Actinomycetal Community Structures in Seawater and Freshwater Examined by DGGE Analysis of 16S rRNA Gene Fragments

The actinomycetal community structures in marine and freshwater environments (the Pacific Ocean, East China Sea, Tokyo Bay, and Arakawa River) were investigated by a culture-independent molecular method to clarify spatial and seasonal distributions. Deoxyribonucleic acid (DNA) was extracted from environmental water samples, and a community analysis was carried out on polymerase chain reaction-amplified 16S ribosomal DNA. The amplified DNA fragments were analyzed by denaturing gradient gel electrophoresis (DGGE) and nonmetric multidimensional scaling analysis, followed by sequencing analysis. The actinomycetal community structures were different at each station in the Pacific Ocean, the East China Sea, Tokyo Bay, and Arakawa River, and different populations predominated in each area. There were vertical variations in actinomycetal communities in the Pacific Ocean and East China Sea between the surface and 100-m depth, but communities were similar from 200- to 1,000-m depths. There were also distinct seasonal variations in communities in Tokyo Bay. Phylogenetic analysis of DNA fragments recovered from DGGE bands revealed that most of the predominant actinomycetal strains were uncultured and were quite different from well known culturable strains, such as the Streptomyces, Micromonospora, Microbispora, Salinispora, and Actinoplanes groups. These results suggest that the marine environment is an attractive target for discovering new actinomycetal populations producing bioactive compounds and that sampling depth and season are important considerations for isolating various populations effectively.     

Effect of Temperature on Structure and Function of the Methanogenic Archaeal Community in an Anoxic Rice Field Soil

Soil temperatures in Italian rice fields typically range between about 15 and 30°C. A change in the incubation temperature of anoxic methanogenic soil slurry from 30°C to 15°C typically resulted in a decrease in the CH₄ production rate, a decrease in the steady-state H₂ partial pressure, and a transient accumulation of acetate. Previous experiments have shown that these changes were due to an alteration of the carbon and electron flow in the methanogenic degradation pathway of organic matter caused by the temperature shift (K. J. Chin and R. Conrad, FEMS Microbiol. Ecol. 18:85–102, 1995). To investigate how temperature affects the structure of the methanogenic archaeal community, total DNA was extracted from soil slurries incubated at 30 and 15°C. The archaeal small-subunit (SSU) rRNA-encoding genes (rDNA) of these environmental DNA samples were amplified by PCR with an archaeal-specific primer system and used for the generation of clone libraries. Representative rDNA clones (n = 90) were characterized by terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis. T-RFLP analysis produced for the clones terminally labeled fragments with a characteristic length of mostly 185, 284, or 392 bp. Sequence analysis allowed determination of the phylogenetic affiliation of the individual clones with their characteristic T-RFLP fragment lengths and showed that the archaeal community of the anoxic rice soil slurry was dominated by members of the families Methanosarcinaceae (185 bp) and Methanosaetaceae (284 bp), the kingdom Crenarchaeota (185 or 284 bp), and a novel, deeply branching lineage of the (probably methanogenic) kingdom Euryarchaeota (392 bp) that has recently been detected on rice roots (R. Großkopf, S. Stubner, and W. Liesack, Appl. Environ. Microbiol. 64:4983–4989, 1998). The structure of the archaeal community changed when the temperature was shifted from 30°C to 15°C. Before the temperature shift, the clones (n = 30) retrieved from the community were dominated by Crenarchaeota (70%), “novel Euryarchaeota” (23%), and Methanosarcinacaeae (7%). Further incubation at 30°C (n = 30 clones) resulted in a relative increase in members of the Methanosarcinaceae (77%), whereas further incubation at 15°C (n = 30 clones) resulted in a much more diverse community consisting of 33% Methanosarcinaceae, 23% Crenarchaeota, 20% Methanosaetaceae, and 17% novel Euryarchaeota. The appearance of Methanosaetaceae at 15°C was conspicuous. These results demonstrate that the structure of the archaeal community in anoxic rice field soil changed with time and incubation temperature.     

Influence of Particle Size on Bacterial Community Structure in Aquatic Sediments as Revealed by 16S rRNA Gene Sequence Analysis

Bacterial communities associated with sediment particles were examined using PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequencing. Particle size influenced community structure, with attached bacterial assemblages separating into 63- to 125-, 125- to 1,000-, and 1,000- to 2,000-μm fractions. Differences were particularly pronounced for the Verrucomicrobia-Planctomycetes, whose numbers were significantly reduced on coarser particles.     

Community Structure and Diversity of Biofilms from a Beer Bottling Plant as Revealed Using 16S rRNA Gene Clone Libraries

The microbial composition of biofilms from a beer bottling plant was analyzed by a cultivation independent analysis of the 16S rRNA genes. Clone libraries were differentiated by amplified 16S rRNA gene restriction analysis and representative clones from each group were sequenced. The diversity of the clone libraries was comparable with the diversity found for environmental samples. No evidences for the presence of strictly anaerobic taxa or important beer spoilers were found, indicating that biofilms developed for more than 6 months at the plant formed no appropriate habitat for those microorganisms. The genus Methylobacterium was one of the dominating groups of the clone libraries. The size of this population was assessed by fluorescence in situ hybridization and fatty acid analysis. In addition, considerable numbers of clones were assigned to uncultivated organisms.     

Comparative Phylogenetic Assignment of Environmental Sequences of Genes Encoding 16S rRNA and Numerically Abundant Culturable Bacteria from an Anoxic Rice Paddy Soil

We used both cultivation and direct recovery of bacterial 16S rRNA gene (rDNA) sequences to investigate the structure of the bacterial community in anoxic rice paddy soil. Isolation and phenotypic characterization of 19 saccharolytic and cellulolytic strains are described in the accompanying paper (K.-J. Chin, D. Hahn, U. Hengstmann, W. Liesack, and P. H. Janssen, Appl. Environ. Microbiol. 65:5042–5049, 1999). Here we describe the phylogenetic positions of these strains in relation to 57 environmental 16S rDNA clone sequences. Close matches between the two data sets were obtained for isolates from the culturable populations determined by the most-probable-number counting method to be large (3 × 10⁷ to 2.5 × 10⁸ cells per g [dry weight] of soil). This included matches with 16S rDNA similarity values greater than 98% within distinct lineages of the division Verrucomicrobia (strain PB90-1) and the Cytophaga-Flavobacterium-Bacteroides group (strains XB45 and PB90-2), as well as matches with similarity values greater than 95% within distinct lines of descent of clostridial cluster XIVa (strain XB90) and the family Bacillaceae (strain SB45). In addition, close matches with similarity values greater than 95% were obtained for cloned 16S rDNA sequences and bacteria (strains DR1/8 and RPec1) isolated from the same type of rice paddy soil during previous investigations. The correspondence between culture methods and direct recovery of environmental 16S rDNA suggests that the isolates obtained are representative geno- and phenotypes of predominant bacterial groups which account for 5 to 52% of the total cells in the anoxic rice paddy soil. Furthermore, our findings clearly indicate that a dual approach results in a more objective view of the structural and functional composition of a soil bacterial community than either cultivation or direct recovery of 16S rDNA sequences alone.     

Yersinia spp. Identification Using Copy Diversity in the Chromosomal 16S rRNA Gene Sequence

API 20E strip test, the standard for Enterobacteriaceae identification, is not sufficient to discriminate some Yersinia species for some unstable biochemical reactions and the same biochemical profile presented in some species, e.g. Yersinia ferderiksenii and Yersinia intermedia, which need a variety of molecular biology methods as auxiliaries for identification. The 16S rRNA gene is considered a valuable tool for assigning bacterial strains to species. However, the resolution of the 16S rRNA gene may be insufficient for discrimination because of the high similarity of sequences between some species and heterogeneity within copies at the intra-genomic level. In this study, for each strain we randomly selected five 16S rRNA gene clones from 768 Yersinia strains, and collected 3,840 sequences of the 16S rRNA gene from 10 species, which were divided into 439 patterns. The similarity among the five clones of 16S rRNA gene is over 99% for most strains. Identical sequences were found in strains of different species. A phylogenetic tree was constructed using the five 16S rRNA gene sequences for each strain where the phylogenetic classifications are consistent with biochemical tests; and species that are difficult to identify by biochemical phenotype can be differentiated. Most Yersinia strains form distinct groups within each species. However Yersinia kristensenii, a heterogeneous species, clusters with some Yersinia enterocolitica and Yersinia ferderiksenii/intermedia strains, while not affecting the overall efficiency of this species classification. In conclusion, through analysis derived from integrated information from multiple 16S rRNA gene sequences, the discrimination ability of Yersinia species is improved using our method.     

Bacterial Community Composition in Central European Running Waters Examined by Temperature Gradient Gel Electrophoresis and Sequence Analysis of 16S rRNA Genes

The bacterial community composition in small streams and a river in central Germany was examined by temperature gradient gel electrophoresis (TGGE) with PCR products of 16S rRNA gene fragments and sequence analysis. Complex TGGE band patterns suggested high levels of diversity of bacterial species in all habitats of these environments. Cluster analyses demonstrated distinct differences among the communities in stream and spring water, sandy sediments, biofilms on stones, degrading leaves, and soil. The differences between stream water and sediment were more significant than those between sites within the same habitat along the stretch from the stream source to the mouth. TGGE data from an entire stream course suggest that, in the upper reach of the stream, a special suspended bacterial community is already established and changes only slightly downstream. The bacterial communities in water and sediment in an acidic headwater with a pH below 5 were highly similar to each other but deviated distinctly from the communities at the other sites. As ascertained by nucleotide sequence analysis, stream water communities were dominated by Betaproteobacteria (one-third of the total bacteria), whereas sediment communities were composed mainly of Betaproteobacteria and members of the Fibrobacteres/Acidobacteria group (each accounting for about 25% of bacteria). Sequences obtained from bacteria from water samples indicated the presence of typical cosmopolitan freshwater organisms. TGGE bands shared between stream and soil samples, as well as sequences found in bacteria from stream samples that were related to those of soil bacteria, demonstrated the occurrence of some species in both stream and soil habitats. Changes in bacterial community composition were correlated with geographic distance along a stream, but in comparisons of different streams and rivers, community composition was correlated only with environmental conditions.     

Sequence versus Structure for the Direct Detection of 16S rRNA on Planar Oligonucleotide Microarrays

A two-probe proximal chaperone detection system consisting of a species-specific capture probe for the microarray and a labeled, proximal chaperone probe for detection was recently described for direct detection of intact rRNAs from environmental samples on oligonucleotide arrays. In this study, we investigated the physical spacing and nucleotide mismatch tolerance between capture and proximal chaperone detector probes that are required to achieve species-specific 16S rRNA detection for the dissimilatory metal and sulfate reducer 16S rRNAs. Microarray specificity was deduced by analyzing signal intensities across replicate microarrays with a statistical analysis-of-variance model that accommodates well-to-well and slide-to-slide variations in microarray signal intensity. Chaperone detector probes located in immediate proximity to the capture probe resulted in detectable, nonspecific binding of nontarget rRNA, presumably due to base-stacking effects. Species-specific rRNA detection was achieved by using a 22-nt capture probe and a 15-nt detector probe separated by 10 to 14 nt along the primary sequence. Chaperone detector probes with up to three mismatched nucleotides still resulted in species-specific capture of 16S rRNAs. There was no obvious relationship between position or number of mismatches and within- or between-genus hybridization specificity. From these results, we conclude that relieving secondary structure is of principal concern for the successful capture and detection of 16S rRNAs on planar surfaces but that the sequence of the capture probe is more important than relieving secondary structure for achieving specific hybridization.     

Novel Euryarchaeotal Lineages Detected on Rice Roots and in the Anoxic Bulk Soil of Flooded Rice Microcosms

Because excised, washed roots of rice (Oryza sativa) immediately produce CH₄ when they are incubated under anoxic conditions (P. Frenzel and U. Bosse, FEMS Microbiol. Ecol. 21:25–36, 1996), we employed a culture-independent molecular approach to identify the methanogenic microbial community present on roots of rice plants. Archaeal small-subunit rRNA-encoding genes were amplified directly from total root DNA by PCR and then cloned. Thirty-two archaeal rice root (ARR) gene clones were randomly selected, and the amplified primary structures of ca. 750 nucleotide sequence positions were compared. Only 10 of the environmental sequences were affiliated with known methanogens; 5 were affiliated with Methanosarcina spp., and 5 were affiliated with Methanobacterium spp. The remaining 22 ARR gene clones formed four distinct lineages (rice clusters I through IV) which were not closely related to any known cultured member of the Archaea. Rice clusters I and II formed distinct clades within the phylogenetic radiation of the orders “Methanosarcinales” and Methanomicrobiales. Rice cluster I was novel, and rice cluster II was closely affiliated with environmental sequences obtained from bog peat in northern England. Rice cluster III occurred on the same branch as Thermoplasma acidophilum and marine group II but was only distantly related to these taxa. Rice cluster IV was a deep-branching crenarchaeotal assemblage that was closely related to clone pGrfC26, an environmental sequence recovered from a temperate marsh environment. The use of a domain-specific oligonucleotide probe in a fluorescent in situ hybridization analysis revealed that viable members of the Archaea were present on the surfaces of rice roots. In addition, we describe a novel euryarchaeotal main line of descent, designated rice cluster V, which was detected in anoxic rice paddy soil. These results indicate that there is an astonishing richness of archaeal diversity present on rice roots and in the surrounding paddy soil.     

Novel Bacterial Lineages at the (Sub)Division Level as Detected by Signature Nucleotide-Targeted Recovery of 16S rRNA Genes from Bulk Soil and Rice Roots of Flooded Rice Microcosms

Using a newly developed 16S rRNA gene (rDNA)-targeted PCR assay with proposed group specificity for planctomycetes, we examined anoxic bulk soil of flooded rice microcosms for the presence of novel planctomycete-like diversity. For comparison, oxic rice roots were included as an additional sample in this investigation. The bacterial diversity detectable by this PCR assay was assessed by using a combined approach that included terminal restriction fragment length polymorphism (T-RFLP) analysis and comparative sequence analysis of cloned 16S rDNA. T-RFLP fingerprint patterns generated from rice roots contained 12 distinct terminal restriction fragments (T-RFs). In contrast, the T-RFLP fingerprint patterns obtained from the anoxic bulk soil contained 33 distinct T-RFs, a clearly higher level of complexity. A survey of 176 bulk soil 16S rDNA clone sequences permitted correlation of 20 T-RFs with phylogenetic information. The other 13 T-RFs remained unidentified. The predominant T-RFs obtained from rice roots could be assigned to members of the genus Pirellula within the Planctomycetales, while most of the T-RFs obtained from the bulk soil corresponded to novel lines of bacterial descent. Using a level of 16S rDNA sequence dissimilarity to cultured microorganisms of approximately 20% as a threshold value, we detected 11 distinct bacterial lineages for which pure-culture representatives are not known. Four of these lineages could be assigned to the order Planctomycetales, while one lineage was affiliated with the division Verrucomicrobia and one lineage was affiliated with the spirochetes. The other five lineages either could not be assigned to any of the main lines of bacterial descent or clearly expanded the known diversity of division level lineages WS3 and OP3. Our results indicate the presence of bacterial diversity at a subdivision and/or division level that has not been detected previously by the so-called universal 16S rDNA PCR assays.     

裸子植物5S rRNA基因序列变异及二级结构特征

在高等植物中,5SrRNA基因一级结构是高度保守的,二级结构也相当一致。通过比较18种裸子植物5SrRNA基因序列和二级结构变异,发现55％的核苷酸位点是可变的,这种变异有68％发生在干区（双链区）,其中一些变异,如双链的互补性核苷酸替代,GU配对等能够维系5SrRNA二级结构的稳定性。环区相对保守,这与5SrRNA三级结构折叠或在转录翻译过程中蛋白质、RNA的结合相关。另外,首次报道了松属环E区核苷酸的变异性,这可能与其他区域的变异一样,是假基因造成的结果。5SrRNA基因信息可反映大分类群的系统进化关系,但由于基因长度短,信息量小,其在近缘种系统分类的应用受到限制。     

Dispersal and Phylogenetic Diversity of Nonmarine Picocyanobacteria, Inferred from 16S rRNA Gene and cpcBA-Intergenic Spacer Sequence Analyses

More than 20 Synechococcus and Cyanobium isolates were obtained from central European subalpine lakes and sequenced for their 16S rRNA gene and part of the phycocyanin operon (cpc), specifically the intergenic spacer (IGS) between cpcB and cpcA, and corresponding flanking regions (cpcBA-IGS). Maximum-likelihood analyses revealed the existence of at least six to seven clusters of nonmarine picocyanobacteria within the picophytoplankton clade and support the conjecture of global dispersal for some closely related picocyanobacterial genotypes.     

Distribution and Diversity of Bacteria in a Saline Meromictic Lake as Determined by PCR-DGGE of 16S rRNA Gene Fragments

The variations in vertical distribution and composition of bacteria in the meromictic Lake Faro (Messina, Italy) were analysed by culture-independent methods in two different mixing conditions. Water samples were collected from a central station from the surface to the bottom (30 m depth) on two different sampling dates—the first characterised by a well-mixed water mass and the second by a marked stratification. A ‘red-water’ layer, caused by a dense growth of photosynthetic sulphur bacteria, was present at a depth of 25 m in December 2005 and at 15 m in August 2006, defining two different zones in terms of their physicochemical properties. The vertical distribution of bacterioplankton showed that the interface zones were more densely populated than others. In both sampling periods, the highest numbers of live cells were observed within ‘red water’ layers. The dominant phylotypes of the bacterial community were determined by sequencing the Denaturing Gradient Gel Electrophoresis (DGGE) bands resulting from PCR amplification of 16S rRNA gene fragments. The number of DGGE bands, considered indicative of the total species richness, did not vary predictably across the two different sampling periods. Proteobacteria (α-, γ-, δ- and ε subclass members), Cytophaga–Flavobacterium–Bacteroides, green sulphur bacteria and Cyanobacteria were retrieved from Lake Faro. Most of the bands showed DNA sequences that did not match with other previously described organisms, suggesting the presence of new indigenous bacterial phylotypes.     

基于线粒体16S rRNA基因探讨鹿科动物系统发生关系

鹿类动物的系统演化关系一直存在争议,特别是獐亚科的设立与否.通过测定獐的线粒体16S rRNA基因,并从GenBank获得鹿类另外14种动物的线粒体16S rRNA基因全序列,以水牛和绵羊作双外群,构建系统进化树,探讨鹿类动物系统发生关系及獐亚科的有效性.结果分析表明:(1)支持鹿科分为鹿亚科、麂亚科、獐亚科和美洲鹿亚科,麝科成立;(2)獐亚科有效,支持獐与原属美洲鹿亚科狍共同组成獐亚科;(3)毛冠鹿的分类地位还有待进一步确定.