橘小实蝇成虫肠道可培养细菌群落结构分析

【目的】研究橘小实蝇(Bactrocera dorsalis) 3个种群(实验室正常喂养种群、实验室无菌糖水喂养种群和野生种群)成虫肠道可培养细菌的群落结构组成。【方法】利用16S rRNA基因的聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)分析技术,结合菌落形态观察和生理生化特征鉴定细菌种类。【结果】从橘小实蝇3个种群成虫肠道600株可培养细菌得到53种不同细菌遗传型,分属于肠杆菌科(Enterobacteriaceae)、肠球菌科(Enterococcaceae)和芽孢杆菌科(Bacillaceae)等3个科。其中肠杆菌科是肠道可培养细菌最优势的细菌种类。同样以序列相似性大于97%的菌株归为相同的细菌种类为标准,找到了橘小实蝇3个种群可培养细菌的共有菌种,结合菌落形态观察和生理生化特征鉴定,确定共有菌种为肠杆菌属5株,克雷伯氏菌属2株,柠檬酸杆菌属1株,泛菌属1株,肠球菌属2株,以及芽孢杆菌属4株。【结论】通过研究橘小实蝇成虫肠道可培养细菌群落结构组成,可为探讨肠道菌群对寄主的生理功能和生态学意义奠定基础,最终为利用微生物防治此类害虫提供新思路。     

HRT and nutrients affect bacterial communities grown on recirculation aquaculture system effluents

In a recirculation aquaculture system the drumfilter effluent can be used as substrate for heterotrophic bacterial production, which can be recycled as feed. Because the bacteria might contain pathogens, which could reduce its suitability as feed, it is important to characterize these communities. Bacteria were produced in growth reactors under different conditions: 7 h hydraulic retention time (HRT) vs. 2 h, sodium acetate vs. molasses, and ammonia vs. nitrate. The community of the drumfilter effluent was different from those found in the reactors. However, all major community components were present in the effluent and reactor broths. HRT influenced the bacteria community, resulting in a DGGE profile dominated by a band corresponding to an Acinetobacter sp.-related population at 2 h HRT compared to 7 h HRT, where bands indicative of alpha-proteobacterial populations most closely related to Rhizobium and Shinella spp. were most abundant. Molasses influenced the bacterial community. It was dominated by an Aquaspirillum serpens-related population. Providing total ammonia nitrogen (TAN) in addition to nitrate led to the occurrence of bacteria close to Sphaerotilus spp., Flavobacterium mizutaii and Jonesia spp. It was concluded from these results that a 6-7 h HRT is recommended, and that the type of substrate is less important, and results in communities with a comparably low pathogenic risk.     

Diversity of endophytic bacterial communities in poplar grown under field conditions

Bacterial endophytes may be important for plant health and other ecologically relevant functions of poplar trees. The composition of endophytic bacteria colonizing the aerial parts of poplar was studied using a multiphasic approach. The terminal restriction fragment length polymorphism analysis of 16S rRNA genes demonstrated the impact of different hybrid poplar clones on the endophytic community structure. Detailed analysis of endophytic bacteria using cultivation methods in combination with cloning of 16S rRNA genes amplified from plant tissue revealed a high phylogenetic diversity of endophytic bacteria with a total of 53 taxa at the genus level that included Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. The community structure displayed clear differences in terms of the presence and relative proportions of bacterial taxa between the four poplar clones studied. The results showed that the genetic background of the hybrid poplar clones corresponded well with the endophytic community structure. Out of the 513 isolates and 209 clones identified, Actinobacteria, in particular the family Microbacteriaceae, made up the largest fraction of the isolates, whereas the clone library was dominated by Alpha- and Betaproteobacteria. The most abundant genera among the isolates were Pseudomonas and Curtobacterium, while Sphingomonas prevailed among the clones.     

利用PCR-DGGE未培养技术对中国白酒高温和中温大曲细菌群落结构的分析

采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术,研究了5种高温和中温白酒大曲细菌群落结构,通过优势条带切胶鉴定确定了大曲中优势细菌种属信息。结果表明,Weissella cibaria、Lactobacillus helveticus、L.fermentum、L.panis等乳酸菌普遍存在于5种大曲中,Ther-moactinomyces sanguinis仅存在于高温曲酱曲中,同时DGGE检测到了传统方法未能分离鉴定的Staphylococcus xylosus、Klebsiella oxytoca。不同工艺大曲细菌群落结构存在明显差异,随着制曲温度的升高,大曲细菌多样性指数有下降趋势。PCR-DGGE技术是一种能够快速有效地研究白酒大曲细菌群落结构的技术。     

现代分子生物学技术在冰川微生物生态研究中的应用

研究冰川中微生物的多样性对于揭示环境气候变迁,研究生物进化,开发微生物资源具有重要意义,现代分子生物学的发展为研究冰川微生物的多样性提供了行之有效的方法.简要综述了16S rDNA文库构建、变性梯度凝胶电泳、限制性片段长度多态性和荧光原位杂交等技术的原理及在冰川微生物生态研究中的应用现状.     

Bacterial Diversity and Community Structure in High Arsenic Aquifers in Hetao Plain of Inner Mongolia,China

The bacterial diversity and community structure of high arsenic (As) aquifers was investigated using an integrated approach adopting both geochemistry and molecular biology (polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analyses). Nine borehole sediments and one groundwater sample from the living place of a villager (affected by arseniasis) and 12 sediments from a control borehole in Hetao Plain were investigated. The As concentrations ranged from 33.6 to 77.6 mg/kg in high As borehole sediments and 1.5 to 5.8 mg/kg in those samples from the control. The As concentration in the groundwater was 744.8 μg/L. Ratios between As(III) and total As in high As sediments increased gradually with depth and ranged from 0.02 to 0.34. Similarly, the Fe(II)/total Fe presented the same increasing trend with depth. The correlation between TOC contents and total As was positive. High concentrations of total As, S, Fe and TOC were found in clay and low in sand samples. Phylogenetic analysis showed significantly different bacterial communities among high As sediments, control sediments and the high As groundwater. Both DGGE and 16S rRNA gene clone library results showed that the high As sediments were dominated by Thiobacillus, Pseudomonas, Brevundimonas, and Hydrogenophaga, with Thiobacillus being distinctly dominant (63.5%). Whereas the low As sediments were dominated by some other genera including Psychrobacter, Massilia and Desulfotalea. The bacterial populations in the high As groundwater mainly included Pseudomonas, Acinetobacter and Aquabacterium. These results improve our understanding of the bacterial diversity in high As aquifers in Hetao Plain and suggest how specific bacterial populations help mediate the mobilization of As into high As groundwaters.     

5株北极微藻藻际环境的细菌多样性

对5株北极微藻,如脆杆藻(Fragilariopsis sp.)、微单胞藻(Micromonas sp.)、四棘藻(Attheya septentrionalis)、海链藻(Thalassiosira sp.)和小球藻(Chlorella sp.)的不同生长时期的粘附细菌和游离细菌的16S rRNA基因进行PCR-DGGE分析,研究藻际环境的细菌多样性。结果表明,5株微藻具有不同的藻际微生物群落结构组成,其中微单胞藻、脆杆藻、四棘藻和海链藻的藻际细菌主要由Cyanobacteria(藻蓝细菌)、α-Proteobacteria(α-变形菌纲)和γ-Proteobacteria(γ-变形菌纲)组成,仅微单胞藻和脆杆藻检测出CFB(Cytophaga-Flexibacter-Bacteroides,噬纤维菌-屈挠杆菌-拟杆菌)。小球藻由Cyanobacteria、CFB、α-Proteobacteria和β-Proteobacteria(β-变形菌纲)组成。微单胞藻的藻际菌群结构稳定,不同生长时期的游离细菌和粘附细菌组成差异不明显。3株硅藻-脆杆藻、四棘藻和海链藻的游离细菌主要由γ-Proteobacteria组成,小球藻的游离细菌主要为β-Proteobacteria,而5株微藻的粘附细菌主要由Cyanobacteria组成。从DGGE图谱来看,在脆杆藻生长的延滞期、指数期和稳定期,其藻际游离细菌和粘附细菌的16S rRNA基因扩增条带数量和位置均有明显差异,但优势扩增条带较稳定;其他4株藻粘附细菌和游离细菌的扩增条带比较稳定,说明藻际关联菌群结构较稳定。藻菌种间特异性关系为不同微藻藻株提供了重要的线索,同时也带来更多的隐藏在藻际环境中的信息。     

Bacterial community composition over a dry winter in meso- and eutrophic Portuguese water bodies

In order to investigate the bacterial diversity in a number of rivers, reservoirs and lakes in northern and central Portugal during the winter of 2004/5 (atypically dry), we applied molecular methodologies, namely denaturing gradient gel electrophoresis with primers targeting fractions of the bacterial 16S rRNA gene. Environmental parameters such as pH, conductivity, inorganic nutrients, total suspended solids and chlorophyll a were determined in order to characterize the trophic status of the studied water bodies. We found water bodies with oligotrophic to hypereutrophic characteristics. Organisms belonging to the Bacteroidetes and Alphaproteobacteria were found at the highest pH environment. Bacteroidetes were also related to high nutrient concentrations. Verrucomicrobia were associated with the most oligotrophic reservoir and low pH values. Actinobacteria were present in all samples from lakes and reservoirs, indicating its preference for lentic water bodies. Cyanobacteria dominance was related to high pH and conductivity levels. In general the conductivity values recorded in winter 2005 were the highest over recent years and chlorophyll a also reached very high levels. The data emphasize an enhanced risk of eutrophication for the studied water bodies, especially in the subsequent months when the temperature rises.     

10种常见细菌基因检测膜条的研制

以16S rRNA基因为检测靶基因,设计10种常见细菌的DNA探针,将探针固定于硝酸纤维素膜条;PCR扩增细菌的16S rRNA基因片段并标记生物素后与膜条杂交;采用碱性磷酸酶标记的链亲和素检测生物素标记,以NBT/BCIP显色。该膜条不仅能单独检测10种细菌中的任何一种,也能同时检测5种细菌。该方法具备高通量、低成本、快速、准确等特点,具有良好的临床应用前景。     

Intracolonial differences in gut bacterial community between worker and soldier castes of Coptotermes formosanus

Abstract The establishment of symbiotic relationships with intestinal microorganisms enables termites to thrive on recalcitrant substrates such as cellulose and wood. A termite colony is composed of several different castes which have distinct feeding habits. The soldiers, for example, cannot feed by themselves and depend on workers, who feed them with digested or semi‐digested foods. To investigate the influence of feeding habits on the bacterial symbionts, a comparative study of gut bacteria between worker and soldier castes of the termite Coptotermes formosanus was conducted. The bacterial communities of both castes were investigated using denaturing gradient gel electrophoresis (DGGE) and clonal analysis of 16S ribosomal DNA (rDNA). Both methods indicated Bacteroidetes was the common predominant group; the common dominant phylotype was affiliated with a reported uncultured Bacteroidetes phylotype (BCf1–03). There were significant differences in Bacteroidetes and Spirochaetes between two castes. Compared to the gut bacteria of workers, those of soldiers were lower in abundance and diversity of Bacteroidetes and slightly higher in Spirochaetes. Two phylotypes (W8, W11) affiliated to Bacteroidetes and two (W26, W29) affiliated to Spirochaetes were exclusively found in the DGGE profile of the worker caste. Bacteroidetes are assumed to be involved in fermentation of sugars and nitrogenous compounds as well as degradation of uric acid. Spirochaetes are supposed to aid in the functions of acetogenesis and N₂‐fixation. The different feeding habits between workers and soldiers of C. formosanus may explain the observed differences in the gut bacterial community.     

Phylogenetic analysis of bacteria associated with Laminaria saccharina

Bacterial communities associated with the brown alga Laminaria saccharina from the Baltic Sea and from the North Sea were investigated using denaturing gradient gel electrophoresis and 16S rRNA gene clone libraries. The rhizoid, cauloid, meristem and phyloid revealed different 16S rRNA gene denaturing gradient gel electrophoresis banding patterns indicating a specific association of bacterial communities with different parts of the alga. Associations with cauloid and meristem were more specific, while less specific associations were obtained from the old phyloid. In addition, seasonal and geographical differences in the associated communities were observed. Results from 16S rRNA gene libraries supported these findings. Bacterial phylotypes associated with the alga were affiliated with the Alphaproteobacteria (nine phylotypes), Gammaproteobacteria (nine phylotypes) and the Bacteroidetes group (four phylotypes). A number of bacteria associated with other algae and other marine macroorganisms were among the closest relatives of phylotypes associated with L. saccharina.     

Occurrence and community composition of fast-growing Mycobacterium in soils contaminated with polycyclic aromatic hydrocarbons

Fast-growing mycobacteria are considered essential members of the polycyclic aromatic hydrocarbons (PAH) degrading bacterial community in PAH-contaminated soils. To study the natural role and diversity of the Mycobacterium community in contaminated soils, a culture-independent fingerprinting method based on PCR combined with denaturing gradient gel electrophoresis (DGGE) was developed. New PCR primers were selected which specifically targeted the 16S rRNA genes of fast-growing mycobacteria, and single-band DGGE profiles of amplicons were obtained for most Mycobacterium strains tested. Strains belonging to the same species revealed identical DGGE fingerprints, and in most cases, but not all, these fingerprints were typical for one species, allowing partial differentiation between species in a Mycobacterium community. Mycobacterium strains inoculated in soil were detected with a detection limit of 10(6) CFU g(-1) of soil using the new primer set as such, or approximately 10(2) CFU g(-1) in a nested PCR approach combining eubacterial and the Mycobacterium specific primers. Using the PCR-DGGE method, different species could be individually recognized in a mixed Mycobacterium community. This approach was used to rapidly assess the Mycobacterium community structure of several PAH-contaminated soils of diverse origin with different overall contamination profiles, pollution concentrations and chemical-physical soil characteristics. In the non-contaminated soil, most of the recovered 16SrRNA gene sequence did not match with previous described PAH-degrading Mycobacterium strains. In most PAH-contaminated soils, mycobacteria were detected which were closely related to fast-growing species such as Mycobacterium frederiksbergense and Mycobacterium austroafricanum, species that are known to include strains with PAH-degrading capacities. Interestingly, 16S rRNA genes related to M. tusciae sequences, a Mycobacterium species so far not reported in relation to biodegradation of PAHs, were detected in all contaminated soils.     

Spatial distribution of sponge-associated bacteria in the Mediterranean sponge Tethya aurantium

The local distribution of the bacterial community associated with the marine sponge Tethya aurantium Pallas 1766 was studied. Distinct bacterial communities were found to inhabit the endosome and cortex. Clear differences in the associated bacterial populations were demonstrated by denaturing gradient gel electrophoresis (DGGE) and analysis of 16S rRNA gene clone libraries. Specifically associated phylotypes were identified for both regions: a new phylotype of Flexibacteria was recovered only from the sponge cortex, while Synechococcus species were present mainly in the sponge endosome. Light conduction via radiate spicule bundles conceivably facilitates the unusual association of Cyanobacteria with the sponge endosome. Furthermore, a new monophyletic cluster of sponge-derived 16S rRNA gene sequences related to the Betaproteobacteria was identified using analysis of 16S rRNA gene clone libraries. Members of this cluster were specifically associated with both cortex and endosome of T. aurantium.