Molecular assessment on impact of uranium ore contamination in soil bacterial diversity

Impact of uranium (U) ore and soluble uranium (at pH 4.0) contamination on agricultural soil bacterial diversity was assessed by using laboratory microcosms for one year. Diversity and abundance of metabolically active bacterial populations in periodically collected microcosm’s samples were analyzed by extracting total RNA and preparation of cDNA followed by analysis of 16S rRNA gene by DGGE and real time PCR. DGGE analysis revealed prominent shift of soil bacterial population due to uranium ore contamination within 12 months while uranium ore along with soluble U completely destroyed the soil bacterial diversity within first six months. Real time PCR based analysis indicated 100–200 folds increase in 16S rRNA gene copies of total as well as individual bacterial taxa in both U ore amended and unamended soils in first six months while increase in incubation period upto 12 months showed reduction of the same only in U ore amended soil. Antagonistic effect of U ore contamination on soil bacterial diversity indicated the severe impact of U mining likely to have on nearby ecosystems. Role of U at acidic pH in destroying the diversity completely is noteworthy as it corroborated the disastrous consequence of acid mine drainage generated from U mine sites.     

Effects of sampling location and time,and host animal on assessment of bacterial diversity and fermentation parameters in the bovine rumen

Aims: To investigate, using culture‐independent methods, whether the ruminal bacterial structure, population and fermentation parameters differed between sampling locations and time. Methods and Results: The detectable bacteria and fermentation parameters in the digesta from five locations in the rumen of three cows at three time points were analysed. The PCR‐denaturing gradient gel electrophoresis (PCR‐DGGE) profiles were similar among digesta samples from five locations (95·4%) and three time points (93·4%) within cows; however, a lower similarity was observed for samples collected from different host animals (85·5%). Rumen pH and concentration of volatile fatty acids (VFA) were affected by time points of sampling relative to feeding. Conclusions: The detectable bacterial structure in the rumen is highly conserved among different locations and over time, while the quantity of individual bacterial species may change diurnally in response to the feeding. Significance and impact of the study: This study supplies the fundamental understanding of the microbial ecology in the rumen, which is essential for manipulation of ruminal microflora and subsequent improvement in animal production.     

一种简单有效且适于土壤微生物多样性分析的DNA提取方法

参照Zhou[11]的方法进行了改进,获得了一种简单、有效的DNA提取方法.此方法操作简单、从大量样品改为小量样品的提取,利用高浓度的PEG沉淀,不作回收纯化,所提DNA片段较大,在23 kb以上,每克土的DNA提取量从3.74～15.28 μg,OD260/OD230比值在0.89～1.21范围内,用真菌和细菌核糖体特异性引物进行PCR扩增,均获得较好的结果,DGGE图谱显示丰富性较高,可用于细菌多样性和真菌多样性的分析.此方法能够从4种不同性质土壤中提取出DNA,但提取盐渍土壤和碱性土壤的效果更好一些,为土壤微生物群落结构的多样性分析奠定良好的基础.     

Processivity and substrate-binding in family 18 chitinases

Enzymatic depolymerisation of polysaccharides is a key technology in the biorefining of biomass. The enzymatic conversion of the abundant insoluble polysaccharides cellulose and chitin is of particular interest and complexity, because of the bi-phasic nature of the process, the seemingly complicated tasks faced by the enzymes, and the importance of these conversions for the future biorefinery. Here we review recent work on family 18 chitinases that sheds light on important aspects of the catalytic action of these depolymerising enzymes, including the structural basis of processivity and its direction, the energies involved in substrate-binding and displacement.     

变性梯度凝胶电泳在实验小鼠细菌检测中的应用

目的 研究变性梯度凝胶电泳(denatured gradient gel electrophoresis, DGGE)在实验小鼠细菌检测中的应用。方法 根据16S rDNA V3区引物,PCR扩增3种实验小鼠(KM小鼠、NIH小鼠和BALB/c小鼠)呼吸道和盲肠段的细菌基因组DNA;扩增产物运用DGGE进行电泳检测,并分析条带数量间差异的统计学意义。结果 KM小鼠盲肠段条带12~18条,呼吸道条带5~10条;NIH小鼠盲肠段条带15~20条,呼吸道条带4~10条;BALB/c小鼠盲肠段条带10~15条,呼吸道条带0~7条。统计分析结果显示,KM小鼠和NIH小鼠在盲肠和呼吸道电泳条带数量上的差异无统计学意义( P >0.05);BALB/c小鼠与KM小鼠、NIH小鼠间的差异均有统计学意义( P <0.05)。结论 DGGE 在实验小鼠盲肠和呼吸道细菌检测中能较好地反映菌群的物种多样性。     

DNA isolation protocols affect the detection limit of PCR approaches of bacteria in samples from the human gastrointestinal tract

A major concern in molecular ecological studies is the lysis efficiency of different bacteria in a complex ecosystem. We used a PCR-based 16S rDNA approach to determine the effect of two DNA isolation protocols (i.e. the bead beating and Triton-X100 method) on the detection limit of seven feces-associated bacterial species of different genera. Glycogen was used in these protocols to improve the precipitation of small concentrations of DNA in ethanol without affecting the sequential procedures. The PCR detection limit of 16S rDNA amplicons on agarose gel from the seven strains tested varied between 8.0 (+/- 1.3) x 10(4) and 4.3 (+/- 1.6) x 10(6) cells for the bead beating method, and between 8.0 (+/- 1.3) x 10(4) and 5.4 (+/- 0.7) x 10(8) cells for the Triton X-100 method. These large differences are most like due to the difference in cell lysis efficiency, since a competitive PCR experiment did not indicate any preference for gram negative, low G+C gram positive or high G+C gram positive bacteria. Denaturing gradient gel electrophoresis (DGGE) analysis was performed to investigate the effect of both DNA isolation protocols on the lysis efficiency of bacteria in fecal samples. A higher diversity in fecal samples was observed with the bead beating method than with the Triton-X100 method. Bands in the bead beating method-derived DGGE profiles corresponding to bands of cloned sequences of the Clostridium coccoides-Eubacterium rectale group and uncultured Fusobacterium prausnitzii were absent or had low intensity in the Triton X-100 method-derived profiles. The applicability of the bead beating method was further investigated by analyzing biopsy samples from the human colon which contain approximately 10(6) cells.     

Molecular biology approaches for understanding microbial polycyclic aromatic hydrocarbons (PAHs) degradation

The known or suspected hazards of polycyclic aromatic hydrocarbons (PAHs) have provoked enormous concentration and endeavours to relieve or eliminate these precarious compounds from miscellaneous environments including soil, water and air. Among various interventions, biodegradation is an appealing approach for its comparative high efficiency and preferable safety. Microorganisms played crucial role in biodegradation of PAHs. Traditional access mainly including culture-dependent procedures has discovered and isolated PAHs-degrading microorganisms which could be subsequently applied to specific contaminated locus. Although certain progress has been achieved owing to traditional methods, much details in PAHs bioremedation leave pending because of the complexity nature of this process. As the rapid development of biology, molecular techniques such as PCR, fingerprinting technique (mainly DGGE), DNA hybridization technique and gene reporters technique have been intensively applied to gain further insight into the mechanism of PAHs degradation. These techniques not only proved the existence and role of uncultivable microorganisms in the whole population of PAHs degrading related microbials, but also made it possible to revealed the otherwise undetectable complex relationships between multi-microorganism concerned in PAHs biodegradation. Application of such techniques in the field of PAHs biodegradation were reviewed in this article.     

Molecular biology approaches for understanding microbial polycyclic aromatic hydrocarbons (PAHs) degradation

The known or suspected hazards of polycyclic aromatic hydrocarbons (PAHs) have provoked enormous concentration and endeavours to relieve or eliminate these precarious compounds from miscellaneous environments including soil, water and air. Among various interventions, biodegradation is an appealing approach for its comparative high efficiency and preferable safety. Microorganisms played crucial role in biodegradation of PAHs. Traditional access mainly including culture-dependent procedures has discovered and isolated PAHs-degrading microorganisms which could be subsequently applied to specific contaminated locus. Although certain progress has been achieved owing to traditional methods, much details in PAHs bioremedation leave pending because of the complexity nature of this process. As the rapid development of biology, molecular techniques such as PCR, fingerprinting technique (mainly DGGE), DNA hybridization technique and gene reporters technique have been intensively applied to gain further insight into the mechanism of PAHs degradation. These techniques not only proved the existence and role of uncultivable microorganisms in the whole population of PAHs degrading related microbials, but also made it possible to revealed the otherwise undetectable complex relationships between multi-microorganism concerned in PAHs biodegradation. Application of such techniques in the field of PAHs biodegradation were reviewed in this article.     

植物几丁质酶及其在抗真菌病害中的应用

植物几丁质酶的研究是抗真菌基因工程的热点之一。几丁质酶能够水解真菌细胞壁的主要成分几丁质,在植物抗真菌病害反应中发挥重要的作用。介绍了几丁质酶的基本生物学特性、基因的诱导表达,并对植物几丁质酶基因在抗真菌病害基因工程中的应用进行了阐述。     

The effects of different vegetation restoration patterns on soil bacterial diversity for sandy land in Hulunbeier

Grassland desertification seriously threatens sustainable economic and social development. Much attention has been paid to the control of grassland desertification, and even to the restoration and reconstruction of the grassland. Vegetation restoration is considered to be a very effective solution. Soil sustains an immense diversity of microbes, and the characteristics of soil microbial communities are sensitive indicators of soil. It is important to understand the relationship between vegetation and soil microbial diversity during the restoration process. Soil microbial, which is the main index to evaluate soil quality, plays a significant role in ecosystem and soil microbial diversity is the important one of global diversity. Exploring the effects of different vegetation patterns on soil microbial diversity can provide scientific bases and technical support for systemic and impersonal assessment of the best vegetation restoration patterns, as well as the vegetation restoration and reconstruction of Hulunbeier sandy land. Based on PCR–DGGE technology, a case study was carried out to investigate the effects of five different vegetation restoration patterns on soil microbial functional diversity after 4 years in sandy land in Hulunbeier, China. The five vegetation restoration patterns included mono-cultivar planting of Agropyron cristatum (UA), mono-cultivar planting of Hedysarum fruticosum (UH), mono-cultivar planting of Caragana korshinskii (UC), mixed-cultivar planting of Agropyron cristatum and Hedysarum fruticosum (AC) and mixed-cultivar planting of Agropyron cristatum, Hedysarum fruticosum, Caragana korshinskii and Elymus nutans (ACHE). Completely degraded sandy land was used as control.The results indicated that the vegetation restoration increased the genetic diversity of soil bacterial community obviously, and the structure of soil bacterial community was changed. The results of phylogenetic analysis suggested that the bacterial community in Hulunbeier sandy land mainly attributed to Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, and Acidobacteria. The dominant groups were Proteobacteria and Bacteroidetes. The effects of different vegetation type on soil bacterial community structures were different.     

Biosynthesis of chitinases by mammals of the order Carnivora

The secretion of chitinases has been examined in six species of Mammals belonging to the order Carnivora. Chitinases were found only in the extracts of the gastric mucosa of two species not adapted to a strictly meat diet [Canidae: dog and fox] while those with exclusive carnivorous habits, [Mustelidae: stoat, ferret, marten; and Felidae: cat, seem not to secrete the enzyme. These observations confirm the existence of a correlation between the ability of a given species to synthetize chitinases in digestive svstem and the feeding habits of the species.     

藏灵菇微生物种群结构的分子特性研究

用PCR—DGGE指纹技术,研究了藏灵菇中微生物多样性及藏灵菇发酵奶发酵过程微生物种群动力学。结果表明,藏灵菇中细菌的种群结构较酵母菌的复杂,不同来源的藏灵菇中细菌种群结构的相似性为78％-84％,酵母菌种群结构的相似性为80％-92％。发酵过程中细菌种群结构变化图谱中的条带B和条带E,以及酵母菌种群结构变化图谱中的条带N贯穿于整个发酵过程,是发酵过程的优势菌。序列分析表明,细菌种群结构的DGGE图谱中的绝大多数条带与乳酸菌相对应,其中最亮条带（条带E）的序列与乳酸乳球菌的相似性为100％。     

利用细胞计数手段和DGGE技术分析松花江干流部分地区的细菌种群多样性

松花江是我国东北地区的重要河流之一,为加强对其水环境微生物状况的了解,对松花江干流部分地区的微生物数量和多样性进行了分析。应用传统平板培养法和流式细胞技术测定水样中的细菌数;直接提取样品中的总DNA,以巢式PCR(Polymerase Chain Reaction)扩增细菌16SrDNA片段,应用聚丙烯酰胺凝胶电泳(Denaturing Gradient Gel Electrophoresis,DGGE)技术对扩增片段进行分离,研究水样和底泥样品细菌的种群多样性。实验结果显示,pH值为影响水环境中微生物总细胞数量的主要因素。水样中细菌群落多样性主要根据上下游分区,分区点在哈尔滨段附近,而底泥中细菌群落多样性的影响因素呈多样化,没有显示出较为明确的分区特征。     

在PCR-DGGE研究土壤微生物多样性中应用GC发卡结构的效应

应用普通细胞裂解法提取 3株实验菌株 (Escherichia coli DH5α,Staphylococcus aureus SA- 1和 A-grobacterium tumerfaciens 1 31 2 9)的基因组 DNA和应用基于高盐和长时高热的细胞裂解法提取 7种不同土壤样品中的微生物的基因组 DNA,两组不同结构的引物 F3 57GC,R51 8(在正向引物的 5′端有 GC发卡结构 )和 F3 57,R51 8,分别对实验菌株和土壤样品中微生物的 1 6Sr RNA基因 V3区进行扩增 ,均得到了目的片段。比较了不同引物扩增的 1 6S r DNA片段在 DGGE中的不同电泳行为 ,结果表明 ,含 GC发卡结构的PCR扩增产物在 DGGE中能够得到很好的分离 ,而无 GC发卡结构的 PCR产物则不能在 DGGE中获得满意分离。引入 GC发卡结构 ,使得对不同微生物的定性和分类更深入细致     

新疆阿克苏地区盐碱地细菌类群多样性及优势菌群分析

【目的】研究新疆阿克苏地区盐碱土样中细菌类群多样性和优势种群,及其与环境因子的相关性。【方法】采用基于16S rDNA的变性梯度凝胶电泳(Denaturing gradient gel electrophoresis,DGGE)、克隆测序和典型相关性分析(CCA)的方法。【结果】优势菌群序列分析表明有29个序列属于未培养的微生物,其他43个序列分属细菌的9个目:粘球菌目(Myxococcales)、假单胞菌目(Pseudomonadales)、根瘤菌目(Rhizobiales)、芽孢杆菌目(Bacillales)、伯克氏菌目(Burkholderiales)、放线菌目(Actinomycetales)、海洋螺菌目(Oceanospirillales)、黄杆菌目(Flavobacteriales)、交替单胞菌目(Alteromonadales),21个属。【结论】新疆阿克苏地区土壤中的微生物丰富度非常高,存在大量的细菌类群,优势菌群不尽相同,盐碱土样中微生物群落结构与环境因子是密切相关的。     

PCR-DGGE analysis reveals a distinct diversity in the bacterial population attached to the rumen epithelium

Bacteria attached to the rumen epithelium (or epimural community) are not well characterised and their role in rumen functioning is not totally understood. There is just one published report of a clone library from one cow that suggests that this epimural community differs from the bacteria associated with the rumen digestive contents. However, this time-consuming approach is not adapted for examining microbial population changes in groups of animals. In in vivo studies, when samples from several animals have to be analysed simultaneously, a simpler technique has to be used. In this study, a genetic fingerprinting technique, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), was used to characterise the structure of the bacterial population attached to the rumen epithelium. This community was compared with that present in the solid and liquid phases of rumen content under two contrasting diets. Rumen samples were obtained from four forage-fed and four high-concentrate-fed (80 : 20, wheat grain : hay) 5-month-old lambs. After slaughter, samples from five epithelial sites and the solid and liquid digesta phases were taken for DNA extraction and analysis. Bacterial communities were profiled by PCR-DGGE using bacterial-specific 16S rDNA primers. Analysis of the fingerprint revealed that the epithelial community differed from those of rumen content in both diets. As expected, the nature of the feed influenced the bacterial communities from the solid and liquid rumen phases but no diet effect was observed in the rumen epithelial profiles suggesting a strong host effect on this bacterial population. Additionally, no differences were observed among the five epithelial sampling sites taken from each animal. The profile of the bacterial population attached to the rumen epithelium presented a high inter-animal variation, whether this difference has an influence in the function of this community remains to be determined.     

Seminested PCR for the detection and analysis of flue-cured tobacco (Nicotiana tabacum) expressing a chitinase transgene

Standard PCR was ineffective in detecting a baculovirus-derived chitinase transgene in the T1 generation of chitinase-expressingNicotiana tabacum cv. CF80 after leaves were flue-cured at high temperatures. Consequently, a seminested PCR method was developed using fresh leaves from T2 generation plants also expressing the chitinase protein. Seminested PCR was highly effective in detecting the chitinase transgene in fresh leaves ofN. tabacum cvs. Xanthi-nc and K326 and in both fresh and flue-cured leaves ofN. tabacum cv. CF80.     

PCR-DGGE analysis of bacterial community dynamics in kava beverages during refrigeration

Aims: Kava beverages are highly perishable even under refrigerated conditions. This study aimed to investigate the bacterial community dynamics in kava beverages during refrigeration. Methods and Results: Four freshly made kava beverages were obtained from kava bars and stored at 4°C. On days 0, 3 and 6, the aerobic plate count (APC), lactic acid bacteria (LAB) count and yeast and mould count (YMC) of the samples were determined. Meanwhile, bacterial DNA was extracted from each sample and subjected to the polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE). Moreover, species‐specific PCR assays were employed to identify predominant Pseudomonas spp. involved in kava spoilage. Over the storage period, the APC, LAB count and YMC of the four kava beverages all increased, whereas their pH values decreased. The DGGE profile revealed diverse bacterial populations in the samples. LAB, such as Weissella soli, Lactobacillus spp. and Lactococcus lactis, were found in the kava beverages. Species‐specific PCR assays detected Pseudomonas putida and Pseudomonas fluorescens in the samples; Ps. fluorescens became dominant during refrigeration. Conclusions: LAB and Pseudomonas may play a significant role in the spoilage of kava beverages. Significance and Impact of the Study: This study provides important information that may be used to extend the shelf life of kava beverages.     

Nematicidal action of chitinases produced by the fungus Monacrosporium thaumasium under laboratorial conditions

Nematophagous fungi produce chitinases that may be important in the process of infection of eggs and larvae of nematodes. This study aimed to produce, purify, characterise and test the nematicidal action of extracellular chitinases produced by Monacrosporium thaumasium on Panagrellus redivivus. Mycelia from M. thaumasium were used to inoculate a solid medium for chitinase production. The enzymes were purified using a specific technique of adsorption for chitinases. The chitinase activity was determined at different pHs and temperatures. NF34 produced two distinct chitinases (27 and 30 kDa). After 72 hours, these enzymes provided a significant reduction (80%; p < 0.01) of the number of P. redivivus larvae, compared to control. It was shown that isolate NF34 produced chitinases with nematicidal activity. Thus, other experimental designs on geohelminths or even arthropods that transmit diseases may become a new aspect of the field of study of biological control using predatory nematophagous fungi.