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Terminal Restriction Fragment Length Polymorphism (terminal + restriction_fragment_length_polymorphism)
Terms modified by Terminal Restriction Fragment Length Polymorphism Selected AbstractsA methane-driven microbial food web in a wetland rice soilENVIRONMENTAL MICROBIOLOGY, Issue 12 2007Jun Murase Summary Methane oxidation is a key process controlling methane emission from anoxic habitats into the atmosphere. Methanotrophs, responsible for aerobic methane oxidation, do not only oxidize but also assimilate methane. Once assimilated, methane carbon may be utilized by other organisms. Here we report on a microbial food web in a rice field soil driven by methane. A thin layer of water-saturated rice field soil was incubated under opposing gradients of oxygen and 13C-labelled methane. Bacterial and eukaryotic communities incorporating methane carbon were analysed by RNA-stable isotope probing (SIP). Terminal restriction fragment length polymorphism (T-RFLP) and cloning showed that methanotrophs were the most prominent group of bacteria incorporating methane carbon. In addition, a few Myxobacteria -related sequences were obtained from the ,heavy' rRNA fraction. Denaturing gradient gel electrophoresis (DGGE) targeting 18S rRNA detected various groups of protists in the ,heavy' rRNA fraction including naked amoeba (Lobosea and Heterolobosea), ciliates (Colpodea) and flagellates (Cercozoa). Incubation of soil under different methane concentrations in air resulted in the development of distinct protozoan communities. These results suggest that methane carbon is incorporated into non-methanotrophic pro- and microeukaryotes probably via grazing, and that methane oxidation is a shaping force of the microeukaryotic community depending on methane availability. [source] Effect of N-fertilization, plant genotype and environmental conditions on nifH gene pools in roots of riceENVIRONMENTAL MICROBIOLOGY, Issue 10 2003Zhiyuan Tan Summary Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified nitrogenase gene (nifH) fragments is a rapid technique for profiling of diazotrophic microbial communities without the necessity of cultures for study. Here, we examined the impact of N-fertilization, plant genotype and environmental conditions on diazotrophic microbial populations in association with roots of rice (Oryza species) by T-RFLP community profiling and found marked effects on the composition of the microbial community. We found a rapid change of the diazotrophic population structure within 15 days after application of nitrogen fertilizer and a strong effect of environmental conditions and plant genotype. Control experiments revealed that phylogenetically distantly related nifH genes were proportionately amplified, and that signal strength reflected the relative abundance of nifH genes in the sample within a 10-fold range of template concentrations. These results clearly demonstrated that our T-RFLP method was suitable to reflect compositional differences in the diazotrophic community in a semiquantitative manner and that the diazotrophic rhizosphere communities of rice are not static but presumably rather highly dynamic. [source] Molecular analysis of bacterial populations in the ileum of broiler chickens and comparison with bacteria in the cecumFEMS MICROBIOLOGY ECOLOGY, Issue 3 2002Jianhua Gong Abstract Bacterial populations in the ileum of broiler chickens were analyzed by molecular analysis of 16S rRNA genes and compared to those in the cecum. Bacteria found in the ileal mucosa were mainly Gram-positive with low G+C content. There were 15 molecular species among 51 cloned sequences. More than 70% of the cloned sequences were related to lactobacilli and Enterococcus cecorum. Two sequences had 95% or less homology to existing database sequences. Terminal restriction fragment length polymorphism (T-RFLP) analysis revealed differences among bacterial populations present in the mucosa and lumen of the ileum. Comparative studies by T-RFLP and sequence analyses of 16S rRNA genes indicated a less diverse bacterial population in the ileum (mucosa and lumen) than in the cecum. Lactobacilli, E. cecorum, and butyrate-producing bacteria related (including both identified and unidentified species) sequences were the three major groups detected in ilea and ceca. Although butyrate-producing bacteria may have good potential in the development of novel probiotics for poultry, verifying the presence of the bacteria in the chicken gut is required to warrant further investigation. [source] Assessment of anaerobic wastewater treatment failure using terminal restriction fragment length polymorphism analysisJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2005C. Scully Abstract Aims:, The suitability of genetic fingerprinting to study the microbiological basis of anaerobic bioreactor failure is investigated. Methods and Results:, Two laboratory-scale anaerobic expanded granular sludge bed bioreactors, R1 and R2, were used for the mesophilic (37°C) treatment of high-strength [10 g chemical oxygen demand (COD) l,1] synthetic industrial-like wastewater over a 100-day trial period. A successful start up was achieved by both bioreactors with COD removal over 90%. Both reactors were operated under identical parameters; however, increased organic loading during the trial induced a reduction in the COD removal of R1, while R2 maintained satisfactory performance (COD removal >90%) throughout the experiment. Specific methanogenic activity measurements of biomass from both reactors indicated that the main route of methane production was hydrogenotrophic methanogenesis. Terminal restriction fragment length polymorphism (TRFLP) analysis was applied to the characterization of microbial community dynamics within the system during the trial. The principal differences between the two consortia analysed included an increased abundance of Thiovulum - and Methanococcus -like organisms and uncultured Crenarchaeota in R1. Conclusions:, The results indicated that there was a microbiological basis for the deviation, in terms of operational performance, of R1 and R2. Significance and Impact of the Study:, High-throughput fingerprinting techniques, such as TRFLP, have been demonstrated as practically relevant for biomonitoring of anaerobic reactor communities. [source] UREASE GENE SEQUENCES FROM ALGAE AND HETEROTROPHIC BACTERIA IN AXENIC AND NONAXENIC PHYTOPLANKTON CULTURES,JOURNAL OF PHYCOLOGY, Issue 3 2009Kristopher M. Baker While urea has long been recognized as an important form of nitrogen in planktonic ecosystems, very little is known about how many or which phytoplankton and bacteria can use urea as a nitrogen source. We developed a method, targeting the gene encoding urease, for the direct detection and identification of ureolytic organisms and tested it on seven axenic phytoplankton cultures (three diatoms, two prymnesiophytes, a eustigmatophyte, and a pelagophyte) and on three nonaxenic Aureococcus anophagefferens Hargraves et Sieburth cultures (CCMP1784 and two CCMP1708 cultures from different laboratories). The urease amplicon sequences from axenic phytoplankton cultures were consistent with genomic data in the three species for which both were available. Seven of 12 phytoplankton species have one or more introns in the amplified region of their urease gene(s). The 63 urease amplicons that were cloned and sequenced from nonaxenic A. anophagefferens cultures grouped into 17 distinct sequence types. Eleven types were related to ,-Proteobacteria, including three types likely belonging to the genus Roseovarius. Four types were related to ,-Proteobacteria, including two likely belonging to the genus Marinobacter, and two types were related to ,-Proteobacteria. Terminal restriction fragment length polymorphism (TRFLP) analyses suggested that the sequenced amplicons represented approximately half of the diversity of bacterial urease genes present in the nonaxenic cultures. While many of the bacterial urease sequence types were apparently lab- or culture-specific, others were found in all three nonaxenic cultures, suggesting the possibility of specific relationships between these bacteria and A. anophagefferens. [source] Comparison of T-RFLP and DGGE techniques to assess denitrifier community composition in soilLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2009K. Enwall Abstract Terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) and subsequent statistical analysis were compared with assess denitrifier community composition in agricultural soil based on the nosZ gene, encoding the nitrous oxide reductase. Analysis of binary or relative abundance-based metric and semi-metric distance matrices provided similar results for DGGE, but not for T-RFLP. Moreover, DGGE had a higher resolution than T-RFLP and binary data was better for discriminating between samples. [source] Bacterioplankton assemblages transforming dissolved organic compounds in coastal seawaterENVIRONMENTAL MICROBIOLOGY, Issue 8 2007Xiaozhen Mou Summary To characterize bacterioplankton functional assemblages that transform specific components of the coastal seawater dissolved organic carbon (DOC) pool, bromodeoxyuridine (BrdU) was used to label the bacterioplankton cells that were active following addition of single-DOC model compounds: two organic osmolytes [dimethylsulfoniopropionate (DMSP) and glycine betaine (GlyB)] and two aromatic monomers [para -hydroxybenzoic acid (pHBA) and vanillic acid (VanA)]. Bacterial populations were analysed based on in situ fluorescent immunodetection of BrdU incorporation followed by fluorescence-activated cell sorting (FACS). Sorted cells were then characterized by 16S rDNA-based analysis. Populations with high BrdU incorporation level (HI) developed within 8 h of introduction of 100 nM model compound. Terminal restriction fragment length polymorphisms (T-RFLP) analysis indicated that the HI populations in all four amendments were composed of bacteria from the same major taxa (phylum and subphylum levels), but the relative abundance of each differed. High-resolution clone libraries (each containing ,200 clones) showed that the HI populations in the GlyB and VanA amendments consisted of both metabolic generalists and specialists within the , -Proteobacteria (mainly members of the Roseobacter clade), , -Proteobacteria and , -Proteobacteria (mainly members of Altermonadaceae, Chromatiaceae, Oceanospirillaceae and Pseudomonadaceae). The presence of members of OM60/241, OM185, SAR11, SAR86 and SAR116 in the HI populations indicated that members of these groups can assimilate the model DOC compounds, providing some of the first glimpses into heterotrophy by members of these poorly understood environmental clusters. [source] In situ measurement of methane fluxes and analysis of transcribed particulate methane monooxygenase in desert soilsENVIRONMENTAL MICROBIOLOGY, Issue 10 2009Roey Angel Summary Aerated soils are a biological sink for atmospheric methane. However, the activity of desert soils and the presence of methanotrophs in these soils have hardly been studied. We studied on-site atmospheric methane consumption rates as well as the diversity and expression of the pmoA gene, coding for a subunit of the particulate methane monooxygenase, in arid and hyperarid soils in the Negev Desert, Israel. Methane uptake was only detected in undisturbed soils in the arid region (,90 mm year,1) and vertical methane profiles in soil showed the active layer to be at 0,20 cm depth. No methane uptake was detected in the hyperarid soils (,20 mm year,1) as well as in disturbed soils in the arid region (i.e. agricultural field and a mini-catchment). Molecular analysis of the methanotrophic community using terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene detected methanotrophs in the active soils, whereas the inactive ones were dominated by sequences of the homologous gene amoA, coding for a subunit of the ammonia monooxygenase. Even in the active soils, methanotrophs (as well as in situ activity) could not be detected in the soil crust, which is the biologically most important layer in desert soils. All pmoA sequences belonged to yet uncultured strains. Transcript analysis showed dominance of sequences clustering within the JR3, formerly identified in Californian grassland soils. Our results show that although active methanotrophs are prevalent in arid soils they seem to be absent or inactive in hyperarid and disturbed arid soils. Furthermore, we postulate that methanotrophs of the yet uncultured JR3 cluster are the dominant atmospheric methane oxidizers in this ecosystem. [source] The role of Variovorax and other Comamonadaceae in sulfur transformations by microbial wheat rhizosphere communities exposed to different sulfur fertilization regimesENVIRONMENTAL MICROBIOLOGY, Issue 6 2008Achim Schmalenberger Summary Sulfonates are a key component of the sulfur present in agricultural soils. Their mobilization as part of the soil sulfur cycle is mediated by rhizobacteria, and involves the oxidoreductase AsfA. In this study, the effect of fertilization regime on rhizosphere bacterial asfA distribution was examined at the Broadbalk long-term wheat experiment, Rothamsted, UK, which was established in 1843, and has included a sulfur-free treatment since 2001. Direct isolation of desulfonating rhizobacteria from the wheat rhizospheres led to the identification of several Variovorax and Polaromonas strains, all of which contained the asfA gene. Rhizosphere DNA was isolated from wheat rhizospheres in plots fertilized with inorganic fertilizer with and without sulfur, with farmyard manure or from unfertilized plots. Genetic profiling of 16S rRNA gene fragments [denaturing gradient gel electrophoresis (DGGE)] from the wheat rhizospheres revealed that the level of inorganic sulfate in the inorganic fertilizer was correlated with changes in the general bacterial community structure and the betaproteobacterial community structure in particular. Community analysis at the functional gene level (asfA) showed that 40% of clones in asfAB clone libraries were affiliated to the genus Variovorax. Analysis of asfAB -based terminal restriction fragment length polymorphism (T-RFLP) fingerprints showed considerable differences between sulfate-free treatments and those where sulfate was applied. The results suggest the occurrence of desulfonating bacterial communities that are specific to the fertilization regime chosen and that arylsulfonates play an important role in rhizobacterial sulfur nutrition. [source] Activity and composition of methanotrophic bacterial communities in planted rice soil studied by flux measurements, analyses of pmoA gene and stable isotope probing of phospholipid fatty acidsENVIRONMENTAL MICROBIOLOGY, Issue 2 2008Minita Shrestha Summary Methanotrophs in the rhizosphere of rice field ecosystems attenuate the emissions of CH4 into the atmosphere and thus play an important role for the global cycle of this greenhouse gas. Therefore, we measured the activity and composition of the methanotrophic community in the rhizosphere of rice microcosms. Methane oxidation was determined by measuring the CH4 flux in the presence and absence of difluoromethane as a specific inhibitor for methane oxidation. Methane oxidation started on day 24 and reached the maximum on day 32 after transplantation. The total methanotrophic community was analysed by terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. The metabolically active methanotrophic community was analysed by stable isotope probing of microbial phospholipid fatty acids (PLFA-SIP) using 13C-labelled CH4 directly added to the rhizospheric region. Rhizospheric soil and root samples were collected after exposure to 13CH4 for 8 and 18 days. Both T-RFLP/cloning and PLFA-SIP approaches showed that type I and type II methanotrophic populations changed over time with respect to activity and population size in the rhizospheric soil and on the rice roots. However, type I methanotrophs were more active than type II methanotrophs at both time points indicating they were of particular importance in the rhizosphere. PLFA-SIP showed that the active methanotrophic populations exhibit a pronounced spatial and temporal variation in rice microcosms. [source] Vertical profiles of methanogenesis and methanogens in two contrasting acidic peatlands in central New York State, USAENVIRONMENTAL MICROBIOLOGY, Issue 8 2006Hinsby Cadillo-Quiroz Summary Northern acidic peatlands are important sources of atmospheric methane, yet the methanogens in them are poorly characterized. We examined methanogenic activities and methanogen populations at different depths in two peatlands, McLean bog (MB) and Chicago bog (CB). Both have acidic (pH 3.5,4.5) peat soils, but the pH of the deeper layers of CB is near-neutral, reflecting its previous existence as a neutral-pH fen. Acetotrophic and hydrogenotrophic methanogenesis could be stimulated in upper samples from both bogs, and phylotypes of methanogens using H2/CO2 (Methanomicrobiales) or acetate (Methanosarcinales) were identified in 16S rRNA gene clone libraries and by terminal restriction fragment length polymorphism (T-RFLP) analyses using a novel primer/restriction enzyme set that we developed. Particularly dominant in the upper layers was a clade in the Methanomicrobiales, called E2 here and the R10 or fen group elsewhere, estimated by quantitative polymerase chain reaction to be present at ,108 cells per gram of dry peat. Methanogenic activity was considerably lower in deeper samples from both bogs. The methanogen populations detected by T-RFLP in deeper portions of MB were mainly E2 and the uncultured euryarchaeal rice cluster (RC)-II group, whereas populations in the less acidic CB deep layers were considerably different, and included a Methanomicrobiales clade we call E1-E1,, as well as RC-I, RC-II, marine benthic group D, and a new cluster that we call the subaqueous cluster. E2 was barely detectable in the deeper samples from CB, further evidence for the associations of most organisms in this group with acidic habitats. [source] DNA extraction procedure: a critical issue for bacterial diversity assessment in marine sedimentsENVIRONMENTAL MICROBIOLOGY, Issue 2 2006Gian Marco Luna Summary In order to evaluate whether different DNA extraction procedures can affect estimates of benthic bacterial diversity, based on 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) fingerprinting technique, we compared two in situ lysis procedures (a SDS-based protocol and a commercial kit for DNA recovery) and one cell-extraction protocol on a variety of marine sediments. Despite the two in situ lysis procedures resulted in significantly different DNA yields (highest with the SDS in situ lysis), estimates of bacterial diversity provided a not significantly different ribotype richness, as well as similar values of the Shannon-Wiener (H,) and Margalef (d) indices of biodiversity and of evenness (Pielou index, J). Conversely, the cell-extraction procedure for DNA extraction resulted always in a significantly lower ribotype richness and diversity. The analysis of similarities (anosim) among the T-RFLP electropherograms allowed concluding that ribotypes composition did not change significantly using different protocols. However, the analysis of ,-diversity (turnover diversity) revealed that a large number of ribotypes was observed exclusively with one of the three protocols utilized. When unshared ribotypes from in situ lysis and cell extraction were pooled together, total ribotype richness resulted much higher (up to 80%). Our results indicate that estimates of ribotype diversity based on a single protocol of DNA extraction can significantly underestimate the total number of bacterial ribotypes present in the benthic domain. We recommend that future studies will not only integrate different DNA extraction procedures, but also will explore the possibility of integrating two or more different genetic markers in order to increase our ability to detect the actual bacterial diversity in environmental samples. [source] Loss of diversity of ammonia-oxidizing bacteria correlates with increasing salinity in an estuary systemENVIRONMENTAL MICROBIOLOGY, Issue 9 2005Anne E. Bernhard Summary Ammonia-oxidizing bacteria (AOB) play an important role in nitrogen cycling in estuaries, but little is known about AOB diversity, distribution and activity in relation to the chemical and physical changes encountered in estuary systems. Although estuarine salinity gradients are well recognized to influence microbial community structure, few studies have examined the influence of varying salinity on the diversity and stability of AOB populations. To investigate these relationships, we collected sediment samples from low-, mid- and high-salinity sites in Plum Island Sound estuary, MA, during spring and late summer over 3 years. Ammonia-oxidizing bacteria distribution and diversity were assessed by terminal restriction fragment length polymorphism (TRFLP) analysis of the ammonia monooxygenase (amoA) gene, and fragments were identified by screening amoA clone libraries constructed from each site. Most striking was the stability and low diversity of the AOB community at the high-salinity site, showing little variability over 3 years. Ammonia-oxidizing bacteria at the high-salinity site were not closely related to any cultured AOB, but were most similar to Nitrosospira spp. Ammonia-oxidizing bacteria at the mid- and low-salinity sites were distributed among Nitrosospira- like sequences and sequences related to Nitrosomonas ureae/oligotropha and Nitrosomonas sp. Nm143. Our study suggests that salinity is a strong environmental control on AOB diversity and distribution in this estuary. [source] Niche heterogeneity determines bacterial community structure in the termite gut (Reticulitermes santonensis)ENVIRONMENTAL MICROBIOLOGY, Issue 7 2005Hong Yang Summary Differences in microenvironment and interactions of microorganisms within and across habitat boundaries should influence structure and diversity of the microbial communities within an ecosystem. We tested this hypothesis using the well characterized gut tract of the European subterranean termite Reticulitermes santonensis as a model. By cloning and sequencing analysis and molecular fingerprinting (terminal restriction fragment length polymorphism), we characterized the bacterial microbiota in the major intestinal habitats , the midgut, the wall of the hindgut paunch, the hindgut fluid and the intestinal protozoa. The bacterial community was very diverse (> 200 ribotypes) and comprised representatives of several phyla, including Firmicutes (mainly clostridia, streptococci and Mycoplasmatales -related clones), Bacteroidetes, Spirochaetes and a number of Proteobacteria, all of which were unevenly distributed among the four habitats. The largest group of clones fell into the so-called Termite group 1 (TG-1) phylum, which has no cultivated representatives. The majority of the TG-1 clones were associated with the protozoa and formed two phylogenetically distinct clusters, which consisted exclusively of clones previously retrieved from the gut of this and other Reticulitermes species. Also the other clones represented lineages of microorganisms that were exclusively recovered from the intestinal tract of termites. The termite specificity of these lineages was underscored by the finding that the closest relatives of the bacterial clones obtained from R. santonensis were usually derived also from the most closely related termites. Overall, differences in diversity between the different gut habitats and the uneven distribution of individual phylotypes support conclusively that niche heterogeneity is a strong determinant of the structure and spatial organization of the microbial community in the termite gut. [source] Effect of temperature change on the composition of the bacterial and archaeal community potentially involved in the turnover of acetate and propionate in methanogenic rice field soilFEMS MICROBIOLOGY ECOLOGY, Issue 2 2010Matthias Noll Abstract The microbial community structure was investigated together with the path of methane production in Italian rice field soil incubated at moderate (35 °C) and high (45 °C) temperature using terminal restriction fragment length polymorphism and stable isotope fractionation. The structure of both the archaeal and bacterial communities differed at 35 °C compared with 45 °C, and acetoclastic and hydrogenotrophic methanogenesis dominated, respectively. Changing the incubation of the 45 °C soil to different temperatures (25, 30, 35, 40, 45, 50 °C) resulted in a dynamic change of both microbial community structure and stable isotope fractionation. In all treatments, acetate first accumulated and then decreased. Propionate was also transiently produced and consumed. It is noteworthy that acetate was also consumed at thermophilic conditions, although archaeal community composition and stable isotope fractionation indicated that acetoclastic methanogenesis did not operate. Instead, acetate must have been consumed by syntrophic acetate oxidizers. The transient accumulation and subsequent consumption of acetate at thermophilic conditions was specifically paralleled by terminal restriction fragments characteristic for clostridial cluster I, whereas those of clostridial clusters I and III, Acidaminococcaceae and Heliobacteraceae, paralleled the thermophilic turnover of both acetate and propionate. [source] Spatial and temporal variability of the bacterial community in different chemotypes of the New Zealand marine sponge Mycale hentscheliFEMS MICROBIOLOGY ECOLOGY, Issue 3 2010Sally A. Anderson Abstract Molecular fingerprinting of 16S rRNA genes using terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) was used to characterize the temporal and spatial variability among sponge-associated bacteria from Mycale hentscheli having distinct bioactive chemotypes. Cluster analysis of T-RFLP and DGGE profiles from M. hentscheli chemotypes largely grouped sponge microbial diversity to their distinct chemotype pattern. Repeat sampling of individual M. hentscheli at one location over a 21-month period showed that the T-RFLP profiles from individual sponges had similarity indices ranging from 60% to 82% and calculated DGGE similarities between 23% and 95%. However, a portion (>35% from DGGE and >19% from T-RFLP) of the microbial community from M. hentscheli appeared to be spatially conserved through all M. hentscheli populations. Sequence analysis of DGGE band fragments showed a similarity among the bands originating from different individuals, different times, and different locations. The sponge-associated relationship of these bands was confirmed, with sequences having similarity to sponge-associated bacteria reported from global locations. This study highlights the spatial and temporal complexity in the distribution of bacterial communities associated with different chemotypes of the marine sponge M. hentscheli. [source] Different bacterial communities associated with the roots and bulk sediment of the seagrass Zostera marinaFEMS MICROBIOLOGY ECOLOGY, Issue 1 2007Sheila Ingemann Jensen Abstract The bacterial community of Zostera marina -inhabited bulk sediment vs. root-associated bacteria was investigated by terminal restriction fragment length polymorphism and sequencing, and the spatial extension of the oxygen loss from roots was determined by oxygen microsensors. Extensive oxygen loss was found in the tip region of the youngest roots, and most of the rhizoplane of Z. marina roots was thus anoxic. A significant difference between the bacterial communities associated with the roots and bulk sediment was found. No significant differences were found between differently aged root-bundles. Terminal restriction fragments (TRFs) assigned to sulfate-reducing Deltaproteobacteria showed a relative mean distribution of 12% and 23% of the PCR-amplified bacterial community in the bulk-sediment at the two sites, but only contributed <2% to the root-associated communities. TRFs assigned to Epsilonproteobacteria showed a relative mean distribution of between 5% and 11% in the root-associated communities of the youngest root bundle, in contrast to the bulk-sediment where this TRF only contributed <1.3%. TRFs assigned to Actinobacteria and Gammaproteobacteria also seemed important first root-colonizers, whereas TRFs assigned to Deltaproteobacteria became increasingly important in the root-associated community of the older root bundles. The presence of the roots thus apparently selects for a distinct bacterial community, stimulating the growth of potential symbiotic Epsilon - and Gammaproteobacteria and/or inhibiting the growth of sulfate-reducing Deltaproteobacteria. [source] Soil parent material is a key determinant of the bacterial community structure in arable soilsFEMS MICROBIOLOGY ECOLOGY, Issue 3 2006Andreas Ulrich Abstract The bacterial community composition in soil and rhizosphere taken from arable field sites, differing in soil parent material and soil texture, was analyzed using terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes. Nine sandy to silty soils from North-East Germany could clearly be distinguished from each other, with a relatively low heterogeneity in the community structure within the field replicates. There was a relationship between the soil parent material, i.e. different glacial and aeolian sediments, and the clustering of the profiles from different sites. A site-specific grouping of T-RFLP profiles was also found for the rhizosphere samples of the same field sites that were planted with potatoes. The branching of the rhizosphere profiles corresponded partly with the soil parent material, whereas the effect of the plant genotype was negligible. Selected terminal restriction fragments differing in their relative abundance within the nine soils were analyzed based on the cloning of the 16S rRNA genes of one soil sample. A high phylogenetic diversity observed to include Acidobacteria, Betaproteobacteria, Bacteroidetes, Verrucomicrobia, and Gemmatimonadetes. The assignment of three out of the seven selected terminal restriction fragments to members of Acidobacteria suggested that this group seems to participate frequently in the shifting of community structures that result from soil property changes. [source] Paleosols in Central Illinois as Potential Sources of Ammonium in GroundwaterGROUND WATER MONITORING & REMEDIATION, Issue 4 2009Justin J. G. Glessner Glacially buried paleosols of pre-Holocene age were evaluated as potential sources for anomalously large concentrations of ammonium in groundwater in East Central Illinois. Ammonium has been detected at concentrations that are problematic to water treatment facilities (greater than 2.0 mg/L) in this region. Paleosols characterized for this study were of Quaternary age, specifically Robein Silt samples. Paleosol samples displayed significant capacity to both store and release ammonium through experiments measuring processes of sorption, ion exchange, and weathering. Bacteria and fungi within paleosols may significantly facilitate the leaching of ammonium into groundwater by the processes of assimilation and mineralization. Bacterial genetic material (DNA) was successfully extracted from the Robein Silt, purified, and amplified by polymerase chain reaction to produce 16S rRNA terminal restriction fragment length polymorphism (TRFLP) community analyses. The Robein Silt was found to have established diverse and viable bacterial communities. 16S rRNA TRFLP comparisons to well-known bacterial species yielded possible matches with facultative chemolithotrophs, cellulose consumers, nitrate reducers, and actinomycetes. It was concluded that the Robein Silt is both a source and reservoir for groundwater ammonium. Therefore, the occurrence of relatively large concentrations of ammonium in groundwater monitoring data may not necessarily be an indication of only anthropogenic contamination. The results of this study, however, need to be placed in a hydrological context to better understand whether paleosols can be a significant source of ammonium to drinking water supplies. [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] Activity and diversity of methanotrophs in the soil,water interface and rhizospheric soil from a flooded temperate rice fieldJOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2009L. Ferrando Abstract Aims:, To combine molecular and cultivation techniques to characterize the methanotrophic community in the soil,water interface (SWI) and rhizospheric soil from flooded rice fields in Uruguay, a temperate region in South America. Methods and Results:, A novel type I, related to the genus Methylococcus, and three type II methanotrophs were isolated from the highest positive dilution steps from the most probable number (MPN) counts. Potential methane oxidation activities measured in slurried samples were higher in the rhizospheric soil compared to the SWI and were stimulated by N-fertilization. PmoA (particulate methane monooxygenase) clone libraries were constructed for both rice microsites. SWI clones clustered in six groups related to cultivated and uncultivated members from different ecosystems of the genera Methylobacter, Methylomonas, Methylococcus and a novel type I sublineage while cultivation and T-RFLP (terminal restriction fragment length polymorphism) analysis confirmed the presence of type II methanotrophs. Conclusions:, Cultivation techniques, cloning analysis and T-RFLP fingerprinting of the pmoA gene revealed a diverse methanotrophic community in the rice rhizospheric soil and SWI. Significance and Impact of the Study:, This study reports, for the first time, the analysis of the methanotrophic diversity in rice SWI and this diversity may be exploited in reducing methane emissions. [source] Genetic identification of morphologically cryptic agricultural pestsAGRICULTURAL AND FOREST ENTOMOLOGY, Issue 1 2009J. S. Ellis Abstract 1,Wireworms are the polyphagous larvae of click beetles and are well known as agricultural pests. Larvae of the genus Agriotes are internationally recognized as economically important pests of potato. Historically associated with crop damage after conversion of grassland, they are an increasing problem even in all-arable rotations. 2,Current studies of Agriotes ecology and behaviour, and consequently control and management, are seriously hampered by the lack of a means of reliably identifying larvae owing to morphological crypsis during this life-stage. 3,Here, sequence data at the mitochondrial 16SrRNA gene are presented for three species of wireworm: Agriotes obscurus, A. lineatus, and A. sputator. A novel terminal restriction fragment length polymorphism (T-RFLP) technique is described that identifies larvae of these species. This technique is shown to be both efficient and reliable. Interestingly, thus far the samples tested have yielded no A. lineatus. Implications for future study of wireworm ecology and control are discussed. [source] Co-existing grass species have distinctive arbuscular mycorrhizal communitiesMOLECULAR ECOLOGY, Issue 11 2003P. Vandenkoornhuyse Abstract Arbuscular mycorrhizal (AM) fungi are biotrophic symbionts colonizing the majority of land plants, and are of major importance in plant nutrient supply. Their diversity is suggested to be an important determinant of plant community structure, but the influence of host-plant and environmental factors on AM fungal community in plant roots is poorly documented. Using the terminal restriction fragment length polymorphism (T-RFLP) strategy, the diversity of AM fungi was assessed in 89 roots of three grass species (Agrostis capillaris, Festuca rubra, Poa pratensis) that co-occurred in the same plots of a field experiment. The impact of different soil amendments (nitrogen, lime, nitrogen and lime) and insecticide application on AM fungal community was also studied. The level of diversity found in AM fungal communities using the T-RFLP strategy was consistent with previous studies based on clone libraries. Our results clearly confirm that an AM fungal host-plant preference exists, even between different grass species. AM communities colonizing A. capillaris were statistically different from the others (P < 0.05). Although grass species evenness changed in amended soils, AM fungal community composition in roots of a given grass species remained stable. Conversely, in plots where insecticide was applied, we found higher AM fungal diversity and, in F. rubra roots, a statistically different AM fungal community. [source] Assessment of intraradicular bacterial composition by terminal restriction fragment length polymorphism analysisMOLECULAR ORAL MICROBIOLOGY, Issue 5 2009D. Saito Background:, The aim of the study was to assess the bacterial community structures associated with endodontic infections using terminal restriction fragment length polymorphism (T-RFLP), and to investigate the correlation of whole community profiles with the manifestation of particular clinical features. Methods:, Intraradicular samples were collected from 34 subjects and classified into three study groups based on the observed clinical symptoms: acute (n = 16), sub-acute (n = 8), and asymptomatic (n = 10). Genomic DNA was extracted from each sample, submitted to polymerase chain reaction using a fluorescently labeled 16S ribosomal DNA forward primer, and digested with two tetrameric endonucleases (HhaI and MspI). The terminal restriction fragments (T-RFs) were subsequently discriminated in an automated DNA sequencer, and the results were filtered using a statistics-based criterion. Results:, Totals of 138 (HhaI) and 145 (MspI) unique T-RFs were detected (means 13.1 and 11.9) and there was high inter-subject variability in the bacterial assemblages. Odds-ratio analysis unveiled the existence of higher order groups of positively associated T-RFs, restating the concept that intricate ecological relationships may take place in the root canal space. A significantly greater T-RF prevalence was detected in acute cases, suggesting a straight correlation between species richness and spontaneous pain. Conclusion:, Overall, no T-RFLP profile representing a specific bacterial consortium could be associated with the manifestation of symptoms of endodontic origin. [source] Use of terminal restriction fragment length polymorphism (T-RFLP) for identification of phytoplasmas in plantsPLANT PATHOLOGY, Issue 3 2007J. Hodgetts A terminal restriction fragment analysis (T-RFLP) technique was developed for the simple and rapid detection and diagnosis of phytoplasmas in plants. The selected primers amplified part of the 23S rRNA gene to provide improved resolution between the taxonomic groups compared to conventional restriction enzyme analysis of the 16S rRNA. Using the restriction enzymes Bsh12361 and MseI on the PCR products, and fragment analysis in the range 68,640 bp, the technique was tested on 37 isolates from 10 of the 16Sr groups. Distinct and unambiguous T-RFLP profiles were produced for nine of the 10 taxonomic groups, such that almost all isolates within a group shared the same profile and could be distinguished from isolates in other groups. The technique also identified the presence of mixtures of phytoplasmas from different groups in samples. Furthermore, the primers were devised to amplify a terminal restriction fragment (TRF) product of a specific defined size (461 bp) from the host plant chloroplast DNA, so that there was a built-in internal control in the procedure to show that the absence of a phytoplasma peak in a sample was the result of no detectable phytoplasma being present, not the result of PCR inhibition. This method offers the possibility of simultaneously detecting and providing a taxonomic grouping for phytoplasmas in test samples using a single PCR reaction. [source] Surveys reveal the occurrence of phytoplasmas in plants at different geographical locations in PeruANNALS OF APPLIED BIOLOGY, Issue 1 2009J. Hodgetts Abstract Two independent surveys were performed in Peru during February and November 2007 to detect the presence of phytoplasmas within any crops showing symptoms resembling those caused by phytoplasmas. Molecular identifications and characterisations were based on phytoplasma 16S and 23S rRNA genes using nested PCR and terminal restriction fragment length polymorphism (T-RFLP). The surveys indicated that phytoplasmas were present in most of the locations sampled in Peru in both cultivated crops, including carrots, maize, native potatoes, improved potato, tomato, oats, papaya and coconut, and in other plants such as dandelion and the ornamental Madagascar periwinkle (Catharanthus roseus). Phylogenetic analysis of the sequences confirmed that while most of the isolates belong to the 16SrI aster yellows group, which is ubiquitous throughout other parts of South America, one isolate from potato belongs to the 16SrII peanut witches' broom group, and one isolate from tomato and one from dandelion belong to the 16SrIII X-disease group. The use of T-RFLP was validated for the evaluation of phytoplasma-affected field samples and provided no evidence for mixed infection of individual plants with more than one phytoplasma isolate. These data represent the first molecular confirmation of the presence of phytoplasmas in a broad range of crops in Peru. [source] Changes in microbial community composition following treatment of methanogenic granules with chloroformENVIRONMENTAL PROGRESS & SUSTAINABLE ENERGY, Issue 1 2009Bo Hu Abstract Eliminating hydrogen consuming bacteria is a critical step in anaerobic fermentation for biohydrogen production. Treatment of anaerobic granular sludge with chloroform was reported as effective in transforming a methane-producing system into a hydrogen-producing system by eliminating methane production. This study, using 16S rRNA gene sequences, further assessed changes in microbial community composition as a result of chloroform treatment and during continuous cultivation of chloroform-treated granules in a continuous upflow reactor employing immobilized cells. Profiles of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes sequences cloned from samples before and after chloroform treatment showed that methanogenic hydrogen consumers and Methanosaeta harundinacea sp. were eliminated. Methanosaeta concilii, however, was not eliminated from the hydrogen-producing system, which might explain, in part, the granulation phenomena in the anaerobic hydrogen fermentation system. The results also showed that Clostridium butyricum dominated the hydrogen-production system. © 2009 American Institute of Chemical Engineers Environ Prog, 2009 [source] Microbial diversity of inflamed and noninflamed gut biopsy tissues in inflammatory bowel diseaseINFLAMMATORY BOWEL DISEASES, Issue 6 2007Shadi Sepehri MD Abstract Background: Inflammatory bowel disease (IBD) is a chronic gastrointestinal condition without any known cause or cure. An imbalance in normal gut biota has been identified as an important factor in the inflammatory process. Methods: Fifty-eight biopsies from Crohn's disease (CD, n = 10), ulcerative colitis (UC, n = 15), and healthy controls (n = 16) were taken from a population-based case-control study. Automated ribosomal intergenic spacer analysis (ARISA) and terminal restriction fragment length polymorphisms (T-RFLP) were used as molecular tools to investigate the intestinal microbiota in these biopsies. Results: ARISA and T-RFLP data did not allow a high level of clustering based on disease designation. However, if clustering was done based on the inflammation criteria, the majority of biopsies grouped either into inflamed or noninflamed groups. We conducted statistical analyses using incidence-based species richness and diversity as well as the similarity measures. These indices suggested that the noninflamed tissues form an intermediate population between controls and inflamed tissue for both CD and UC. Of particular interest was that species richness increased from control to noninflamed tissue, and then declined in fully inflamed tissue. Conclusions: We hypothesize that there is a recruitment phase in which potentially pathogenic bacteria colonize tissue, and once the inflammation sets in, a decline in diversity occurs that may be a byproduct of the inflammatory process. Furthermore, we suspect that a better knowledge of the microbial species in the noninflamed tissue, thus before inflammation sets in, holds the clues to the microbial pathogenesis of IBD. (Inflamm Bowel Dis 2007) [source] | ||||||||||||||||||||||||||||||||||