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Microorganisms catalyze carbon cycling and biogeochemical reactions in the deep subsurface and thus may be expected to influence the fate of injected supercritical (sc) CO₂ following geological carbon sequestration (GCS). We hypothesized that natural subsurface scCO₂ reservoirs, which serve as analogs for the long-term fate of sequestered scCO₂, harbor a ‘deep carbonated biosphere’ with carbon cycling potential. We sampled subsurface fluids from scCO₂-water separators at a natural scCO₂ reservoir at McElmo Dome, Colorado for analysis of 16S rRNA gene diversity and metagenome content. Sequence annotations indicated dominance of Sulfurospirillum, Rhizobium, Desulfovibrio and four members of the Clostridiales family. Genomes extracted from metagenomes using homology and compositional approaches revealed diverse mechanisms for growth and nutrient cycling, including pathways for CO₂ and N₂ fixation, anaerobic respiration, sulfur oxidation, fermentation and potential for metabolic syntrophy. Differences in biogeochemical potential between two production well communities were consistent with differences in fluid chemical profiles, suggesting a potential link between microbial activity and geochemistry. The existence of a microbial ecosystem associated with the McElmo Dome scCO₂ reservoir indicates that potential impacts of the deep biosphere on CO2fate and transport should be taken into consideration as a component of GCS planning and modelling., National Institute of Environmental Health Sciences (Grant P30-ES002109), National Institute of General Medical Sciences (U.S.) (Grant GMS T32GM008334)

Sequence alignment of geosmin synthase (geoA) a key enzyme in the pathway for geosmin biosynthesis. Nucleotide alignment was conducted using MUSCLE [49] implemented in Geneious R9 [50]. The height of letters in the sequence logo indicated the degree to which the sequence is conserved. While the annotated geoA sequence from MRB 220 reveals partial nucleotide homology, strictly conserved Mg2+ binding sites at position 90 (DDHFLE) and position 476 (DDYFP) are not shared by MRB 220 indicating the enzyme is likely inactive. (PDF 2328Â kb)

Sequence alignment of geosmin synthase (geoA) a key enzyme in the pathway for geosmin biosynthesis. Nucleotide alignment was conducted using MUSCLE [49] implemented in Geneious R9 [50]. The height of letters in the sequence logo indicated the degree to which the sequence is conserved. While the annotated geoA sequence from MRB 220 reveals partial nucleotide homology, strictly conserved Mg2+ binding sites at position 90 (DDHFLE) and position 476 (DDYFP) are not shared by MRB 220 indicating the enzyme is likely inactive. (PDF 2328Â kb)

Neighbor-joining tree of the 16Sâ 23S rRNA internal transcribed spacer of MRB220 and selected gene sequences extracted from sequence genomes of Stigonematales. All sequences were aligned with MUSCLE [49], and alignment column

Average nucleotide identity (ANI) between genome of MRB 220 and those of other Stigonematales. Note: Average nucleotide identity was computed using the JGI IMG pipeline [32]. (DOC 38Â kb)

We have characterized the molecular and genomic diversity of the microbiota of the starlet sea anemone Nematostella vectensis, a cnidarian model for comparative developmental and functional biology and a year-round inhabitant of temperate salt marshes. Molecular phylogenetic analysis of 16S rRNA gene clone libraries revealed four ribotypes associated with N. vectensis at multiple locations and times. These associates include two novel ribotypes within the ε-Proteobacterial order Campylobacterales and the Spirochetes, respectively, each sharing 99% 16S rRNA identity with Endozoicomonas elysicola and Pseudomonas oleovorans, respectively. Species-specific PCR revealed that these populations persisted in N. vectensis asexually propagated under laboratory conditions. cDNA indicated expression of the Campylobacterales and Endozoicomonas 16S rRNA in anemones from Sippewissett Marsh, MA. A collection of bacteria from laboratory raised N. vectensis was dominated by isolates from P. oleovorans and Rhizobium radiobacter. Isolates from field-collected anemones revealed an association with Limnobacter and Stappia isolates. Genomic DNA sequencing was carried out on 10 cultured bacterial isolates representing field- and laboratory-associates, i.e., Limnobacter spp., Stappia spp., P. oleovorans and R. radiobacter. Genomes contained multiple genes identified as virulence (host-association) factors while S. stellulata and L. thiooxidans genomes revealed pathways for mixotrophic sulfur oxidation. A pilot metatranscriptome of laboratory-raised N. vectensis was compared to the isolate genomes and indicated expression of ORFs from L. thiooxidans with predicted functions of motility, nutrient scavenging (Fe and P), polyhydroxyalkanoate synthesis for carbon storage, and selective permeability (porins). We hypothesize that such activities may mediate acclimation and persistence of bacteria in a N. vectensis holobiont defined by both internal and external gradients of chemicals and nutrients in a dynamic coastal habitat., Massachusetts Institute of Technology. Department of Civil and Environmental Engineering, National Science Foundation (U.S.). Graduate Research Fellowship, National Research Foundation of Korea (Fellowship)

We report draft genome sequences of Bacillus subterraneus MITOT1 and Bacillus cereus MIT0214 isolated through enrichment of samples from geologic sequestration sites in pressurized bioreactors containing a supercritical (sc) CO[subscript 2] headspace. Their genome sequences expand the phylogenetic range of sequenced bacilli and allow characterization of molecular mechanisms of scCO[subscript 2] tolerance., United States. Dept. of Energy. Office of Fossil Energy (Award DE-FE0002128), MIT Energy Initiative

Cyanobacterial harmful algal blooms (cyanoHABs) appear to be increasing in frequency on a global scale. The Cyanobacteria in blooms can produce toxic secondary metabolites that make freshwater dangerous for drinking and recreation. To characterize microbial activities in a cyanoHAB, transcripts from a eutrophic freshwater reservoir in Singapore were sequenced for six samples collected over one day-night period. Transcripts from the Cyanobacterium Microcystis dominated all samples and were accompanied by at least 533 genera primarily from the Cyanobacteria, Proteobacteria, Bacteroidetes and Actinobacteria. Within the Microcystis population, abundant transcripts were from genes for buoyancy, photosynthesis and synthesis of the toxin microviridin, suggesting that these are necessary for competitive dominance in the Reservoir. During the day, Microcystis transcripts were enriched in photosynthesis and energy metabolism while at night enriched pathways included DNA replication and repair and toxin biosynthesis. Microcystis was the dominant source of transcripts from polyketide and non-ribosomal peptide synthase (PKS and NRPS, respectively) gene clusters. Unexpectedly, expression of all PKS/NRPS gene clusters, including for the toxins microcystin and aeruginosin, occurred throughout the day-night cycle. The most highly expressed PKS/NRPS gene cluster from Microcystis is not associated with any known product. The four most abundant phyla in the reservoir were enriched in different functions, including photosynthesis (Cyanobacteria), breakdown of complex organic molecules (Proteobacteria), glycan metabolism (Bacteroidetes) and breakdown of plant carbohydrates, such as cellobiose (Actinobacteria). These results provide the first estimate of secondary metabolite gene expression, functional partitioning and functional interplay in a freshwater cyanoHAB., Singapore. National Research Foundation (Singapore MIT Alliance for Research and Technology (SMART), Center for Environmental Sensing and Modeling (CENSAM) research program), National Science Foundation (U.S.) (Postdoctoral Research Fellowship in Biology, Grant No. DBI-1202865), National Institute of Environmental Health Sciences (NIEHS Grant P30-ES002109 to the MIT Center for Environmental Health Sciences), MIT International Science and Technology Initiatives (MISTI-Hayashi fund)

Members of the genus Vibrio are important marine and aquaculture pathogens. Hemolytic activity has been identified as a virulence factor in many pathogenic vibrios including V. cholerae, V. parahaemolyticus, V. alginolyticus, V. harveyi and V. vulnificus. We have used transposon mutagenesis to identify genes involved in the hemolytic activity of shrimp-pathogenic V. harveyi strain PSU3316. Out of 1,764 mutants screened, five mutants showed reduced hemolytic activity on sheep blood agar and exhibited virulence attenuation in shrimp (Litopenaeus vannamei). Mutants were identified by comparing transposon junction sequences to a draft of assembly of the PSU3316 genome. Surprisingly none of the disrupted open reading frames or gene neighborhoods contained genes annotated as hemolysins. The gene encoding RseB, a negative regulator of the sigma factor (σE), was interrupted in 2 out of 5 transposon mutants, in addition, the transcription factor CytR, a threonine synthetase, and an efflux-associated cytoplasmic protein were also identified. Knockout mutations introduced into the rpoE operon at the rseB gene exhibited low hemolytic activity in sheep blood agar, and were 3-to 7-fold attenuated for colonization in shrimp. Comparison of whole cell extracted proteins in the rseB mutant (PSU4030) to the wild-type by 2-D gel electrophoresis revealed 6 differentially expressed proteins, including two down-regulated porins (OmpC-like and OmpN) and an upregulated protease (DegQ) which have been associated with σE in other organisms. Our study is the first report linking hemolytic activity to the σE regulators in pathogenic Vibrio species and suggests expression of this virulence-linked phenotype is governed by multiple regulatory pathways within the V. harveyi., Thailand Research Fund (Royal Golden Jubilee Ph.D. program (Grant No. HD/0153/2547)), Harvard University, Massachusetts Institute of Technology