‐MBM showed the highest diversity (H′ = 3·89) compared with those of G‐MBM, P‐MBM and A‐BM (H′ = 3·59, 3·23 and 3·39, respectively). Although previously uncultured rumen bacteria were isolated from all media used, the ratio of previously uncultured bacteria to total isolates was increased in A‐MBM, P‐MBM and G‐MBM.     

Ecogeochemistry of the subsurface food web at pH 0–2.5 in Iron Mountain,California, U.S.A.

Pyrite oxidation in the underground mining environment of Iron Mountain, California, has created the most acidic pH values ever reported in aquatic systems. Sulfate values as high as 120000 mg l^–1 and iron as high as 27600 mg l^–1 have been measured in the mine water, which also carries abundant other dissolved metals including Al, Zn, Cu, Cd, Mn, Sb and Pb. Extreme acidity and high metal concentrations apparently do not preclude the presence of an underground acidophilic food web, which has developed with bacterial biomass at the base and heliozoans as top predators. Slimes, oil-like films, flexible and inflexible stalactites, sediments, water and precipitates were found to have distinctive communities. A variety of filamentous and non-filamentous bacteria grew in slimes in water having pH values <1.0. Fungal hyphae colonize stalactites dripping pH 1.0 water; they may help to form these drip structures. Motile hypotrichous ciliates and bdelloid rotifers are particularly abundant in slimes having a pH of 1.5. Holdfasts of the iron bacterium Leptothrix discophora attach to biofilms covering pools of standing water having a pH of 2.5 in the mine. The mine is not a closed environment – people, forced air flow and massive flushing during high intensity rainfall provide intermittent contact between the surface and underground habitats, so the mine ecosystem probably is not a restricted one.