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Clone Library Analysis (clone + library_analysis)
Selected AbstractsMicrobial succession of nitrate-reducing bacteria in the rhizosphere of Poa alpina across a glacier foreland in the Central AlpsENVIRONMENTAL MICROBIOLOGY, Issue 9 2006K. Deiglmayr Summary Changes in community structure and activity of the dissimilatory nitrate-reducing community were investigated across a glacier foreland in the Central Alps to gain insight into the successional pattern of this functional group and the driving environmental factors. Bulk soil and rhizosphere soil of Poa alpina was sampled in five replicates in August during the flowering stage and in September after the first snowfalls along a gradient from 25 to 129 years after deglaciation and at a reference site outside the glacier foreland (> 2000 years deglaciated). In a laboratory-based assay, nitrate reductase activity was determined colorimetrically after 24 h of anaerobic incubation. In selected rhizosphere soil samples, the community structure of nitrate-reducing microorganisms was analysed by restriction fragment length polymorphism (RFLP) analysis using degenerate primers for the narG gene encoding the active site of the membrane-bound nitrate reductase. Clone libraries of the early (25 years) and late (129 years) succession were constructed and representative clones sequenced. The activity of the nitrate-reducing community increased significantly with age mainly due to higher carbon and nitrate availability in the late succession. The community structure, however, only showed a small shift over the 100 years of soil formation with pH explaining a major part (19%) of the observed variance. Clone library analysis of the early and late succession pointed to a trend of declining diversity with progressing age. Presumably, the pressure of competition on the nitrate reducers was relatively low in the early successional stage due to minor densities of microorganisms compared with the late stage; hence, a higher diversity could persist in this sparse environment. These results suggest that the nitrate reductase activity is regulated by environmental factors other than those shaping the genetic structure of the nitrate-reducing community. [source] Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methodsFEMS MICROBIOLOGY ECOLOGY, Issue 2 2007Blaire Steven Abstract A combination of culture-dependent and culture-independent methodologies (Bacteria and Archaea 16S rRNA gene clone library analyses) was used to determine the microbial diversity present within a geographically distinct high Arctic permafrost sample. Culturable Bacteria isolates, identified by 16S rRNA gene sequencing, belonged to the phyla Firmicutes, Actinobacteria and Proteobacteria with spore-forming Firmicutes being the most abundant; the majority of the isolates (19/23) were psychrotolerant, some (11/23) were halotolerant, and three isolates grew at ,5°C. A Bacteria 16S rRNA gene library containing 101 clones was composed of 42 phylotypes related to diverse phylogenetic groups including the Actinobacteria, Proteobacteria, Firmicutes, Cytophaga , Flavobacteria , Bacteroides, Planctomyces and Gemmatimonadetes; the bacterial 16S rRNA gene phylotypes were dominated by Actinobacteria- and Proteobacteria -related sequences. An Archaea 16S rRNA gene clone library containing 56 clones was made up of 11 phylotypes and contained sequences related to both of the major Archaea domains (Euryarchaeota and Crenarchaeota); the majority of sequences in the Archaea library were related to halophilic Archaea. Characterization of the microbial diversity existing within permafrost environments is important as it will lead to a better understanding of how microorganisms function and survive in such extreme cryoenvironments. [source] Succession of microbial communities during a biostimulation process as evaluated by DGGE and clone library analysesJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2001A. Ogino Aims:,The objective of this study was to investigate the changes in the indigenous bacterial community structure for assessing the impact of biostimulation on spilled oil. Methods and Results:,Changes in the bacterial community structure were monitored by denaturing gradient gel electrophoresis (DGGE) and clone library methods based on 16S rRNA gene (rDNA) sequences. The results of DGGE, coupled with the use of the Shannon index and principal component analysis (PCA) and clone library analyses, were consistent. In the treated (fertilized) area, one operational taxonomic unit (OTU) became dominant during the fertilization period, and it was most closely related to Pseudomonas putida. Conclusions:,The bacterial community structure in the treated area was markedly different from that in the control (non-fertilized) area during the fertilization period, but in the two areas it became similar at 14 weeks after the end of fertilization. Significance and Impact of the Study:,The results suggest that the bacterial community structure was disrupted by the biostimulation treatment, but that it recovered immediately after the end of fertilization. [source] Active bacterial community structure along vertical redox gradients in Baltic Sea sedimentENVIRONMENTAL MICROBIOLOGY, Issue 8 2008Anna Edlund Summary Community structures of active bacterial populations were investigated along a vertical redox profile in coastal Baltic Sea sediments by terminal-restriction fragment length polymorphism (T-RFLP) and clone library analysis. According to correspondence analysis of T-RFLP results and sequencing of cloned 16S rRNA genes, the microbial community structures at three redox depths (179, ,64 and ,337 mV) differed significantly. The bacterial communities in the community DNA differed from those in bromodeoxyuridine (BrdU)-labelled DNA, indicating that the growing members of the community that incorporated BrdU were not necessarily the most dominant members. The structures of the actively growing bacterial communities were most strongly correlated to organic carbon followed by total nitrogen and redox potentials. Bacterial identification by sequencing of 16S rRNA genes from clones of BrdU-labelled DNA and DNA from reverse transcription polymerase chain reaction showed that bacterial taxa involved in nitrogen and sulfur cycling were metabolically active along the redox profiles. Several sequences had low similarities to previously detected sequences, indicating that novel lineages of bacteria are present in Baltic Sea sediments. Also, a high number of different 16S rRNA gene sequences representing different phyla were detected at all sampling depths. [source] Microbial communities in a porphyry copper tailings impoundment and their impact on the geochemical dynamics of the mine wasteENVIRONMENTAL MICROBIOLOGY, Issue 2 2007Nouhou Diaby Summary The distribution and diversity of acidophilic bacteria of a tailings impoundment at the La Andina copper mine, Chile, was examined. The tailings have low sulfide (1.7% pyrite equivalent) and carbonate (1.4% calcite equivalent) contents and are stratified into three distinct zones: a surface (0-70-80 cm) ,oxidation zone' characterized by low-pH (2.5,4), a ,neutralization zone' (70,80 to 300,400 cm) and an unaltered ,primary zone' below 400 cm. A combined cultivation-dependent and biomolecular approach (terminal restriction enzyme fragment length polymorphism and 16S rRNA clone library analysis) was used to characterize the indigenous prokaryotic communities in the mine tailings. Total cell counts showed that the microbial biomass was greatest in the top 125 cm of the tailings. The largest numbers of bacteria (109 g,1 dry weight of tailings) were found at the oxidation front (the junction between the oxidation and neutralization zones), where sulfide minerals and oxygen were both present. The dominant iron-/sulfur-oxidizing bacteria identified at the oxidation front included bacteria of the genus Leptospirillum (detected by molecular methods), and Gram-positive iron-oxidizing acidophiles related to Sulfobacillus (identified both by molecular and cultivation methods). Acidithiobacillus ferrooxidans was also detected, albeit in relatively small numbers. Heterotrophic acidophiles related to Acidobacterium capsulatum were found by molecular methods, while another Acidobacterium -like bacterium and an Acidiphilium sp. were isolated from oxidation zone samples. A conceptual model was developed, based on microbiological and geochemical data derived from the tailings, to account for the biogeochemical evolution of the Piuquenes tailings impoundment. [source] Molecular characterization of microbial community in nitrate-removing activated sludgeFEMS MICROBIOLOGY ECOLOGY, Issue 2 2002Han-Woong Lee Abstract The microbial community composition and dominant denitrifying populations in high-nitrate-removing (CR-I) and low-nitrate-removing (CR-II) activated sludge from continuous bioreactors were investigated with most probable number (MPN) enumeration, fluorescence in situ hybridization (FISH) and 16S rDNA characterization. MPNs of nitrate-reducing bacteria of sludge CR-I and sludge CR-II were 2.82×107 and 2.69×104 colony-forming units ml,1, respectively. Eight denitrifying bacteria and two nitrate-reducing bacteria were isolated from sludge CR-I, and four denitrifying bacteria and three nitrate-reducing bacteria from sludge CR-II. Small subunit rDNA characterization of the isolates showed that the majority belonged to the genus Pseudomonas. By using FISH up to 76% (CR-I) and 52% (CR-II) of total 4,6-diamidino-2-phenylindole cell counts hybridized to the bacterial probe EUB338. Members of ,-Proteobacteria were the most abundant proteobacterial group in both sludges, accounting for up to 41.6% and 37.1% of those detected by EUB338, respectively, whereas a higher number of Cytophaga,Flexibacter cluster members were observed in CR-I sludge compared to CR-II sludge. In contrast with culture-based results, the numbers of rRNA group I Pseudomonads accounted for less than 0.01% of those detected by EUB338 in both sludges. Ribosomal DNA clone library analysis showed that the ,-Proteobacteria were also dominant in both sludges. In CR-I sludge, they were related to Zooglorea ramigera, Alcaligenes defragrans, denitrifying Fe-oxidizing bacteria and Dechlorimonas sp., whereas in CR-II sludge, they were related to Nitrosomonas sp. and Dechlorimonas agitatus. When this reactor was operated under anaerobic and anoxic conditions, nitrifying bacteria could adapt to the anoxic environment. We inferred that anaerobic ammonium oxidation and nitrite oxidation may occur in low-nitrate-removing sludge CR-II and inhibit denitrification. [source] Phylogenetic analysis of intestinal bacteria in the Chinese mitten crab (Eriocheir sinensis)JOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2007K. Li Abstract Aims:, To identify the dominant intestinal bacteria in the Chinese mitten crab, and to investigate the differences in the intestinal bacteria between pond-raised and wild crabs. Methods and Results:, The diversity of intestinal bacteria in the Chinese mitten crabs was investigated by denaturing gradient gel electrophoresis (DGGE) fingerprinting, 16S rRNA gene clone library analysis and real-time quantitative PCR. The principal component analysis of DGGE profiles indicated that substantial intersubject variations existed in intestinal bacteria in pond-raised crab. The sequencing of 16S rRNA genes revealed that 90,95% of the phylotypes in the clone libraries were affiliated with Proteobacteria and Bacteroidetes. Some genera were identified as unique in wild crabs and in pond-raised crabs, whereas Bacteroidetes was found to be common in all sampled crab groups. Real-time quantitative PCR indicated that the abundance of Bacteroides and the total bacterial load were approximately four-to-10 times higher in pond-raised crabs than in wild crabs. A significant portion of the phylotypes shared low similarity with previously sequenced organisms, indicating that the bacteria in the gut of Chinese mitten crabs are yet to be described. Conclusions:, The intestinal bacteria of pond-raised crabs showed higher intersubject variation, total diversity and abundance than that observed in wild crabs. The high proportion of the clones of Proteobacteria and Bacteroidetes in the clone library is an indication that these bacteria may be the dominant population in the gut of the Chinese mitten crab. Significance and Impact of the Study:, This study demonstrated obvious differences in the intestinal bacterial composition of pond-raised crabs and wild crabs. This knowledge will increase our understanding of the effects of aquaculture operations on bacterial community composition in the crab gut and provide necessary data for the development of probiotic products for crab cultivation. [source] Molecular analysis of the root canal microbiota associated with endodontic treatment failuresMOLECULAR ORAL MICROBIOLOGY, Issue 4 2008M. Sakamoto Introduction:, The failure of endodontic treatment is usually caused by persistent/secondary intraradicular infections and Enterococcus faecalis has been considered to be the main pathogen involved. Nevertheless, the breadth of bacterial diversity involved with endodontic treatment failures remains to be consistently explored by culture-independent approaches. Methods:, This study determined the intraradicular microbiota of root-canal-treated teeth with post-treatment apical periodontitis using 16S ribosomal RNA gene clone library analysis. Results:, Bacteria were present in all cases, confirming the infectious etiology of post-treatment disease. Seventy-four bacterial taxa belonging to six phyla were found in the nine cases investigated. Of these, 55% were identified as as-yet-uncultivated phylotypes, which also made up a significant proportion of the microbiota in many cases. Twenty-five new phylotypes were identified. Most teeth harbored a mixed consortium, with a mean number of 10 taxa per case. Only 11 taxa were found in more than one case, revealing a high interindividual variability in the composition of the microbiota. Conclusion:, The current findings revealed new candidate endodontic pathogens, including as-yet-uncultivated bacteria and taxa other than E. faecalis, which may participate in the mixed infections associated with post-treatment apical periodontitis. [source] | ||||||||||