Bacterial diversity associated with the tunic of the model chordate Ciona intestinalis

The sea squirt Ciona intestinalis is a well-studied model organism in developmental biology, yet little is known about its associated bacterial community. In this study, a combination of 454 pyrosequencing of 16S ribosomal RNA genes, catalyzed reporter deposition-fluorescence in situ hybridization and bacterial culture were used to characterize the bacteria living inside and on the exterior coating, or tunic, of C. intestinalis adults. The 454 sequencing data set demonstrated that the tunic bacterial community structure is different from that of the surrounding seawater. The observed tunic bacterial consortium contained a shared community of <10 abundant bacterial phylotypes across three individuals. Culture experiments yielded four bacterial strains that were also dominant groups in the 454 sequencing data set, including novel representatives of the classes Alphaproteobacteria and Flavobacteria. The relatively simple bacterial community and availability of dominant community members in culture make C. intestinalis a promising system in which to investigate functional interactions between host-associated microbiota and the development of host innate immunity.     

Genetic diversity of Acacia tortilis ssp. raddiana rhizobia in Tunisia assessed by 16S and 16S-23S rDNA genes analysis

AIMS: In order to understand the genetic diversity of Acacia tortilis ssp. raddiana-rhizobia in Tunisia, isolates from nine geographical locations were obtained and analysed. METHODS AND RESULTS: Characterization using restriction fragment length polymorphism analysis (RFLP) of PCR-amplified 16S rRNA gene and the intergenic spacer (IGS) between the 16S and 23S rRNA genes was undertaken. Symbiotic efficiency of the strains was also estimated. Analysis of the 16S rRNA by PCR-RFLP showed that the isolates were phylogenetically related to Ensifer ssp., Rhizobium tropicii-IIA, and Rhizobium tumefaciens species. Analysis of 16S-23S spacer by PCR-RFLP showed a high diversity of these rhizobia and revealed eleven additional groups, which indicates that these strains are genetically very diverse. Full 16S rRNA gene-sequencing showed that the majority of strains form a new subdivion inside the genera Ensifer, with Ensifer meliloti being its nearest neighbour. Nodulation test performed on the plant host demonstrated differences in the infectivity among the strains. CONCLUSION: Rhizobial populations that nodulate specifically and efficiently Acacia tortilis ssp. raddiana in representative soils of Tunisia is dominated by E. meliloti-like genomospecies. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides the first clear characterization and symbiotic efficiency data of rhizobia strains nodulating A. tortilis in Tunisia.     

Novel diversity within Roseofilum (Desertifilaceae,Cyanobacteria) from marine benthic mats with description of four new species

Benthic cyanobacterial mats (BCMs) are natural phenomena in marine environments. Reports of BCMs occurring across coastal marine environments have increased, partly driven by nutrient loading and climate change; thus, there is a need to understand the diversity involved in the proliferations and potential toxicity of the BCMs. Furthermore, marine cyanobacterial mats are observed growing on and affecting the health of corals with one specific cyanobacterial genus, Roseofilum, dominating the microbial mats associated with black band disease (BBD), a destructive polymicrobial disease that affects corals. To explore the diversity of Roseofilum, cyanobacterial mats from various marine habitats were sampled, and individual isolates were identified based on morphology, 16S rRNA gene phylogenies, 16S–23S ITS rRNA region sequence dissimilarities, and phylogenomics. Four novel species of Roseofilum were isolated from benthic marine mats, three from the coasts of Florida, United States (R. capinflatum sp. nov., R. casamattae sp. nov., and R. acuticapitatum sp. nov.) and one from the coast of France (R. halophilum sp. nov.). Our analyses revealed that Roseofilum associated with coral BBD and those not associated with corals but rather from coastal benthic mats are systematically distinct based on both phylogenetic and phylogenomic analyses. Enzyme-linked immunosorbent assay (ELISA) and LC–MS data indicated that microcystin production was found in one of the four species.     

Integrating metagenomic and amplicon databases to resolve the phylogenetic and ecological diversity of the Chlamydiae

In the era of metagenomics and amplicon sequencing, comprehensive analyses of available sequence data remain a challenge. Here we describe an approach exploiting metagenomic and amplicon data sets from public databases to elucidate phylogenetic diversity of defined microbial taxa. We investigated the phylum Chlamydiae whose known members are obligate intracellular bacteria that represent important pathogens of humans and animals, as well as symbionts of protists. Despite their medical relevance, our knowledge about chlamydial diversity is still scarce. Most of the nine known families are represented by only a few isolates, while previous clone library-based surveys suggested the existence of yet uncharacterized members of this phylum. Here we identified more than 22 000 high quality, non-redundant chlamydial 16S rRNA gene sequences in diverse databases, as well as 1900 putative chlamydial protein-encoding genes. Even when applying the most conservative approach, clustering of chlamydial 16S rRNA gene sequences into operational taxonomic units revealed an unexpectedly high species, genus and family-level diversity within the Chlamydiae, including 181 putative families. These in silico findings were verified experimentally in one Antarctic sample, which contained a high diversity of novel Chlamydiae. In our analysis, the Rhabdochlamydiaceae, whose known members infect arthropods, represents the most diverse and species-rich chlamydial family, followed by the protist-associated Parachlamydiaceae, and a putative new family (PCF8) with unknown host specificity. Available information on the origin of metagenomic samples indicated that marine environments contain the majority of the newly discovered chlamydial lineages, highlighting this environment as an important chlamydial reservoir.     

Feedbacks of plant identity and diversity on the diversity and community composition of rhizosphere microbiomes from a long‐term biodiversity experiment

Soil microbes are known to be key drivers of several essential ecosystem processes such as nutrient cycling, plant productivity and the maintenance of plant species diversity. However, how plant species diversity and identity affect soil microbial diversity and community composition in the rhizosphere is largely unknown. We tested whether, over the course of 11 years, distinct soil bacterial communities developed under plant monocultures and mixtures, and if over this time frame plants with a monoculture or mixture history changed in the bacterial communities they associated with. For eight species, we grew offspring of plants that had been grown for 11 years in the same field monocultures or mixtures (plant history in monoculture vs. mixture) in pots inoculated with microbes extracted from the field monoculture and mixture soils attached to the roots of the host plants (soil legacy). After 5 months of growth in the glasshouse, we collected rhizosphere soil from each plant and used 16S rRNA gene sequencing to determine the community composition and diversity of the bacterial communities. Bacterial community structure in the plant rhizosphere was primarily determined by soil legacy and by plant species identity, but not by plant history. In seven of the eight plant species the number of individual operational taxonomic units with increased abundance was larger when inoculated with microbes from mixture soil. We conclude that plant species richness can affect below‐ground community composition and diversity, feeding back to the assemblage of rhizosphere bacterial communities in newly establishing plants via the legacy in soil.     

Comparison of prokaryotic diversity at offshore oceanic locations reveals a different microbiota in the Mediterranean Sea

The bacterial and archaeal assemblages at two offshore sites located in polar (Greenland Sea; depth: 50 and 2000 m) and Mediterranean (Ionian Sea; depth 50 and 3000 m) waters were studied by PCR amplification and sequencing of the last 450-500 bp of the 16S rRNA gene. A total of 1621 sequences, together with alignable 16S rRNA gene fragments from the Sargasso Sea metagenome database, were analysed to ascertain variations associated with geographical location and depth. The Ionian 50 m sample appeared to be the most diverse and also had remarkable differences in terms of the prokaryotic groups retrieved; surprisingly, however, many similarities were found at the level of large-scale diversity between the Sargasso database fragments and the Greenland 50 m sample. Most sequences with more than 97% sequence similarity, a value often taken as indicative of species delimitation, were only found at a single location/depth; nevertheless, a few examples of cosmopolitan sequences were found in all samples. Depth was also an important factor and, although both deep-water samples had overall similarities, there were important differences that could be due to the warmer waters at depth of the Mediterranean Sea.     

DNA条形码技术在海洋贝类鉴定中的实践: 以大亚湾生态监控区为例

为提高物种鉴定的准确性, 本研究采用DNA条形码技术对大亚湾生态监控区冬季采集的贝类样品进行了种类鉴定。结果表明, 26个形态种中, 有15个可以通过线粒体COI和16S rRNA基因的系统发育分析鉴定到种的水平。部分形态上难以鉴定的种类, 如线缝摺塔螺(Ptychobela suturalis)和区系螺(Funa sp.)可以通过条形码实现有效鉴定。锯齿巴非蛤(Paphia gallus)、西格织纹螺(Nassarius siquijorensis)、爪哇拟塔螺(Turricula javana)等种类存在相当大的种内遗传距离, 有存在隐存种的可能性。尽管基于线粒体COI和16S rRNA基因的种内遗传距离和属内种间的遗传距离发生重合, 无明显的条形码间隙, 但通过系统树的方法仍能有效鉴定物种。可见, DNA条形码技术能有效提高海洋贝类物种鉴定的准确性并发现隐存种。     

Ascaris suum infection was associated with a worm-independent reduction in microbial diversity and altered metabolic potential in the porcine gut microbiome

The effect of infection of pigs with Ascaris suum on the microbial composition in the proximal colon and fecal matter was investigated using 16S rRNA gene sequencing. The infection significantly decreased various microbial diversity indices including Chao1 richness, but the effect on Chao1 in the colon luminal contents was worm burden-independent. The abundance of 49 genera present in colon contents, such as Prevotella and Faecalibacterium, and 179 operational taxonomic units was significantly changed as a result of infection. Notably, infection was also associated with a significant shift in the metabolic potential of the proximal colon microbiome, where the relative abundance of at least 30 metabolic pathways including carbohydrate metabolism and amino acid metabolism was reduced, while the abundance of 28 pathways was increased by infection. Furthermore, the microbial co-occurrence network in infected pigs was highly modular. Two of 52 modules or subnetworks were negatively correlated with fecal butyrate concentrations (r?<??0.7; P?<?0.05) while one module with 18 members was negatively correlated with fecal acetate, propionate and total short-chain fatty acids. A partial Mantel test identified a strong positive correlation between node connectivity of the operational taxonomic units assigned to β-Proteobacteria (especially the family Alcaligenaceae) and fecal acetate and propionate levels (r?=?0.82 and 0.74, respectively), while that of the family Porphyromonadaceae was positively correlated with fecal egg counts. Overall, Ascaris infection was associated with a profound change in the gut microbiome, especially in the proximity of the initial site of larval infection, and should facilitate our understanding of the pathophysiological consequence of gastrointestinal nematode infections.     

Influence of petroleum contamination and biostimulation treatment on the diversity of Pseudomonas spp. in soil microcosms as evaluated by 16S rRNA based-PCR and DGGE

AIMS: The aim of this study was to apply a group specific PCR system followed by denaturing gradient gel electrophoresis (DGGE) analysis to evaluate the effect of oil contamination and the biostimulation process on the diversity of Pseudomonas populations in soil ecosystems. METHODS AND RESULTS: Direct DNA extraction from biostimulated- and oil-contaminated soil samples was performed. Primers specific for the genus Pseudomonas spp. were used to amplify 16S rRNA genes and then a semi-nested PCR reaction was applied to obtain smaller fragments for comparing the PCR products by DGGE. Whether in bulk, oil-contaminated or biostimulated soils, the DGGE profiles revealed little change in Pseudomonas community throughout the 270 days of experiment. The presence of a few additional bands observed only in treated samples indicated that a bacterial shift occurred with the addition of nutrients and with oil contamination. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of semi-nested PCR and DGGE was found to be a rapid and sensitive technique to study the diversity within the genus Pseudomonas and may be suitable for further studies concerning the role of this bacterial group in large-scale oil-contaminated areas.     

Molecular phylogenetic diversity of the microbial community associated with a high-temperature petroleum reservoir at an offshore oilfield

The microbial community and its diversity in production water from a high-temperature, water-flooded petroleum reservoir of an offshore oilfield in China were characterized by 16S rRNA gene sequence analysis. The bacterial and archaeal 16S rRNA gene clone libraries were constructed from the community DNA and, using sequence analysis, 388 bacterial and 220 archaeal randomly selected clones were clustered with 60 and 28 phylotypes, respectively. The results showed that the 16S rRNA genes of bacterial clones belonged to the divisions Firmicutes, Thermotogae, Nitrospirae and Proteobacteria, whereas the archaeal library was dominated by methanogen-like rRNA genes (Methanothermobacter, Methanobacter, Methanobrevibacter and Methanococcus), with a lower percentage of clones belonging to Thermoprotei. Thermophilic microorganisms were found in the production water, as well as mesophilic microorganisms such as Pseudomonas and Acinetobacter-like clones. The thermophilic microorganisms may be common inhabitants of geothermally heated specialized subsurface environments, which have been isolated previously from a number of high-temperature petroleum reservoirs worldwide. The mesophilic microorganisms were probably introduced into the reservoir as it was being exploited. The results of this work provide further insight into the composition of microbial communities of high-temperature petroleum reservoirs at offshore oilfields.     

Population structure and the impact of regional and local habitat isolation upon levels of genetic diversity of the endangered damselfly Coenagrion mercuriale (Odonata: Zygoptera)

1. Coenagrion mercuriale is one of Europe's most threatened damselflies. There is concern for the long‐term persistence of many of its U.K. colonies because adult lifetime movement is limited, making isolated populations susceptible to extinction. 2. Using 14 microsatellite loci we characterised levels of genetic diversity, evidence for a recent decline and the spatial genetic structure for C. mercuriale population in Wales, U.K. 3. Spatial isolation is not an absolute predictor of low genetic diversity at either local or regional scales. 4. One population inhabiting a remote, edge of range site is genetically impoverished with levels of variability (at microsatellite loci) among the lowest reported for any insect species. 5. Agricultural land and high ground are physical barriers to dispersal by adults. 6. Consistent with work from elsewhere, movement by mature C. mercuriale in Pembrokeshire is sufficient to prevent significant genetic differentiation throughout a habitat matrix of some 3–4 km if the suitable habitat sites are <2 km apart and lack barriers to movement. Even within a good habitat matrix, however, genetic isolation by distance develops within 10 km.     

Increased genetic diversity in mitochondrial genes is correlated with the evolution of parasitism in the Hymenoptera

A higher AT content and rate of mtDNA sequence divergence was found in parasitic wasps (Apocrita) compared with nonparasitic wasps (Symphyta). The compositional bias was reflected in extreme codon bias for a cytochrome oxidase I protein coding gene fragment as well as in the types of amino acid substitutions that have occurred during the evolution of this gene fragment. In some instances, compositional bias influenced the definition of a conservative amino acid change. The increased rate of mtDNA sequence evolution probably arose during the early Jurassic, coincident with the first appearance of parasitic wasps in the fossil record. Our results suggest a causal link between the rate of sequence divergence and the parasitic lifestyle.Abbreviations AT adenosine-thymine - CO-1 cytochrome oxidase 1 - mtDNA mitochondrial DNA - Myr million years Correspondence to: M. Dowton     

转几丁质酶和葡聚糖酶双价基因棉花对土壤细菌种群多样性的影响

以转几丁质酶和葡聚糖酶双价基因棉花为研究对象,非转基因受体棉花为对照,通过比较可培养细菌数量和基于16S rRNA克隆文库细菌种群分析,评价外源双价基因的导入在苗期、蕾期、花铃期和吐絮期对棉花根际细菌群落多样性的影响。结果表明,可培养细菌的数量不受外源双价基因的影响,随着棉花生育期的交替而变化,以代谢旺盛的花铃期最多。构建的转基因和非转基因不同生育期根际土壤细菌16S rRNA文库容量为2400个克隆,涵盖了细菌的283个属。其中,Acidobacterium是最大优势类群,共包括624个克隆,其次为未知细菌种群和Flavisolibacter。比较转基因和非转基因棉花根际土壤细菌的种群结构,结果显示,同一生育期内前者种群的多样性显著低于后者,二者的共有类群随着生长发育的进行而增多。研究结果说明几丁质酶基因和葡聚糖酶基因对棉花根际细菌种群多样性有着不同程度的削减作用,但是随着种植时间的延长,该差异呈现逐渐缩小的趋势。     

Phylogenetic diversity of sediment bacteria from the southern Cretan margin,Eastern Mediterranean Sea

This study is the first culture-independent report on the regional variability of bacterial diversity in oxic sediments from the unexplored southern Cretan margin (SCM). Three main deep basins (water column depths: 2670–3603 m), located at the mouth of two submarine canyons (Samaria Gorge and Paximades Channel) and an adjacent slope system, as well as two shallow upper-slope stations (water column depths: 215 and 520 m), were sampled. A total of 454 clones were sequenced and the bacterial richness, estimated through five clone libraries using rarefaction analysis, ranged from 71 to 296 unique phylotypes. The average sequence identity of the retrieved Cretan margin sequences compared to the >1,000,000 known rRNA sequences was only 93.5%. A diverse range of prokaryotes was found in the sediments, which were represented by 15 different taxonomic groups at the phylum level. The phylogenetic analysis revealed that these new sequences grouped with the phyla Acidobacteria, Planctomycetes, Actinobacteria, Gamma-, Alpha- and Delta-proteobacteria. Only a few bacterial clones were affiliated with Chloroflexi, Bacteroidetes, Firmicutes, Gemmatimonadetes, Verrucomicrobia, Nitrospirae, Beta-proteobacteria, Lentisphaerae and Dictyoglomi. A large fraction of the retrieved sequences (12%) did not fall into any taxonomic division previously characterized by molecular criteria, whereas four novel division-level lineages, termed candidate division SCMs, were identified. Bacterial community composition demonstrated significant differences in comparison to previous phylogenetic studies. This divergence was mainly triggered by the dominance of Acidobacteria and Actinobacteria and reflected a bacterial community different from that currently known for oxic and pristine marine sediments.     

Molecular bacterial diversity of a forest soil under residue management regimes in subtropical Australia

A major operational change in exotic pine plantations of subtropical Australia has been the decision to retain postharvest residues on site. A long-term field experiment was established in February 1996 to examine the impacts of residue management regimes [i.e. the postharvest residues removed (G0R), natural amount of residues retained (G1R) and residue quantity doubled and retained (G2R)] on tree growth (F1 hybrid pine) and sustainable soil management. Twelve soil samples, which included the above three residue regimes with four replicates, were collected at plantation age 6.4 years. A 16S rRNA gene clone library was established following soil community DNA extraction, polymerase chain reaction amplification and cloning. A total of 324 clones, including 27 from each sample, were randomly selected and sequenced to represent the bacterial composition and diversity of the clone library and thus the soil bacterial community under the residue management regimes. Phylogenetic analyses indicated that Acidobacteria (37.6%) and Proteobacteria (35.6%) were the dominant components of the soil bacterial community, followed by Actinobacteria (14.7%), Chlamydiae/Verrucomicrobia (7.3%), Unclassified Bacteria (3.8%) and Gemmatimonadetes (1.0%). Analysis of molecular variance revealed that there was no significant difference in bacterial composition and diversity among the residue management regimes or their replicated samples.     

Assessing the genetic diversity and population structure of Culter alburnus in China based on mitochondrial 16S rRNA and COI gene sequences

The genetic diversity and population genetic structure of Culter alburnus were investigated using mitochondrial cytochrome c oxidase subunit I (COI) and ribosomal 16S subunit (16S rRNA) gene sequences. A total of 89 individuals from four localities were included in the analysis. Overall, 12 polymorphic sites were observed and 10 haplotypes were defined. The C. alburnus populations were characterized by high haplotype diversity (0.587 ± 0.047) and low nucleotide diversity (0.00197 ± 0.00073). Pairwise fixation index (F_ST) analysis indicated significant genetic differentiation among different populations. The hierarchical analysis of molecular variance (AMOVA) analysis also showed significant genetic divergence (φ_ST = 0.3792, P < 0.01) among these populations. The present results suggest that subdivisions exist among four C. alburnus populations, and should be considered as different management unit for effective conservation and management purposes. This study provides new information for genetic assessment and will be crucial for establishing fisheries management and strategies for this species.     

Comparative 16S rDNA sequence analysis of some alkaliphilic bacilli and the establishment of a sixth rRNA group within the genus Bacillus

Abstract Comparative sequence analysis of the 16S rDNA of 14 alkaliphilic or alkalitolerant, Gram-positive, aerobic, endo-spore forming bacterial strains was performed. Bacillus alcalophilus DSM 485^T and Bacillus cohnii DSM 6307^T were included to represent the two validly described alkaliphiles assigned to the genus Bacillus . The majority of isolates (8 strains) clustered with B. alcalophilus DSM 485^T forming a distinct phylogenetic group (rRNA group 6) within the radiation of the genus Bacillus and related taxa. Bacillus cohnii DSM 6307^T and two of the isolates, DSM 8719 and DSM 8723, grouped with B. fastidiosus and B. megaterium and are allocated to rRNA group 1. The remaining two strains DSM 8720 and DSM 8721 show an equidistant relationship to both groups.