The prokaryotic diversity of biological soil crusts in the Sonoran Desert (Organ Pipe Cactus National Monument, AZ)

We studied prokaryotic community structure and composition in biological soil crusts (BSCs) from the Sonoran Desert, and their variability over space and time, using statistically analyzed, PCR-based molecular surveys of environmental 16S rRNA genes. Four sites, tens of km apart, were sampled, 3 times over a 1 year period, collecting 10 duplicate samples every 50 m in each site. Denaturing gradient gel electrophoresis (DGGE) revealed communities much less diverse than those of typical soil assemblages, displaying dominance of some bacterial types. No differences in crust microbial diversity or composition were detected between crusts under plant canopies and those in plant interspaces, indicating a likely crust independence from higher plant resources. However, statistically significant variability with space and time could be detected, and samples within a site were more similar than samples between sites. Both temporal and spatial variability in community composition involved non-dominant members of the community. Extensive sequencing and phylogenetic analysis revealed a large array of bacterial types, many novel. The most common included members of Cyanobacteria, Proteobacteria, Actinobacteria and Acidobacteria. Bacteriodetes, Chloroflexi and Gemmatimonadetes were not seen in high numbers, but were present in all sites, and Deinococci were also detected. Archaea were present, but as minor components. Sonoran BSC communities were distinct in rough compositional character from those in bulk arid soils or agricultural soils, and contained reoccurring, uncultured microbes.     

Methanotrophic diversity in an agricultural soil as evaluated by denaturing gradient gel electrophoresis profiles of pmoA, mxaF and 16S rDNA sequences

Molecular methods were used to characterize the diversity of a methanotrophic population in an agricultural soil. For this purpose we have used DGGE analysis of functional and phylogenetic markers. Functional markers utilised comprised the pmoA-gene coding for the -subunit of the particulate methane monooxygenase (pMMO) present in all known methanotrophs and the mxaF-gene coding for the -subunit of methanol dehydrogenase (MDH) present in all Gram-negative methylotrophs. In addition, we have used 16S rDNA as a phylogenetic marker. DGGE patterns of an enrichment culture, and sequencing of major DGGE bands obtained with the bacterial specific primers showed that the community structure was dominated by methanotrophic populations related to Methylobacter sp. and Methylomicrobium sp. The PCR products amplified with the functional primer sets were related to both type I and type II methanotrophs. We also designed a new pmoA-targeting primer set which could be used in a nested protocol to amplify PCR-products from DNA extracted directly from the soil.     

Spatial differences in bacterioplankton composition along the Catalan coast (NW Mediterranean) assessed by molecular fingerprinting

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments was used to compare surface bacterioplankton assemblages along the Catalan coast (NW Mediterranean). Samples from three coastal stations were compared with samples taken inside the Barcelona harbour and open sea samples taken during a cruise. The bacterial assemblage of each sample showed a characteristic and reproducible DGGE fingerprint. Between 17 and 35 bands were detected in each sample, and about 40% of the bands accounted for more than 80% of the band intensity in each sample. The presence of bands as well as their relative intensity was used to compare bacterial assemblages. Clear differences between the harbour samples and the coastal samples were evident during all periods. Marked temporal changes in the bacterial assemblages were detectable for the coastal sites, suggesting seasonal succession of coastal bacterioplankton. During each season, two stations presented a very similar bacterial composition (Barcelona and Masnou) whereas bacterial assemblages in Blanes were slightly different. These differences were consistent with the different hydrography of the area. Diversity indices calculated from DGGE fingerprints were relatively similar for all samples analysed, even though harbour samples were expected to present lower diversity values.     

DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil

A denaturing gradient gel electrophoresis (DGGE) method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil is presented. Five specific primers for 16S rDNA of methanogenic archaea, which were modified from the primers for archaea, were first evaluated by polymerase chain reaction and DGGE using genomic DNAs of 13 pure culture strains of methanogenic archaea. The DGGE analysis was possible with two primer pairs (0348aF-GC and 0691R; 0357F-GC and 0691R) of the five pairs tested although 16S rDNA of some non-methanogenic archaea was amplified with 0348aF-GC and 0691R. These two primer pairs were further evaluated for use in analysis of methanogenic archaeal community in Japanese paddy field soil. Good separation and quality of patterns were obtained in DGGE analysis with both primer pairs. A total of 41 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic archaea. These results indicate that the procedure of DGGE analysis with the primer pair 0357F-GC and 0691R is suitable for investigating methanogenic archaeal community in paddy field soil.     

Methanogenic profiles by denaturing gradient gel electrophoresis using order-specific primers in anaerobic sludge digestion

In the present study, the diversity of methanogenic populations was monitored for 25 days, together with the process data for an anaerobic batch reactor treating waste-activated sludge. To understand this microbial diversity and dynamics, 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting was conducted at two different taxonomic levels: the domain and order levels. The DGGE profiles of the domain Archaea and the three orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales were comparatively analyzed after each DGGE band was sequenced to enable identification. The DGGE profiles of the three orders showed methanogens belonging to each order that were not detected in the DGGE profile of the Archaea. This discrepancy may have resulted from PCR bias or differences in the abundances of the three microbial orders in the anaerobic bioreactor. In conclusion, to fully understand the detailed methanogenic diversity and dynamics in an anaerobic bioreactor, it is necessary to conduct DGGE analysis with 16S rRNA gene primers that target lower taxonomic groups.     

Phylogenetic diversity of nitrogen-fixing bacteria in mangrove sediments assessed by PCR-denaturing gradient gel electrophoresis

Culture-independent PCR–denaturing gradient gel electrophoresis (DGGE) was employed to assess the composition of diazotroph species from the sediments of three mangrove ecosystem sites in Sanya, Hainan Island, China. A strategy of removing humic acids prior to DNA extraction was conducted, then total community DNA was extracted using the soil DNA kit successfully for nifH PCR amplification, which simplified the current procedure and resulted in good DGGE profiles. The results revealed a novel nitrogen-fixing bacterial profile and fundamental diazotrophic biodiversity in mangrove sediments, as reflected by the numerous bands present DGGE patterns. Canonical correspondence analysis (CCA) revealed that the sediments organic carbon concentration and available soil potassium accounted for a significant amount of the variability in the nitrogen-fixing bacterial community composition. The predominant DGGE bands were sequenced, yielding 31 different nifH sequences, which were used in phylogenetic reconstructions. Most sequences were from Proteobacteria, e.g. α, γ, β, δ-subdivisions, and characterized by sequences of members of genera Azotobacter, Desulfuromonas, Sphingomonas, Geobacter, Pseudomonas, Bradyrhizobium and Derxia. These results significantly expand our knowledge of the nitrogen-fixing bacterial diversity of the mangrove environment.     

Methane and sulfate profiles within the subsurface of a tidal flat are reflected by the distribution of sulfate-reducing bacteria and methanogenic archaea

The anoxic layers of marine sediments are dominated by sulfate reduction and methanogenesis as the main terminal oxidation processes. The aim of this study was to analyze the vertical succession of microbial populations involved in these processes along the first 4.5 m of a tidal-flat sediment. Therefore, a quantitative PCR approach was applied using primers targeting the domains of Bacteria and Archaea, and key functional genes for sulfate reduction (dsrA) and methanogenesis (mcrA). The sampling site was characterized by an unusual sulfate peak at 250 cm depth resulting in separate sulfate-methane transition zones. Methane and sulfate profiles were diametrically opposed, with a methane maximum in the sulfate-depleted zone showing high numbers of archaea and methanogens. The methane-sulfate interfaces harbored elevated numbers of sulfate reducers, and revealed a slight increase in mcrA and archaeal 16S rRNA genes, suggesting sulfate-dependent anaerobic oxidation of methane. A diversity analysis of both functional genes by PCR-denaturing gradient gel electrophoresis revealed a vertical succession of subpopulations that were governed by geochemical and sedimentologic conditions. Along the upper 200 cm, sulfate-reducing populations appeared quite uniform and were dominated by the Deltaproteobacteria. In the layers beneath, an apparent increase in diversity and a shift to the Firmicutes as the predominant group was observed.     

Direct profiling of the yeast dynamics in wine fermentations

We present a method to directly characterize the yeast diversity present in wine fermentations by employing denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 26S ribosomal RNA (rRNA) genes. PCR-DGGE of a portion of the 26S rRNA gene was shown to distinguish most yeast genera associated with the production of wine. With this method the microbial dynamics in several model wine fermentations were profiled. PCR-DGGE provided a qualitative assessment of the yeast diversity in these fermentations accurately identifying populations as low as 1000 cells ml(-1). PCR-DGGE represents an attractive alternative to traditional plating schemes for analysis of the microbial successions inherent in the fermentation of wine.     

Monitoring exogenous and indigenous bacteria by PCR-DGGE technology during the process of microbial enhanced oil recovery

A field experiment was performed to monitor changes in exogenous bacteria and to investigate the diversity of indigenous bacteria during a field trial of microbial enhanced oil recovery (MEOR). Two wells (26-195 and 27-221) were injected with three exogenous strains and then closed to allow for microbial growth and metabolism. After a waiting period, the pumps were restarted and the samples were collected. The bacterial populations of these samples were analyzed by denaturing gradient gel electrophoresis (DGGE) with PCR-amplified 16S rRNA fragments. DGGE profiles indicated that the exogenous strains were retrieved in the production water samples and indigenous strains could also be detected. After the pumps were restarted, average oil yield increased to 1.58 and 4.52 tons per day in wells 26-195 and 27-221, respectively, compared with almost no oil output before the injection of exogenous bacteria. Exogenous bacteria and indigenous bacteria contributed together to the increased oil output. Sequence analysis of the DGGE bands revealed that Proteobacteria were a major component of the predominant bacteria in both wells. Changes in the bacteria population in the reservoirs during MEOR process were monitored by molecular analysis of the 16S rRNA gene sequence. DGGE analysis was a successful approach to investigate the changes in microorganisms used for enhancing oil recovery. The feasibility of MEOR technology in the petroleum industry was also demonstrated.     

Nitrification and occurrence of salt-tolerant nitrifying bacteria in the Negev desert soils

Ammonia oxidation potential, major ammonia oxidizers and occurrence of salt-tolerant nitrifying bacteria were studied in soil samples collected from diverse ecosystems along the northern Negev desert. Great diversity in ammonia oxidation potential was observed among the soil samples, and ammonia oxidizers were the rate-limiting step of nitrification. Denaturing gradient gel electrophoresis and partial 16S rRNA gene sequences indicate that members of the genus Nitrosospira are the major ammonia oxidizers in the natural desert soil samples. Upon enrichment with different salt concentrations, salt-tolerant nitrifying enrichments were established from several soil samples. In two enrichments, nitrification was not inhibited by 400 mM NaCl. Electrophoretic analysis and partial 16S rRNA gene sequences indicate that Nitrosomonas species were dominant in the 400 mM salt enrichment. The results point towards the potential of the desert ecosystem as a source of stress-tolerant nitrifying bacteria or other microorganisms with important properties.     

Quantitation and diversity analysis of ruminal methanogenic populations in response to the antimethanogenic compound bromochloromethane

Methyl coenzyme-M reductase A (mcrA) clone libraries were generated from microbial DNA extracted from the rumen of cattle fed a roughage diet with and without supplementation of the antimethanogenic compound bromochloromethane. Bromochloromethane reduced total methane emissions by c. 30%, with a resultant increase in propionate and branched chain fatty acids. The mcrA clone libraries revealed that Methanobrevibacter spp. were the dominant species identified. A decrease in the incidence of Methanobrevibacter spp. from the clone library generated from bromochloromethane treatment was observed. In addition, a more diverse methanogenic population with representatives from Methanococcales, Methanomicrobiales and Methanosacinales orders was observed for the bromochloromethane library. Sequence data generated from these libraries aided in the design of an mcrA-targeted quantitative PCR (qPCR) assay. The reduction in methane production by bromochloromethane was associated with an average decrease of 34% in the number of methanogenic Archaea when monitored with this qPCR assay. Dissociation curve analysis of mcrA amplicons showed a clear difference in melting temperatures for Methanobrevibacter spp. (80-82 degrees C) and all other methanongens (84-86 degrees C). A decrease in the intensity of the Methanobrevibacter spp. specific peak and an increase for the other peak in the bromochloromethane-treated animals corresponded with the changes within the clone libraries.     

Dominant bacterioplankton populations in the meromictic Lake Suigetsu as determined by denaturing gradient gel electrophoresis of 16S rRNA gene fragments

Lake Suigetsu is a typical meromictic lake in Japan characterized by a permanent chemocline at a depth of between 3 and 8 m separating the oxic freshwater mixolimnion from anoxic saline sulfidogenic monimolimnion. Dominant bacterioplankton populations in Lake Suigetsu were investigated using PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. The bacterial population was vertically stratified, and temporal shifts in the microbial communities were observed in both the oxic and anoxic layers of Lake Suigetsu during the sampling period. Several dominant DGGE bands were excised and sequenced. In the chemocline, green sulfur bacteria phylogenetically related to the genera Prosthecochloris, Pelodyctyon, and Chlorobium within the phylum Chlorobi were dominant; the colorless sulfur bacteria closely related to the genus Thiomicrospira were detected. These sulfur bacterial groups appear to be important in the biogeochemical cycling of sulfur and/or carbon in Lake Suigetsu. Bacterial sequences affiliated with the Bacteroidetes phylum were frequent among the dominant fragments in the DGGE profiles throughout the water column. Populations possessing a fermentative metabolism exist in Bacteroidetes, suggesting they may contribute to the degradation of organic matter in the anoxic environment of Lake Suigetsu.     

Phylogenetic analysis of intestinal bacteria in the Chinese mitten crab (Eriocheir sinensis)

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.     

肠道菌群变化对实验小鼠肠黏膜免疫的影响

目的探讨肠道菌群变化对肠黏膜相关淋巴组织的影响。方法通过变性梯度凝胶电泳（Denatu-ring gradient gel electrophoresis,DGGE）法研究了三种不同级别实验小鼠即清洁级小鼠、SPF小鼠和普通小鼠肠道菌群的组成,并用免疫组织化学（immunohistochemistry,IHC）方法研究了此三种不同级别的实验小鼠肠黏膜相关淋巴组织sIgA阳性细胞分布情况。结果普通小鼠肠道细菌种类最多,其sIgA阳性细胞分布最多,肠道不同部位之间sIgA分布情况差异有显著性（P〈0.05）,小肠和大肠之间的阳性细胞分布差异极显著（P〈0.01）;其次是清洁级小鼠,其肠道不同部位之间菌种组成差异无显著性,小肠和大肠之间的阳性细胞分布差异有显著性（P〈0.05）;SPF小鼠肠道细菌种类最少,故其sIgA阳性细胞分布最少,且其肠道不同部位之间菌种组成差异无显著性,小肠和大肠之间的阳性细胞分布差异无显著性（P〉0.05）。结论随着动物微生物控制级别的增高,肠道微生物多样性递减;sIgA阳性细胞与肠道细菌种类正相关。