Biotechnological significance of toxic marine dinoflagellates

Dinoflagellates are microalgae that are associated with the production of many marine toxins. These toxins poison fish, other wildlife and humans. Dinoflagellate-associated human poisonings include paralytic shellfish poisoning, diarrhetic shellfish poisoning, neurotoxic shellfish poisoning, and ciguatera fish poisoning. Dinoflagellate toxins and bioactives are of increasing interest because of their commercial impact, influence on safety of seafood, and potential medical and other applications. This review discusses biotechnological methods of identifying toxic dinoflagellates and detecting their toxins. Potential applications of the toxins are discussed. A lack of sufficient quantities of toxins for investigational purposes remains a significant limitation. Producing quantities of dinoflagellate bioactives requires an ability to mass culture them. Considerations relating to bioreactor culture of generally fragile and slow-growing dinoflagellates are discussed. Production and processing of dinoflagellates to extract bioactives, require attention to biosafety considerations as outlined in this review.     

胆管结扎对大鼠肠道双歧杆菌组成的影响

目的分析胆管结扎对SD大鼠肠道双歧杆菌菌群结构组成的影响。方法采集手术前3 d和手术后2周5只模型组和5只对照组大鼠的粪便样品,提取粪便样品中微生物的混合DNA进行双歧杆菌类群特异性PCR-DGGE,结合主成分分析技术比较2组大鼠在手术前后肠道内双歧杆菌结构组成的变化。结果DGGE图谱及其主成分分析表明手术后对照组和模型组大鼠肠道双歧杆菌菌群的结构组成明显不同,主要表现在假长双歧杆菌的数量在模型组显著增加,而动物双歧杆菌却明显减少。结论胆管结扎导致SD大鼠肠道双歧杆菌结构发生异常变化。     

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.     

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

Biohydrogen production has been concerned ex-tremely as a new technology of energy resource pro-duction by many scientists[1—4]. Enhancement of hy-drogen production efficiency and cutting down the operating cost are very important problems, which are the limiting factors for the industrialization of hydro-gen production process. The fermentation hydrogen production technology offers a new method to resolve these difficulties[5—8]. Compared with photosynthetic hydrogen production possesses, f…     

贡嘎蝠蛾幼虫肠道细菌多样性分析

[目的]对实验室养殖条件下的重要经济昆虫冬虫夏草寄主-贡嘎蝠蛾(Hepialus gonggaensis,Hg)幼虫肠道微生物群落的多样性进行了研究.[方法]采用常规分离培养与分子鉴定的方法和基于16S rRNA作为分子标记的变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)的方法.[结果]用常规分离与分子鉴定方法获得8个属的细菌类群,其中肠杆菌属(Enterobacter)是优势菌群,肉食杆菌属(Carnobacterium)是次优势菌群.对通过DGGE方法得到的11条16S rRNA优势条带序列进行了比对和系统进化树分析,结果表明肉食杆菌属(Carnobacterium)的丰度最高,是肠道细菌中主要的优势菌群,芽孢杆菌属(Bacillus)是次优势菌群.DGGE图谱还显示Hg幼虫不同虫龄肠道细菌菌群的结构存在差异,推测可能与其发育生理状态的差异有关系.[结论]结合常规分离法与DGGE法能够更有效的分析肠道微生物的多样性,获得更多更全面的微生物多样性信息.     

Transgenic tobacco revealing altered bacterial diversity in the rhizosphere during early plant development

The rhizosphere constitutes a complex niche that may be exploited by a wide variety of bacteria. Bacterium–plant interactions in this niche can be influenced by factors such as the expression of heterologous genes in the plant. The objective of this work was to describe the bacterial communities associated with the rhizosphere and rhizoplane regions of tobacco plants, and to compare communities from transgenic tobacco lines (CAB1, CAB2 and TRP) with those found in wild-type (WT) plants. Samples were collected at two stages of plant development, the vegetative and flowering stages (1 and 3 months after germination). The diversity of the culturable microbial community was assessed by isolation and further characterization of isolates by amplified ribosomal RNA gene restriction analysis (ARDRA) and 16S rRNA sequencing. These analyses revealed the presence of fairly common rhizosphere organisms with the main groups Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Bacilli. Analysis of the total bacterial communities using PCR-DGGE (denaturing gradient gel electrophoresis) revealed that shifts in bacterial communities occurred during early plant development, but the reestablishment of original community structure was observed over time. The effects were smaller in rhizosphere than in rhizoplane samples, where selection of specific bacterial groups by the different plant lines was demonstrated. Clustering patterns and principal components analysis (PCA) were used to distinguish the plant lines according to the fingerprint of their associated bacterial communities. Bands differentially detected in plant lines were found to be affiliated with the genera Pantoea, Bacillus and Burkholderia in WT, CAB and TRP plants, respectively. The data revealed that, although rhizosphere/rhizoplane microbial communities can be affected by the cultivation of transgenic plants, soil resilience may be able to restore the original bacterial diversity after one cycle of plant cultivation.     

Methanogenic profiles by denaturing gradient gel electrophoresis using order-specific primers in anaerobic sludge digestion

In the present study, the diversity of methanogenic populations was monitored for 25 days, together with the process data for an anaerobic batch reactor treating waste-activated sludge. To understand this microbial diversity and dynamics, 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting was conducted at two different taxonomic levels: the domain and order levels. The DGGE profiles of the domain Archaea and the three orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales were comparatively analyzed after each DGGE band was sequenced to enable identification. The DGGE profiles of the three orders showed methanogens belonging to each order that were not detected in the DGGE profile of the Archaea. This discrepancy may have resulted from PCR bias or differences in the abundances of the three microbial orders in the anaerobic bioreactor. In conclusion, to fully understand the detailed methanogenic diversity and dynamics in an anaerobic bioreactor, it is necessary to conduct DGGE analysis with 16S rRNA gene primers that target lower taxonomic groups.     

Comparative metabolomic analysis of <Emphasis Type="Italic">Sinorhizobium</Emphasis> sp. C4 during the degradation of phenanthrene

We describe a quantitative analysis of the genetic diversity of phenol-degrading potential in bacterial communities from laboratory-scale activated sludge. Genomic DNA extracted from activated sludge from two sequential batch reactors fed with synthetic sewage plus phenol was amplified using conserved primers for the major subunit of the phenol hydroxylase (LmPH) gene and used to generate clone libraries. Following phylogenetic analysis, 59 sequences containing a 470-bp fragment clustered into six distinct subgroups with a genetic distance of 8%, most likely representing ecologically relevant variants of the enzyme. Seven sets of primers were designed to target the six clusters and used to obtain quantitative information on the dynamics of LmPH gene diversity using real-time PCR assays throughout 9 months of bioreactors operation. Total LmPH gene copy number remained approximately steady in phenol-amended and control reactors. However, a significant increase in phenol-degrading activity in the phenol-amended sludge was accompanied by a parallel increase in LmPH gene diversity, suggesting that phenol degradation in the activated sludge depends on the combined activity of a number of redundant species.     

贵州烟草根围AM真菌多样性的初步研究

从贵州省内烟区不同土壤生态环境下采集烟草根际土样,湿筛离心法分离丛枝菌根（AM）真菌孢子,鉴定出烟草AM真菌4属20种,其中球囊霉属9种,无梗囊霉属7种,巨孢囊霉属3种,盾巨孢囊霉属1种。从土壤样品DNA中扩增AM真菌特异性片段并采用DGGE技术对AM真菌多样性进行分析。测序结果显示烟草根际土壤中菌根真菌主要菌群为球囊霉属,与湿筛离心法的鉴定结果一致。为进一步研究贵州地区AM真菌多样性以及开发应用提供了依据。     

Screening of alpha-1-antitrypsin gene by denaturing gradient gel electrophoresis (DGGE)

Alpha-1-antitrypsin (AAT) is a serine protease inhibitor whose deficiency could cause emphysema and liver disease and, as recently described, could be a risk factor for lung cancer development. Alpha-1-antitrypsin inhibits a variety of proteases but its primary target is neutrophil elastase, an extracellular endopeptidase capable of degrading most protein components of the extracellular matrix. Inhibition of neutrophil elastase by AAT has an important role in maintaining the integrity of connective tissue. The gene encoding for AAT spans over 12.2 kb, consists of seven exons and is highly polymorphic. Therefore several methods for mutation screening of alpha-1-antitrypsin gene have been developed. Method described here is based on denaturing gradient gel electrophoresis (DGGE). This method is highly efficient and reliable and allows rapid analysis of entire coding region of alpha-1-antitrypsin gene, including splice junction sites. Previously described DGGE based analysis of AAT gene included overnight electrophoresis of individually amplified fragments. The optimization of the method described in this paper is directed towards the shortening of the duration of electrophoresis and amplification of fragments in multiplex reaction in order to make the analysis less time-consuming and therefore more efficient.