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Clone Library (clone + library)
Kinds of Clone Library Terms modified by Clone Library Selected AbstractsCharacterization of bacterial communities in four freshwater lakes differing in nutrient load and food web structureFEMS MICROBIOLOGY ECOLOGY, Issue 2 2005Katleen van der Gucht Abstract The phylogenetic composition of bacterioplankton communities in the water column of four shallow eutrophic lakes was analyzed by partially sequencing cloned 16S rRNA genes and by PCR-DGGE analysis. The four lakes differed in nutrient load and food web structure: two were in a clearwater state and had dense stands of submerged macrophytes, while two others were in a turbid state characterized by the occurrence of phytoplankton blooms. One turbid and one clearwater lake had very high nutrient levels (total phosphorus > 100 ,g/l), while the other lakes were less nutrient rich (total phosphorus < 100,g/l). Cluster analysis, multidimensional scaling and ANOSIM (analysis of similarity) were used to investigate differences among the bacterial community composition in the four lakes. Our results show that each lake has its own distinct bacterioplankton community. The samples of lake Blankaart differed substantially from those of the other lakes; this pattern was consistent throughout the year of study. The bacterioplankton community composition in lake Blankaart seems to be less diverse and less stable than in the other three lakes. Clone library results reveal that Actinobacteria strongly dominated the bacterial community in lake Blankaart. The relative abundance of Betaproteobacteria was low, whereas this group was dominant in the other three lakes. Turbid lakes had a higher representation of Cyanobacteria, while clearwater lakes were characterized by more representatives of the Bacteroidetes. Correlating our DGGE data with environmental parameters, using the BIOENV procedure, suggests that differences are partly related to the equilibrium state of the lake. [source] Sulfate-reducing bacteria in marine sediment (Aarhus Bay, Denmark): abundance and diversity related to geochemical zonationENVIRONMENTAL MICROBIOLOGY, Issue 5 2009Julie Leloup Summary In order to better understand the main factors that influence the distribution of sulfate-reducing bacteria (SRB), their population size and their metabolic activity in high- and low-sulfate zones, we studied the SRB diversity in 3- to 5-m-deep sediment cores, which comprised the entire sulfate reduction zone and the upper methanogenic zone. By combining EMA (ethidium monoazide that can only enter damaged/dead cells and may also bind to free DNA) treatment with real-time PCR, we determined the distributions of total intact bacteria (16S rDNA genes) and intact SRB (dsrAB gene), their relative population sizes, and the proportion of dead cells or free DNA with depth. The abundance of SRB corresponded in average to 13% of the total bacterial community in the sulfate zone, 22% in the sulfate,methane transition zone and 8% in the methane zone. Compared with the total bacterial community, there were relatively less dead/damaged cells and free DNA present than among the SRB and this fraction did not change systematically with depth. By DGGE analysis, based on the amplification of the dsrA gene (400 bp), we found that the richness of SRB did not change with depth through the geochemical zones; but the clustering was related to the chemical zonation. A full-length clone library of the dsrAB gene (1900 bp) was constructed from four different depths (20, 110, 280 and 500 cm), and showed that the dsrAB genes in the near-surface sediment (20 cm) was mainly composed of sequences close to the Desulfobacteraceae, including marine complete and incomplete oxidizers such as Desulfosarcina, Desulfobacterium and Desulfococcus. The three other libraries were predominantly composed of Gram-positive SRB. [source] Fungal rDNA signatures in coronary atherosclerotic plaquesENVIRONMENTAL MICROBIOLOGY, Issue 12 2007Stephan J. Ott Summary Bacterial DNA has been found in coronary plaques and it has therefore been concluded that bacteria may play a role as trigger factors in the chronic inflammatory process underlying coronary atherosclerosis. However, the microbial spectrum is complex and it is not known whether microorganisms other than bacteria are involved in coronary disease. Fungal 18S rDNA signatures were systematically investigated in atherosclerotic tissue obtained through catheter-based atherectomy of 38 patients and controls (unaffected coronary arteries) using clone libraries, denaturating gradient gel analysis (DGGE), in situ hybridization and fluorescence in situ hybridization (FISH). Fungal DNA was found in 35 of 38 (92.11%) coronary heart disease patients by either polymerase chain reaction (PCR) with universal primers or in situ hybridization analysis (n = 5), but not in any control sample. In a clone library with more than 350 sequenced clones from pooled patient DNA, an overall richness of 19 different fungal phylotypes could be observed. Fungal profiles of coronary heart disease patients obtained by DGGE analysis showed a median richness of fungal species of 5 (range from 2 to 9) with a high interindividual variability (mean similarity 18.83%). For the first time, the presence of fungal components in atherosclerotic plaques has been demonstrated. Coronary atheromatous plaques harbour diverse and variable fungal communities suggesting a polymicrobial contribution to the chronic inflammatory aetiology. [source] Crash of a population of the marine heterotrophic flagellate Cafeteria roenbergensis by viral infectionENVIRONMENTAL MICROBIOLOGY, Issue 11 2007Ramon Massana Summary Viruses are known as important mortality agents of marine microorganisms. Most studies focus on bacterial and algal viruses, and few reports exist on viruses infecting marine heterotrophic protists. Here we show results from several incubations initiated with a microbial assemblage from the central Indian Ocean and amended with different amounts of organic matter. Heterotrophic flagellates developed up to 30 000 cells ml,1 in the most enriched incubation. A 18S rDNA clone library and fluorescent in situ hybridization counts with newly designed probes indicated that the peak was formed by Cafeteria roenbergensis and Caecitellus paraparvulus (90% and 10% of the cells respectively). Both taxa were below detection in the original sample, indicating a strong positive selective bias during the enrichment. During the peak, C. roenbergensis cells were observed with virus-like particles in the cytoplasm, and 4 days later this taxa could not be detected. Transmission electron microscopy confirmed the viral nature of these particles, which were large (280 nm), had double-stranded DNA, and were produced with a burst size of ,70. This virus was specific of C. roenbergensis as neither C. paraparvulus that was never seen infected, nor other flagellate taxa that developed in later stages of the incubation, appeared attacked. This is one of the few reports on a heterotrophic flagellate virus and the implications of this finding in the Indian Ocean are discussed. [source] Prokaryotic diversity and metabolically active microbial populations in sediments from an active mud volcano in the Gulf of MexicoENVIRONMENTAL MICROBIOLOGY, Issue 10 2006Robert J. Martinez Summary In this study, ribosomes and genomic DNA were extracted from three sediment depths (0,2, 6,8 and 10,12 cm) to determine the vertical changes in the microbial community composition and identify metabolically active microbial populations in sediments obtained from an active seafloor mud volcano site in the northern Gulf of Mexico. Domain-specific Bacteria and Archaea 16S polymerase chain reaction primers were used to amplify 16S rDNA gene sequences from extracted DNA. Complementary 16S ribosomal DNA (crDNA) was obtained from rRNA extracted from each sediment depth that had been subjected to reverse transcription polymerase chain reaction amplification. Twelve different 16S clone libraries, representing the three sediment depths, were constructed and a total of 154 rDNA (DNA-derived) and 142 crDNA (RNA-derived) Bacteria clones and 134 rDNA and 146 crDNA Archaea clones obtained. Analyses of the 576 clones revealed distinct differences in the composition and patterns of metabolically active microbial phylotypes relative to sediment depth. For example, ,- Proteobacteria rDNA clones dominated the 0,2 cm clone library whereas ,-Proteobacteria dominated the 0,2 cm crDNA library suggesting , to be among the most active in situ populations detected at 0,2 cm. Some microbial lineages, although detected at a frequency as high as 9% or greater in the total DNA library (i.e. Actinobacteria, ,- Proteobacteria), were markedly absent from the RNA-derived libraries suggesting a lack of in situ activity at any depth in the mud volcano sediments. This study is one of the first to report the composition of the microbial assemblages and physiologically active members of archaeal and bacterial populations extant in a Gulf of Mexico submarine mud volcano. [source] Bacterial diversity in the bacterioneuston (sea surface microlayer): the bacterioneuston through the looking glassENVIRONMENTAL MICROBIOLOGY, Issue 5 2005Mark P. Franklin Summary The bacterioneuston is defined as the community of bacteria present within the neuston or sea surface microlayer. Bacteria within this layer were sampled using a membrane filter technique and bacterial diversity was compared with that in the underlying pelagic coastal seawater using molecular ecological techniques. 16S rRNA gene libraries of , 500 clones were constructed from both bacterioneuston and the pelagic water samples and representative clones from each library were sequenced for comparison of bacterial diversity. The bacterioneuston was found to have a significantly lower bacterial diversity than the pelagic seawater, with only nine clone types (ecotaxa) as opposed to 46 ecotaxa in the pelagic seawater library. Surprisingly, the bacterioneuston clone library was dominated by 16S rRNA gene sequences affiliated to two groups of organisms, Vibrio spp. which accounted for over 68% of clones and Pseudoalteromonas spp. accounting for 21% of the library. The dominance of these two 16S rRNA gene sequence types within the bacterioneuston clone library was confirmed in a subsequent gene probing experiment. 16S rRNA gene probes specific for these groups of bacteria were designed and used to probe new libraries of 1000 clones from both the bacterioneuston and pelagic seawater DNA samples. This revealed that 57% of clones from the bacterioneuston library hybridized to a Vibrio sp.-specific 16S rRNA gene probe and 32% hybridized to a Pseudoalteromonas sp.-specific 16S rRNA gene probe. In contrast, the pelagic seawater library resulted in only 13% and 8% of 16S rRNA gene clones hybridizing to the Vibrio sp. and Pseudoalteromonas sp. probes respectively. Results from this study suggest that the bacterioneuston contains a distinct population of bacteria and warrants further detailed study at the molecular level. [source] Physiological and molecular characterization of anaerobic benzene-degrading mixed culturesENVIRONMENTAL MICROBIOLOGY, Issue 2 2003Ania C. Ulrich Summary Nine distinct anaerobic benzene-degrading cultures were enriched from sediment samples from four different sites. These cultures used nitrate, sulphate or CO2 as electron acceptors. The shortest doubling times were observed in nitrate-reducing cultures, although cell yield was lowest in these cultures. The highest substrate concentration utilized and maximum absolute rates of benzene degraded (in µM day,1) were observed in methanogenic cultures. The microbial compositions of a methanogenic and nitrate-reducing culture were determined from a clone library of 16S rRNA genes. Five Bacterial 16S rRNA sequences, one of which resembled a clone previously found in a sulphate-reducing, benzene-degrading culture and four Archaeal 16S rRNA sequences were identified in a methanogenic culture. Four Bacterial and no Archaeal 16S rRNA sequences were identified in a nitrate-reducing culture. The relative abundance of the four nitrate-reducing putative species was determined by slot blot hybridization. Two green sulphur bacteria together formed 52% of the clone library, but were found to be less than 4% of the culture by slot blot analysis. One of the cloned 16S rRNA gene sequences comprised 70% of the culture and was phylogenetically 93% similar to both Azoarcus and Dechloromonas species, which have been shown to degrade aromatic compounds, including benzene, under nitrate-reducing conditions. [source] Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone librariesENVIRONMENTAL MICROBIOLOGY, Issue 11 2002Andreas Schramm Summary A method is presented for fluorescence in situ hybridization (FISH) of 16S rRNA gene clones targeting in vivo transcribed plasmid inserts (Clone-FISH). Several different cloning approaches and treatments to generate target-rRNA in the clones were compared. Highest signal intensities of Clone-FISH were obtained using plasmids with a T7 RNA polymerase promoter and host cells with an IPTG-inducible T7 RNA polymerase. Combined IPTG-induction and chloramphenicol treatment of those clones resulted in FISH signals up to 2.8-fold higher than signals of FISH with probe EUB338 to cells of Escherichia coli. Probe dissociation curves for three oligonucleotide probes were compared for reference cells containing native (FISH) or cloned (Clone-FISH) target sequences. Melting behaviour and calculated Td values were virtually identical for clones and cells, providing a format to use 16S rRNA gene clones instead of pure cultures for probe validation and optimization of hybridization conditions. The optimized Clone-FISH protocol was also used to screen an environmental clone library for insert sequences of interest. In this application format, 13 out of 82 clones examined were identified to contain sulphate-reducing bacterial rRNA genes. In summary, Clone-FISH is a simple and fast technique, compatible with a wide variety of cloning vectors and hosts, that should have general utility for probe validation and screening of clone libraries. [source] High bacterial diversity of a waste gas-degrading community in an industrial biofilter as shown by a 16S rDNA clone libraryENVIRONMENTAL MICROBIOLOGY, Issue 11 2002Udo Friedrich Summary The bacterial diversity of an industrial biofilter used for waste gas abatement in an animal-rendering plant was investigated. A 16S rDNA clone library was generated and 444 clones were screened using computer-aided amplified ribosomal DNA restriction analysis (ARDRA). Of the screened clones, 60.8% showed unique ARDRA patterns and the remaining 174 clones were clustered into 65 groups. Almost full-length 16S rDNA sequences of 106 clones were determined and 90.5% of the clones were affiliated with the two phyla Proteobacteria and Bacteroidetes. Alpha -, Beta -, and Gammaproteobacteria accounted for 22.1, 17.6 and 18.6% respectively. Minor portions were affiliated with the Actinobacteria (2.0%), Firmicutes and Verrucomicrobia (both 1.0%), and the Deltaproteobacteria and Thermomicrobia (each 0.5%). Only six out of the 106 16S rDNA sequences exhibited similarities of more than 97% to classified bacterial species indicating that a substantial fraction of the clone sequences were derived from unknown taxa. It was also evaluated whether a database containing 281 computer-simulated bacterial rDNA fragment patterns generated from published reference sequences can be used for identification purposes. The data analysis demonstrated that this was possible only for a small number of clones, which were closely related to described bacterial strains. Rarefaction analysis of ARDRA clusters demonstrated that the 444 clones screened are insufficient to describe the entire diversity of the clone library. [source] Bacterial and archaeal populations associated with freshwater ferromanganous micronodules and sedimentsENVIRONMENTAL MICROBIOLOGY, Issue 1 2001Lisa Y. Stein Biology is believed to play a large role in the cycling of iron and manganese in many freshwater environments, but specific microbial groups indigenous to these systems have not been well characterized. To investigate the populations of Bacteria and Archaea associated with metal-rich sediments from Green Bay, WI, we extracted nucleic acids and analysed the phylogenetic relationships of cloned 16S rRNA genes. Because nucleic acids have not been routinely extracted from metal-rich samples, we investigated the bias inherent in DNA extraction and gene amplification from pure MnO2 using defined populations of whole cells or naked DNA. From the sediments, we screened for manganese-oxidizing bacteria using indicator media and found three isolates that were capable of manganese oxidation. In the phylogenetic analysis of bacterial 16S rRNA gene clones, we found two groups related to known metal-oxidizing genera, Leptothrix of the ,-Proteobacteria and Hyphomicrobium of the ,-Proteobacteria, and a Fe(III)-reducing group related to the Magnetospirillum genus of the ,-Proteobacteria. Groups related to the metal-reducing ,-Proteobacteria constituted 22% of the gene clones. In addition, gene sequences from one group of methanogens and a group of Crenarchaeota, identified in the archaeal gene clone library, were related to those found previously in Lake Michigan sediments. [source] Genetic diversity of the toxic cyanobacterium Microcystis in Lake MikataENVIRONMENTAL TOXICOLOGY, Issue 3 2005Mitsuhiro Yoshida Abstract The aim of the present study was to clarify the bloom dynamics and community composition of hepatotoxin microcystin-producing and non-microcystin-producing Microcystis genotypes in the environment. In Lake Mikata (Fukui, Japan) from April 2003 to January 2004, seasonal variation in the number of cells with microcystin (mcy) genotypes and the genetic diversity of the total population were investigated using quantitative competitive PCR and a 16S rDNA clone library, respectively. Using competitive PCR, cells with mcyA genotypes were quantified in August and October, and the ratio of the number of these mcyA genotypes to colony-forming Microcystis cells was 0.37 and 2.37, respectively. The 16S rDNA clones obtained could be divided into 12 ribotypes: a,l. Sixty-one Microcystis strains isolated from Lake Mikata during the sampling period were subjected to toxicity tests using HPLC and ELISA, PCR-based detection of the mcyA gene, and sequence analysis of the 16S rDNA. All isolates could be differentiated into 11 ribotypes (a, b, d, f, h, i, and m,q). Ribotypes b, f, i, m, n, and p had at least one strain that was a microcystin producer. In natural communities ribotypes b and f accounted for 85% of the 16S rDNA clones in August, and ribotypes b and i accounted for 24% of the clones in October. Thus, in some bloom stages the presence of microcystin genotypes identified using the 16S rDNA clone library correlated with that of mcy genotypes determined using competitive PCR. © 2005 Wiley Periodicals, Inc. Environ Toxicol 20: 229,234, 2005. [source] Diversity of endophytic bacterial communities in poplar grown under field conditionsFEMS MICROBIOLOGY ECOLOGY, Issue 2 2008Kristina Ulrich Abstract 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. [source] High prokaryote diversity and analysis of community structure in mobile mud deposits off French Guiana: identification of two new bacterial candidate divisionsFEMS MICROBIOLOGY ECOLOGY, Issue 3 2001Vanessa M. Madrid Abstract Bacterial and archaeal community compositions in highly mobile nearshore muds typical of the Guiana coastline of South America were examined by sequence analysis of a 16S rDNA clone library. DNA was extracted from a subsurface sediment layer (10,30 cm) collected at a subtidal (,1 m deep) mud wave site between Kourou and Sinnamary, French Guiana. Analysis of 96 non-chimeric sequences showed the majority to be bacteria (98%), that diversity was high with 64 unique sequences, and that proteobacteria were dominant (46%). Two crenarchaeota sequences were found (2%). Bacterial sequences belonged to the Cytophaga-Flexibacter-Bacteroides (18%), Actinobacteria (11.5%), Planctomycetes (6.3%), Cyanobacteria (3.2%), low-GC Gram-positive (1%), ,, , and , subdivisions of Proteobacteria (27%, 16%, and 9%, respectively). Additional bacterial sequences belonged to the candidate division TM6 (1%) and to two newly proposed candidate divisions: KS-A (2%) and KS-B (3%). A sizeable fraction (22%) of sequences from the Kourou,Sinnamary library are normally found in water column populations, reflecting frequent entrainment of suspended debris into physically reworked underlying sediments. Dominant sequences (56%) were related to Gelidibacter algens (Cytophaga-Flexibacter-Bacteroides group), Actinobacteria, Sulfitobacter and Ruegeria spp. (,-proteobacteria), all of which are chemoorganotrophs, consistent with abundant labile organic carbon. The presence of sequences from potential sulfate reducers and sulfide oxidizers suggests the likelihood of sulfur cycling in these sediments, despite the dominance of suboxic (iron-reducing), non-sulfidic diagenetic properties. Rarefaction analysis indicated that bacterial diversity in the French Guiana library is not only unusually high in comparison with other marine sedimentary environments, but among the most diverse of all environments reported to date. [source] Ribosomal RNA gene fragments from fossilized cyanobacteria identified in primary gypsum from the late Miocene, ItalyGEOBIOLOGY, Issue 2 2010G. PANIERI Earth scientists have searched for signs of microscopic life in ancient samples of permafrost, ice, deep-sea sediments, amber, salt and chert. Until now, evidence of cyanobacteria has not been reported in any studies of ancient DNA older than a few thousand years. Here, we investigate morphologically, biochemically and genetically primary evaporites deposited in situ during the late Miocene (Messinian) Salinity Crisis from the north-eastern Apennines of Italy. The evaporites contain fossilized bacterial structures having identical morphological forms as modern microbes. We successfully extracted and amplified genetic material belonging to ancient cyanobacteria from gypsum crystals dating back to 5.910,5.816 Ma, when the Mediterranean became a giant hypersaline brine pool. This finding represents the oldest ancient cyanobacterial DNA to date. Our clone library and its phylogenetic comparison with present cyanobacterial populations point to a marine origin for the depositional basin. This investigation opens the possibility of including fossil cyanobacterial DNA into the palaeo-reconstruction of various environments and could also be used to quantify the ecological importance of cyanobacteria through geological time. These genetic markers serve as biosignatures providing important clues about ancient life and begin a new discussion concerning the debate on the origin of late Miocene evaporites in the Mediterranean. [source] Comparison of DNA- and RNA-based bacterial community structures in soil exposed to 2,4-dichlorophenolJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2009L. Lillis Abstract Aims:, To examine the effect of the pollutant 2,4-dichlorophenol on DNA- and RNA-based bacterial communities in soil. Methods and Results:, Soil was exposed to 100 mg kg,1 of 2,4-dichlorophenol (2,4-DCP), and degradation was monitored over 35 days. DNA and RNA were coextracted, and terminal restriction fragment length polymorphism (T-RFLP) was used to report changes in bacterial communities in response to the presence of the chlorophenol. The phylogenetic composition of the soil during degradation was determined by creating a clone library of amplified 16S rRNA sequences from both DNA and reverse-transcribed RNA from exposed soil. Resulting clones were sequenced, and putative identities were assigned. Conclusions:, A significant difference between active (RNA-based) and total (DNA-based) bacterial community structure was observed for both T-RFLP and phylogenetic analyses in response to 2,4-DCP, with more pronounced changes seen in RNA-based communities. Phylogenetic analysis indicated the dominance of Proteobacteria in both profiles. Significance and Impact of the Study:, This study describes the response of soil bacterial communities to the addition of the xenobiotic compound 2,4-DCP, and highlights the importance of including RNA-based 16S rRNA analysis to complement any molecular study in a perturbed soil. [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] Diversity and abundance of Bacteria and Archaea in the Bor Khlueng Hot Spring in ThailandJOURNAL OF BASIC MICROBIOLOGY, Issue 6 2004Pattanop Kanokratana The prokaryotic diversity in the Bor Khlueng hot spring in Ratchaburi province, Thailand was investigated by a culture-independent molecular approach. This hydrothermal pool is located in the central part of Thailand and contains sulfide-rich mineral water that is believed to relieve muscle ache and pain. The water flow year-round with temperature ranging between 50,57 °C. Community DNA was extracted directly from sediment samples by coring to depth of ,20,30 cm. Small-subunit rRNA genes (16S rDNA) were amplified by PCR using primers specific for the domains Archaea and Bacteria. The PCR products were cloned and sequenced. For the bacterial rDNA clone library, 200 clones were randomly selected for further analyses. After restriction fragment length polymorphism (RFLP) analysis of rDNA clones and exclusion of chimeric sequences 36 phylotypes were obtained. The Bor Khlueng phylotypes spanned a wide range within the domain Bacteria, occupying eleven major lineages (phyla). Almost a quarter (23%) of the clones were classifed as Acidobacteria. The other clones were grouped into the Bacteriodetes (19%), Nitrospirae (13%), Proteobacteria (12%), Deinococcus-Thermus lineage (11%), planctomycetes (6%), and Verrucomicrobia (5%). The four remaining phyla, 5% each, were assigned to Actinobacteria, Chloroflexi, Cyanobacteria, and the candidate division "OP10". For the archaeal 16S rRNA gene sequence library, 25 distinct phylotypes were obtained, 17 clones were found to be associated with Crenarahaeota and 8 clones were associated with Euryarachaeota. The findings of the molecular survey of this so far not investigated site showed that Bor Khlueng hot spring is a potential rich source of unique bacterial and archaeal species. The great majority (,80%) of the prokaryotic sequences detected in Bor Khlueng were unknown. (© 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] PHYLOGENY OF FOUR DINOPHYSIACEAN GENERA (DINOPHYCEAE, DINOPHYSIALES) BASED ON rDNA SEQUENCES FROM SINGLE CELLS AND ENVIRONMENTAL SAMPLES,JOURNAL OF PHYCOLOGY, Issue 5 2009Sara M. Handy Dinoflagellates are a highly diverse and environmentally important group of protists with relatively poor resolution of phylogenetic relationships, particularly among heterotrophic species. We examined the phylogeny of several dinophysiacean dinoflagellates using samples collected from four Atlantic sites. As a rule, 3.5 kb of sequence including the nuclear ribosomal genes SSU, 5.8S, LSU, plus their internal transcribed spacer (ITS) 1 and 2 regions were determined for 26 individuals, including representatives of two genera for which molecular data were previously unavailable, Ornithocercus F. Stein and Histioneis F. Stein. In addition, a clone library targeting the dinophysiacean ITS2 and LSU sequences was constructed from bulk environmental DNA from three sites. Three phylogenetic trees were inferred from the data, one using data from this study for cells identified to genus or species (3.5 kb, 28 taxa); another containing dinoflagellate SSU submissions from GenBank and the 12 new dinophysiacean sequences (1.9 kb, 56 taxa) from this study; and the third tree combing data from identified taxa, dinophysiacean GenBank submissions, and the clone libraries from this study (2.1 kb, 136 taxa). All trees were congruent and indicated a distinct division between the genera Phalacroma F. Stein and Dinophysis Ehrenb. The cyanobionts containing genera Histioneis and Ornithocercus were also monophyletic. This was the largest molecular phylogeny of dinophysoid taxa performed to date and was consistent with the view that the genus Phalacroma may not be synonymous with Dinophysis. [source] Marine sponge Craniella austrialiensis -associated bacterial diversity revelation based on 16S rDNA library and biologically active Actinomycetes screening, phylogenetic analysisLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2006Z.-Y. Li Abstract Aims:, The aim of this study was to investigate the bacterial diversity associated with the sponge Craniella australiensis using a molecular strategy and isolating Actinomycetes with antimicrobial potentials. Methods and Results:, The bacterial diversity associated with South China Sea sponge C. austrialiensis was assessed using a 16S rDNA clone library alongside restriction fragment length polymorphism and phylogenetic analysis. It was found that the C. austrialiensis -associated bacterial community consisted of alpha, beta and gamma- Proteobacteria, Firmicutes, Bacteroidetes as well as Actinobacterium. Actinomycetes were isolated successfully using seawater medium with sponge extracts. According to the BLAST and phylogenetic analysis based on about 600-bp 16S rDNA sequences, 11 of the representative 23 isolates closely matched the Streptomyces sp. while the remaining 12 matched the Actinomycetales. Twenty Actinomycetes have antimicrobial potentials, of which 15 are found to possess broad-spectrum antimicrobial potentials. Conclusions:, The sponge C. austrialiensis -associated bacterial community is very abundant including Proteobacteria, Firmicutes, Bacteroidetes and Actinobacterium while Actinomycetes is not predominant. Artificial seawater medium with sponge extracts is suitable for Actinomycetes isolation. Most of the isolated C. austrialiensis -associated Actinomycetes have a broad spectrum of antimicrobial activity. Significance and Impact of the Study:, This study revealed the diversity of the bacterial community and the isolated Actinomycetes with antimicrobial potentials associated with sponge C. australiensis. [source] Phylogenetic analysis of the fecal flora of the wild pygmy lorisAMERICAN JOURNAL OF PRIMATOLOGY, Issue 8 2010Xu Bo Abstract The bacterial diversity in fecal samples from the wild pygmy loris was examined with a 16S rDNA clone library and restriction fragment length polymorphism analysis. The clones were classified as Firmicutes (43.1%), Proteobacteria (34.5%), Actinobacteria (5.2%), and Bacteroidetes (17.2%). The 58 different kinds of 16S rDNA sequences were classified into 16 genera and 20 uncultured bacteria. According to phylogenetic analysis, the major genera within the Proteobacteria was Pseudomonas, comprising 13.79% of the analyzed clone sequences. Many of the isolated rDNA sequences did not correspond to known microorganisms, but had high homology to uncultured clones found in human feces. Am. J. Primatol. 72:699,706, 2010. © 2010 Wiley-Liss, Inc. [source] Bacterial diversity in various coastal mariculture ponds in Southeast China and in diseased eels as revealed by culture and culture-independent molecular techniquesAQUACULTURE RESEARCH, Issue 9 2010Yonghui Zeng Abstract Mariculture ponds are widely distributed in Chinese coasts and have become a threat to the health of coastal ecosystems. In order to improve our understanding on the microbial composition in mariculture environments, we sampled a variety of ponds farming different animals or plants around the Dongshan Island and Xiamen Island in Southeast China and isolated cultures from the tissues of diseased eels. Analysis by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE), clone library and direct culturing methods revealed highly diverse bacterial communities in these samples. Bacterial communities in the Dongshan samples were dominated by Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes. The Gracilaria verrucosa pond harbours the most abundant species (20 DGGE bands), followed by Epinephelus diacanthus pond (18 bands), Haliotis diversicolor supertexta pond I (18 bands) and Penaeus vannamei pond (11 bands). In comparison with surface waters, Penacus orientalis pond sediment showed a much more complex bacterial community, from which only sequences affiliated with Deltaproteobacteria, Firmicutes, Acidobacteria and candidate phylum TM6 were found. Bacterial cultures in diseased eels were closely related to two pathogenic genera, Aeromonas in Gammaproteobacteria and Bacillus, in Firmicutes. Clones affiliated with another two genera, Escherichia and Vibrio, that have pathogenic potentials were also identified. Phylogenetic analysis of a total of 131 sequences showed that 48.9% of the sequences were clustered into Gammaproteobacteria and formed the most abundant group, followed by Alphaproteobacteria (19.1%), Firmicutes (7.6%), Bacteroidetes (5.3%), Deltaproteobacteria (5.3%), Actinobacteria (4.6%), Chloroplast (3.8%), Acidobacteria (2.3%), Cyanobacteria (1.5%), Betaproteobacteria (0.7%) and TM6 (0.7%). 43.7% (28/64) of the phylogenetic clusters cannot be classified into any known genus and 44.3% (58/131) of the sequences show <95% similarity to public database records, suggesting that abundant novel species exist in mariculture ponds. Gathering bacterial diversity data in mariculture ponds and diseased fish is meaningful for the prevention and control of fish diseases and for the improvement of our understanding of microbial ecology in a pond environment. [source] Microbial community structure in a biofilm anode fed with a fermentable substrate: The significance of hydrogen scavengersBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2010Prathap Parameswaran Abstract We compared the microbial community structures that developed in the biofilm anode of two microbial electrolysis cells fed with ethanol, a fermentable substrate,one where methanogenesis was allowed and another in which it was completely inhibited with 2-bromoethane sulfonate. We observed a three-way syntrophy among ethanol fermenters, acetate-oxidizing anode-respiring bacteria (ARB), and a H2 scavenger. When methanogenesis was allowed, H2 -oxidizing methanogens were the H2 scavengers, but when methanogenesis was inhibited, homo-acetogens became a channel for electron flow from H2 to current through acetate. We established the presence of homo-acetogens by two independent molecular techniques: 16S rRNA gene based pyrosequencing and a clone library from a highly conserved region in the functional gene encoding formyltetrahydrofolate synthetase in homo-acetogens. Both methods documented the presence of the homo-acetogenic genus, Acetobacterium, only with methanogenic inhibition. Pyrosequencing also showed a predominance of ethanol-fermenting bacteria, primarily represented by the genus Pelobacter. The next most abundant group was a diverse community of ARB, and they were followed by H2 -scavenging syntrophic partners that were either H2 -oxidizing methanogens or homo-acetogens when methanogenesis was suppressed. Thus, the community structure in the biofilm anode and suspension reflected the electron-flow distribution and H2 -scavenging mechanism. Biotechnol. Bioeng. 2010;105: 69,78. © 2009 Wiley Periodicals, Inc. [source] Microbiological and geochemical dynamics in simulated-heap leaching of a polymetallic sulfide oreBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2008Kathryn Wakeman Abstract The evolution of microbial populations involved in simulated-heap leaching of a polymetallic black schist sulfide ore (from the recently-commissioned Talvivaara mine, Finland) was monitored in aerated packed bed column reactors over a period of 40 weeks. The influence of ore particle size (2-6.5 mm and 6.5-12 mm) on changes in composition of the bioleaching microflora and mineral leaching dynamics in columns was investigated and compared to fine-grain (<2 µm) ore that was bioprocessed in shake flask cultures. Both column reactors and shake flasks were inoculated with 24 different species and strains of mineral-oxidizing and other acidophilic micro-organisms, and maintained at 37°C. Mineral oxidation was most rapid in shake flask cultures, with about 80% of both manganese and nickel and 68% of zinc being leached within 6 weeks, though relatively little of the copper present in the ore was solubilised. The microbial consortium that emerged from the original inoculum was relatively simple in shake flasks, and was dominated by the iron-oxidizing autotroph Leptospirillum ferriphilum, with smaller numbers of Acidimicrobium ferrooxidans, Acidithiobacillus caldus and Leptospirillum ferrooxidans. Both metal recovery and (for the most part) total numbers of prokaryotes were greater in the column reactor containing the medium-grain than that containing the coarse-grain ore. The bioleaching communities in the columns displayed temporal changes in composition and differed radically from those in shake flask cultures. While iron-oxidizing chemoautotrophic bacteria were always the most numerically dominant bacteria in the medium-grain column bioreactor, there were major shifts in the most abundant species present, with the type strain of Acidithiobacillus ferrooxidans dominating in the early phase of the experiment and other bacteria (At. ferrooxidans NO37 and L. ferriphilum) dominating from week 4 to week 40. With the coarse-grain column bioreactor, similar transitions in populations of iron-oxidizing chemoautotrophs were observed, though heterotrophic acidophiles were often the most abundant bacteria found in mineral leach liquors. Four bacteria not included in the mixed culture used to inoculate the columns were detected by biomolecular techniques and three of these (all Alicyclobacillus -like Firmicutes) were isolated as pure cultures. The fourth bacterium, identified from a clone library, was related to the Gram-positive sulfate reducer Desulfotomaculum salinum. All four were considered to have been present as endospores on the dried ore, which was not sterilized in the column bioreactors. Two of the Alicyclobacillus -like isolates were found, transiently, in large numbers in mineral leachates. The data support the hypothesis that temporal and spatial heterogeneity in mineral heaps create conditions that favour different mineral-oxidizing microflora, and that it is therefore important that sufficient microbial diversity is present in heaps to optimize metal extraction. Biotechnol. Bioeng. 2008;101: 739,750. © 2008 Wiley Periodicals, Inc. [source] Effect of the applied organic load rate on biodegradable polymer production by mixed microbial cultures in a sequencing batch reactorBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Davide Dionisi Abstract This article studies the operation of a new process for the production of biopolymers (polyhydroxyalkanoates, PHAs) at different applied organic load rates (OLRs). The process is based on the aerobic enrichment of activated sludge to obtain mixed cultures able to store PHAs at high rates and yields. A mixture of acetic, lactic, and propionic acids at different concentrations (in the range 8.5,31.25 gCOD/L) was fed every 2 h in a sequencing batch reactor (SBR). The resulting applied OLR was in the range 8.5,31.25 gCOD/L/day. Even though, as expected, the increase in the OLR caused an increase in biomass concentration (up to about 8.7 g COD/L), it also caused a relevant decrease of maximal polymer production rate. This decrease in polymer production rate was related to the different extent of "feast and famine" conditions, as function of the applied OLR and of the start-up conditions. As a consequence the best performance of the process was obtained at an intermediate OLR (20 gCOD/L/day) where both biomass productivity and PHA storage were high enough. However, at this high OLR the process was unstable and sudden decrease of performance was also observed. The sludge characterized by the highest PHA storage response was investigated by 16S rDNA clone library. The clone library contained sequences mostly from PHA producers (e.g., Alcaligenes and Comamonas genera); however many genera and among them, one of the dominant (Thauera), were never described before in relation to PHA storage response. © 2005 Wiley Periodicals, Inc. [source] | |||||||||||||||||||||||||