Stefan M. Sievert

Insights into the phylogeny and coding potential of microbial dark matter

Genome sequencing enhances our understanding of the biological world by providing blueprints for the evolutionary and functional diversity that shapes the biosphere. However, microbial genomes that are currently available are of limited phylogenetic breadth, owing to our historical inability to cultivate most microorganisms in the laboratory. We apply single-cell genomics to target and sequence 201 uncultivated archaeal and bacterial cells from nine diverse habitats belonging to 29 major mostly uncharted branches of the tree of life, so-called ‘microbial dark matter’. With this additional genomic information, we are able to resolve many intra- and inter-phylum-level relationships and to propose two new superphyla. We uncover unexpected metabolic features that extend our understanding of biology and challenge established boundaries between the three domains of life. These include a novel amino acid use for the opal stop codon, an archaeal-type purine synthesis in Bacteria and complete sigma factors in Archaea similar to those in Bacteria. The single-cell genomes also served to phylogenetically anchor up to 20% of metagenomic reads in some habitats, facilitating organism-level interpretation of ecosystem function. This study greatly expands the genomic representation of the tree of life and provides a systematic step towards a better understanding of biological evolution on our planet.

Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea

Ammonia-oxidizing archaea are ubiquitous in marine and terrestrial environments and now thought to be significant contributors to carbon and nitrogen cycling. The isolation of Candidatus “Nitrosopumilus maritimus” strain SCM1 provided the opportunity for linking its chemolithotrophic physiology with a genomic inventory of the globally distributed archaea. Here we report the 1,645,259-bp closed genome of strain SCM1, revealing highly copper-dependent systems for ammonia oxidation and electron transport that are distinctly different from known ammonia-oxidizing bacteria. Consistent with in situ isotopic studies of marine archaea, the genome sequence indicates N. maritimus grows autotrophically using a variant of the 3-hydroxypropionate/4-hydroxybutryrate pathway for carbon assimilation, while maintaining limited capacity for assimilation of organic carbon. This unique instance of archaeal biosynthesis of the osmoprotectant ectoine and an unprecedented enrichment of multicopper oxidases, thioredoxin-like proteins, and transcriptional regulators points to an organism responsive to environmental cues and adapted to handling reactive copper and nitrogen species that likely derive from its distinctive biochemistry. The conservation of N. maritimus gene content and organization within marine metagenomes indicates that the unique physiology of these specialized oligophiles may play a significant role in the biogeochemical cycles of carbon and nitrogen.

Characterization of an Autotrophic Sulfide-Oxidizing Marine Arcobacter sp. That Produces Filamentous Sulfur

ABSTRACT A coastal marine sulfide-oxidizing autotrophic bacterium produces hydrophilic filamentous sulfur as a novel metabolic end product. Phylogenetic analysis placed the organism in the genus Arcobacter in the epsilon subdivision of the Proteobacteria. This motile vibrioid organism can be considered difficult to grow, preferring to grow under microaerophilic conditions in flowing systems in which a sulfide-oxygen gradient has been established. Purified cell cultures were maintained by using this approach. Essentially all 4′,6-diamidino-2-phenylindole dihydrochloride-stained cells in a flowing reactor system hybridized with Arcobacter-specific probes as well as with a probe specific for the sequence obtained from reactor-grown cells. The proposed provisional name for the coastal isolate is “Candidatus Arcobacter sulfidicus.” For cells cultured in a flowing reactor system, the sulfide optimum was higher than and the CO2 fixation activity was as high as or higher than those reported for other sulfur oxidizers, such as Thiomicrospira spp. Cells associated with filamentous sulfur material demonstrated nitrogen fixation capability. No ribulose 1,5-bisphosphate carboxylase/oxygenase could be detected on the basis of radioisotopic activity or by Western blotting techniques, suggesting an alternative pathway of CO2 fixation. The process of microbial filamentous sulfur formation has been documented in a number of marine environments where both sulfide and oxygen are available. Filamentous sulfur formation by “Candidatus Arcobacter sulfidicus” or similar strains may be an ecologically important process, contributing significantly to primary production in such environments.

Anaerobic oxidation of short-chain hydrocarbons by marine sulphate-reducing bacteria

The short-chain hydrocarbons ethane, propane and butane are constituents of natural gas. They are usually assumed to be of thermochemical origin, but biological formation of ethane and propane has been also observed. Microbial utilization of short-chain hydrocarbons has been shown in some aerobic species but not in anaerobic species of bacteria. On the other hand, anaerobic utilization of short-chain hydrocarbons would in principle be expected because various anaerobic bacteria grow with higher homologues (≥C6). Indeed, chemical analyses of hydrocarbon-rich habitats with limited or no access of oxygen indicated in situ biodegradation of short-chain hydrocarbons. Here we report the enrichment of sulphate-reducing bacteria (SRB) with such capacity from marine hydrocarbon seep areas. Propane or n-butane as the sole growth substrate led to sediment-free sulphate-reducing enrichment cultures growing at 12, 28 or 60 °C. With ethane, a slower enrichment with residual sediment was obtained at 12 °C. Isolation experiments resulted in a mesophilic pure culture (strain BuS5) that used only propane and n-butane (methane, isobutane, alcohols or carboxylic acids did not support growth). Complete hydrocarbon oxidation to CO2 and the preferential oxidation of 12C-enriched alkanes were observed with strain BuS5 and other cultures. Metabolites of propane included iso- and n-propylsuccinate, indicating a subterminal as well as an unprecedented terminal alkane activation with involvement of fumarate. According to 16S ribosomal RNA analyses, strain BuS5 affiliates with Desulfosarcina/Desulfococcus, a cluster of widespread marine SRB. An enrichment culture with propane growing at 60 °C was dominated by Desulfotomaculum-like SRB. Our results suggest that diverse SRB are able to thrive in seep areas and gas reservoirs on propane and butane, thus altering the gas composition and contributing to sulphide production.

Evidence for Autotrophic CO2 Fixation via the Reductive Tricarboxylic Acid Cycle by Members of the ε Subdivision of Proteobacteria

Based on 16S rRNA gene surveys, bacteria of the epsilon subdivision of proteobacteria have been identified to be important members of microbial communities in a variety of environments, and quite a few have been demonstrated to grow autotrophically. However, no information exists on what pathway of autotrophic carbon fixation these bacteria might use. In this study, Thiomicrospira denitrificans and Candidatus Arcobacter sulfidicus, two chemolithoautotrophic sulfur oxidizers of the epsilon subdivision of proteobacteria, were examined for activities of the key enzymes of the known autotrophic CO(2) fixation pathways. Both organisms contained activities of the key enzymes of the reductive tricarboxylic acid cycle, ATP citrate lyase, 2-oxoglutarate:ferredoxin oxidoreductase, and pyruvate:ferredoxin oxidoreductase. Furthermore, no activities of key enzymes of other CO(2) fixation pathways, such as the Calvin cycle, the reductive acetyl coenzyme A pathway, and the 3-hydroxypropionate cycle, could be detected. In addition to the key enzymes, the activities of the other enzymes involved in the reductive tricarboxylic acid cycle could be measured. Sections of the genes encoding the alpha- and beta-subunits of ATP citrate lyase could be amplified from both organisms. These findings represent the first direct evidence for the operation of the reductive tricarboxylic acid cycle for autotrophic CO(2) fixation in epsilon-proteobacteria. Since epsilon-proteobacteria closely related to these two organisms are important in many habitats, such as hydrothermal vents, oxic-sulfidic interfaces, or oilfields, these results suggest that autotrophic CO(2) fixation via the reductive tricarboxylic acid cycle might be more important than previously considered.

Spatiotemporal Distribution of Marine Magnetotactic Bacteria in a Seasonally Stratified Coastal Salt Pond

ABSTRACT The occurrence and distribution of magnetotactic bacteria (MB) were studied as a function of the physical and chemical conditions in meromictic Salt Pond, Falmouth, Mass., throughout summer 2002. Three dominant MB morphotypes were observed to occur within the chemocline. Small microaerophilic magnetite-producing cocci were present at the top of the chemocline, while a greigite-producing packet-forming bacterium occurred at the base of the chemocline. The distributions of these groups displayed sharp changes in abundance over small length scales within the water column as well as strong seasonal fluctuations in population abundance. We identified a novel, greigite-producing rod in the sulfidic hypolimnion that was present in relatively constant abundance over the course of the season. This rod is the first MB that appears to belong to the γ-Proteobacteria, which may suggest an iron- rather than sulfur-based respiratory metabolism. Its distribution and phylogenetic identity suggest that an alternative model for the ecological and physiological role of magnetotaxis is needed for greigite-producing MB.

The genome of deep-sea vent chemolithoautotroph Thiomicrospira crunogena XCL-2.

Presented here is the complete genome sequence of Thiomicrospira crunogena XCL-2, representative of ubiquitous chemolithoautotrophic sulfur-oxidizing bacteria isolated from deep-sea hydrothermal vents. This gammaproteobacterium has a single chromosome (2,427,734 base pairs), and its genome illustrates many of the adaptations that have enabled it to thrive at vents globally. It has 14 methyl-accepting chemotaxis protein genes, including four that may assist in positioning it in the redoxcline. A relative abundance of coding sequences (CDSs) encoding regulatory proteins likely control the expression of genes encoding carboxysomes, multiple dissolved inorganic nitrogen and phosphate transporters, as well as a phosphonate operon, which provide this species with a variety of options for acquiring these substrates from the environment. Thiom. crunogena XCL-2 is unusual among obligate sulfur-oxidizing bacteria in relying on the Sox system for the oxidation of reduced sulfur compounds. The genome has characteristics consistent with an obligately chemolithoautotrophic lifestyle, including few transporters predicted to have organic allocrits, and Calvin-Benson-Bassham cycle CDSs scattered throughout the genome.

Comparative metagenomics of microbial communities inhabiting deep-sea hydrothermal vent chimneys with contrasting chemistries

Deep-sea hydrothermal vent chimneys harbor a high diversity of largely unknown microorganisms. Although the phylogenetic diversity of these microorganisms has been described previously, the adaptation and metabolic potential of the microbial communities is only beginning to be revealed. A pyrosequencing approach was used to directly obtain sequences from a fosmid library constructed from a black smoker chimney 4143-1 in the Mothra hydrothermal vent field at the Juan de Fuca Ridge. A total of 308 034 reads with an average sequence length of 227 bp were generated. Comparative genomic analyses of metagenomes from a variety of environments by two-way clustering of samples and functional gene categories demonstrated that the 4143-1 metagenome clustered most closely with that from a carbonate chimney from Lost City. Both are highly enriched in genes for mismatch repair and homologous recombination, suggesting that the microbial communities have evolved extensive DNA repair systems to cope with the extreme conditions that have potential deleterious effects on the genomes. As previously reported for the Lost City microbiome, the metagenome of chimney 4143-1 exhibited a high proportion of transposases, implying that horizontal gene transfer may be a common occurrence in the deep-sea vent chimney biosphere. In addition, genes for chemotaxis and flagellar assembly were highly enriched in the chimney metagenomes, reflecting the adaptation of the organisms to the highly dynamic conditions present within the chimney walls. Reconstruction of the metabolic pathways revealed that the microbial community in the wall of chimney 4143-1 was mainly fueled by sulfur oxidation, putatively coupled to nitrate reduction to perform inorganic carbon fixation through the Calvin–Benson–Bassham cycle. On the basis of the genomic organization of the key genes of the carbon fixation and sulfur oxidation pathways contained in the large genomic fragments, both obligate and facultative autotrophs appear to be present and contribute to biomass production.

Identification of 16S ribosomal DNA-defined bacterial populations at a shallow submarine hydrothermal vent near Milos Island (Greece)

ABSTRACT In a recent publication (S. M. Sievert, T. Brinkhoff, G. Muyzer, W. Ziebis, and J. Kuever, Appl. Environ. Microbiol. 65:3834–3842, 1999) we described spatiotemporal changes in the bacterial community structure at a shallow-water hydrothermal vent in the Aegean Sea near the isle of Milos (Greece). Here we describe identification and phylogenetic analysis of the predominant bacterial populations at the vent site and their distribution at the vent site as determined by sequencing of DNA molecules (bands) excised from denaturing gradient gels. A total of 36 bands could be sequenced, and there were representatives of eight major lineages of the domainBacteria. Cytophaga-Flavobacterium andAcidobacterium were the most frequently retrieved bacterial groups. Less than 33% of the sequences exhibited 90% or more identity with cultivated organisms. The predominance of putative heterotrophic populations in the sequences retrieved is explained by the input of allochthonous organic matter at the vent site.

Genomic and enzymatic evidence for acetogenesis among multiple lineages of the archaeal phylum Bathyarchaeota widespread in marine sediments.

Members of the archaeal phylum Bathyarchaeota are widespread and abundant in the energy-deficient marine subsurface sediments. However, their life strategies have remained largely elusive. Here, we provide genetic evidence that some lineages of Bathyarchaeota are acetogens, being capable of homoacetogenesis, a metabolism so far restricted to the domain Bacteria. Metabolic reconstruction based on genomic bins assembled from the metagenome of deep-sea subsurface sediments shows that the metabolism of some lineages of Bathyarchaeota is similar to that of bona fide bacterial homoacetogens, by having pathways for acetogenesis and for the fermentative utilization of a variety of organic substrates. Heterologous expression and activity assay of the acetate kinase gene ack from Bathyarchaeota, demonstrate further the capability of these Bathyarchaeota to grow as acetogens. The presence and expression of bathyarchaeotal genes indicative of active acetogenesis was also confirmed in Peru Margin subsurface sediments where Bathyarchaeota are abundant. The analyses reveal that this ubiquitous and abundant subsurface archaeal group has adopted a versatile life strategy to make a living under energy-limiting conditions. These findings further expand the metabolic potential of Archaea and argue for a revision of the role of Archaea in the carbon cycle of marine sediments.