共查询到20条相似文献,搜索用时 31 毫秒
1.
Denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) were used to characterise the changes that occurred in Bacillus cereus group strains present in the phylloplane of clover Trifolium hybridum over 4 months. These strains had previously been analysed by multiple locus sequence typing (MLST). DGGE displayed many equally intense bands which indicated many equally abundant ribotypes. The bacterial community composition was variable and the leaves sampled as little as a week apart were found to have some temporal variability, indicating that diverse phylloplane bacterial communities follow sequential patterns from time to time. The B. cereus group community clearly clustered into early, mid and late branches, possibly due to multiple successional sequences occurring during growing seasons. The functionally and phylogenetically diverse microbial communities appeared to exhibit predictable successional patterns over shorter time scales. DGGE analysis with the molecular marker rpoB gave better resolution than 16S rRNA amplicons. There were no strong similarities between the dendrograms produced by DGGE, MLST and T-RFLP and the clustering produced by the automated T-RFLP method was variable even between the three restriction enzymes used. The DGGE–MLST method emerged as a superior method to T-RFLP–MLST for rapid typing of bacterial communities. 相似文献
2.
Optimization of Terminal-Restriction Fragment Length Polymorphism Analysis for Complex Marine Bacterioplankton Communities and Comparison with Denaturing Gradient Gel Electrophoresis 总被引:16,自引:8,他引:16
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Markus M. Moeseneder Jesús M. Arrieta Gerard Muyzer Christian Winter Gerhard J. Herndl 《Applied microbiology》1999,65(8):3518-3525
The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of operational taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE). A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1,632 bp by using CE and laser-induced fluorescence detection. Additionally, we compared T-RFLP fingerprinting to DGGE optimized for detection of less abundant OTUs. Similar results were obtained with both fingerprinting techniques, although the T-RFLP approach and CE detection of OTUs was more sensitive, as indicated by the higher number of OTUs detected. We tested the T-RFLP fingerprinting technique on complex marine bacterial communities by using the 16S rRNA gene and 16S rRNA as templates for PCR. Samples from the Northern and Middle Adriatic Sea and from the South and North Aegean Sea were compared. Distinct clusters were identifiable for different sampling sites. Thus, this technique is useful for rapid evaluation of the biogeographical distribution and relationships of bacterioplankton communities. 相似文献
3.
M M Moeseneder J M Arrieta G Muyzer C Winter G J Herndl 《Applied and environmental microbiology》1999,65(8):3518-3525
The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of operational taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE). A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1, 632 bp by using CE and laser-induced fluorescence detection. Additionally, we compared T-RFLP fingerprinting to DGGE optimized for detection of less abundant OTUs. Similar results were obtained with both fingerprinting techniques, although the T-RFLP approach and CE detection of OTUs was more sensitive, as indicated by the higher number of OTUs detected. We tested the T-RFLP fingerprinting technique on complex marine bacterial communities by using the 16S rRNA gene and 16S rRNA as templates for PCR. Samples from the Northern and Middle Adriatic Sea and from the South and North Aegean Sea were compared. Distinct clusters were identifiable for different sampling sites. Thus, this technique is useful for rapid evaluation of the biogeographical distribution and relationships of bacterioplankton communities. 相似文献
4.
目的建立能快速筛查和比较大规模人群肠道中柔嫩梭菌类群组成结构的T-RFLP(末端限制性片段长度多态性)。方法采用T-RFLP技术特异性地分析柔嫩梭菌类群的组成,考察该方法的重复性和灵敏度。用该方法对15个志愿者肠道中的柔嫩梭菌类群进行分析,并与DGGE方法分析的结果进行对比。结果T-RFLP方法重复性高,最低能检测到群落中1%的细菌,其结果与DGGE的分析结果具有较好的一致性。结论本研究建立的柔嫩梭菌类群特异性的T-RFLP方法能够对大量肠道样品中柔嫩梭菌类群的结构进行快速、有效的筛查和比较。 相似文献
5.
DGGE and T-RFLP Analysis of Bacterial Succession during Mushroom Compost Production and Sequence-aided T-RFLP Profile of Mature Compost 总被引:6,自引:0,他引:6
The amount of button mushroom (Agaricus bisporus) harvested from compost is largely affected by the microbial processes taking place during composting and the microbes inhabiting
the mature compost. In this study, the microbial changes during the stages of this specific composting process were monitored,
and the dominant bacteria of the mature compost were identified to reveal the microbiological background of the favorable
properties of the heat-treated phase II mushroom compost. 16S ribosomal deoxyribonucleic acid (rDNA)-based denaturing gradient
gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) molecular fingerprinting methods
were used to track the succession of microbial communities in summer and winter composting cycles. DNA from individual DGGE
bands were reamplified and subjected to sequence analysis. Principal component analysis of fingerprints of the composting
processes showed intensive changes in bacterial community during the 22-day procedure. Peak temperature samples grouped together
and were dominated by Thermus thermophilus. Mature compost patterns were almost identical by both methods (DGGE, T-RFLP). To get an in-depth analysis of the mature
compost bacterial community, the sequence data from cultivation of the bacteria and cloning of environmental 16S rDNA were
uniquely coupled with the output of the environmental T-RFLP fingerprints (sequence-aided T-RFLP). This method revealed the
dominance of a supposedly cellulose-degrading consortium composed of phylotypes related to Pseudoxanthomonas, Thermobifida, and Thermomonospora. 相似文献
6.
Nunan N Daniell TJ Singh BK Papert A McNicol JW Prosser JI 《Applied and environmental microbiology》2005,71(11):6784-6792
Molecular analysis of grassland rhizosphere soil has demonstrated complex and diverse bacterial communities, with resultant difficulties in detecting links between plant and bacterial communities. These studies have, however, analyzed "bulk" rhizosphere soil, rather than rhizoplane communities, which interact most closely with plants through utilization of root exudates. The aim of this study was to test the hypothesis that plant species was a major driver for bacterial rhizoplane community composition on individual plant roots. DNA extracted from individual roots was used to determine plant identity, by analysis of the plastid tRNA leucine (trnL) UAA gene intron, and plant-related bacterial communities. Bacterial communities were characterized by analysis of PCR-amplified 16S rRNA genes using two fingerprinting methods: terminal restriction fragment length polymorphisms (T-RFLP) and denaturing gradient gel electrophoresis (DGGE). Links between plant and bacterial rhizoplane communities could not be detected by visual examination of T-RFLP patterns or DGGE banding profiles. Statistical analysis of fingerprint patterns did not reveal a relationship between bacterial community composition and plant species but did demonstrate an influence of plant community composition. The data also indicated that topography and other, uncharacterized, environmental factors are important in driving bacterial community composition in grassland soils. T-RFLP had greater potential resolving power than DGGE, but findings from the two methods were not significantly different. 相似文献
7.
Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to characterize and compare human fecal microbiota among individuals. T-RFLP patterns of fecal 16S ribosomal DNA (rDNA) PCR products from three adults revealed host-specific bacterial communities and were in good agreement with those reported in our previous study. In addition, we applied T-RFLP analysis for the analysis of complex bifidobacterial communities in human fecal samples. The developed method based on Bifidobacterium genus-specific PCR and T-RFLP could identify more than one bifidobacterial species. T-RFLP patterns of Bifidobacterium genus-specific PCR products from the fecal samples were host-specific as well as those of fecal 16S rDNA PCR products. These results were confirmed by PCR-denaturing gradient gel electrophoresis (DGGE) with primers specific for the genus Bifidobacterium and Bifidobacterium species- and group-specific PCR. Our study demonstrates that T-RFLP analysis is useful for assessment of the diversity of the human fecal microbiota and rapid comparison of the community structure among individuals, and that the applied method is useful for rapid and sensitive analysis of bifidobacterial community. 相似文献
8.
Links between Plant and Rhizoplane Bacterial Communities in Grassland Soils, Characterized Using Molecular Techniques 总被引:1,自引:2,他引:1
下载免费PDF全文
![点击此处可从《Applied microbiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Naoise Nunan Timothy J. Daniell Brajesh K. Singh Artemis Papert James W. McNicol James I. Prosser 《Applied microbiology》2005,71(11):6784-6792
Molecular analysis of grassland rhizosphere soil has demonstrated complex and diverse bacterial communities, with resultant difficulties in detecting links between plant and bacterial communities. These studies have, however, analyzed “bulk” rhizosphere soil, rather than rhizoplane communities, which interact most closely with plants through utilization of root exudates. The aim of this study was to test the hypothesis that plant species was a major driver for bacterial rhizoplane community composition on individual plant roots. DNA extracted from individual roots was used to determine plant identity, by analysis of the plastid tRNA leucine (trnL) UAA gene intron, and plant-related bacterial communities. Bacterial communities were characterized by analysis of PCR-amplified 16S rRNA genes using two fingerprinting methods: terminal restriction fragment length polymorphisms (T-RFLP) and denaturing gradient gel electrophoresis (DGGE). Links between plant and bacterial rhizoplane communities could not be detected by visual examination of T-RFLP patterns or DGGE banding profiles. Statistical analysis of fingerprint patterns did not reveal a relationship between bacterial community composition and plant species but did demonstrate an influence of plant community composition. The data also indicated that topography and other, uncharacterized, environmental factors are important in driving bacterial community composition in grassland soils. T-RFLP had greater potential resolving power than DGGE, but findings from the two methods were not significantly different. 相似文献
9.
Aquatic hyphomycetes are the main fungal decomposers of plant litter in streams. We compared the importance of substrate (three leaf species, wood) and season on fungal colonization. Substrates were exposed for 12 4-week periods. After recovery, mass loss, fungal biomass and release of conidia by aquatic hyphomycetes were measured. Fungal communities were characterized by counting and identifying released conidia and by extracting and amplifying fungal DNA (ITS2), which was subdivided into phylotypes by denaturing gradient gel electrophoresis (DGGE) and terminal-restriction fragment length polymorphism (T-RFLP). Mass loss, fungal biomass and reproduction were positively correlated with stream temperature. Conidial diversity was highest between May and September. Numbers of different phylotypes were more stable. Principal coordinate analyses (PCO) and canonical analyses of principal coordinates (CAP) of presence/absence data (DGGE bands, T-RFLP peaks and conidial species) showed a clear seasonal trend (Por=0.88). Season was also a significant factor when proportional similarities of conidial communities or relative intensities of DGGE bands were evaluated (P相似文献
10.
Smalla K Oros-Sichler M Milling A Heuer H Baumgarte S Becker R Neuber G Kropf S Ulrich A Tebbe CC 《Journal of microbiological methods》2007,69(3):470-479
Bacterial communities of four arable soils--pelosol, gley, para brown soil, and podsol brown soil--were analysed by fingerprinting of 16S rRNA gene fragments amplified from total DNA of four replicate samples for each soil type. Fingerprints were generated in parallel by denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP), and single strand conformation polymorphism (SSCP) to test whether these commonly applied techniques are interchangeable. PCR amplicons could be separated with all three methods resulting in complex ribotype patterns. Although the fragments amplified comprised different variable regions and lengths, DGGE, T-RFLP and SSCP analyses led to similar findings: (a) a clustering of fingerprints which correlated with soil physico-chemical properties, (b) little variability between the four replicates of the same soil, (c) the patterns of the two brown soils were more similar to each other than to those of the other two soils, and (d) the fingerprints of the different soil types revealed significant differences in a permutation test, which was recently developed for this purpose. 相似文献
11.
Community Structure of Actively Growing Bacterial Populations in Plant Pathogen Suppressive Soil 总被引:2,自引:0,他引:2
The bacterial community in soil was screened by using various molecular approaches for bacterial populations that were activated
upon addition of different supplements. Plasmodiophora
brassicae spores, chitin, sodium acetate, and cabbage plants were added to activate specific bacterial populations as an aid in screening
for novel antagonists to plant pathogens. DNA from growing bacteria was specifically extracted from the soil by bromodeoxyuridine
immunocapture. The captured DNA was fingerprinted by terminal restriction fragment length polymorphism (T-RFLP). The composition
of the dominant bacterial community was also analyzed directly by T-RFLP and by denaturing gradient gel electrophoresis (DGGE).
After chitin addition to the soil, some bacterial populations increased dramatically and became dominant both in the total
and in the actively growing community. Some of the emerging bands on DGGE gels from chitin-amended soil were sequenced and
found to be similar to known chitin-degrading genera such as Oerskovia, Kitasatospora, and Streptomyces species. Some of these sequences could be matched to specific terminal restriction fragments on the T-RFLP output. After
addition of Plasmodiophora spores, an increase in specific Pseudomonads could be observed with Pseudomonas-specific primers for DGGE. These results demonstrate the utility of microbiomics, or a combination of molecular approaches,
for investigating the composition of complex microbial communities in soil. 相似文献
12.
Casamayor EO Massana R Benlloch S Øvreås L Díez B Goddard VJ Gasol JM Joint I Rodríguez-Valera F Pedrós-Alió C 《Environmental microbiology》2002,4(6):338-348
Microbial communities inhabiting a multipond solar saltern were analysed and compared using SSU rRNA polymerase chain reaction (PCR)-based fingerprintings carried out in parallel by four laboratories. A salinity gradient from seawater (3.7%) to NaCl precipitation (37%) was studied for Bacteria, Archaea and Eukarya, and laboratories applied their own techniques and protocols on the same set of samples. Members of all three domains were retrieved from all salt concentrations. Three fingerprinting techniques were used: denaturing gradient gel electrophoresis (DGGE), ribosomal internal spacer analysis (RISA), and terminal-restriction fragments length polymorphism (T-RFLP). In addition, each laboratory used its own biomass collection method and DNA extraction protocols. Prokaryotes were addressed using DGGE and RISA with different 'domain-specific' primers sets. Eukaryotes were analysed by one laboratory using DGGE and T-RFLP, but targeting the same 18S rDNA site. Fingerprints were compared through cluster analysis and non-metric multidimensional scaling plots. This exercise allowed fast comparison of microbial assemblages and determined to what extent the picture provided by each laboratory was similar to those of others. Formation of two main, salinity-based groups of samples in prokaryotes (4-15% and 22-37% salinity) was consistent for all the laboratories. When other clusters appeared, this was a result of the particular technique and the protocol used in each case, but more affected by the primers set used. Eukaryotic microorganisms changed more from pond to pond; 4-5% and 8-37% salinity were but the two main groups detected. Archaea showed the lowest number of bands whereas Eukarya showed the highest number of operational taxonomic units (OTUs) in the initial ponds. Artefacts appeared in the DGGE from ponds with extremely low microbial richness. On the other hand, different 16S rDNA fragments with the same restriction or internal transcribed spacer (ITS) length were the main limitations for T-RFLP and RISA analyses, respectively, in ponds with the highest OTUs richness. However, although the particular taxonomic composition could vary among protocols, the general structure of the microbial assemblages was maintained. 相似文献
13.
Correlations between molecular and operational parameters in continuous lab-scale anaerobic reactors
Carballa M Smits M Etchebehere C Boon N Verstraete W 《Applied microbiology and biotechnology》2011,89(2):303-314
In this study, the microbial community characteristics in continuous lab-scale anaerobic reactors were correlated to reactor
functionality using the microbial resource management (MRM) approach. Two molecular techniques, denaturing gradient gel electrophoresis
(DGGE) and terminal-restriction fragment length polymorphism (T-RFLP), were applied to analyze the bacterial and archaeal
communities, and the results obtained have been compared. Clustering analyses showed a similar discrimination of samples with
DGGE and T-RFLP data, with a clear separation between the meso- and thermophilic communities. Both techniques indicate that
bacterial and mesophilic communities were richer and more even than archaeal and thermophilic communities, respectively. Remarkably,
the community composition was highly dynamic for both Bacteria and Archaea, with a rate of change between 30% and 75% per 18 days, also in stable performing periods. A hypothesis to explain the latter
in the context of the converging metabolism in anaerobic processes is proposed. Finally, a more even and diverse bacterial
community was found to be statistically representative for a well-functioning reactor as evidenced by a low Ripley index and
high biogas production. 相似文献
14.
Nathalie J.A. Curlevski Susan M. Chambers Ian C. Anderson & John W.G. Cairney 《FEMS microbiology ecology》2009,67(3):411-420
Assemblages of fungi associated with roots of cooccurring Epacris pulchella ( Ericaceae ) and Leptospermum polygalifolium ( Myrtaceae ) seedlings at a sclerophyll forest site in New South Wales, Australia, were investigated by direct DNA extraction and analysis of rRNA gene internal transcribed spacer (ITS) products by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analyses. While ordination of the DGGE data suggested that the assemblages did not differ significantly between the two plant taxa, T-RFLP data provided marginal statistical support for the presence of different assemblages. Fungi isolated from roots of both plants were identified by ITS sequence comparisons largely as ascomycetes, several of which had close sequence identity to Helotiales ericoid mycorrhizal (ERM) fungi. One isolate morphotype from E. pulchella had close sequence similarity to ectomycorrhizal fungi in the Cenococcum geophilum complex, and neighbour-joining analysis grouped this strongly with other Australian C. geophilum- like sequences. Distribution of genotypes of an ERM Helotiales ascomycete in root systems of the two plant taxa was also investigated using inter-simple sequence repeat (ISSR)-PCR. Nineteen ISSR genotypes were identified, two of which were present in roots of both plant taxa. The results are discussed in the context of potential mycelial connections between Ericaceae and non- Ericaceae plants. 相似文献
15.
Methanotrophic bacteria play a crucial role in regulating the emission of CH4 from rice fields into the atmosphere. We investigated the CH4 oxidation activity together with the diversity of methanotrophic bacteria in ten rice field soils from different geographic locations. Upon incubation of aerated soil slurries under 7% CH4, rates of CH4 oxidation increased after a lag phase of 1-4 days and reached values of 3-10 micromol d(-1) g-dw(-1) soil. The methanotrophic community was assayed by retrieval of the pmoA gene which encodes the a subunit of the particulate methane monooxygenase. After extraction of DNA from actively CH4-oxidizing soil samples and PCR-amplification of the pmoA, the community was analyzed by Denaturant Gradient Gel Electrophoresis (DGGE) and Terminal Restriction Fragment Length Polymorphism (T-RFLP). DGGE bands were excised, the pmoA re-amplified, sequenced and the encoded amino acid sequence comparatively analyzed by phylogenetic treeing. The analyses allowed the detection of pmoA sequences related to the following methanotrophic genera: the type-I methanotrophs Methylobacter, Methylomicrobium, Methylococcus and Methylocaldum, and the type-II methanotrophs Methylocystis and Methylosinus. T-RFLP analysis detected a similar diversity, but type-II pmoA more frequently than DGGE. All soils but one contained type-II in addition to type-I methanotrophs. Type-I Methylomonas was not detected at all. Different combinations of methanotrophic genera were detected in the different soils. However, there was no obvious geographic pattern of the distribution of methanotrophs. 相似文献
16.
The bacterial community in a partial nitrification reactor was analyzed on the basis of 16S rRNA gene by cloning–sequencing
method, and the percentages of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the activated sludge
were quantified by three independent methods, namely, denaturing gradient gel electrophoresis (DGGE), terminal restriction
fragment length polymorphism (T-RFLP) and Double Monod modeling. The clone library results suggested that there were only
a dominant AOB and a dominant NOB species in the reactor, belonging to Nitrosomonas genus and Nitrospira genus, respectively. The percentages of NOB in total bacterial community increased from almost 0% to 30% when dissolved oxygen
(DO) levels were changed from 0.15 mg/L to 0.5 mg/L, coinciding with the accumulation and conversion of nitrite, while the
percentages of AOB changed little in the two phases. The results confirmed the importance of low DO level for inhibiting NOB
to achieve partial nitrification. Furthermore, the percentages of AOB and NOB in the total bacteria community were estimated
based on the results of batch experiments using Double Monod model, and the results were comparable with those determined
according to profiles of DGGE and T-RFLP. 相似文献
17.
Tegan N. Evans Garth Watson Gavin N. Rees Robert J. Seviour 《Antonie van Leeuwenhoek》2014,105(3):559-569
We compared the relative values of denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) for profiling fungal communities in wastewater treatment plants using both ITS and 18S rRNA gene fragments as phylogenetic markers. A similar number of fungal ribotypes was obtained with both methods for the same treatment plant when the ITS primer set was used, while a greater number of ribotypes was obtained with T-RFLP compared to DGGE with the 18S rRNA primer set. Non-metric multi-dimensional scaling of presence/absence data and analysis of similarity showed that both methods could distinguish between the different plant communities at a statistically significant level (p < 0.05), regardless of which phylogenetic marker was used. The data suggest that both methods can be used preferably together to profile activated sludge fungal communities. A comparison of profiles generated with both these phylogenetic markers based on the number of ribotypes/bands, suggests that the 18S rRNA region is more discriminatory than the ITS region. Detected differences in fungal community compositions between plants probably reflect differences in their influent compositions and operational parameters. 相似文献
18.
Comparison of molecular fingerprinting methods for analysis of soil microbial community structure 总被引:1,自引:0,他引:1
Denaturing gradient gel electrophoresis (DGGE), terminal-restriction fragment length polymorphism (T-RFLP) analysis, and automated
ribosomal intergenic spacer analysis (ARISA) have been widely used as molecular fingerprinting methods for analysis of microbial
communities. To find suitable methods, we compared the three fingerprinting methods by analyzing soil fungal communities in
four differing land-use types: bare ground, crop fields, grasslands, and forests. We also examined optimal primer pairs for
DGGE analysis by comparing single and mixed DNA samples of cultured fungal populations. Principal coordinate analysis (PCO),
nonmetric multidimensional scaling method (NMDS), and analysis of similarities (ANOSIM), which are major multivariate statistical
analyses for quantifying fingerprint patterns, were compared. All three fingerprinting methods yielded clear discrimination
of soil fungal communities among the four land-use types, irrespective of statistical methods. The advantages and disadvantages
of the three fingerprinting methods were discussed. 相似文献
19.
Determining diversity of freshwater fungi on decaying leaves: comparison of traditional and molecular approaches 总被引:4,自引:0,他引:4
Traditional microscope-based estimates of species richness of aquatic hyphomycetes depend upon the ability of the species in the community to sporulate. Molecular techniques which detect DNA from all stages of the life cycle could potentially circumvent the problems associated with traditional methods. Leaf disks from red maple, alder, linden, beech, and oak as well as birch wood sticks were submerged in a stream in southeastern Canada for 7, 14, and 28 days. Fungal biomass, estimated by the amount of ergosterol present, increased with time on all substrates. Alder, linden, and maple leaves were colonized earlier and accumulated the highest fungal biomass. Counts and identifications of released conidia suggested that fungal species richness increased, while community evenness decreased, with time (up to 11 species on day 28). Conidia of Articulospora tetracladia dominated. Modifications of two molecular methods-denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analysis-suggested that both species richness and community evenness decreased with time. The dominant ribotype matched that of A. tetracladia. Species richness estimates based on DGGE were consistently higher than those based on T-RFLP analysis and exceeded those based on spore identification on days 7 and 14. Since traditional and molecular techniques assess different aspects of the fungal organism, both are essential for a balanced view of fungal succession on leaves decaying in streams. 相似文献
20.
Effect of soil ammonium concentration on N2O release and on the community structure of ammonia oxidizers and denitrifiers 总被引:3,自引:0,他引:3
The effect of ammonium addition (6.5, 58, and 395 microg of NH4+-N g [dry weight] of soil(-1)) on soil microbial communities was explored. For medium and high ammonium concentrations, increased N2O release rates and a shift toward a higher contribution of nitrification to N2O release occurred after incubation for 5 days at 4 degrees C. Communities of ammonia oxidizers were assayed after 4 weeks of incubation by denaturant gradient gel electrophoresis (DGGE) of the amoA gene coding for the small subunit of ammonia monooxygenase. The DGGE fingerprints were invariably the same whether the soil was untreated or incubated with low, medium, or high ammonium concentrations. Phylogenetic analysis of cloned PCR products from excised DGGE bands detected amoA sequences which probably belonged to Nitrosospira 16S rRNA clusters 3 and 4. Additional clones clustered with Nitrosospira sp. strains Ka3 and Ka4 and within an amoA cluster from unknown species. A Nitrosomonas-like amoA gene was detected in only one clone. In agreement with the amoA results, community profiles of total bacteria analyzed by terminal restriction fragment length polymorphism (T-RFLP) showed only minor differences. However, a community shift occurred for denitrifier populations based on T-RFLP analysis of nirK genes encoding copper-containing nitrite reductase with incubation at medium and high ammonia concentrations. Major terminal restriction fragments observed in environmental samples were further described by correspondence to cloned nirK genes from the same soil. Phylogenetic analysis grouped these clones into clusters of soil nirK genes. However, some clones were also closely related to genes from known denitrifiers. The shift in the denitrifier community was probably the consequence of the increased supply of oxidized nitrogen through nitrification. Nitrification activity increased upon addition of ammonium, but the community structure of ammonium oxidizers did not change. 相似文献