DGGE技术在环境微生物多样性研究中的应用

微生物是污水净化的主要作用者之一.采用变性梯度凝胶电泳(denaturing gradient gel electrophresis,DGGE)方法培育和鉴定土壤微生物具有可靠性强、重复性好、方便快捷等优点,已被广泛应用于环境科学和污染防治研究领域.综述了基于PCR-DGGE技术的基本原理、关键环节及其在微生物多样性研究中的应用,同时就其自身存在的不足进行了评价并提出了解决方案.     

PCR-DGGE技术在土壤微生物多样性研究中的应用

DGGE是一种有效的微生物多样性研究技术。本文简要介绍了DGGE(denaturing gradient gel electrophoresis)的基本原理,及其在研究土壤微生物类群多样性中的应用,并对该技术自身存在的缺陷进行了评价。     

DGGE技术在森林土壤微生物多样性研究中的应用

微生物在森林土壤物质转化中扮演着重要角色,与森林的林型、土壤理化性质存在着密切关系.森林土壤微生物多样性及其变化在一定程度上反映了土壤环境的生产力和稳定性,对表征森林演替,土壤生态修复等有重要意义.变性梯度凝胶电泳(DGGE)技术测定微生物多样性具有快捷、高效和可重复性高等优点.简要介绍DGGE技术的原理,分析这一技术的局限性和优化方法.重点以实例说明该技术在森林土壤微生物多样性研究中的应用现状,展望该技术的发展前景,以期能为今后这一领域的研究提供科学依据.     

微生物分子生态学技术在湖泊微生物多样性研究中的应用

微生物是湖泊生物圈物质循环和能量流动的主要参与者,在湖泊的生态系统中起着重要的作用。但是,湖泊中存在着大量不可培养的细菌,利用传统的培养技术,无法对湖泊微生物的多样性进行深入而全面的研究,而不依赖培养的分子生物学技术的发展为此方面研究开辟了新的路径。微生物分子生态学作为分子生物学与微生物生态学交叉产生的学科,在研究湖泊微生物多样性方面已经得到了广泛的应用。主要综述了变性梯度凝胶电泳(PCR-DGGE)技术,末端限制性酶切片段长度多态性技术(T-RFLP),16SrDNA克隆文库技术等微生物分子生态学技术在研究湖泊微生物多样性方面的应用情况。     

分子生物学方法在微生物多样性研究中的应用

微生物多样性是生物多样性的重要组成部分。由于微生物和大生物（动、植物）相比,存在着多种显著差异,因此其多样性,保护及利用也有所不同,尤其是研究方法亟待完善,提高。近年来,分子生物学方法广泛用于微生物多样性的研究并取得了一系列研究成果。本文从四个方面加以介绍：１）微生物总ＤＮＡ制备及其遗传多样性检测方法;２）１６ＳｒＲＮＡ基因序列研究;３）核酸杂交分析技术;４）ＤＮＡ动力学的研究。今后的发展趋势是加     

分子生物学在石油微生物多样性研究中的应用

该文较系统地概述了目前以16SrRNA为基础的核酸探针检测技术、利用引物的PCR技术、DNA序列分析技术和电泳分离及显示技术在油藏微生物群落多样性和石油环境污染研究中的研究进展,并探讨了未来分子生物学技术在石油微生物研究中的发展方向。     

PCR-DGGE技术在农田土壤微生物多样性研究中的应用

变性梯度凝胶电泳技术(DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组DNA,并以此基因组DNA为模板,选择特异性引物F357GC和R515对16S rRNA基因的V3区进行扩增,长约230bp的PCR产物经变性梯度凝胶电泳(DGGE)进行分离后,得到不同数目且分离效果较好的电泳条带。结果说明,DGGE能够对土壤样品中的不同微生物的16S rRNA基因的V3区的DNA扩增片断进行分离,为这些DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比,变性梯度凝胶电泳(DGGE)技术能够更精确的反映出土壤微生物多样性,它是一种有效的微生物多样性研究技术。     

DNA指纹图谱技术在土壤微生物多样性研究中的应用

土壤中的微生物多样性是十分丰富的,传统培养方法对土壤微生物多样性的研究有很大局限性。近年来,各种基于16S rDNA基因的指纹图谱分析技术取得了长足的进步,并广泛应用于土壤微生物多样性的研究。这些技术主要有变性梯度凝胶电泳(DGGE)/温度梯度凝胶电泳(TGGE)、单链构象多态性(SSCP)、随机引物扩增多态性DNA(RAPD)、限制性片段长度多态性(RFLP)和扩增核糖体DNA限制性分析(ARDRA)等。对这些技术近年来在土壤微生物多样性研究领域的应用予以简短综述,并初步探讨未来几年土壤微生物分子生态学发展的方向。     

青藏高原微生物多样性研究

正青藏高原被誉为世界屋脊,其内部除平原外还有许多山峰、冰川、高山湖泊和高山沼泽,是生态环境最为奇特、生物资源最为丰富的自然资源宝库之一。同时,青藏高原的微生物群落结构及其多样性与其他区域存在巨大差异,因而具有极高的科学研究价值,并逐渐被人们所关注。研究发现气候变化对青藏高原高寒草地生态系统草丛-地境界面微生物会产生重要的影响[1]。冰川雪藻的研究主要在南部的Yala冰川开展,     

分子生态学方法在微生物多样性研究中的应用

现代的核酸技术正在给生物学的研究带来新的革命,它使人们对于生命现象的研究更为深人,成为揭示生物科学规律的有力手段。自１９８５年Ｐａｔｅ等以核酸测序技术研究微生物的生态和进化问题以对］,对微生物多样性的研究进入了一个新阶段．以往对微生物的认识基于对微生物的培养和纯种分离技术,而在自然环境中的微生物９９．５％～９９．９％的种类至今尚是不可培养的［‘］,成为正确认识微生物生态系的严重障碍。分子生态技术的应用克服了培养技术的限制,对样品进行客观的分析,更精确地揭示微生物种类和遗传的多样性。因此,该技术与…     

湖光岩玛珥湖可培养浮游细菌的BOX-PCR图谱及生物多样性分析

玛珥湖是一类具有独特地质构造特征的火山口湖泊, 目前国内外对玛珥湖内微生物多样性的研究还鲜有报道。为了解具有典型玛珥特征的湖光岩湖泊中可培养浮游细菌的种群资源特征, 采用盒式PCR (BOX-PCR) 筛选技术, 通过通用贫营养和原位湖水培养基研究在冬夏两季各水层内获得的可培养细菌种群的差异。结果表明: 不同培养基在不同水层上表现的细菌数量的变化相一致, 为夏季的5 m>1 m>13 m, 冬季的1 m>5 m>13 m, 变形菌占据优势地位, 其它为放线菌、厚壁菌和拟杆菌; 两类培养基上所获培养菌BOX-PCR图谱的多样性相似, 但菌群结构不同; 而细菌多样性在季节上的变化表现为冬季高于夏季, 这与相关湖泊内细菌微生物多样性在夏季会发生减少的特点相似; 相同季节同一水层上不同培养基上获得的培养菌门类不同, 表明此类通用性培养基在培养该环境微生物中的局限。研究的结果为探讨此类型湖泊微生物多样性与环境的关系及分离该环境下特定微生物菌株提供参考。     

Genetic relationships of Bacillus anthracis and closely related species based on variable-number tandem repeat analysis and BOX-PCR genomic fingerprinting

Variable-number tandem repeats (VNTR) analysis and BOX-repeat-based PCR (BOX-PCR) genomic fingerprinting were performed on 25 Bacillus strains to investigate the genetic relatedness of Bacillus anthracis to the closely related species. Based on VNTR analysis, all B. anthracis strains could be assigned to (VNTR)(4), which is the most commonly found type in the world. Interestingly, a (VNTR)(2) was also observed in Bacillus cereus KCTC 1661 and with an exact match to the tandem repeats found in B. anthracis. This finding has never been reported before in the closely related species. According to the BOX-PCR, B. anthracis strains clustered together and separated reliably from the closely related species. However, B. cereus KCTC 1661 was linked to the B. anthracis cluster and showed close relationships with B. anthracis strains. These results indicated that there was a strong correlation between VNTR analysis and BOX-PCR genomic fingerprinting.     

BOX-PCR is an Adequate Tool for Typing <Emphasis Type="Italic">Aeromonas</Emphasis> spp.

PCR-based methods of fingerprinting take advantage of the presence of repetitive sequences that are interspersed throughout the genome of diverse bacterial species. They include the repetitive extragenic palindromic (REP) sequence, the enterobacterial repetitive intergenic consensus sequence (ERIC) and the 154-bp BOX element. The combination of the three methods is used for fine discrimination of strains and is designated as rep-polymerase chain reaction (PCR). REP-PCR and ERIC-PCR have been shown to be useful for typing Aeromonas strains. To our knowledge, rep-PCR fingerprinting method using the BOXA1R primer has never been tested on aeromonads. In this study, the BOX-PCR fingerprinting technique was evaluated for the discrimination of strains of some Aeromonas species. All strains were typeable and the majority showed unique banding patterns. Four strains from culture collections were used to investigate the reproducibility of the method. According to our results, BOX-PCR fingerprinting is applicable for typing of Aeromonas strains and can be considered as a useful complementary tool for epidemiological studies of members of this genus.     

Diversity and Functional Analysis of Soil Culturable Microorganisms Using a Keratin Baiting Technique

Microbiology - Using chicken feathers as bait, the microbiota of soil samples collected from 20 zoos in China were enriched for keratin-utilizing microorganisms, which were cultured in the current...     

Diversity of endophytic enterobacteria associated with different host plants

Fifty-three endophytic enterobacteria isolates from citrus, cocoa, eucalyptus, soybean, and sugar cane were evaluated for susceptibility to the antibiotics ampicillin and kanamycin, and cellulase production. Susceptibility was found on both tested antibiotics. However, in the case of ampicillin susceptibility changed according to the host plant, while all isolates were susceptible to kanamycin. Cellulase production also changed according to host plants. The diversity of these isolates was estimated by employing BOX-PCR genomic fingerprints and 16S rDNA sequencing. In total, twenty-three distinct operational taxonomic units (OTUs) were identified by employing a criterion of 60% fingerprint similarity as a surrogate for an OTU. The 23 OTUs belong to the Pantoea and Enterobacter genera, while their high diversity could be an indication of paraphyletic classification. Isolates representing nine different OTUs belong to Pantoea agglomerans, P. ananatis, P. stewartii, Enterobacter sp., and E. homaechei. The results of this study suggest that plant species may select endophytic bacterial genotypes. It has also become apparent that a review of the Pantoea/Enterobacter genera may be necessary.     

Diversity of hydrocarbon-degrading Klebsiella strains isolated from hydrocarbon-contaminated estuaries

Aims:  To investigate the diversity and the catabolic capacity of oil-degrading Klebsiella strains isolated from hydrocarbon-contaminated sediments in Santos–São Vicente estuary systems in Brazil.
Methods and Results:  Klebsiella strains obtained from the estuary were characterized using 16S rRNA gene sequencing and BOX-PCR patterns, testing their catabolic capacity to degrade toluene, xylene, naphthalene and nonane, and identifying the catabolic genes present in the oil-degrading strains. Results show that Klebsiella strains were widespread in the estuary. Twenty-one isolates from the Klebsiella genus were obtained; 14 had unique BOX patterns and were further investigated. Among four distinct catabolic genes tested ( todC 1, ndoB , xylE and alkB 1), only the todC 1 gene could be amplified in two Klebsiella strains. The biodegradation assay showed that most of the strains had the ability to degrade all of the tested hydrocarbons; however, the strains displayed different efficiencies.
Conclusions:  The oil-degrading Klebsiella isolates obtained from the estuary were closely related to Klebsiella pneumoniae and Klebsiella ornithinolytica . The isolates demonstrated a substantial degree of catabolic plasticity for hydrocarbon degradation.
Significance and Impact of the Study:  The results of this study show that several strains from the Klebsiella genus are able to degrade diverse hydrocarbon compounds. These findings indicate that Klebsiella spp. can be an important part of the oil-degrading microbial community in estuarine areas exposed to sewage.     

Diversity of Microorganisms Isolated from Amber

Abstract Claims that organisms can be cultured from amber, if substantiated, would be significant contributions to our understanding of the evolution, tenacity, and potential spread of life. Three reports on the isolation of organisms from amber have been published. Cano and Borucki recently reported the isolation of Bacillus sphaericus and Lambert et al. have described a new species designated Staphylococcus succinus from 25–40 million year old Dominican amber. These characterized organisms were phylogenetically distant from extant relatives and the Staphylococcus sp. sufficiently far removed from other extant staphylococci to be considered a new species. Here we report the culture of bacteria from Dominican and previously untested 120 million year old Israeli (Lebanese lode) amber. Twenty-seven isolates from the amber matrix have been characterized by fatty-acid profiles (FAME) and/or 16S rRNA sequencing. We also performed a terminal restriction fragment pattern (TRF) analysis of the original amber before prolonged culture by consensus primer amplification of the 16S rRNA followed by restriction enzyme digestion of the amplicons. Sample TRFs were consistent with a sparse bacterial assemblage and included at least five of the isolated organisms. Finally, we microscopically mapped the internal topography of an amber slice. Received: 14 December 1998; Accepted: 16 March 1999     

Riboprinting: A Tool for the Study of Genetic Diversity in Microorganisms1

ABSTRACT. Classical morphology-based methods of taxonomic and phylogenetic analysis are inadequate in many groups of structurally simple eukaryotes. Molecular methods can generate data independently of the complexity of the organisms’ morphology. Riboprinting is one such technique, and involves restriction enzyme analysis of polymerase chain reaction amplified small subunit ribosomal RNA genes. The utility of the method is illustrated with examples from several genera of intestinal and bloodstream parasites. Among the applications of riboprinting are the detection of cryptic genetic variation within species, organism misidentifications and culture mix-ups, independent verification of DNA sequences, and the rapid generation of data useful in phylogenetic analyses.     

The Diversity and Functions of Choline Sulphatases in Microorganisms

Choline sulphates have two putative roles in microorganisms: as a reservoir of C, N and S and as osmoprotectants. Although there is no established connection to date regarding the relative distribution of these two functions in microbial communities, this information is crucial in determining the role of choline sulphate in soils, particularly in cultivated soils where S is limiting. Therefore, in order to establish such a connection, the diversity of choline sulphatase (betC) genes was investigated in this study using numerous fully sequenced microbes available in GenBank. Our genomic analyses revealed unequivocally that the betICBA operon is restricted to Rhizobiaceae family members, which live under symbiotic conditions that prevent elemental depletion. Together with the uniform genetic organisation of the betICBA operon in Rhizobiaceae, BetC appears to be both utilised for osmoprotection or S replenishment. In contrast, betC in a wide variety of free-living microbes (including fungi, archaea and bacteria) was found in a cassette encoding only BetC and a choline sulphate transporter, a configuration that appears to be responsible for fulfilling elemental S requirements. Lastly, the relatively high number of BetC sequences available allowed the identification of a specific signature sequence that discriminates between these two functions and also globally defines some conserved motifs in microbial choline sulphatases. Due to the widespread presence of BetC in microbes and the wide repartition of the betC cassette system, the potential importance of choline sulphatase in global S recycling requires further clarification.     

Diversity of the Degradation of Panthenol by Microorganisms

Several microorganisms isolated from soil were found to grow in the medium containing panthenol. The results of the investigation of the degradative metabolism of this compound demonstrated that there are two different inducible pathways.

Strain 1041 produced 3-aminopropanol and β-alanine when grown with panthenol. 3-Aminopropanol plus pantoate, as well as panthenol, supported the growth of induced culture. Both washed cells and cell extract of the organism also produced 3-aminopropanol, which was then oxidized to β-alanine. No oxidation of panthenol to pantothenic acid was observed. Isolation and identification of the products were performed. These results led to the conclusion that panthenol is hydrolyzed to pantoic acid and 3-aminopropanol as the first step, which is then followed by oxidation to β-alanine.

Strain 1091 produced pantothenic acid, but not 3-aminopropanol, from panthenol. 3-Aminopropanol plus pantoate did not support the growth of the induced culture. No degradation of 3-aminopropanol was observed. Isolation and identification of pantothenic acid and a 3-methyl-2-benzothiazolone hydrazone derivative of the aldehyde form panthenol were performed. From the results, it was confirmed that panthenol is first oxidized to pantothenic acid, which is then hydrolyzed to β-alanine and pantoic acid.

Panthenol was also oxidized to pantothenic acid by Bacillus roseus AKU 0208. The enzyme was not induced in the presence of panthenol.