Project description:Magnetotactic bacteria comprise a phylogenetically diverse group that is capable of synthesizing intracellular magnetic particles. Although various morphotypes of magnetotactic bacteria have been observed in the environment, bacterial strains available in pure culture are currently limited to a few genera due to difficulties in their enrichment and cultivation. In order to obtain genetic information from uncultured magnetotactic bacteria, a genome preparation method that involves magnetic separation of cells, flow cytometry, and multiple displacement amplification (MDA) using phi29 polymerase was used in this study. The conditions for the MDA reaction using samples containing 1 to 100 cells were evaluated using a pure-culture magnetotactic bacterium, "Magnetospirillum magneticum AMB-1," whose complete genome sequence is available. Uniform gene amplification was confirmed by quantitative PCR (Q-PCR) when 100 cells were used as a template. This method was then applied for genome preparation of uncultured magnetotactic bacteria from complex bacterial communities in an aquatic environment. A sample containing 100 cells of the uncultured magnetotactic coccus was prepared by magnetic cell separation and flow cytometry and used as an MDA template. 16S rRNA sequence analysis of the MDA product from these 100 cells revealed that the amplified genomic DNA was from a single species of magnetotactic bacterium that was phylogenetically affiliated with magnetotactic cocci in the Alphaproteobacteria. The combined use of magnetic separation, flow cytometry, and MDA provides a new strategy to access individual genetic information from magnetotactic bacteria in environmental samples.
Project description:We investigated the in situ gene expression profile of sulfur-turf microbial mats dominated by an uncultured large sausage-shaped Aquificae bacterium, a key metabolic player in sulfur-turfs in sulfidic hot springs. A reverse transcription-PCR analysis revealed that the genes responsible for sulfide, sulfite, and thiosulfate oxidation and carbon fixation via the reductive TCA cycle were continuously expressed in sulfur-turf mats taken at different sampling points, seasons, and years. These results suggest that the uncultured large sausage-shaped bacterium has the ability to grow chemolithoautotrophically and plays key roles as a primary producer in the sulfidic hot spring ecosystem in situ.
Project description:Advances in single-cell and mini-metagenome sequencing have enabled important investigations into uncultured bacteria. In this study, we applied the mini-metagenome sequencing method to assemble genome drafts of the uncultured causative agents of epitheliocystis, an emerging infectious disease in the Mediterranean aquaculture species gilthead seabream. We sequenced multiple cyst samples and constructed 11 genome drafts from a novel beta-proteobacterial lineage, Candidatus Ichthyocystis. The draft genomes demonstrate features typical of pathogenic bacteria with an obligate intracellular lifestyle: a reduced genome of up to 2.6 Mb, reduced G + C content, and reduced metabolic capacity. Reconstruction of metabolic pathways reveals that Ca Ichthyocystis genomes lack all amino acid synthesis pathways, compelling them to scavenge from the fish host. All genomes encode type II, III, and IV secretion systems, a large repertoire of predicted effectors, and a type IV pilus. These are all considered to be virulence factors, required for adherence, invasion, and host manipulation. However, no evidence of lipopolysaccharide synthesis could be found. Beyond the core functions shared within the genus, alignments showed distinction into different species, characterized by alternative large gene families. These comprise up to a third of each genome, appear to have arisen through duplication and diversification, encode many effector proteins, and are seemingly critical for virulence. Thus, Ca Ichthyocystis represents a novel obligatory intracellular pathogenic beta-proteobacterial lineage. The methods used: mini-metagenome analysis and manual annotation, have generated important insights into the lifestyle and evolution of the novel, uncultured pathogens, elucidating many putative virulence factors including an unprecedented array of novel gene families.
Project description:Bacterial vaginosis-associated bacterium 1 (BVAB1) is an as-yet uncultured bacterial species found in the human vagina that belongs to the family Lachnospiraceae within the order Clostridiales. As its name suggests, this bacterium is often associated with bacterial vaginosis (BV), a common vaginal disorder that has been shown to increase a woman's risk for HIV, Chlamydia trachomatis, and Neisseria gonorrhoeae infections as well as preterm birth. BVAB1 has been further associated with the persistence of BV following metronidazole treatment, increased vaginal inflammation, and adverse obstetrics outcomes. There is no available complete genome sequence of BVAB1, which has made it difficult to mechanistically understand its role in disease. We present here a circularized metagenome-assembled genome (cMAG) of BVAB1 as well as a comparative analysis including an additional six metagenome-assembled genomes (MAGs) of this species. These sequences were derived from cervicovaginal samples of seven separate women. The cMAG was obtained from a metagenome sequenced with long-read technology on a PacBio Sequel II instrument while the others were derived from metagenomes sequenced on the Illumina HiSeq platform. The cMAG is 1.649 Mb in size and encodes 1,578 genes. We propose to rename BVAB1 to "Candidatus Lachnocurva vaginae" based on phylogenetic analyses, and provide genomic and metabolomic evidence that this candidate species may metabolize D-lactate, produce trimethylamine (one of the chemicals responsible for BV-associated odor), and be motile. The cMAG and the six MAGs are valuable resources that will further contribute to our understanding of the heterogeneous etiology of bacterial vaginosis.
Project description:Using metagenome sequencing, a nearly complete genome sequence was retrieved for the uncultured Methyloceanibacter sp. strain A49, recovered from an activated sludge system used for landfill leachate treatment at a closed landfill site. The total size and encoded sequences are 3,407,434?bp and 3,280 genes, respectively.
Project description:Filamentous tropical marine cyanobacteria such as Moorea producens strain JHB possess a rich community of heterotrophic bacteria on their polysaccharide sheaths; however, these bacterial communities have not yet been adequately studied or characterized.Through efforts to sequence the genome of this cyanobacterial strain, the 5.99 MB genome of an unknown bacterium emerged from the metagenomic information, named here as Mor1. Analysis of its genome revealed that the bacterium is heterotrophic and belongs to the phylum Acidobacteria, subgroup 22; however, it is only 85 % identical to the nearest cultured representative. Comparative genomics further revealed that Mor1 has a large number of genes involved in transcriptional regulation, is completely devoid of transposases, is not able to synthesize the full complement of proteogenic amino acids and appears to lack genes for nitrate uptake. Mor1 was found to be present in lab cultures of M. producens collected from various locations, but not other cyanobacterial species. Diverse efforts failed to culture the bacterium separately from filaments of M. producens JHB. Additionally, a co-culturing experiment between M. producens JHB possessing Mor1 and cultures of other genera of cyanobacteria indicated that the bacterium was not transferable.The data presented support a specific relationship between this novel uncultured bacterium and M. producens, however, verification of this proposed relationship cannot be done until the "uncultured" bacterium can be cultured.
Project description:BACKGROUND:Current understanding of the carbon cycle in methanogenic environments involves trophic interactions such as interspecies H2 transfer between organotrophs and methanogens. However, many metabolic processes are thermodynamically sensitive to H2 accumulation and can be inhibited by H2 produced from co-occurring metabolisms. Strategies for driving thermodynamically competing metabolisms in methanogenic environments remain unexplored. RESULTS:To uncover how anaerobes combat this H2 conflict in situ, we employ metagenomics and metatranscriptomics to revisit a model ecosystem that has inspired many foundational discoveries in anaerobic ecology-methanogenic bioreactors. Through analysis of 17 anaerobic digesters, we recovered 1343 high-quality metagenome-assembled genomes and corresponding gene expression profiles for uncultured lineages spanning 66 phyla and reconstructed their metabolic capacities. We discovered that diverse uncultured populations can drive H2-sensitive metabolisms through (i) metabolic coupling with concurrent H2-tolerant catabolism, (ii) forgoing H2 generation in favor of interspecies transfer of formate and electrons (cytochrome- and pili-mediated) to avoid thermodynamic conflict, and (iii) integration of low-concentration O2 metabolism as an ancillary thermodynamics-enhancing electron sink. Archaeal populations support these processes through unique methanogenic metabolisms-highly favorable H2 oxidation driven by methyl-reducing methanogenesis and tripartite uptake of formate, electrons, and acetate. CONCLUSION:Integration of omics and eco-thermodynamics revealed overlooked behavior and interactions of uncultured organisms, including coupling favorable and unfavorable metabolisms, shifting from H2 to formate transfer, respiring low-concentration O2, performing direct interspecies electron transfer, and interacting with high H2-affinity methanogenesis. These findings shed light on how microorganisms overcome a critical obstacle in methanogenic carbon cycles we had hitherto disregarded and provide foundational insight into anaerobic microbial ecology. Video Abstract.
Project description:An uncultured bacterium associated with the ectomycorrhizal fungus Tuber borchii Vittad. was identified as a novel member of the Cytophaga-Flexibacter-Bacteroides group. Utilizing a quantitative PCR targeting the 16S rRNA gene, we relatively quantified this bacterium in the host. The estimated number of bacteria was found to be approximately 10(6) cells per 30-day-old T. borchii mycelium culture. This represents the first molecular attempt to enumerate an uncultured bacterium associated with a mycorrhizal fungus.