Gene Terminal Restriction Fragment Length Polymorphism Research Articles

Ice formation and growth shape bacterial community structure in Baltic Sea drift ice.

Drift ice, open water and under-ice water bacterial communities covering several developmental stages from open water to thick ice were studied in the northern Baltic Sea. The bacterial communities were assessed with 16S rRNA gene terminal-restriction fragment length polymorphism and cloning, together with bacterial abundance and production measurements. In the early stages, open water and pancake ice were dominated by Alphaproteobacteria and Actinobacteria, which are common bacterial groups in Baltic Sea wintertime surface waters. The pancake ice bacterial communities were similar to the open-water communities, suggesting that the parent water determines the sea-ice bacterial community in the early stages of sea-ice formation. In consolidated young and thick ice, the bacterial communities were significantly different from water bacterial communities as well as from each other, indicating community development in Baltic Sea drift ice along with ice-type changes. The thick ice was dominated by typical sea-ice genera from classes Flavobacteria and Gammaproteobacteria, similar to those in polar sea-ice bacterial communities. Since the thick ice bacterial community was remarkably different from that of the parent seawater, results indicate that thick ice bacterial communities were recruited from the rarer members of the seawater bacterial community.

Evaluating T-RFLP protocols to sensitively analyze the genetic diversity and community changes of soil alkane degrading bacteria

Due to the enormous complexity of soil bacterial communities, functional gene markers, like the alkB gene coding for the bacterial alkane monooxygenase, might be more suitable to monitor specific ecosystem functions, i.e. alkane degradation, compared to phylogenetic gene markers which include species not providing the respective service. However, the routine application of such functional fingerprints needs careful method development and evaluation. This study aimed at identifying a reliable, highly resolving fingerprinting method to monitor alkane degrading communities in soil. Therefore, we (i) evaluated various available terminal restriction fragment length polymorphism (T-RFLP) protocols for the alkB gene, (ii) comparatively analyzed the same soil communities targeting either a functional (alkB) or a phylogenetic marker gene (16S rRNA gene) in the T-RFLP, and (iii) judged on the applicability of diversity indices to follow diversity changes in the degrader community during alkane input. In silico restriction of 83 alkB gene sequences from public databases identified HpyCH4V as the enzyme yielding the highest T-RF richness for alkB. Analyzing DNA extracts from microcosms of two different agricultural soils (sandy and silty, amended with either maize or pea litter as alkane source) revealed a higher sensitivity of the alkB T-RFLP compared to 16S rRNA gene T-RFLP for following spatiotemporal changes in the alkane degrader community. As expected, the diversity indices based on either phylogenetic or functional fingerprinting profiles deviated significantly, but both resulted in lower richness estimates compared to an alkane degrading enrichment culture obtained from the same soil samples. The latter indicated that alkane degradation is a very common capability of soil bacteria. However, as the two fingerprinting methods monitor different kinds of diversity, they might provide complementary answers (i.e. phylogenetic and functional) to the same research questions, i.e. which is the robustness of an ecosystem function in a given system.

Performance and microbial community profiles in an anaerobic reactor treating with simulated PTA wastewater: From mesophilic to thermophilic temperature

Performance and microbial community profiles in a hybrid anaerobic reactor treating synthetic PTA wastewater (contained the major pollutants terephthalate and benzoate) were studied over 220 days from 33 °C to 52 °C. Results indicated that PTA treatment process was highly sensitive to temperature variations in terms of COD removal. Operation at 37 °C showed the best performance as well as the most diverse microbial community revealed by 16S rRNA gene clone library and T-RFLP (terminal restriction fragment length polymorphism). Finally, the anaerobic process achieved a total COD removal of 77.4%, 91.9%, 87.4% and 66.1% at 33, 37, 43 and 52 °C. While the corresponding TA removal were 77.6%, 94.0%, 89.1% and 60.8%, respectively. Sequence analyses revealed acetoclastic Methanosaeta was preponderant at 37 °C, while hydrogenotrophic genera including Methanobrevibacter and Methanofollis were more abundant at other temperatures. For bacterial community, 16 classes were identified. The largely existent Syntrophorhabdus members (belonging to δ-Proteobacteria) at 37 °C was likely to play an important role in mesophilic anaerobic wastewater treatment system contained terephthalate. Meanwhile, β-Proteobacteria seemed to be favored in an anaerobic system higher than 43 °C.

象山港网箱养殖区沉积物的古菌空间分布

PDF HTML阅读 XML下载 导出引用 引用提醒 象山港网箱养殖区沉积物的古菌空间分布 DOI: 10.5846/stxb201212041745 作者: 作者单位: 宁波大学海洋学院,宁波大学海洋学院,宁波大学海洋学院,宁波大学海洋学院,宁波大学海洋学院,宁波大学海洋学院 作者简介: 通讯作者: 中图分类号: 基金项目: 国家自然科学基金（41101234）；浙江省教育厅科研项目（Y201121499）；宁波大学学科项目（xkl11090）；国家863资助项目（2012AA092001） The archaeal spatial distribution in sediments of cage culture area in Xiangshan Bay, Zhejiang, China Author: Affiliation: School of Marine Science, Ningbo University,School of Marine Science,Ningbo University,School of Marine Science,Ningbo University,School of Marine Science,Ningbo University,School of Marine Science,Ningbo University,School of Marine Science,Ningbo University Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:对象山港网箱养殖区及其周边沉积物中古菌群落的空间分布进行研究，应用基于16S rRNA基因的T-RFLP（末端限制性片段多态性分析）技术分析象山港网箱养殖区及其周边不同深度沉积物中古菌的群落结构和多样性，并构建克隆文库进行系统发育学分析。测定沉积物各项理化因子，通过PCA和RDA分析了古菌群落分布及其与环境因子之间的关系。结果表明，泉古菌是港口沉积物中的优势古菌群，占古菌群落的50%以上。网箱养殖区沉积物的古菌群落结构较非养殖区简单，多样性降低。非养殖区古菌群落随深度呈现有规律的变化。营养盐类和pH是造成养殖区域古菌群落结构区别于非养殖区域的主要环境因素。 Abstract:Archaea was distributed widely in marine environments. It plays an important role in nutrient and energy cycling in marine ecosystems. For studying the archaeal spatial distribution in sediment of cage culture area, archaeal community structures in sediments were investigated in July 2011, in Xiangshan Bay, Zhejiang Province, China. Sediments used in this paper were as the same as described previously[1]: a control or check site (CK), an intersection site 6-8 km from the aquaculture site (IS), and a cage culture site (AC). For sub-sampling, sediments were cut into several sections throughout their depth: a (0-5 cm), b (5-10 cm), c (10-15 cm), d (15-20 cm), e (20-25 cm) and f (50-55 cm). Physiochemical factors (NH4+, NO2-, NO3-, SiO32- and P) of sediments were analyzed in laboratory after transport with liquid nitrogen. Based on 16S rRNA gene, the structure and diversity of archaeal communities in sediments were analyzed by T-RFLP (Terminal Restriction Fragment Length Polymorphism), which was conducted after amplification of the archaeal 16S rRNA partial gene using a fluorescent primer pair 109F /934R-FAM and digestion by TaqI (T^CGA). Archaeal 16S rRNA gene clone library was constructed using DNA extracted from layer 0-5 cm sediment of the CK site, and the following phylogenetic analysis was carried out using ARB software. In agreement with other studies, Crenarchaeota is the dominant archaea in marine sediment. T-RFLP profiles showed that the dominant T-RF of 188 bp, affiliated to Crenarchaeota, occupied 80.3% in the cage culture site, more than it in the check site (61.2%). While, the kingdoms Euryarchaeota and Crenarchaeota were present equally in clone library and methanogens constituted a small proportion of the archaeal community. Because the check site is near to East Sea with a higher salinity, T-RF of 78 bp, belonged Halobacteriales, emerged in its upper layers (0-20 cm) sediments and was more abundant than it in other sites. Principal component analysis for the archaeal 16S rRNA gene T-RFLP using R software indicated a complete separation of archaeal communities of the cage culture sit from the check site. And archaeal communities of the intersection site were in the middle. An obvious difference of archaeal communities in the check site sediments at various depths was indicated by PCA. T-RFs of 78 bp, 88 bp, 383 bp and 188 bp related archaea may be the main microorganisms who made the difference among horizontal and vertical distribution. Although calculation of Shannon diversity index H' and evenness index E' showed large errors in parallels, comparing with the check site, the diversity of archaeal communities in cage culture sediments displayed a dropping tendency. Redundancy analysis of archaeal community compositions and environmental factors of sediments in different sites and depths suggested that nutritive salt and pH mainly affected the distribution of archaeal in horizontal by aquaculture. Eh and ammonium were tend to have a relation with archaeal vertical distribution, especially in the check site. In short, aquaculture affected the spatial distribution of uncultivated archaea. 参考文献 相似文献 引证文献

16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota.

A novel non-culture based 16S rRNA Terminal Restriction Fragment Length Polymorphism (T-RFLP) method using the restriction enzymes Tsp509I and Hpy166II was developed for the characterization of the nasopharyngeal microbiota and validated using recently published 454 pyrosequencing data. 16S rRNA gene T-RFLP for 153 clinical nasopharyngeal samples from infants with acute otitis media (AOM) revealed 5 Tsp509I and 6 Hpy166II terminal fragments (TFs) with a prevalence of >10%. Cloning and sequencing identified all TFs with a prevalence >6% allowing a sufficient description of bacterial community changes for the most important bacterial taxa. The conjugated 7-valent pneumococcal polysaccharide vaccine (PCV-7) and prior antibiotic exposure had significant effects on the bacterial composition in an additive main effects and multiplicative interaction model (AMMI) in concordance with the 16S rRNA 454 pyrosequencing data. In addition, the presented T-RFLP method is able to discriminate S. pneumoniae from other members of the Mitis group of streptococci, which therefore allows the identification of one of the most important human respiratory tract pathogens. This is usually not achieved by current high throughput sequencing protocols. In conclusion, the presented 16S rRNA gene T-RFLP method is a highly robust, easy to handle and a cheap alternative to the computationally demanding next-generation sequencing analysis. In case a lot of nasopharyngeal samples have to be characterized, it is suggested to first perform 16S rRNA T-RFLP and only use next generation sequencing if the T-RFLP nasopharyngeal patterns differ or show unknown TFs.

733 Bacterial 16S R RNA Genetic Markers for Fecal Samples to Differentiate Choledochal Cyst from Biliary Atresia

Background and AimsMicrobiota in fecal content from choledochal cyst (CC) and biliary atresia (BA) individuals at the operation were compared using 16S rRNA gene libraries and terminal restriction fragment length...

Characterization of the microbial community from the marine sediment of the Venice lagoon capable of reductive dechlorination of coplanar polychlorinated biphenyls (PCBs)

The native microbial community of a contaminated sediment from Brentella Canal (Venice Lagoon, Italy) was enriched in slurry microcosms consisting of sterile sediment suspended in sterile site water in the presence of 3,3',4,4'-tetrachlorobiphenyl, 3,3',4,4',5- and 2,3',4,4',5-pentachlorobiphenyls, 3,3',4,4',5,5'- and 2,3,3',4,4',5-hexachlorobiphenyls. The enrichment cultures were characterized at each subculturing step by 16S rRNA gene Terminal-Restriction Fragment Length Polymorphism (T-RFLP) and Denaturing Gradient Gel Electrophoresis (DGGE) analysis. About 90% of spiked polychlorinated biphenyls (PCBs) were stoichiometrically converted into di- and tri-chlorinated congeners by each enriched culture via dechlorination of flanked para chlorines and ortho-flanked meta chlorines. A 2-fold increase in PCB-dechlorination rate, the disappearance of lag phase, as well as a remarkable increase of sulfate consumption and a decline of methanogenic activity, were observed throughout subculturing. A reduction of complexity of the archaeal community, which was composed by Methanomicrobiales and Methanosarcinales, was also observed as a result of culture enrichment. The bacterial community included members of the Alpha, Gamma, Delta and Epsilon divisions of Proteobacteria, Firmicutes and Chloroflexi. Two sequence phylotypes related to the genus Sulforovum and the species Desulfococcus multivorans and two Chloroflexi enriched throughout subculturing, thus suggesting that these bacteria were involved in PCB dechlorination in the marine sediments of Brentella canal.

Chitinase genes revealed and compared in bacterial isolates, DNA extracts and a metagenomic library from a phytopathogen-suppressive soil

Soil that is suppressive to disease caused by fungal pathogens is an interesting source to target for novel chitinases that might be contributing towards disease suppression. In this study, we screened for chitinase genes, in a phytopathogen-suppressive soil in three ways: (1) from a metagenomic library constructed from microbial cells extracted from soil, (2) from directly extracted DNA and (3) from bacterial isolates with antifungal and chitinase activities. Terminal restriction fragment length polymorphism (T-RFLP) of chitinase genes revealed differences in amplified chitinase genes from the metagenomic library and the directly extracted DNA, but approximately 40% of the identified chitinase terminal restriction fragments (TRFs) were found in both sources. All of the chitinase TRFs from the isolates were matched to TRFs in the directly extracted DNA and the metagenomic library. The most abundant chitinase TRF in the soil DNA and the metagenomic library corresponded to the TRF(103) of the isolate Streptomyces mutomycini and/or Streptomyces clavifer. There were good matches between T-RFLP profiles of chitinase gene fragments obtained from different sources of DNA. However, there were also differences in both the chitinase and the 16S rRNA gene T-RFLP patterns depending on the source of DNA, emphasizing the lack of complete coverage of the gene diversity by any of the approaches used.

Fine-scale vertical distribution of bacteria in the East Pacific deep-sea sediments determined via 16S rRNA gene T-RFLP and clone library analyses

Although the deep-sea sediments harbor diverse and novel bacteria with important ecological and environmental functions, a comprehensive view of their community characteristics is still lacking, considering the vast area and volume of the deep-sea sedimentary environments. Sediment bacteria vertical distribution and community structure were studied of the E272 site in the East Pacific Ocean with the molecular methods of 16S rRNA gene T-RFLP (terminal restriction fragment length polymorphism) and clone library analyses. Layered distribution of the bacterial assemblages was detected by both methods, indicating that the shallow sediments (40 cm in depth) harbored a diverse and distinct bacterial composition with fine-scale spatial heterogeneity. Substantial bacterial diversity was detected and nine major bacterial lineages were obtained, including Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Nitrospirae, Planctomycetes, Proteobacteria, and the candidate divisions OP8 and TM6. Three subdivisions of the Proteobacteria presented in our libraries, including the α-, γ- and δ-Proteobacteria. Most of our sequences have low similarity with known bacterial 16S rRNA genes, indicating that these sequences may represent as-yet-uncultivated novel bacteria. Most of our sequences were related to the GenBank nearest neighboring sequences retrieved from marine sediments, especially from deep-sea methane seep, gas hydrate or mud volcano environments. Several sequences were related to the sequences recovered from the deep-sea hydrothermal vent or basalt glasses-bearing sediments, indicating that our deep-sea sampling site might be influenced to certain degree by the nearby hydrothermal field of the East Pacific Rise at 13°N.

Vegetation cover of forest, shrub and pasture strongly influences soil bacterial community structure as revealed by 16S rRNA gene T-RFLP analysis

Bacterial community structure is influenced by vegetation, climate and soil chemical properties. To evaluate these influences, terminal restriction fragment length polymorphism (T-RFLP) and cloning of the 16S rRNA gene were used to analyze the soil bacterial communities in different ecosystems in southwestern China. We compared (1) broad-leaved forest, shrub and pastures in a high-plateau region, (2) three broad-leaved forests representing a climate gradient from high-plateau temperate to subtropical and tropical regions and (3) the humus and mineral soil layers of forests, shrub lands and pastures with open and restricted grazing activities, having varied soil carbon and nutrient contents. Principal component analysis of the T-RFLP patterns revealed that soil bacterial communities of the three vegetation types were distinct. The broad-leaved forests in different climates clustered together, and relatively minor differences were observed between the soil layers or the grazing regimes. Acidobacteria dominated the broad-leaved forests (comprising 62% of the total clone sequences), but exhibited lower relative abundances in the soils of shrub (31%) and pasture (23%). Betaproteobacteria was another dominant taxa of shrub land (31%), whereas Alpha- (19%) and Gammaproteobacteria (13%) and Bacteriodetes (16%) were major components of pasture. Vegetation exerted more pronounced influences than climate and soil chemical properties.

Response of Estuarine Biofilm Microbial Community Development to Changes in Dissolved Oxygen and Nutrient Concentrations

The information content and responsiveness of microbial biofilm community structure, as an integrative indicator of water quality, was assessed against short-term changes in oxygen and nutrient loading in an open-water estuarine setting. Biofilms were grown for 7-day periods on artificial substrates in the Pensacola Bay estuary, Florida, in the vicinity of a wastewater treatment plant (WWTP) outfall and a nearby reference site. Substrates were deployed floating at the surface and near the benthos in 5.4 m of water. Three sampling events covered a 1-month period coincident with declining seasonal WWTP flow and increasing dissolved oxygen (DO) levels in the bottom waters. Biomass accumulation in benthic biofilms appeared to be controlled by oxygen rather than nutrients. The overriding effect of DO was also seen in DNA fingerprints of community structure by terminal restriction fragment length polymorphism (T-RFLP) of amplified 16S rRNA genes. Ribotype diversity in benthic biofilms at both sites dramatically increased during the transition from hypoxic to normoxic. Terminal restriction fragment length polymorphism patterns showed pronounced differences between benthic and surface biofilm communities from the same site in terms of signal type, strength, and diversity, but minor differences between sites. Sequencing of 16S rRNA gene clone libraries from benthic biofilms at the WWTP site suggested that low DO levels favored sulfate-reducing prokaryotes (SRP), which decreased with rising oxygen levels and increasing overall diversity. A 91-bp ribotype in the CfoI-restricted 16S rRNA gene T-RFLP profiles, indicative of SRP, tracked the decrease in relative SRP abundance over time.

Role of eukaryotic microbiota in soil survival and catabolic performance of the 2,4-D herbicide degrading bacteria Cupriavidus necator JMP134

Cupriavidus necator (formerly Ralstonia eutropha) JMP134, harbouring the catabolic plasmid pJP4, is the best-studied 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide degrading bacterium. A study of the survival and catabolic performance of strain JMP134 in agricultural soil microcosms exposed to high levels of 2,4-D was carried out. When C. necator JMP134 was introduced into soil microcosms, the rate of 2,4-D removal increased only slightly. This correlated with the poor survival of the strain, as judged by 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) profiles, and the semi-quantitative detection of the pJP4-borne tfdA gene sequence, encoding the first step in 2,4-D degradation. After 3 days of incubation in irradiated soil microcosms, the survival of strain JMP134 dramatically improved and the herbicide was completely removed. The introduction of strain JMP134 into native soil microcosms did not produce detectable changes in the structure of the bacterial community, as judged by 16S rRNA gene T-RFLP profiles, but provoked a transient increase of signals putatively corresponding to protozoa, as indicated by 18S rRNA gene T-RFLP profiling. Accordingly, a ciliate able to feed on C. necator JMP134 could be isolated after soil enrichment. In native soil microcosms, C. necator JMP134 survived better than Escherichia coli DH5alpha (pJP4) and similarly to Pseudomonas putida KT2442 (pJP4), indicating that species specific factors control the survival of strains harbouring pJP4. The addition of cycloheximide to soil microcosms strongly improved survival of these three strains, indicating that the eukaryotic microbiota has a strong negative effect in bioaugmentation with catabolic bacteria.

Community structure of Archaea and Bacteria in a profundal lake sediment Lake Kinneret (Israel)

The microbial community structure of an anoxic profundal lake sediment, i.e., subtropical Lake Kinneret, was analysed with respect to its composition by culture-independent molecular methods including terminal restriction fragment length polymorphism (T-RFLP) analysis, comparative sequence analysis, and quantitative real-time PCR. In particular we were interested in the structure, species composition, and relative abundance of the overall microbial community in the methanogenic sediment layer (0–10 cm depth). Pairwise comparison of archaeal and bacterial 16S rRNA gene T-RFLP profiles obtained from three independent samplings indicated stability of the microbial community. The numbers of Archaea and Bacteria, quantified by real-time PCR, amounted to about 10 8 and 10 10 16S rRNA gene copies cm −3 sediment, respectively, suggesting that Archaea may account for only a minor fraction (approximately 1%) of the total prokaryotic community. Hydrogenotrophic Methanomicrobiales and acetoclastic Methanosaeta spp. dominated T-RFLP profiles of the archaeal community. T-RFLP profiles of the bacterial community were dominated by Deltaproteobacteria, sulphate reducers and syntrophs in particular. The second most abundant group was assigned to the Bacteroidetes–Chlorobi-group. Only one bacterial group, which was affiliated with halorespiring bacteria of subphylum II of the Chloroflexi, showed variation in abundance within the sediment samples investigated. Our study gives a comprehensive insight into the structure of the bacterial and archaeal community of a profundal lake sediment, indicating that sulphate reducers, syntrophs, bacteroidetes, halorespirers and methanogens are of particular importance in Lake Kinneret sediment.