Detection of WWE2-related Lentisphaerae by 16S rRNA gene sequencing and fluorescence in situ hybridization in landfill leachate

We collected samples of anaerobic landfill leachate from municipal solid waste landfill (Vert-le-Grand, France) and constructed 16S rRNA clone libraries using primers targeting Planctomycetes and relatives (Pla46F and 1390R). Analyses of 16S rRNA gene sequences resulted in the abundant representation of WWE2-related Lentisphaerae, members of the phylum Lentisphaerae, in the clone library (98% of the retrieved sequences). Although the sequences that are phylogenetically affiliated with the cultured isolate Victivallis vadensis were identified (WWE2 subgroup II), the majority of the sequences were affiliated with an uncultured Lentisphaerae lineage (WWE2 subgroup I). We designed oligonucleotides probes targeting the specific 16S rRNA gene regions of those 2 subgroups. Fluorescence in situ hybridization confirmed the abundance of the uncultivated WWE2 subgroup I in our leachate samples.     

A new black Aspergillus species, A. vadensis, is a promising host for homologous and heterologous protein production

A new species of the group of black aspergilli, Aspergillus vadensis, was analyzed for its potential as a host for homologous and heterologous protein production. Unlike the other black aspergilli, this strain does not acidify the culture medium when nitrate is the nitrogen source and only produces very low levels of extracellular proteases, mainly serine metalloproteases. The stability of A. tubingensis feruloyl esterase A (FaeA) was compared upon production in wild-type A. vadensis, A. tubingensis, and an A. niger strain in which the three main protease-encoding genes were disrupted. The production of FaeA in A. vadensis resulted in larger amounts of intact protein than production in A. tubingensis and was similar to production in an A. niger protease disruptant, confirming in vivo the low proteolytic activity of A. vadensis. The protoplast formation and transformation efficiencies of A. vadensis were much higher than those of A. niger. These characteristics make A. vadensis a very promising candidate for homologous, and possibly heterologous, protein production.     

Newly isolated but uncultivated magnetotactic bacterium of the phylum Nitrospirae from Beijing, China

Magnetotactic bacteria (MTB) in the phylum Nitrospirae synthesize up to hundreds of intracellular bullet-shaped magnetite magnetosomes. In the present study, a watermelon-shaped magnetotactic bacterium (designated MWB-1) from Lake Beihai in Beijing, China, was characterized. This uncultivated microbe was identified as a member of the phylum Nitrospirae and represents a novel phylogenetic lineage with ≥6% 16S rRNA gene sequence divergence from all currently described MTB. MWB-1 contained 200 to 300 intracellular bullet-shaped magnetite magnetosomes and showed a helical swimming trajectory under homogeneous magnetic fields; its magnetotactic velocity decreased with increasing field strength, and vice versa. A robust phylogenetic framework for MWB-1 and all currently known MTB in the phylum Nitrospirae was constructed utilizing maximum-likelihood and Bayesian algorithms, which yielded strong evidence that the Nitrospirae MTB could be divided into four well-supported groups. Considering its population densities in sediment and its high numbers of magnetosomes, MWB-1 was estimated to account for more than 10% of the natural remanent magnetization of the surface sediment. Taken together, the results of this study suggest that MTB in the phylum Nitrospirae are more diverse than previously realized and can make important contributions to the sedimentary magnetization in particular environments.     

Characterization of Melioribacter roseus gen. nov., sp. nov., a novel facultatively anaerobic thermophilic cellulolytic bacterium from the class Ignavibacteria,and a proposal of a novel bacterial phylum Ignavibacteriae

A novel moderately thermophilic, facultatively anaerobic chemoorganotrophic bacterium strain P3M‐2^T was isolated from a microbial mat developing on the wooden surface of a chute under the flow of hot water (46°C) coming out of a 2775‐m‐deep oil exploration well (Tomsk region, Russia). Strain P3M‐2^T is a moderate thermophile and facultative anaerobe growing on mono‐, di‐ or polysaccharides by aerobic respiration, fermentation or by reducing diverse electron acceptors [nitrite, Fe(III), As(V)]. Its closest cultivated relative (90.8% rRNA gene sequence identity) is Ignavibacterium album, the only chemoorganotrophic member of the phylum Chlorobi. New genus and species Melioribacter roseus are proposed for isolate P3M‐2^T. Together with I. album, the new organism represents the class Ignavibacteria assigned to the phylum Chlorobi. The revealed group includes a variety of uncultured environmental clones, the 16S rRNA gene sequences of some of which have been previously attributed to the candidate division ZB1. Phylogenetic analysis of M. roseus and I. album based on their 23S rRNA and RecA sequences confirmed that these two organisms could represent an even deeper, phylum‐level lineage. Hence, we propose a new phylum Ignavibacteriae within the Bacteroidetes–Chlorobi group with a sole class Ignavibacteria, two families Ignavibacteriaceae and Melioribacteraceae and two species I. album and M. roseus. This proposal correlates with chemotaxonomic data and phenotypic differences of both organisms from other cultured representatives of Chlorobi. The most essential differences, supported by the analyses of complete genomes of both organisms, are motility, facultatively anaerobic and obligately organotrophic mode of life, the absence of chlorosomes and the apparent inability to grow phototrophically.     

Genome sequence of the verrucomicrobium Opitutus terrae PB90-1, an abundant inhabitant of rice paddy soil ecosystems

Bacteria of the deeply branching phylum Verrucomicrobia are rarely cultured yet commonly detected in metagenomic libraries from aquatic, terrestrial, and intestinal environments. We have sequenced the genome of Opitutus terrae PB90-1, a fermentative anaerobe within this phylum, isolated from rice paddy soil and capable of propionate production from plant-derived polysaccharides.     

Genome sequence of "Pedosphaera parvula" Ellin514, an aerobic Verrucomicrobial isolate from pasture soil

"Pedosphaera parvula" Ellin514 is an aerobically grown verrucomicrobial isolate from pasture soil. It is one of the few cultured representatives of subdivision 3 of the phylum Verrucomicrobia. Members of this group are widespread in terrestrial environments.     

An assessment of actinobacterial diversity in the marine environment

The 16S rRNA gene sequence diversity within the Phylum Actinobacteria was assessed from four sources: PCR-generated V6 sequence tags derived from seawater samples, metagenomic data from the Global Ocean Sampling (GOS) expedition, marine-derived sequences maintained in the Ribosomal Database Project (RDP), and select cultured strains for which sequence data is not yet available in the RDP. This meta-analysis revealed remarkable levels of phylogenetic diversity and confirms the existence of major, deeply rooted, and as of yet uncharacterized lineages within the phylum. A dramatic incongruence among cultured strains and those detected using culture-independent techniques was also revealed. Redundancy among the actinobacteria detected using culture-independent techniques suggests that greater sequence coverage or improved DNA extraction efficiencies may be required to detect the rare phylotypes that can be readily cultured from marine samples. Conversely, new strategies need to be developed for the cultivation of frequently observed but yet to be cultured marine actinobacteria.     

Lentisphaera araneosa gen. nov., sp. nov, a transparent exopolymer producing marine bacterium, and the description of a novel bacterial phylum, Lentisphaerae

Two phylogenetically distinct marine strains producing transparent exopolymers (TEP), designated HTCC2155(T) and HTCC2160, were cultivated from Oregon coast seawater by dilution to extinction in a high throughput culturing format. When cultured in low-nutrient seawater media, these strains copiously produced Alcian Blue-stainable viscous TEP. Growing cells were attached to each other by the TEP in a three dimensional network. Polymerase chain reaction employing 16S rDNA primers specific for the novel isolates indicated that they are indigenous to the water column of the Atlantic and Pacific oceans. The abundance of the isolates as determined by 16S rRNA dot blots, however, indicated that they are less than 1% of the total bacterial community. In phylogenetic analyses, the strains consistently formed a new phylum-level lineage within the domain Bacteria, together with members of the candidate phylum VadinBE97, which consists of Victivallis, the first cultured genus in the candidate phylum, and 16S rRNA gene clones from DNA extracted from marine or anaerobic terrestrial habitats. Five putative subgroups were delineated within this phylum-level lineage, including a marine group and an anaerobic group. The isolates are Gram negative, strictly aerobic, chemoheterotrophic, and facultatively oligotrophic sphere-shaped bacteria. The DNA G+C content of strain HTCC2155(T) was 48.3 mol% and the genome size was 2.9 mb. It is proposed from these observations that the strains be placed into a new genus and a new species named Lentisphaera araneosa (type strain HTCC2155(T) = ATCC BAA-859(T) = KCTC 12141(T)) gen. nov., sp. nov., the cultured marine representative of the Lentisphaerae phyl. nov., and the phylum be divided into two novel orders named the Lentisphaerales ord. nov. and the Victivallales ord. nov.     

Refining the phylum Chlorobi by resolving the phylogeny and metabolic potential of the representative of a deeply branching,uncultivated lineage

Recent studies have expanded the phylum Chlorobi, demonstrating that the green sulfur bacteria (GSB), the original cultured representatives of the phylum, are a part of a broader lineage whose members have more diverse metabolic capabilities that overlap with members of the phylum Bacteroidetes. The 16S rRNA gene of an uncultivated clone, OPB56, distantly related to the phyla Chlorobi and Bacteroidetes, was recovered from Obsidian Pool in Yellowstone National Park; however, the detailed phylogeny and function of OPB56 and related clones have remained unknown. Culturing of thermophilic bacterial consortia from compost by adaptation to grow on ionic-liquid pretreated switchgrass provided a consortium in which one of the most abundant members, NICIL-2, clustered with OPB56-related clones. Phylogenetic analysis using the full-length 16S rRNA gene from NICIL-2 demonstrated that it was part of a monophyletic clade, referred to as OPB56, distinct from the Bacteroidetes and Chlorobi. A near complete draft genome (>95% complete) was recovered from metagenomic data from the culture adapted to grow on ionic-liquid pretreated switchgrass using an automated binning algorithm, and this genome was used for marker gene-based phylogenetic analysis and metabolic reconstruction. Six additional genomes related to NICIL-2 were reconstructed from metagenomic data sets obtained from thermal springs at Yellowstone National Park and Nevada Great Boiling Spring. In contrast to the 16S rRNA gene phylogenetic analysis, protein phylogenetic analysis was most consistent with the clustering of the Chlorobea, Ignavibacteria and OPB56 into a single phylum level clade. Metabolic reconstruction of NICIL-2 demonstrated a close linkage with the class Ignavibacteria and the family Rhodothermaceae, a deeply branching Bacteroidetes lineage. The combined phylogenetic and functional analysis of the NICIL-2 genome has refined the membership in the phylum Chlorobi and emphasized the close evolutionary and metabolic relationship between the phyla Chlorobi and the Bacteroidetes.     

四川冬菜中细菌群落组成及多样性

【目的】了解腌制4年的四川南充冬菜中细菌群落组成及多样性。【方法】通过16S rDNA多样性分析样品细菌落组成;采用16S rDNA-RFLP方法分析从样品中分离出的纯培养细菌。【结果】16S rDNA多样性分析结果表明,样品中细菌主要属于变形杆菌门(Proteobacteria)和厚壁菌门(Firmicutes),分别占克隆文库的87.9%、7.1%,其中包括Virgibacillus kekensis,Marinococcus albus,Salinicoccus sp.,Lactobacillus halophilus和Halomonas等中度嗜盐菌,仅有5%属于放线菌门(Actinobacteria)。通过纯培养方法从冬菜中分离到35株菌,16S rDNA-RFLP分析结果表明,34株属于厚壁菌门(Firmicutes),包括Virgibacillus,Bacillus megaterium和Gracilibacillus saliphilus等中度嗜盐菌,1株属于放线菌门(Actinobacteria)。【结论】冬菜中细菌群落多样性较低,以中度嗜盐菌为主。     

Arginine kinase from the Tardigrade, Macrobiotus occidentalis: molecular cloning, phylogenetic analysis and enzymatic properties

Arginine kinase (AK), which catalyzes the reversible transfer of phosphate from ATP to arginine to yield phosphoarginine and ADP, is widely distributed throughout the invertebrates. We determined the cDNA sequence of AK from the tardigrade (water bear) Macrobiotus occidentalis, cloned the sequence into pET30b plasmid, and expressed it in Escherichia coli as a 6x His-tag—fused protein. The cDNA is 1377 bp, has an open reading frame of 1080 bp, and has 5′- and 3′-untranslated regions of 116 and 297 bp, respectively. The open reading frame encodes a 359-amino acid protein containing the 12 residues considered necessary for substrate binding in Limulus AK. This is the first AK sequence from a tardigrade. From fragmented and non-annotated sequences available from DNA databases, we assembled 46 complete AK sequences: 26 from arthropods (including 19 from Insecta), 11 from nematodes, 4 from mollusks, 2 from cnidarians and 2 from onychophorans. No onychophoran sequences have been reported previously. The phylogenetic trees of 104 AKs indicated clearly that Macrobiotus AK (from the phylum Tardigrada) shows close affinity with Epiperipatus and Euperipatoides AKs (from the phylum Onychophora), and therefore forms a sister group with the arthropod AKs. Recombinant 6x His-tagged Macrobiotus AK was successfully expressed as a soluble protein, and the kinetic constants (K(m), K(d), V(ma) and k(cat)) were determined for the forward reaction. Comparison of these kinetic constants with those of AKs from other sources (arthropods, mollusks and nematodes) indicated that Macrobiotus AK is unique in that it has the highest values for k(cat) and K(d)K(m) (indicative of synergistic substrate binding) of all characterized AKs.     

Members of the Candidate Division OP10 are spread in a variety of environments

Our planet holds a huge bacterial diversity. Most of these bacteria have only been detected by their 16S rRNA gene sequences remaining to be cultured. Many are classified within Candidate Divisions. One them is the Candidate Division OP10. Analysis of environmental 16S rRNA gene sequences available in public repositories revealed the existence of numerous sequences clustering within the Candidate Division OP10 but currently unclassified or assigned to other bacterial phyla. Newly proposed 16S rRNA sequences multiply several fold the reported sequences for the Candidate Division OP10. This study showed that the Candidate Division OP10 is a diverse and broadly distributed bacterial phylum and represents a stable microbial component in different natural environments.     

High-throughput sequencing-based analysis of bacterial communities associated with Barbour's seahorses (Hippocampus barbouri) from Surigao del Norte,Philippines

This study identified the bacterial community composition associated with Barbour's seahorses (Hippocampus barbouri). Seahorses and samples from the surrounding environment (sediment and water) were collected from Cantiasay Island in Surigao del Norte, Philippines. Genomic DNA was isolated from these samples, and the V1–V3 region of the 16S rRNA gene was amplified and sequenced on the Illumina MiSeq platform. There were 929 803 sequence reads corresponding to 859 operational taxonomic units (OTUs), which were obtained from the six genomic libraries. The results showed that the most abundant OTUs were affiliated to the phylum Proteobacteria, particularly those belonging to the genera Shewanella, Pseudomonas, Acinetobacter and Aeromonas, which were found to be dominant in seahorse gut samples. The only exception was for the skin of male seahorses, which was dominated by members belonging to the genus Bacillus (phylum Firmicutes). These findings on bacterial community structure and composition may shed light on therapeutic claims of Barbour's seahorses and help in the conservation of this species.     

Correction: Phylogenetic placement of the enigmatic parasite, Polypodium hydriforme, within the Phylum Cnidaria

Correction to Evans, N.M., Lindner, A., Raikova, E.V., Collins, A.G. and Cartwright, P. Phylogenetic placement of the enigmatic parasite, Polypodium hydriforme, within the phylum Cnidaria. BMC Evol Biol, 2008, 8:139.     

Candidate OP Phyla: Importance,Ecology and Cultivation Prospects

OP phyla were created in the domain bacteria, based on the group of 16S rRNA gene sequences recovered from the Obsidian Pool. However, due to the lack of cultured representative it is referred to as candidate phyla. Wider ecological occurrence was predicted for the OP phyla, especially OP3, OP10 and OP11. Recently, members of phylum OP5 and OP10 were cultured, providing clues to their cultivation prospects. At last the bioprospecting potentials of the OP members are discussed herein.     

Structural and phylogenetic analysis of a conserved actinobacteria-specific protein (ASP1; SCO1997) from Streptomyces coelicolor

Background  

The Actinobacteria phylum represents one of the largest and most diverse groups of bacteria, encompassing many important and well-characterized organisms including Streptomyces, Bifidobacterium, Corynebacterium and Mycobacterium. Members of this phylum are remarkably diverse in terms of life cycle, morphology, physiology and ecology. Recent comparative genomic analysis of 19 actinobacterial species determined that only 5 genes of unknown function uniquely define this large phylum [1]. The cellular functions of these actinobacteria-specific proteins (ASP) are not known.     

Laboratory cultivation of widespread and previously uncultured soil bacteria

Most soil bacteria belong to family-level phylogenetic groups with few or no known cultivated representatives. We cultured a collection of 350 isolates from soil by using simple solid media in petri dishes. These isolates were assigned to 60 family-level groupings in nine bacterial phyla on the basis of a comparative analysis of their 16S rRNA genes. Ninety-three (27%) of the isolates belonged to 20 as-yet-unnamed family-level groupings, many from poorly studied bacterial classes and phyla. They included members of subdivisions 1, 2, 3, and 4 of the phylum Acidobacteria, subdivision 3 of the phylum Verrucomicrobia, subdivision 1 of the phylum Gemmatimonadetes, and subclasses Acidimicrobidae and Rubrobacteridae of the phylum ACTINOBACTERIA: In addition, members of 10 new family-level groupings of subclass Actinobacteridae of the phylum Actinobacteria and classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria of the phylum Proteobacteria were obtained. The high degree of phylogenetic novelty and the number of isolates affiliated with so-called unculturable groups show that simple cultivation methods can still be developed further to obtain laboratory cultures of many phylogenetically novel soil bacteria.     

Thermal resistance, developmental rate and heat shock proteins in Artemia franciscana, from San Francisco Bay and southern Vietnam

Cysts (encysted gastrula embryos) of Artemia franciscana collected from salterns in San Francisco Bay, California, USA (SF) were inoculated into much warmer growth ponds in the Mekong Delta region of Vietnam (V) in 1996. V adults arising directly from these cysts during 17 April to 15 May produced their own cysts, which were collected, processed and stored until shipped to the USA for study. Adults grown in the laboratory from SF cysts (those used for the inoculation) were less resistant to high temperature than adults cultured from V cysts. V cysts produced heat-resistant adults, even though cultured under the same laboratory conditions as SF animals, at much lower temperatures than they ever experienced in Vietnam. Differences in thermal performance between SF and V adults were retained in the second generation, cultured from cysts produced in the laboratory by first generation adults, suggesting a genetic basis for the better heat resistance of V adults. We propose that the operation of natural selection in the Vietnam growth ponds produced adults with improved thermal tolerance, and that the basis for this tolerance was incorporated into the developmental program of their cysts. Surprisingly, differences in heat resistance of laboratory reared animals were not reflected in constitutive levels of the hsp70 family which were similar in first generation SF and V adults. A conditioning heat shock (HS, 37 degrees C, 30 min) led to the same level of induced thermotolerance in SF and V first generation adults when evaluated 24 h post-HS. Levels of hsp70 were also up-regulated at that time, but to about the same extent in SF and V adults. Developmental rates of SF cysts used for the inoculation were faster than those of cysts produced in Vietnam when both were incubated at 21+/-1 degrees C, suggesting that V cysts have become adapted to develop at higher temperatures.     

Acidobacteria, Rubrobacteridae and Chloroflexi are abundant among very slow-growing and mini-colony-forming soil bacteria

Easily visible colonies of bacteria continued to form on plates inoculated with soil and incubated for 24 weeks. Using two different media, 13% and 29% of easily visible colonies appeared after more than 12 weeks. In addition, 10% and 18% of all colonies had diameters of 25-200 μm (mini-colonies), which could not be readily seen with the unaided eye. Members of soil bacterial groups that are only rarely cultured, such as members of the subclass Rubrobacteridae of the phylum Actinobacteria, members of subdivisions 1 and 2 of the phylum Acidobacteria and members of three subphyla of the phylum Chloroflexi, were more abundant among the easily visible colonies and mini-colonies that developed after > 12 weeks of incubation. Our results indicate that there is a hidden culturable diversity of soil bacteria that may require laboratory study at colony sizes and incubation periods outside those commonly anticipated by most microbiologists. Working at these scales increases the likelihood of obtaining cultures from groups of soil bacteria that have generally eluded laboratory study by cultivation methods.     

Phylogenetic Profiling and Diversity of Bacterial Communities in the Death Valley,an Extreme Habitat in the Atacama Desert

The Atacama Desert, one of the driest deserts in the world, represents a unique extreme environmental ecosystem to explore the bacterial diversity as it is considered to be at the dry limit for life. A 16S rRNA gene (spanning the hyper variable V3 region) library was constructed from an alkaline sample of unvegetated soil at the hyperarid margin in the Atacama Desert. A total of 244 clone sequences were used for MOTHUR analysis, which revealed 20 unique phylotypes or operational taxonomic units (OTUs). V3 region amplicons of the 16S rRNA were suitable for distinguishing the bacterial community to the genus and specie level. We found that all OTUs were affiliated with taxa representative of the Firmicutes phylum. The extremely high abundance of Firmicutes indicated that most bacteria in the soil were spore-forming survivors. In this study we detected a narrower diversity as compared to other ecological studies performed in other areas of the Atacama Desert. The reported genera were Oceanobacillus (representing the 69.5 % of the clones sequenced), Bacillus, Thalassobacillus and Virgibacillus. The present work shows physical and chemical parameters have a prominent impact on the microbial community structure. It constitutes an example of the communities adapted to live in extreme conditions caused by dryness and metal concentrations .

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0539-3) contains supplementary material, which is available to authorized users.