Characterization of depth-related microbial communities in lake sediment by denaturing gradient gel electrophoresis of amplified 16S rRNA fragments

The characterization of microbial communities of different depth sediment samples was examined by a culture-independent method and compared with physicochemical parameters, those are organic matter （OM）, total nitrogen （TN）, total phosphorus （TP）, pH and redox potential （Eh）. Total genomic DNA was extracted from samples derived from different depths. After they were amplified with the GC-341 f/907r primer sets of partial bacterial 16S rRNA genes, the products were separated by denaturing gradient gel electrophoresis （DGGE）. The profile of DGGE fingerprints of different depth sediment samples revealed that the community structure remained relatively stable along the entire 45 cm sediment core, however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident. The principle coordinates analysis suggested that the bacterial communities along the sediment core could be separated into two groups, which were located 0-20 cm and 21-45 cm, respectively. The sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belonged to Bacillus, Bacterium, Brevibacillus, Exiguobacterium, γ-Proteobacterium, Acinetobacter sp. and some uncultured or unidentified bacteria. The results indicated the existence of highly diverse bacterial community in the lake sediment core.

Analysis of interspecies adherence of oral bacteria using a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis profiling

Information on co-adherence of different oral bacterial species is important for understanding interspecies interactions within oral microbial community. Current knowledge on this topic is heavily based on pariwise coaggregation of known, cultivable species. In this study, we employed a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） to systematically analyze the co-adherence profiles of oral bacterial species, and achieved a more profound knowledge beyond pairwise coaggregation. Two oral bacterial species were selected to serve as ＂bait＂： Fusobacterium nucleatum （F. nucleatum） whose ability to adhere to a multitude of oral bacterial species has been extensively studied for pairwise interactions and Streptococcus mutans （S. mutans） whose interacting partners are largely unknown. To enable screening of interacting partner species within bacterial mixtures, cells of the ＂bait＂ oral bacterium were immobilized on nitrocellulose membranes which were washed and blocked to prevent unspecific binding. The ＂prey＂ bacterial mixtures （including known species or natural saliva samples） were added, unbound ceils were washed off after the incubation period and the remaining cells were eluted using 0.2 mol.L1 glycine. Genomic DNA was extraeted, subjeeted to 16S rRNA PCR amplification and separation of the resulting PCR produets by DGGE. Selected bands were recovered from the gel, sequenced and identified via Nucleotide BLAST searches against different databases. While few bacterial species bound to S. mutans, consistent with previous findings F.. nucleatum adhered to a variety of bacterial species including uncultivable and uneharacterized onesl This new approach can more effectively analyze the co-adherence profiles of oral bacteria, and could facilitate the systematic study of interbacterial binding of oral microbial species.

Establishment and Comparison of Pulsed-field Gel Electrophoresis,Multiple-locus Variable Number Tandem Repeat Analysis and Automated Ribotyping Methods for Subtyping of Citrobacter Strains

Objective To establish and compare the pulsed-field gel electrophoresis （PFGE）, multiple-locus variable number tandem repeat analysis （MLVA） and automated ribotyping for subtyping of Citrobacter strains. Methods PFGE protocol was optimized in terms of plug preparation procedure, restriction enzymes and configuration of electrophoretic parameters. MLVA method was evaluated by finding variable number tandem repeats in two genomes of Citrobacter strains. The ribotyping was performed by using the automated RiboPrinter system. Results We optimized the plug preparation procedure, focused on the cell suspension concentration （turbidity of 2.5 to 3.5）, SDS addition （no SDS needed） and lysis time （1 h）, and selected the appropriate restriction enzyme （Xbal） and the electrophoretic parameters （1.0 s-20.0 s for 19 h） of PFGE. There was nearly no discriminatory power of MLVA between Citrobacter strains. For 51 Citrobacter strains, automated ribotyping gave a D-value of 0.9945, while PFGE gave a D-value of 0.9969. Both PFGE and automated ribotyping clustered strains from the same sources （with the same species from the same place at the same time identified as the same source） and divided strains from different sources （from different years, places and hosts） into different subtypes. Conclusion PFGE protocol established in this paper and automated ribotyping are suitable for application in Citrobacter subtyping.

Variations in Laboratory-Scale Actinomycete Communities Exposed to Cadmium as Assessed by Denaturing Gradient Gel Electrophoresis Profiles

The actinomycete populations and functions in cadmium （Cd） contaminated soil were investigated by the cultivation- independent molecular methods. The genomic DNA was extracted and purified from soil adulterated with various con- centrations of Cd in the laboratory. The partial 16S rDNA genes were amplified by polymerase chain reaction （PCR） using specific primers bound to evolutionarily conserved regions within these actinomycete genes. The diversity in PCR- amplified products, as measured by denaturing gradient gel electrophoresis （EGGE）, was used as a genetic fingerprint of the population. Principle component analysis and Shannon-Weaver diversity index （H） analyses were used to analyze the DGGE results. Results showed that the two principal components accounted for only a low level of the total variance. The value H in contaminated soil was lower than that in the control at later stages of cultivation, whereas at earlier stages it was higher. Among the six sampling time points, the first, fifth and sixth weeks had the highest values of H. Significantly negative correlations between bioavallable Cd concentration and H values existed in the samples from weeks 2 （R = 0.929, P 〈 0.05） and 4 （R = 0.909, P 〈 0.05）. These results may shed light on the effect of Cd on the soil environment and the chemical behavior and toxicity of Cd to actinomycetes.

Optimization of Pulsed-field Gel Electrophoresis Procedure for Bacillus cereus

In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of intact genomic DNA, and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation, were controlled. Optimal cellular growth （Luria-Bertani agar plates for 12-18 h） and lysis conditions （4 h incubation with 500 μg/mL lysozyme） produced sharp bands on the gel.

Antimicrobial resistance, genotypic characterization and pulsed-field gel electrophoresis typing of extended spectrum β-lactamases-producing clinical Escherichia coli strains in Macao, China

Background The rise of the production of CTX-M class extended spectrum β-lactamases （ESBLs） has been well documented in traveling countries but no data are found for Macao, an international travel city. The objectives of this study were to identify the antimicrobial resistance pattern, and determine the prevalence, genotype and clonal relationship of ESBLs in 209 clinical Escherichia coli strains from Macao, China. Methods Antimicrobial susceptibility test was performed to determine the resistance patterns of the isolates using the disk diffusion method with 17 antimicrobial agents. Phenotypic detection was screened and confirmed according to the Clinical and Laboratory Standards Institute. Genotypic characterization was detected by isoelectric focusing analysis, polymerase chain reaction and sequencing. The clonal relationship between the different ESBL isolates was studied by pulsed-field gel electrophoresis （PFGE）. Results Imipenem and meropenem exhibited 100% susceptible among 209 strains. Overall, 82.3%, 67.3%, 52.9%, 51.2% and 51.0% of the isolates displayed resistance to ampicillin, tetracylcline, ciprofloxacin, sulfamethoxazole trimethoprin and gentamycin. The prevalence rate of ESBLs was 30.1%. Antibiotic resistances were found to be significantly higher among the ESBL producing group compared to non-ESBL producing group. We detected CTX-M-14 to be the major genotypic characterization of ESBLs （76.2%）. Two strains showed indistinguishable patterns by PFGE. Conclusions The prevalence of antimicrobial resistance is alarming high in Macao. Antimicrobial resistance is significantly higher among the ESBL producing group. This study documented CTX-M-14 as the predominant ESBL type. Although indistinguishable pattern was found between two strains, it was too small to decide whether any of the investigated strains was epidemic. Our findings may be also pertinent for other geographic areas undergoing similar travel characteristics to understand the corresponding effects on bacterial populations.

Monitoring of microbial community structure and succession in the biohydrogen production reactor by denaturing gradient gel electrophoresis(DGGE)

To study the structure of microbial communities in the biological hydrogen produc-tion reactor and determine the ecological function of hydrogen producing bacteria,anaerobic sludge was obtained from the continuous stirred tank reactor(CSTR)in different periods of time,and the diversity and dynamics of microbial communities were investigated by denaturing gra-dient gel electrophoresis(DGGE).The results of DGGE demonstrated that an obvious shift of microbial population happened from the beginning of star-up to the 28th day,and the ethanol type fermentation was established.After 28 days the structure of microbial community became stable,and the climax community was formed.Comparative analysis of 16S rDNA sequences from reamplifying and sequencing the prominent bands indicated that the dominant population belonged to low G+C Gram-positive bacteria(Clostridium sp.and Ethanologenbacterium sp.),β-proteobacteria(Acidovorax sp.),γ-proteobacteria(Kluyvera sp.),Bacteroides(uncultured bacte-rium SJA-168),and Spirochaetes(uncultured eubacterium E1-K13),respectively.The hydrogen production rate increased obviously with the increase of Ethanologenbacterium sp.,Clostridium sp.and uncultured Spirochaetes after 21 days,meanwhile the succession of ethanol type fer-mentation was formed.Throughout the succession the microbial diversity increased however it decreased after 21 days.Some types of Clostridium sp.Acidovorax sp.,Kluyvera sp.,and Bac-teroides were dominant populations during all periods of time.These special populations were essential for the construction of climax community.Hydrogen production efficiency was de-pendent on both hydrogen producing bacteria and other populations.It implied that the co-metabolism of microbial community played a great role of biohydrogen production in the reactors.

Denaturing gradient gel electrophoresis fingerprinting of soil bacteria in the vicinity of the Chinese Great Wall Station, King George Island, Antarctica

Bacterial diversity was investigated in soil samples collected from 13 sites around the Great Wall Station, Fildes Peninsula, King George Island, Antarctica, using denaturing gradient gel electrophoresis （DGGE） of 16S rRNA genes. The classes α-, β-, and γ-Proteobacteria, as well as the phylum Actinobacteria, were found to be the dominant bacteria in the soils around the Great Wall Station. Although the selected samples were not contaminated by oil, a relationship between soil parameters, microbial biodiversity, and human impact was still seen. Sample sites in human impacted areas showed lower bacterial biodiversity （average H′= 2.65） when compared to nonimpacted sites （average H′= 3.05）. There was no statistically significant correlation between soil bacterial diversity and total organic carbon （TOC）, total nitrogen, or total phosphorus contents of the soil. Canonical correlation analysis showed that TOC content was the most important factor determining bacterial community profiles among the measured soil parameters. In conclusion, microbial biodiversity and community characteristics within relatively small scales （1.5 km） were determined as a function of local environment parameters and anthropogenic impact.

3种植物精油对调理牛排优势腐败菌的抑菌性

为明确调理牛排的优势腐败菌并筛选出抑菌性、专一性强的植物精油,通过传统培养基法结合聚合酶链式反应-变性梯度凝胶电泳技术筛选出调理牛排的优势腐败菌,并通过抑菌圈、最小抑菌浓度、最小杀菌浓度及细菌生长曲线,从丁香精油、豆蔻精油、黑胡椒精油中选出抑菌性最强的植物精油。结果表明:优势腐败菌为深蓝紫色杆菌(Janthinobacterium lividum)、恶臭假单胞菌(Pseudomonas putida)和热杀索丝菌(Brochothrix thermosphacta);丁香精油的抑菌圈直径均大于10 mm,2~4μL/mL的精油添加量就能明显抑制优势腐败菌的生长繁殖;豆蔻精油抑菌性稍弱;黑胡椒精油对3株优势腐败菌均无明显抑菌效果。

不同性状窖泥的细菌群落结构与酸酯含量分析

为了解不同性状窖泥细菌群落结构及酸酯代谢的差异,分别选取新窖泥、趋老熟窖泥和老熟窖泥,对其细菌16S r DNA的V3区进行变性梯度凝胶电泳分析和同源性比较,并结合窖泥主要有机酸及有机酸酯含量进行了典型相关分析。结果表明,老熟窖泥的细菌多样性指数及均匀度指数高于新窖泥和趋老熟窖泥,得到的39个优势条带,进行细菌DNA测序可分为14类;Clostridium XIVa、Aminobacterium均只在老熟窖泥中检测到;新窖泥和趋老熟窖泥与Lactococcus、Lactobacillus、乳酸、乳酸乙酯含量正相关,老熟窖泥与Clostridiales、己酸、己酸乙酯和丁酸含量正相关。

多功能农药降解基因工程菌株m-CDS-1环境释放安全评价

【目的】为了评价多功能农药降解基因工程菌株m-CDS-1的环境释放中间试验水平的安全性。【方法】通过农药检测、平板计数、Most probable number-PCR(MPN-PCR)和Denaturing Gradient Gel Electrophoresis(DGGE)等方法在江苏大丰进行了工程菌株m-CDS-1田间降解农药效果、定殖动态和对土壤微生物群落结构影响的研究。【结果】投加1.01×107CFU/g干土的工程菌株m-CDS-1在30d时均能完全降解10.71mg/kg的甲基对硫磷和1.29mg/kg的呋喃丹。平板计数表明工程菌株m-CDS-1在土壤中快速下降;MPN-PCR检测结果显示,在4d,15d和30d时,0～10cm混合土壤中该工程菌株的数目分别为2.15±0.98×106CFU/g干土,3.70±4.66×104CFU/g干土和检测不出。工程菌株m-CDS-1的投加不会对土壤可培养三大微生物菌群数量产生显著影响;基于细菌16S rDNA V3区的DGGE分析结果表明,施加农药对细菌菌落结构有显著影响,4d,11d,30d时农药施用区与空白对照区的图谱相似性分别为17.16%,49.81%和75.01%,但到60d时的相似性为98.62%。工程菌株m-CDS-1释放在前期对细菌群落结构有一定影响,4d,11d和30d工程菌株释放区相对于空白的相似性分别为49.57%,38.30%和83.30%。在60d时,空白、施药和施菌小区的图谱相似性都在90%以上。【结论】工程菌株m-CDS-1不仅可同时高效降解甲基对硫磷和呋喃丹,仍保持了实验室内的原有特性,而且不会成为优势菌群长期在土壤环境中存在,也不会对土壤微生物群落结构造成长期影响。

高效毛细管线性聚丙烯酰胺凝胶电泳柱的制备、柱性能表征及操作条件考察

发展一种简便快速的线性聚丙烯酰胺毛细管凝胶电泳（ＣＧＥ）柱的新型制备方法，用于分离Ｐｏｌｙ ｄＡ（４０～６０）和双链ＤＮＡ，柱效达 ６百万理论板／米。提出“筛分能力”作为 ＣＧＥ柱评价指标，对样品迁移行为随操作条件的变化规律进行考察，为毛细管凝胶电泳的理论研究和实验条件优化奠定了基础。

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 R518对 1 6Sr RNA基因的 V3区进行扩增 ,长约 2 30 bp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 1 6Sr RNA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。

堆肥化过程中微生物群落的动态

通过变性梯度凝胶电泳（DGGE）和平板计数法对堆肥化过程中微生物的区系动态变化进行了研究.结果表明,在堆肥化过程中,微生物数量总的趋势是细菌的数量最多,放线菌次之,真菌的数量最少,中温微生物的数量始终高于高温微生物.当发酵结束后,中温微生物的数量低于发酵初始水平,高温放线菌和高温真菌的数量试验结束后高于初始水平,高温细菌的数量在整个堆肥化过程中变化不大.通过DGGE分析表明,发酵过程中细菌的种类发生了明显的更迭现象.发酵初期Bdellovibrio、Clostridiabacterium、Bacillus、Clostridium等占优势,中期Beta proteobacterium、Petrobacter succinimandens、Nitrospiraebacterium、Clostridium等占优势,后期Clostridium、Beta proteobacterium、Paenibacillus等占优势,而在整个堆肥化过程中Clostridium都是优势种.

变性梯度凝胶电泳技术在微生物多样性研究中的应用

变性梯度凝胶电泳是不依赖于培养的、依据DNA分子的大小和所带电荷分析微生物多样性和动态变化的分子生物学技术,具有检测极限低、分析速度快及重复性好等优点。主要对变性梯度凝胶电泳原理、特点及其在微生物多样性应用方面进行综述。

秸秆还田后土壤微生物群落结构变化的初步研究

通过实验室条件下的小麦秸秆粉和油菜秸秆粉的还土试验,结合常规的分析手段和基于DNA的分子技术(变性梯度凝胶电泳(DGGE)及测序技术),初步研究了秸秆粉还田后土壤物理化学特性及生物学特性的变化。结果表明,培养60d后,秸秆还田土壤的肥力明显提高,其纤维素酶活性明显增强。DGGE图谱表明,对照土壤(S)以及处理土壤(SW和SR),在培养过程中,β-Proteobacteria类细菌组成都在发生变化。对其中一个样品的DGGE条带进行了割胶测序,测序结果表明,大部分条带代表的微生物是未培养的或不可培养的。采用传统分离培养技术,从秸秆还田土壤中分离了2株纤维素降解菌。以上结果说明,秸秆还田具有良好的土壤综合效应。

PCR-DGGE技术用于湖泊沉积物中微生物群落结构多样性研究

采用PCR-DGGE分子指纹图谱技术比较南京市玄武湖、奠愁湖和太湖不同位置的表层沉积物微生物群落结构，研究结果表明，三湖泊沉积物微生物的16SrDNA的PCR扩增结果约为626bp，为16S rDNA V3～V5区特异性片段。玄武湖和莫愁湖表层沉积物中大约有20种优势菌群，且同一湖泊不同采样点DGGE图谱的差异性不大，细菌群落结构具有较高的相似性，而太湖样品DGGE条带的数目和位置表现出明显差异，且不同采样点图谱的差异性较大。三湖泊除具有特征性的微生物种属外，还分布约5个相同的细菌种群，可能与沉积物的理化性质和水生植被的影响相关。对DGGE图谱中7条主带进行回收、扩增和测序，结果显示其优势菌群具有不同的序列组成，其中5个序列与Genebank中已登录的细菌种群的同源性≥99％，2个序列的同源性为96％和93％，其中2个相似的细菌类群目前尚未获得纯培养。

应用PCR-DGGE技术解析白酒大曲细菌群落结构

应用DGGE技术分析不同批次的大曲细菌群落结构,采用SDS+酶法提取大曲基因组DNA,以细菌通用引物进行16SrRNA基因V3高变异区PCR扩增,将产物进行DGGE分离,获得表征大曲细菌群落结构的DGGE指纹图谱,结果表明,不同批次的大曲细菌群落结构组成类似,系统发育分析结果显示大曲中细菌主要属于厚壁菌门(Firmicutes),包括乳杆菌科(Lactobacillaceae)、芽孢杆菌科(Bacillaceae)和放线菌科(Thermoactino-mycetaceae)等。另外在大曲中除了存在酒醅中多有发现的细菌外,还存在着不可培养的微生物。

浓香型白酒窖泥放线菌的群落结构及其多样性

以泸州老窖1、50、100和400年窖泥为研究对象,采用变性梯度凝胶电泳(DGGE)研究浓香型白酒窖泥放线菌的群落结构及其多样性。DGGE图谱显示,除1年样品外,其余窖底泥多样性指数(H)均低于同窖龄窖壁泥,但均匀度指数(EH)较高。不同窖池相同部位窖泥的群落结构变化趋势为:随窖龄的延长,窖壁泥H值逐渐上升,为1.74—2.28;窖底泥下降,为1.73—2.07。EH值均为波动下降,分别在0.986—0.991和0.971—0.994之间。窖底泥相似性系数(SC)逐渐上升,为0.46—0.82;窖壁泥为0.31—0.62。DGGE条带测序结果显示,窖泥放线菌归于Olsenella、Atopobium、Streptomyces和Corynebacterium 4个属。Olsenella和Atopobium属为共有的优势属,且在窖壁泥中的优势度(di)均随窖龄延长而降低,在窖底泥中升高。实验结果表明,浓香型白酒窖泥蕴藏着丰富的放线菌资源,群落结构和多样性存在差异,菌群演替呈现一定规律性。

成年和老年小鼠脑蛋白质组双向电泳图谱比较

使用双向电泳 (2 DE)比较成年和老年小鼠脑蛋白质差异 ,从分子水平初步探索老年脑蛋白整体变化规律 .以固相 pH梯度等电聚焦为第一向 ,SDS 聚丙烯酰胺凝胶水平电泳 (PAGE)为第二向进行 2 DE .图象分析软件Imagemaster 2DElite分析电泳图谱 .重复性实验结果显示 ,同组样品在三次不同实验中所得蛋白质斑点数目的相对标准差 (变异系数 )为 4 43%± 0 2 5 % ;同一蛋白质斑点在三次实验中等电点、分子质量和蛋白质量的相对标准差分别为 8 76 %± 5 14% ,13 0 0 %± 4 2 2 %和 10 84%± 9 16 % .成年和老年小鼠脑组织 2 DE图谱分别获得 996和 12 5 6个蛋白质斑点 ,其中 8个蛋白质在老年脑组织中含量降低 ,2 0个蛋白质斑点含量增加 .另至少有 4个蛋白质斑点在老年脑组织中缺失 ,14个蛋白质点为老年脑特有 .