Bacterial diversity,community structure and potential growth rates along an estuarine salinity gradient

Very little is known about growth rates of individual bacterial taxa and how they respond to environmental flux. Here, we characterized bacterial community diversity, structure and the relative abundance of 16S rRNA and 16S rRNA genes (rDNA) using pyrosequencing along the salinity gradient in the Delaware Bay. Indices of diversity, evenness, structure and growth rates of the surface bacterial community significantly varied along the transect, reflecting active mixing between the freshwater and marine ends of the estuary. There was no positive correlation between relative abundances of 16S rRNA and rDNA for the entire bacterial community, suggesting that abundance of bacteria does not necessarily reflect potential growth rate or activity. However, for almost half of the individual taxa, 16S rRNA positively correlated with rDNA, suggesting that activity did follow abundance in these cases. The positive relationship between 16S rRNA and rDNA was less in the whole water community than for free-living taxa, indicating that the two communities differed in activity. The 16S rRNA:rDNA ratios of some typically marine taxa reflected differences in light, nutrient concentrations and other environmental factors along the estuarine gradient. The ratios of individual freshwater taxa declined as salinity increased, whereas the 16S rRNA:rDNA ratios of only some typical marine bacteria increased as salinity increased. These data suggest that physical and other bottom-up factors differentially affect growth rates, but not necessarily abundance of individual taxa in this highly variable environment.     

新疆泥火山细菌遗传多样性

为了解新疆乌苏泥火山细菌多样性,从泥火山泥浆样品中直接提取总DNA,构建了含150个有效转化子的泥火山细菌16S rDNA基因文库,转化子经菌液PCR及HaeⅢ酶切后获得16个不同带型,克隆测序结果表明,其分属于16个不同的分类单元.一部分序列与已知细菌类群的16S rDNA序列相似性较高,归属变形菌门(Proteobacteria),厚壁菌门(Firmicutes),梭杆菌门(Fusobacteria),放线菌门(Actinobacteria);另外一部分序列与已知细菌类群的16S rDNA序列同源性较低,可能代表新的分类单位.研究结果显示,泥火山环境中微生物种群丰富,值得进一步研究.     

Novel endophytes of rice form a taxonomically distinct subgroup of Serratia marcescens

Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDS-PAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens (more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens (> or =86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.     

Novel Endophytes of Rice form a Taxonomically Distinct Subgroup of Serratia marcescens

Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDS-PAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens(more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens(≥86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.     

Relationship between genome similarity and DNA-DNA hybridization among closely related bacteria

DNA-DNA hybridization has been established as an important technology in bacterial species taxonomy and phylogenetic analysis. In this study, we analyzed how the efficiency with which the genomic DNA from one species hybridizes to the genomic DNA of another species (DNA-DNA hybridization) in microarray analysis relates to the similarity between two genomes. We found that the predicted DNA-DNA hybridization based on genome sequence similarity correlated well with the experimentally determined microarray hybridization. Between closely related strains, significant numbers of highly divergent genes (<55% identity) and/or the accumulation of mismatches between conserved genes lowered the DNA-DNA hybridization signal, and this reduced the hybridization signals to below 70% for even bacterial strains with over 97% 16S rRNA gene identity. In addition, our results also suggest that a DNA-DNA hybridization signal intensity of over 40% indicates that two genomes at least shared 30% conserved genes (>60% gene identity). This study may expand our knowledge of DNA-DNA hybridization based on genomic sequence similarity comparison and further provide insights for bacterial phylogeny analyses.     

Bacterial diversity in deep-sea sediment from northeastern Pacific Ocean

16S rDNA sequencing method is one of the effectively used culture-independent techniques in recent years. In this study, 16S rDNA sequencing method was used to investigate the bacterial diversity in deep-sea sediment from northeastern Pacific polymetallic nodule province. Total DNAs were extracted by using 2 different methods (chemical method and DNA extracting kit method). After purification, genomic DNA was amplified by using 2 universal primers (27F and 1492R). Clones were selected and sequenced randomly. After the sequences were checked by using the Chimera Check Program of the RDP database, a bacterial 16S rRNA gene library of 79 clones was established. Phylogenetic analysis indicated that 79 clones could be divided into 11 phylotypes. Gamma Proteobacteria (22.8%) and alpha Proteobacteria (16.5%) were the dominant components of the sediment bacterial community, followed by Planctomycetacia (7.6%), delta Proteobacteria (6.3%), Nitrospira (6.3%), Actinobacteria (6.3%), beta Proteobacteria (5%), Acidobacteria (5.1%), Sphingobacteria (3.8%), Firmicutes (2.5%) and uncultured bacteria (17.7%). Gamma Proteobacteria also dominated at slices 0–2 cm and 4–6 cm. Different slices had different types of bacteria, alpha Proteobacteria, gamma Proteobacteria, delta Proteobacteria, Planctomycetacia, Nitrospira, Actinobacteria and Acidobacteria, however, appeared in all slices. Pseudomonas is common in many different deep-sea environments. In this study, it accounted for 22.2% of the total gamma Proteobacteria.     

喀斯特原生土壤与退化生态系统土壤细菌群落结构

运用PCR-RFLP技术,对桂西北喀斯特原生土壤和退化生态系统土壤细菌16S rDNA基因多样性及系统发育关系进行了研究.结果表明：原生土壤比退化生态系统土壤具有更丰富的16S rDNA基因型和更高的多样性指数,两样地共有的基因型仅有2个.从每种基因型中随机选择一个克隆子作为代表进行测序分析,所有序列与GenBank 数据库中序列的同源性为87%～100%,且两样地中均有超过一半的基因型序列与数据库中已知序列同源性低于97%,属于分类在“种”地位上的新发现细菌;通过系统发育研究将两样地的细菌分为10大类群,两样地共同拥有5大类群,但两样地的细菌优势类群明显不同,原生土壤为Proteobacteria,含39种基因型,占总克隆子数的58.0%,退化生态系统土壤为Acidobacteria和Proteobacteria,分别含19种和15种基因型,占总克隆子数的32.5%和30.5%;与原生土壤细菌类群相比,退化生态系统土壤Proteobacteria类群明显减少,Acidobacteria类群明显增加.土壤理化性质及土壤环境因素的差异是引起两类型土壤细菌多样性差异的原因.     

Bacterial community structure associated with a dimethylsulfoniopropionate-producing North Atlantic algal bloom

The bacteria associated with oceanic algal blooms are acknowledged to play important roles in carbon, nitrogen, and sulfur cycling, yet little information is available on their identities or phylogenetic affiliations. Three culture-independent methods were used to characterize bacteria from a dimethylsulfoniopropionate (DMSP)-producing algal bloom in the North Atlantic. Group-specific 16S rRNA-targeted oligonucleotides, 16S ribosomal DNA (rDNA) clone libraries, and terminal restriction fragment length polymorphism analysis all indicated that the marine Roseobacter lineage was numerically important in the heterotrophic bacterial community, averaging >20% of the 16S rDNA sampled. Two other groups of heterotrophic bacteria, the SAR86 and SAR11 clades, were also shown by the three 16S rRNA-based methods to be abundant in the bloom community. In surface waters, the Roseobacter, SAR86, and SAR11 lineages together accounted for over 50% of the bacterial rDNA and showed little spatial variability in abundance despite variations in the dominant algal species. Depth profiles indicated that Roseobacter phylotype abundance decreased with depth and was positively correlated with chlorophyll a, DMSP, and total organic sulfur (dimethyl sulfide plus DMSP plus dimethyl sulfoxide) concentrations. Based on these data and previous physiological studies of cultured Roseobacter strains, we hypothesize that this lineage plays a role in cycling organic sulfur compounds produced within the bloom. Three other abundant bacterial phylotypes (representing a cyanobacterium and two members of the alpha Proteobacteria) were primarily associated with chlorophyll-rich surface waters of the bloom (0 to 50 m), while two others (representing Cytophagales and delta Proteobacteria) were primarily found in deeper waters (200 to 500 m).     

Molecular phylogenetic diversity of bacteria associated with the leachate of a closed municipal solid waste landfill

A 16S rDNA-based molecular study was performed to determine the nature of the bacterial constituents of the leachate from a closed municipal solid waste landfill. Total community DNA was extracted and bacterial 16S rRNA genes were subsequently amplified and cloned. Recombinant rDNA clones in the library were randomly selected, and they were sequenced for a single run and then grouped. A total of 76 sequence types representing 138 randomly selected nonchimeric clones were identified. Full-length sequencing and phylogenetic analysis of the sequence types revealed that more than 90% of the screened clones were affiliated with low-G+C gram-positive bacteria (38.4%), Proteobacteria (35.5%), the Cytophaga Flexibacter Bacteroides group (11.6%), and Spirochaetes (5.1%). Minor portions were affiliated with Verrucomicrobia (2.9%), candidate division OP11 (2.2%), and the green nonsulfur bacteria, Cyanobacteria and the Deinococcus Thermus group (each <1.0%). Although some rDNA sequences clustered with genera or taxa that were classically identified within anaerobic treatment systems and expected with known functions, a substantial fraction of the clone sequences showed relatively low levels of similarity with any other reported rDNA sequences and thus were derived from unknown taxa. These results suggest that bacterial communities in landfill environment are far more complex than previously expected and remain largely unexplored.     

Phylogenetic characterization of a new thermoacidophilic bacterium isolated from hot springs in Japan

Three strains of thermophilic-acidophilic bacteria isolated previously from different hot springs in Japan were characterized by molecular genetic methods. The strategy taken involved PCR amplification, sequencing and restriction pattern analysis of 16S rDNA, 16S-23S rDNA spacer polymorphism analysis and genomic DNA-DNA hybridization. A phylogenetic analysis based on 16S rDNA sequences showed that the new thermoacidophilic isolates formed a genetically coherent group at the species level and fell into a major cluster together with members of the genera Alicyclobacillus and Sulfobacillus with A. acidocaldarius and A. acidoterrestris as their closest relatives. The levels of binary sequence similarity between the isolates and the two Alicyclobacillus species were 97.6 to 97.9%, values considered low enough to warrant placement of the isolates in a distinct species of the genus Alicyclobacillus. The 16S rDNA restriction pattern analysis, but not 16S-23S rDNA spacer polymorphism analysis, was useful for differentiating the isolates from the established Alicyclobacillus species. DNA-DNA hybridization assays demonstrated a distinct phylogenetic position of our isolates as a genospecies within the genus Alicyclobacillus. On the basis of these results, the thermoacidophilic isolates should be classified into a new species of Alicyclobacillus. The results of this study suggest that this new genospecies of Alicyclobacillus is widely distributed in hot springs in Japan.     

Global dinucleotide signatures and analysis of genomic heterogeneity

Early biochemical experiments measuring nearest neighbor frequencies established that the set of dinucleotide relative abundance values (dinucleotide biases) is a remarkably stable property of the DNA of an organism. Analyses of currently available genomic sequence data have extended these earlier results, showing that the dinucleotide biases evaluated for successive 50 kb segments of a genome are significantly more similar to each other than to those of sequences from more distant organisms. From this perspective, the set of dinucleotide biases constitutes a 'genomic signature' that can discriminate sequences from different organisms. The dinucleotide biases appear to reflect species-specific properties of DNA stacking energies, modification, replication, and repair mechanisms. The genomic signature is useful for detecting pathogenicity islands in bacterial genomes.     

A preliminary report of phylogenetic diversity of bacterial strains isolated from marine creatures

Bacterial diversity among marine creatures, especially molluscs, as a source for searching out novel lineages of bacteria, was studied. Marine creatures were collected at the coasts of the Kanto area in Japan. A total of 116 strains of bacteria were isolated from the intestines of 19 species of marine creatures includings molluscs, pisces and protochordata. Partial sequencing of 16S rDNA revealed that most of the isolates belonged to the gamma subclass of the Proteobacteria and Cytophaga-Flavobacterium-Bacteroides group. The BLAST searches revealed that the complete 16S rDNA sequence of 17 strains out of 116 isolates showed less than 94% similarity with 16S rDNA sequences deposited in the database. Four strains out of the 17 isolates belonged to the Rhodobacter group, 8 strains to the Alteromonas group, and the remaining 5 strains to the Cytophaga-Flavobacterium-Bacteroides group. Phylogenetic positions of 6 strains belonging to the Alteromonas group, which were isolated from different marine creatures, were close to each other, and represented a novel 16S rDNA lineage within the gamma subclass of Proteobacteria. Therefore, it may be inferred that these 6 strains belong to a new genus of Proteobacteria. Phylogenetic positions of the other strains are also independent from neighboring taxa, and they were suggested to respectively form a novel lineage. From these results, it is clear that the biodiversity of bacteria in marine creatures is much wider than was previously thought, and unknown microbiological resources are buried in these organisms.     

Endophytic Bacterial Diversity in Rice (Oryza sativa L.) Roots Estimated by 16S rDNA Sequence Analysis

The endophytic bacterial diversity in the roots of rice (Oryza sativa L.) growing in the agricultural experimental station in Hebei Province, China was analyzed by 16S rDNA cloning, amplified ribosomal DNA restriction analysis (ARDRA), and sequence homology comparison. To effectively exclude the interference of chloroplast DNA and mitochondrial DNA of rice, a pair of bacterial PCR primers (799f–1492r) was selected to specifically amplify bacterial 16S rDNA sequences directly from rice root tissues. Among 192 positive clones in the 16S rDNA library of endophytes, 52 OTUs (Operational Taxonomic Units) were identified based on the similarity of the ARDRA banding profiles. Sequence analysis revealed diverse phyla of bacteria in the 16S rDNA library, which consisted of alpha, beta, gamma, delta, and epsilon subclasses of the Proteobacteria, Cytophaga/Flexibacter/Bacteroides (CFB) phylum, low G+C gram-positive bacteria, Deinococcus-Thermus, Acidobacteria, and archaea. The dominant group was Betaproteobacteria (27.08% of the total clones), and the most dominant genus was Stenotrophomonas. More than 14.58% of the total clones showed high similarity to uncultured bacteria, suggesting that nonculturable bacteria were detected in rice endophytic bacterial community. To our knowledge, this is the first report that archaea has been identified as endophytes associated with rice by the culture-independent approach. The results suggest that the diversity of endophytic bacteria is abundant in rice roots.     

Genome comparisons in the genus Dipodascus de Lagerheim

Phenotypic characteristics of physiology and morphology of 71 strains belonging to the genus Dipodascus de Lagerheim were examined. The GC contents of genomic DNAs of 46 strains were calculated from the thermal denaturation curves using the spectrophotometric method. The first derivatives of the melting curves revealed that the DNAs of these strains are heterogeneous; four categories could be recognized. However, DNA similarity values calculated by using DNA-DNA reassociation kinetics showed that each category could be subdivided further. Two categories were separated into four subgroups each; the other two yielded five subgroups each. Strains belonging to the same subgroup exhibited high levels of DNA similarity ranging from 82 to 100%. The 18 subgroups represented 13 currently accepted Dipodascus species and five anamorphic Geotrichum species, four representing novel taxa. A phenotypic key to distinguish the taxa of Dipodascus, Galactomyces and Geotrichum is presented.     

青藏铁路沿线唐古拉山口土壤微生物的ARDRA分析

通过构建16S rDNA文库及文库的限制性片段长度多态性分析（ARDRA）,对青藏铁路沿线唐古拉山口的土壤微生物多样性进行了研究。采用限制性内切酶HaeIII和RsaI对克隆文库中的90个克隆子进行了酶切分型,根据ARDRA酶切图谱的不同,可将其分为23个OTUs。16SrDNA序列分析结果表明,该克隆文库中主要包括变形菌门（Proteobacteria）的alpha、beta、detla亚类、厚壁菌门（Firmicutes）、放线菌门（Actinobacteria）、拟杆菌门（Bacteroidetes）、酸杆菌门（Acidobacteria）及浮霉菌门（Planctomycetes）等8类细菌及未培养细菌。Alpha变形细菌为该文库中的主要菌群,占克隆总数的33.3%;其次为未培养细菌,占克隆总数的22.2%,Bradyrhizobium为优势菌属。研究结果揭示,青藏铁路唐古拉山口的土壤微生物种群不仅具有丰富的多样性,还存在丰富的潜在新菌种。     

High bacterial diversity of a waste gas-degrading community in an industrial biofilter as shown by a 16S rDNA clone library

The bacterial diversity of an industrial biofilter used for waste gas abatement in an animal-rendering plant was investigated. A 16S rDNA clone library was generated and 444 clones were screened using computer-aided amplified ribosomal DNA restriction analysis (ARDRA). Of the screened clones, 60.8% showed unique ARDRA patterns and the remaining 174 clones were clustered into 65 groups. Almost full-length 16S rDNA sequences of 106 clones were determined and 90.5% of the clones were affiliated with the two phyla Proteobacteria and Bacteroidetes. Alpha-, Beta-, and Gammaproteobacteria accounted for 22.1, 17.6 and 18.6% respectively. Minor portions were affiliated with the Actinobacteria (2.0%), Firmicutes and Verrucomicrobia (both 1.0%), and the Deltaproteobacteria and Thermomicrobia (each 0.5%). Only six out of the 106 16S rDNA sequences exhibited similarities of more than 97% to classified bacterial species indicating that a substantial fraction of the clone sequences were derived from unknown taxa. It was also evaluated whether a database containing 281 computer-simulated bacterial rDNA fragment patterns generated from published reference sequences can be used for identification purposes. The data analysis demonstrated that this was possible only for a small number of clones, which were closely related to described bacterial strains. Rarefaction analysis of ARDRA clusters demonstrated that the 444 clones screened are insufficient to describe the entire diversity of the clone library.     

Vibrio tasmaniensis sp. nov., isolated from Atlantic salmon (Salmo salar L.)

We describe the polyphasic characterization of four Vibrio isolates which formed a tight AFLP group in a former study. The group was closely related to V. cyclitrophicus, V. lentus and V. splendidus (98.2-98.9% similarity) on the basis of the 16S rDNA sequence analysis, but by DNA-DNA hybridisation experiments it had at maximum 61% DNA similarity towards V. splendidus. Thus, we propose that the isolates represent a new Vibrio species i.e. V. tasmaniensis (LMG 20012T; EMBL under the accession numbers AJ316192; mol% G+C of DNA of the type strain is 44.7). Useful phenotypical features for discrimination of V. tasmaniensis from other Vibrio species include gelatinase and beta-galactosidase activity, fatty acid composition (particularly 14:0), utilisation and fermentation of different compounds (e.g. sucrose, melibiose and D-galactose) as sole carbon source.     

基于16S-23S rDNA 间隔区序列（ITS）、REP序列的基因指纹图谱及UPGMA聚类法的拟诺卡菌属分类方法

拟诺卡菌属（Nocardiopsis）是拟诺卡菌科（Nocardiopsaceae） 的唯一属．该属内进行物种鉴别时通常是在多相分类方法基础上,以全基 因组杂交同源性在70%以下的为不同物种,此为国际公认的定种标准;但在 进行大量菌株的比对时操作比较复杂,于是多种基于DNA的基因图谱技术发 展起来．本实验利用适宜引物,对拟诺卡菌属15株基准株基因组DNA的16S -23S rDNA 间隔区序列（ITS）和REP序列进行了扩增,获得了两种基因指 纹图谱,同时根据UPGMA聚类法构建了相应的进化距离树图．结果表明,对 于拟诺卡菌属中不同物种的区分,两种基因图谱技术的分辨力相当,均可 以较好的呈现物种间差异,可以作为拟诺卡菌属菌株多相分类的组成部分 ,应用于物种水平的分类与鉴定．     

腾格里沙漠东南缘不同生物土壤结皮细菌多样性及其季节动态特征

微生物多样性对于生物土壤结皮在沙漠生态系统中改善局部环境以及提升生态功能具有重要作用。本研究对腾格里沙漠东南缘沙坡头地区藻结皮、藓结皮及其下层的四季样品进行了16S rDNA高通量测序, 以期阐明细菌多样性及其在生物土壤结皮演替过程中的季节变化规律。结果表明4种类型样品的细菌丰富度在夏季显著低于其他3个季节。4种类型样品中主要的细菌类群为变形菌门、放线菌门、绿弯菌门、酸杆菌门、蓝细菌门等, 其中变形菌门和放线菌门为优势类群, 夏季时变形菌门的相对多度显著高于春季、秋季、冬季, 且在结皮层中相对多度显著高于结皮下层。放线菌门的相对多度在春季、夏季显著高于秋季、冬季, 且结皮下层相对多度高于结皮层。生物土壤结皮演替过程中细菌多样性及其相对多度季节动态变化表明其对沙漠土壤局部环境的变化作出了响应, 这为深入理解生物土壤结皮在沙漠生态系统中的生态功能提供了微生物多样性数据。     

Bacterial diversity of the digestive gland of Sydney rock oysters,Saccostrea glomerata infected with the paramyxean parasite,Marteilia sydneyi

Aims: To determine whether the infestation by the protozoan paramyxean parasite, Marteilia sydneyi, changes the bacterial community of the digestive gland of Sydney rock oysters, Saccostrea glomerata. Methods and Results: Six 16S rDNA clone libraries were established from three M. sydneyi‐infected and three un‐infected oysters. Restriction enzyme analysis followed by sequencing representative clones revealed a total of 23 different operational taxonomic units (OTUs) in un‐infected oysters, comprising the major phyla: Firmicutes, Proteobacteria, Cyanobacteria and Spirocheates, where the clone distribution was 44, 36, 7 and 5%, respectively. Close to half of the OTUs are not closely related to any other hitherto determined sequence. In contrast, S. glomerata infected by M. sydneyi had only one OTU present in the digestive gland. Phylogenetic analysis of the 16S rDNA sequence reveals that this dominant OTU, belonging to the α‐Proteobacteria, is closely related to a Rickettsiales‐like prokaryote (RLP). Conclusions: The microbiota of the digestive gland of Sydney rock oysters is changed by infection by M. sydneyi, becoming dominated by a RLP, and generally less diverse. The bacterial community of un‐infected S. glomerata differs from previous studies in that we identified the dominant taxa as Firmicutes and α‐Proteobacteria, rather than heterotrophic γ‐Proteobacteria. Significance and Impact of the Study: This is the first culture‐independent study of the microbiota of the digestive glands of edible oysters to the species level. The commercial viability of the Sydney rock oyster industry in Australia is currently threatened by Queensland Unknown disease and the changes in the bacterial community of S. glomerata corresponding with infection by M. sydneyi sheds further light on the link between parasite infection and mortality in this economically damaging disease.