Phylogenetic analysis of particle-attached and free-living bacterial communities in the Columbia river, its estuary, and the adjacent coastal ocean.

The Columbia River estuary is a dynamic system in which estuarine turbidity maxima trap and extend the residence time of particles and particle-attached bacteria over those of the water and free-living bacteria. Particle-attached bacteria dominate bacterial activity in the estuary and are an important part of the estuarine food web. PCR-amplified 16S rRNA genes from particle-attached and free-living bacteria in the Columbia River, its estuary, and the adjacent coastal ocean were cloned, and 239 partial sequences were determined. A wide diversity was observed at the species level within at least six different bacterial phyla, including most subphyla of the class Proteobacteria. In the estuary, most particle-attached bacterial clones (75%) were related to members of the genus Cytophaga or of the alpha, gamma, or delta subclass of the class Proteobacteria. These same clones, however, were rare in or absent from either the particle-attached or the free-living bacterial communities of the river and the coastal ocean. In contrast, about half (48%) of the free-living estuarine bacterial clones were similar to clones from the river or the coastal ocean. These free-living bacteria were related to groups of cosmopolitan freshwater bacteria (beta-proteobacteria, gram-positive bacteria, and Verrucomicrobium spp.) and groups of marine organisms (gram-positive bacteria and alpha-proteobacteria [SAR11 and Rhodobacter spp.]). These results suggest that rapidly growing particle-attached bacteria develop into a uniquely adapted estuarine community and that free-living estuarine bacteria are similar to members of the river and the coastal ocean microbial communities. The high degree of diversity in the estuary is the result of the mixing of bacterial communities from the river, estuary, and coastal ocean.     

Archaeaplankton in the Columbia River, its estuary and the adjacent coastal ocean, USA

PCR-amplified 16S rRNA genes from particle-attached and free-living Archaea in the Columbia River estuary, particle-attached Archaea in the river, and Archaea in the adjacent coastal ocean were cloned, and 43 partial sequences were determined. There was a high diversity of Archaea in the estuary, especially among the particle-attached Archaea, with representatives from four major phylogenetic clusters. Eighteen of 21 estuarine clones were closely related to clones from the river and the coastal ocean or to clusters of marine and soil clones identified in other studies. This contrasts with a similar study of the estuarine bacterial community that found 62% of bacterial 16S rRNA clones to be unique to the estuary. Archaea in the estuary were primarily allochthonous, and therefore, unlike the bacteria, probably do not form a native estuarine community.     

Denaturing Gradient Gel Electrophoresis Can Rapidly Display the Bacterial Diversity Contained in 16S rDNA Clone Libraries

Two different strategies for molecular analysis of bacterial diversity, 16S rDNA cloning and denaturing gradient gel electrophoresis (DGGE), were combined into a single protocol that took advantage of the best attributes of each: the ability of cloning to package DNA sequence information and the ability of DGGE to display a community profile. In this combined protocol, polymerase chain reaction products from environmental DNA were cloned, and then DGGE was used to screen the clone libraries. Both individual clones and pools of randomly selected clones were analyzed by DGGE, and these migration patterns were compared to the conventional DGGE profile produced directly from environmental DNA. For two simple bacterial communities (biofilm from a humics-fed laboratory reactor and planktonic bacteria filtered from an urban freshwater pond), pools of 35–50 clones produced DGGE profiles that contained most of the bands visible in the conventional DGGE profiles, indicating that the clone pools were adequate for identifying the dominant genotypes. However, DGGE profiles of two different pools of 50 clones from a lawn soil clone library were distinctly different from each other and from the conventional DGGE profile, indicating that this small number of clones poorly represented the bacterial diversity in soil. Individual clones with the same apparent DGGE mobility as prominent bands in the humics reactor community profiles were sequenced from the clone plasmid DNA rather than from bands excised from the gel. Because a longer fragment was cloned (∼1500 bp) than was actually analyzed in DGGE (∼350 bp), far more sequence information was available using this approach that could have been recovered from an excised gel band. This clone/DGGE protocol permitted rapid analysis of the microbial diversity in the two moderately complex systems, but was limited in its ability to represent the diversity in the soil microbial community. Nonetheless, clone/DGGE is a promising strategy for fractionating diverse microbial communities into manageable subsets consisting of small pools of clones.     

Microbial biogeography along an estuarine salinity gradient: combined influences of bacterial growth and residence time

Shifts in bacterioplankton community composition along the salinity gradient of the Parker River estuary and Plum Island Sound, in northeastern Massachusetts, were related to residence time and bacterial community doubling time in spring, summer, and fall seasons. Bacterial community composition was characterized with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA. Average community doubling time was calculated from bacterial production ([(14)C]leucine incorporation) and bacterial abundance (direct counts). Freshwater and marine populations advected into the estuary represented a large fraction of the bacterioplankton community in all seasons. However, a unique estuarine community formed at intermediate salinities in summer and fall, when average doubling time was much shorter than water residence time, but not in spring, when doubling time was similar to residence time. Sequencing of DNA in DGGE bands demonstrated that most bands represented single phylotypes and that matching bands from different samples represented identical phylotypes. Most river and coastal ocean bacterioplankton were members of common freshwater and marine phylogenetic clusters within the phyla Proteobacteria, Bacteroidetes, and ACTINOBACTERIA: Estuarine bacterioplankton also belonged to these phyla but were related to clones and isolates from several different environments, including marine water columns, freshwater sediments, and soil.     

Phylogeny of Proteobacteria and Bacteroidetes from oxic habitats of a tidal flat ecosystem

Bacteria of the phyla Proteobacteria and Bacteroidetes are known to be the most prominent heterotrophic organisms in marine surface waters. In order to investigate the occurrence of these phyla in a coastal environment, the tidal flat ecosystem German Wadden Sea, we analyzed a clone library of PCR-amplified and sequenced 16S rRNA gene fragments and isolated 46 new strains affiliated with these phyla from the water column with various polymers and complex media as substrates. The phylogenetic affiliation of these strains was analyzed on the basis of sequenced 16S rRNA gene fragments. Subsequently, a comprehensive phylogenetic analysis of Proteobacteria and Bacteroidetes including available sequences from oxic habitats of earlier studies of this ecosystem was performed. Sequences of the earlier studies were derived from isolation approaches and from denaturing gradient gel electrophoresis (DGGE) analyses of environmental samples and high dilution steps of MPN (most probable number) cultures. The majority of the 265 sequences included in this analysis affiliated with alpha-Proteobacteria (45.3%), gamma-Proteobacteria (31.7%), and Bacteroidetes (16.2%). Almost 7% belong to the delta-Proteobacteria and several of these clones affiliated with the Myxococcales, a group comprising obligate aerobic organisms. Within the alpha- and gamma-Proteobacteria specific clusters were identified including isolates from high dilution steps of dilution cultures and/or clones from the clone library or DGGE gels, implying a high abundance of some of these organisms. Within the gamma-Proteobacteria a new cluster is proposed, which consists of marine surface-attached organisms. This SAMMIC (Surface Attached Marine MICrobes) cluster comprises only uncultured phylotypes and exhibits a global distribution. Overall, the analysis indicates that Proteobacteria and Bacteroidetes of the Wadden Sea have a surprisingly high diversity, presumably a result of the signature of this ecosystem as a melting pot at the land-sea interface and comprising a great habitat variety.     

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.     

Succession of bacterial community structure along the Changjiang River determined by denaturing gradient gel electrophoresis and clone library analysis

Bacterial community structure along the Changjiang River (which is more than 2,500 km long) was studied by using denaturing gradient gel electrophoresis (DGGE) and clone library analysis of PCR-amplified 16S ribosomal DNA (rDNA) with universal bacterial primer sets. DGGE profiles and principal-component analysis (PCA) demonstrated that the bacterial community gradually changed from upstream to downstream in both 1998 and 1999. Bacterial diversity, as determined by the Shannon index (H'), gradually decreased from upstream to downstream. The PCA plots revealed that the differences in the bacterial communities among riverine stations were not appreciable compared with the differences in two adjacent lakes, Lake Dongting and Lake Poyang. The relative stability of the bacterial communities at the riverine stations was probably due to the buffering action of the large amount of water flowing down the river. Clone library analysis of 16S rDNA revealed that the dominant bacterial groups changed from beta-proteobacteria and the Cytophaga-Flexibacter-Bacteroides group upstream to high-G+C-content gram-positive bacteria downstream and also that the bacterial community structure differed among the stations in the river and the lakes. The results obtained in this study should provide a reference for future changes caused by construction of the Three Gorges Dam.     

Succession of Bacterial Community Structure along the Changjiang River Determined by Denaturing Gradient Gel Electrophoresis and Clone Library Analysis

Bacterial community structure along the Changjiang River (which is more than 2,500 km long) was studied by using denaturing gradient gel electrophoresis (DGGE) and clone library analysis of PCR-amplified 16S ribosomal DNA (rDNA) with universal bacterial primer sets. DGGE profiles and principal-component analysis (PCA) demonstrated that the bacterial community gradually changed from upstream to downstream in both 1998 and 1999. Bacterial diversity, as determined by the Shannon index (H′), gradually decreased from upstream to downstream. The PCA plots revealed that the differences in the bacterial communities among riverine stations were not appreciable compared with the differences in two adjacent lakes, Lake Dongting and Lake Poyang. The relative stability of the bacterial communities at the riverine stations was probably due to the buffering action of the large amount of water flowing down the river. Clone library analysis of 16S rDNA revealed that the dominant bacterial groups changed from β-proteobacteria and the Cytophaga-Flexibacter-Bacteroides group upstream to high-G+C-content gram-positive bacteria downstream and also that the bacterial community structure differed among the stations in the river and the lakes. The results obtained in this study should provide a reference for future changes caused by construction of the Three Gorges Dam.     

Seasonality in bacterial diversity in north-west Mediterranean coastal waters: assessment through clone libraries, fingerprinting and FISH

We combined denaturing gradient gel electrophoresis (DGGE), catalysed reporter deposition-FISH (CARD-FISH) and clone libraries to investigate the seasonality of the bacterial assemblage composition in north-west Mediterranean coastal waters. DGGE analysis indicated that bacterial diversity changed gradually throughout the year, although with a clear distinction of the summer period. Alphaproteobacteria were the dominant group on an annual basis [29% of the DAPI (4',6-diamidino-2-phenylindole) counts by CARD-FISH, and 70% of the bacterial clones]. The SAR11 clade was most abundant during spring and summer (>20% of DAPI counts), while the Roseobacter clade was abundant primarily in winter and spring (up to 7% of DAPI counts). The phylum Bacteroidetes constituted the second most important group and was quantitatively uniform throughout the year (average 11% of the DAPI counts). Gammaproteobacteria showed a peak during summer (8% of DAPI counts), when most of them belonged to the NOR5 cluster. Clone libraries and CARD-FISH showed reasonable agreement in the quantitative proportions of Bacteroidetes and Gammaproteobacteria, but Alphaproteobacteria were overrepresented in clone libraries. Sequencing of the most predominant DGGE bands failed to detect the SAR11 group despite their high abundance. The combination of the three molecular approaches allowed a comprehensive assessment of seasonal changes in bacterial diversity.     

Phylogenetic 16S rRNA analysis reveals the presence of complex and partly unknown bacterial communities in Tito Bustillo cave,Spain, and on its Palaeolithic paintings

Tito Bustillo cave (Ribadesella, Spain) contains valuable Palaeolithic paintings, which date back 15 000-20 000 years. Since 1969, the cave has been open to the public. Rock wall surfaces, spelaeothems and soils are covered by apparent biofilms of phototrophic microorganisms, which develop under artificial lighting. In addition, rock surfaces present conspicuous bacterial growth in the form of round colonies of different colours and about 1-2 mm in diameter. Even the famous Paintings Panel shows some evident microbial growth. In the present study, bacterial communities on the paintings and on the rock surfaces near the paintings were analysed by culture-independent techniques, including polymerase chain reaction (PCR) amplification of bacterial 16S rRNA genes (16S rDNA), phylogenetic sequence analyses and genetic community fingerprinting by denaturing gradient gel electrophoresis (DGGE). DGGE fingerprints showed complex bacterial community patterns. Forty-one clones matching DGGE bands of the community fingerprints were sequenced, representing about 39% of DNA fragments in the DGGE patterns. Phylogenetic sequence analyses revealed a high number of phylogenetically novel 16S rDNA sequence types and a high diversity of putatively chemotrophic and heterotrophic bacteria. Sequences were phylogenetically most closely related to the Proteobacteria (20 clones), green non-sulphur bacteria (three clones), Planctomycetales order (one clone), Cytophaga-Flexibacter- Bacteroides division (one clone) and the Actinobacteria (four clones). Furthermore, we report the presence of members of the Acidobacterium division (12 clones) in a karstic hypogean environment. Members of this phylum have not so far been detected in these particular environments.     

Characterization of the prokaryotic diversity in cold saline perennial springs of the Canadian high Arctic

The springs at Gypsum Hill and Colour Peak on Axel Heiberg Island in the Canadian Arctic originate from deep salt aquifers and are among the few known examples of cold springs in thick permafrost on Earth. The springs discharge cold anoxic brines (7.5 to 15.8% salts), with a mean oxidoreduction potential of -325 mV, and contain high concentrations of sulfate and sulfide. We surveyed the microbial diversity in the sediments of seven springs by denaturing gradient gel electrophoresis (DGGE) and analyzing clone libraries of 16S rRNA genes amplified with Bacteria and Archaea-specific primers. Dendrogram analysis of the DGGE banding patterns divided the springs into two clusters based on their geographic origin. Bacterial 16S rRNA clone sequences from the Gypsum Hill library (spring GH-4) were classified into seven phyla (Actinobacteria, Bacteroidetes, Firmicutes, Gemmatimonadetes, Proteobacteria, Spirochaetes, and Verrucomicrobia); Deltaproteobacteria and Gammaproteobacteria sequences represented half of the clone library. Sequences related to Proteobacteria (82%), Firmicutes (9%), and Bacteroidetes (6%) constituted 97% of the bacterial clone library from Colour Peak (spring CP-1). Most GH-4 archaeal clone sequences (79%) were related to the Crenarchaeota while half of the CP-1 sequences were related to orders Halobacteriales and Methanosarcinales of the Euryarchaeota. Sequences related to the sulfur-oxidizing bacterium Thiomicrospira psychrophila dominated both the GH-4 (19%) and CP-1 (45%) bacterial libraries, and 56 to 76% of the bacterial sequences were from potential sulfur-metabolizing bacteria. These results suggest that the utilization and cycling of sulfur compounds may play a major role in the energy production and maintenance of microbial communities in these unique, cold environments.     

Analysis of bacterial communities in soil by use of denaturing gradient gel electrophoresis and clone libraries, as influenced by different reverse primers

To assess soil bacterial diversity, PCR systems consisting of several slightly different reverse primers together with forward primer F968-GC were used along with subsequent denaturing gradient gel electrophoresis (DGGE) or clone library analyses. In this study, a set of 13 previously used and novel reverse primers was tested with the canonical forward primer as to the DGGE fingerprints obtained from grassland soil. Analysis of these DGGE profiles by GelCompar showed that they all fell into two main clusters separated by a G/A alteration at position 14 in the reverse primer used. To assess differences between the dominant bacteria amplified, we then produced four (100-membered) 16S rRNA gene clone libraries by using reverse primers with either an A or a G at position 14, designated R1401-1a, R1401-1b, R1401-2a, and R1401-2b. Subsequent sequence analysis revealed that, on the basis of the about 410-bp sequence information, all four primers amplified similar, as well as different (including novel), bacterial groups from soil. Most of the clones fell into two main phyla, Firmicutes and Proteobacteria. Within Firmicutes, the majority of the clones belonged to the genus Bacillus. Within Proteobacteria, the majority of the clones fell into the alpha or gamma subgroup whereas a few were delta and beta proteobacteria. The other phyla found were Actinobacteria, Acidobacteria, Verrucomicrobia, Chloroflexi, Gemmatimonadetes, Chlorobi, Bacteroidetes, Chlamydiae, candidate division TM7, Ferribacter, Cyanobacteria, and Deinococcus. Statistical analysis of the data revealed that reverse primers R1401-1b and R1401-1a both produced libraries with the highest diversities yet amplified different types. Their concomitant use is recommended.     

Stratification and seasonal stability of diverse bacterial communities in a Pinus merkusii (pine) forest soil in central Java,Indonesia

In Java, Indonesia, many nutrient-poor soils are intensively reforested with Pinus merkusii (pine). Information on nutrient cycles and microorganisms involved in these cycles will benefit the management of these important forests. Here, seasonal effects on the stratification of bacterial community structure in the soil profile of a tropical pine forest are described, and differences in bacterial communities are related to chemical and physical soil parameters. Culture-independent community profiles of litter, fragmented litter and mineral soil layers were made by denaturing gradient gel electrophoresis (DGGE) of 16S rDNA-specific polymerase chain reaction (PCR) fragments. The community profiles of the different soil layers clustered separately, correlating with significant differences in organic matter content between the three layers. The bacterial communities appeared to be stable during the wet season of 1998. The drought in 1997, caused by the El Ni?o climatic effect, did not influence the bacterial communities in fragmentation and mineral soil, although moisture content and other soil parameters were markedly lower than in the wet season. However, communities in litter were influenced by drought. In the litter layer, the moisture content was significantly lower than in the fragmentation and mineral layers during the dry season. A clone library was made from a litter sample taken during the wet season. Partial sequencing of 74 clones and linking the DGGE banding positions of these clones to bands in the DGGE profile of the sample from which the clone library was derived showed considerable bacterial diversity. Alpha-proteobacteria (40.5% of the clones, of which 57% belonged to the Rhizobium-Agrobacterium group) and high-G+C content, Gram-positive bacteria (36.5%) dominated the clone library.     

Bacterial populations colonizing and degrading rice straw in anoxic paddy soil

Rice straw is a major substrate for the production of methane, a greenhouse gas, in flooded rice fields. The bacterial community degrading rice straw under anoxic conditions was investigated with molecular methods. Rice straw was incubated in paddy soil anaerobically for 71 days. Denaturing gradient gel electrophoresis (DGGE) of the amplified bacterial 16S rRNA genes showed that the composition of the bacterial community changed during the first 15 days but then was stable until the end of incubation. Fifteen DGGE bands with different signal intensities were excised, cloned, and sequenced. In addition, DNA was extracted from straw incubated for 1 and 29 days and the bacterial 16S rRNA genes were amplified and cloned. From these clone libraries 16 clones with different electrophoretic mobilities on a DGGE gel were sequenced. From a total of 31 clones, 20 belonged to different phylogenetic clusters of the clostridia, i.e., clostridial clusters I (14 clones), III (1 clone), IV (1 clone), and XIVa (4 clones). One clone fell also within the clostridia but could not be affiliated to one of the clostridial clusters. Ten clones grouped closely with the genera Bacillus (3 clones), Nitrosospira (1 clone), Fluoribacter (1 clones), and Acidobacterium (2 clones) and with clone sequences previously obtained from rice field soil (3 clones). The relative abundances of various phylogenetic groups in the rice straw-colonizing community were determined by fluorescence in situ hybridization (FISH). Bacteria were detached from the incubated rice straw with an efficiency of about 80 to 90%, as determined by dot blot hybridization of 16S rRNA in extract and residue. The number of active (i.e., a sufficient number of ribosomes) Bacteria detected with a general eubacterial probe (Eub338) after 8 days of incubation was 61% of the total cell counts. This percentage decreased to 17% after 29 days of incubation. Most (55%) of the active cells on day 8 belonged to the genus Clostridium, mainly to clostridial clusters I (24%), III (6%), and XIVa (24%). An additional 5% belonged to the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides phylum, 4% belonged to the alpha, beta, and gamma Proteobacteria, and 1.3% belonged to the Bacillus subbranch of the gram-positive bacteria with a low G+C content. The results show that the bacterial community colonizing and decomposing rice straw developed during the first 15 days of incubation and was dominated by members of different clostridial clusters, especially clusters I, III, and XIVa.     

Evidence for Selective Bacterial Community Structuring in the Freshwater Sponge Ephydatia fluviatilis

To understand the functioning of sponges, knowledge of the structure of their associated microbial communities is necessary. However, our perception of sponge-associated microbiomes remains mainly restricted to marine ecosystems. Here, we report on the molecular diversity and composition of bacteria in the freshwater sponge Ephydatia fluviatilis inhabiting the artificial lake Vinkeveense Plassen, Utrecht, The Netherlands. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints revealed that the apparent diversities within the domain Bacteria and the phylum Actinobacteria were lower in E. fluviatilis than in bulk water. Enrichment of specific PCR-DGGE bands in E. fluviatilis was detected. Furthermore, sponge- and bulk water-derived bacterial clone libraries differed with respect to bacterial community composition at the phylum level. E. fluviatilis-derived sequences were affiliated with six recognized phyla, i.e., Proteobacteria, Planctomycetes, Actinobacteria, Bacteroidetes, Chlamydiae and Verrucomicrobia, in order of relative abundance; next to the uncultured candidate phylum TM7 and one deeply rooted bacterial lineage of undefined taxonomy (BLUT). Actinobacteria, Proteobacteria, and Bacteroidetes were the dominant bacterial phyla in the freshwater clone library whereas sequences affiliated with Planctomycetes, Verrucomicrobia, Acidobacteria and Armatimonadetes were found at lower frequencies. Fine-tuned phylogenetic inference showed no or negligible overlaps between the E. fluviatilis and water-derived phylotypes within bacterial taxa such as Alphaproteobacteria, Bacteroidetes and Actinobacteria. We also ascertained the status of two alphaproteobacterial lineages as freshwater sponge-specific phylogenetic clusters, and report on high distinctiveness of other E. fluviatilis specific phylotypes, especially within the Bacteroidetes, Planctomycetes and Chlamydia taxa. This study supports the contention that the composition and diversity of bacteria in E. fluviatilis is partially driven by the host organism.     

Phylogenetic analysis of the gut bacterial microflora of the fungus-growing termite Odontotermes formosanus

We constructed a bacterial 16S rRNA gene clone library from the gut microbial community of O. formosanus and phylogenetically analyzed it in order to contribute to the evolutional study of digestive symbiosis and method development for termite control. After screening by restriction fragment length polymorphism (RFLP) analysis, 56 out of 280 clones with unique RFLP patterns were sequenced and phylogenetically analyzed. The representative phylotypes were affiliated to four phylogenetic groups, Firmicutes, the Bacteroidetes/Chlorobi group, Proteobacteria, and Actinobacteria of the domain Bacteira. No one clone affiliated with the phylum Spirochaetes was identified, in contrast to the case of wood-feeding termites. The phylogenetic analysis revealed that nearly half of the representative clones (25 phylotypes) formed monophyletic clusters with clones obtained from other termite species, especially with the sequences retrieved from fungus-growing termites. These results indicate that the presence of termite-specific bacterial lineages implies a coevolutional relationship of gut microbes and host termites.     

新疆艾比湖湿地博乐河入口处土壤细菌多样性分析

【目的】了解新疆艾比湖湿地国家级自然保护区非培养土壤细菌群落组成及多样性。【方法】采用非培养法直接从湿地土壤提取总DNA进行16S r RNA基因扩增,构建细菌16S r RNA基因克隆文库。使用MspⅠ和AfaⅠ限制性内切酶对阳性克隆进行16S r RNA基因扩增片段的限制性酶切分析(Amplified r DNA restriction analysis,ARDRA),挑取具有不同双酶切图谱的克隆进行测序,序列比对并构建16S r RNA基因系统发育树。【结果】从土壤细菌的16S r RNA基因文库中随机挑取75个不同谱型的克隆子,共得到58个OTUs,系统发育归类为8个细菌类群:绿弯菌门(Chloroflexi)、蓝藻门(Cyanobacteria)、变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、疣微菌门(Verrucomicrob)和芽单胞菌门(Gemmatimonadetes)。其中,变形菌门为第一优势菌群,拟杆菌门为第二优势菌群,两者约占总克隆的65%。【结论】艾比湖湿地博乐河入口处土壤细菌多样性丰富,且存在一定数量的潜在微生物新种。     

基于PCR-DGGE技术的红树林区微生物群落结构

【目的】为了解红树林沉积物中细菌的群落结构特征。【方法】应用PCR-DGGE技术对福建浮宫红树林的16个采样站位样品细菌的群落结构进行了研究。根据DGGE指纹图谱,对它们的遗传多样性进行了分析。【结果】各站位样品细菌多样性指数(H)、丰度(S)和均匀度(EH)均有所不同,这些差异与它们所处站位的不同有关,红树林区细菌多样性高于非红树林区细菌多样性。对不同站位细菌群落相似性分析,它们的相似性系数也存在一定的规律,同一断面的细菌群落结构相近性较高。对DGGE的优势条带序列分析,同源性最高的微生物分别属于变形菌门(Proteobacteria)、酸菌门(Acidobacteria)和绿菌门(Chlorobi),它们均为未培养微生物,分别来自于河口海岸沉积物。【结论】应用PCR-DGGE技术更能客观地反映红树林沉积物中真实的细菌群落结构信息。另外,研究也表明红树林区微生物多样性丰富,在红树林区研究开发未知微生物资源具有巨大的潜力。     

Community Dynamics of Free-living and Particle-associated Bacterial Assemblages during a Freshwater Phytoplankton Bloom

Bacterial community dynamics were followed in a 19-day period during an induced diatom bloom in two freshwater mesocosms. The main goal was to compare diversity and succession among free-living (<10 MM) AND PARTICLE-ASSOCIATED (>10 mm) bacteria. Denaturing gradient gel electrophoresis (DGGE) of PCR amplified 16S rDNA showed the highest number of bands among free-living bacteria, but with a significant phylogenetic overlap in the two size fractions indicating that free-living bacteria were also important members of the particle-associated bacterial assemblage. Whereas the number of bands in the free-living fraction decreased during the course of the bloom, several phylotypes unique to particles appeared towards the end of the experiment. Besides the primer set targeting Bacteria, a primer set targeting most members of the Cytophaga-Flavobacterium (CF)-cluster of the Cytophaga-Flavobacterium-Bacteroides group and a primer set mainly targeting a-Proteobacteria were applied. PCR-DGGE analyses revealed that a number of phylotypes targeted by those primer sets were found solely on particles. Almost all sequenced bands from the bacterial DGGE gel were related to phylogenetic groups commonly found in freshwater: a-Proteobacteria, CF, and Firmicutes. Despite the use of primers intended to be specific mainly for a-Proteobacteria most bands sequenced from the a-proteobacterial DGGE gel formed a cluster within the Verrucomicrobiales subdivision of the Verrucomicrobia division and were not related to a-Proteobacteria. Bands sequenced from the CF DGGE gel were related to members of the CF cluster. From the present study, we suggest that free-living and particle-associated bacterial communities should not be perceived as separate entities, but rather as interacting assemblages, where the extent of phylogenetic overlap is dependent on the nature of the particulate matter.     

Comparison of the Cecal Microbiota of Domestic and Wild Turkeys

The extent to which production methods alter intestinal microbial communities of livestock is currently unknown. As the intestinal microbiota may affect animal health, nutrition, and food safety, a baseline comparison of the cecal communities of domestic and wild turkeys was performed. Oligonucleotide fingerprinting of ribosomal RNA (rRNA) genes (OFRG) of 2,990 16S rRNA clones and dot blot quantification of dominant populations were used to identify the dominant bacterial taxa. Seventy-three percent of all the clones belonged to as yet uncultured genera. However, at a higher phylogenetic level, the OFRG library was composed of 54% Bacteroidetes clones (52% of the domestic library clones, 56% of the wild library clones), 30% Firmicutes clones (33% of the domestic library clones, 32% of the wild library clones), 3% Proteobacteria clones (5% domestic, 2% wild), and 3% Deferribacteres clones (4% domestic, 1% wild). Seven percent of the clones were unidentifiable (6% domestic, 9% wild). Bacteroidetes clones included the genera Alistipes, Prevotella, Megamonas, and Bacteroides. Of the Clostridiales clones, groups IV, IX, and XIV including genera Faecalibacterium, Megasphaera, Phascolarctobacterium, and Papillibacter were predominant. Lactobacillus, Enterococcus, and Streptococcus bacilli were also identified. beta- delta- and gamma-proteobacterial genera included Acinetobacter, Sutterella, and Escherichia. Deferribacteres clones showed high similarity to Mucispirillum schaedleri. Statistical comparison of the domestic and wild turkey clone libraries indicated similar levels of community richness and evenness despite the fact that the two libraries shared only 30% of the total clone operational taxonomic units. Together these results indicate that although high level taxonomic community structure is similar, high-density turkey production causes considerable divergence of the genera found in the ceca of commercial birds from those of their wild counterparts.