Brett J. Baker

Microbial communities in acid mine drainage

The dissolution of sulfide minerals such as pyrite (FeS2), arsenopyrite (FeAsS), chalcopyrite (CuFeS2), sphalerite (ZnS), and marcasite (FeS2) yields hot, sulfuric acid-rich solutions that contain high concentrations of toxic metals. In locations where access of oxidants to sulfide mineral surfaces is increased by mining, the resulting acid mine drainage (AMD) may contaminate surrounding ecosystems. Communities of autotrophic and heterotrophic archaea and bacteria catalyze iron and sulfur oxidation, thus may ultimately determine the rate of release of metals and sulfur to the environment. AMD communities contain fewer prokaryotic lineages than many other environments. However, it is notable that at least two archaeal and eight bacterial divisions have representatives able to thrive under the extreme conditions typical of AMD. AMD communities are characterized by a very limited number of distinct species, probably due to the small number of metabolically beneficial reactions available. The metabolisms that underpin these communities include organoheterotrophy and autotrophic iron and sulfur oxidation. Other metabolic activity is based on anaerobic sulfur oxidation and ferric iron reduction. Evidence for physiological synergy in iron, sulfur, and carbon flow in these communities is reviewed. The microbial and geochemical simplicity of these systems makes them ideal targets for quantitative, genomic-based analyses of microbial ecology and evolution and community function.

Community Proteomics of a Natural Microbial Biofilm

One proposed strategy for controlling the transmission of insect-borne pathogens uses a drive mechanism to ensure the rapid spread of transgenes conferring disease refractoriness throughout wild populations. Here, we report the creation of maternal-effect selfish genetic elements in Drosophila that drive population replacement and are resistant to recombination-mediated dissociation of drive and disease refractoriness functions. These selfish elements use microRNA-mediated silencing of a maternally expressed gene essential for embryogenesis, which is coupled with early zygotic expression of a rescuing transgene.The phosphoinositide phosphatase PTEN is mutated in many human cancers. Although the role of PTEN has been studied extensively, the relative contributions of its numerous potential downstream effectors to deregulated growth and tumorigenesis remain uncertain. We provide genetic evidence in Drosophila melanogaster for the paramount importance of the protein kinase Akt [also called protein kinase B (PKB)] in mediating the effects of increased phosphatidylinositol 3,4,5-trisphosphate (PIP3) concentrations that are caused by the loss of PTEN function. A mutation in the pleckstrin homology (PH) domain of Akt that reduces its affinity for PIP3 sufficed to rescue the lethality of flies devoid of PTEN activity. Thus, Akt appears to be the only critical target activated by increased PIP3 concentrations in Drosophila.Using genomic and mass spectrometry-based proteomic methods, we evaluated gene expression, identified key activities, and examined partitioning of metabolic functions in a natural acid mine drainage (AMD) microbial biofilm community. We detected 2033 proteins from the five most abundant species in the biofilm, including 48% of the predicted proteins from the dominant biofilm organism, Leptospirillum group II. Proteins involved in protein refolding and response to oxidative stress appeared to be highly expressed, which suggests that damage to biomolecules is a key challenge for survival. We validated and estimated the relative abundance and cellular localization of 357 unique and 215 conserved novel proteins and determined that one abundant novel protein is a cytochrome central to iron oxidation and AMD formation.

A new view of the tree of life

The tree of life is one of the most important organizing principles in biology1. Gene surveys suggest the existence of an enormous number of branches2, but even an approximation of the full scale of the tree has remained elusive. Recent depictions of the tree of life have focused either on the nature of deep evolutionary relationships3–5 or on the known, well-classified diversity of life with an emphasis on eukaryotes6. These approaches overlook the dramatic change in our understanding of lifes diversity resulting from genomic sampling of previously unexamined environments. New methods to generate genome sequences illuminate the identity of organisms and their metabolic capacities, placing them in community and ecosystem contexts7,8. Here, we use new genomic data from over 1,000 uncultivated and little known organisms, together with published sequences, to infer a dramatically expanded version of the tree of life, with Bacteria, Archaea and Eukarya included. The depiction is both a global overview and a snapshot of the diversity within each major lineage. The results reveal the dominance of bacterial diversification and underline the importance of organisms lacking isolated representatives, with substantial evolution concentrated in a major radiation of such organisms. This tree highlights major lineages currently underrepresented in biogeochemical models and identifies radiations that are probably important for future evolutionary analyses.

Community-wide analysis of microbial genome sequence signatures

BackgroundAnalyses of DNA sequences from cultivated microorganisms have revealed genome-wide, taxa-specific nucleotide compositional characteristics, referred to as genome signatures. These signatures have far-reaching implications for understanding genome evolution and potential application in classification of metagenomic sequence fragments. However, little is known regarding the distribution of genome signatures in natural microbial communities or the extent to which environmental factors shape them.ResultsWe analyzed metagenomic sequence data from two acidophilic biofilm communities, including composite genomes reconstructed for nine archaea, three bacteria, and numerous associated viruses, as well as thousands of unassigned fragments from strain variants and low-abundance organisms. Genome signatures, in the form of tetranucleotide frequencies analyzed by emergent self-organizing maps, segregated sequences from all known populations sharing < 50 to 60% average amino acid identity and revealed previously unknown genomic clusters corresponding to low-abundance organisms and a putative plasmid. Signatures were pervasive genome-wide. Clusters were resolved because intra-genome differences resulting from translational selection or protein adaptation to the intracellular (pH ~5) versus extracellular (pH ~1) environment were small relative to inter-genome differences. We found that these genome signatures stem from multiple influences but are primarily manifested through codon composition, which we propose is the result of genome-specific mutational biases.ConclusionsAn important conclusion is that shared environmental pressures and interactions among coevolving organisms do not obscure genome signatures in acid mine drainage communities. Thus, genome signatures can be used to assign sequence fragments to populations, an essential prerequisite if metagenomics is to provide ecological and biochemical insights into the functioning of microbial communities.

EMIRGE: reconstruction of full-length ribosomal genes from microbial community short read sequencing data

Recovery of ribosomal small subunit genes by assembly of short read community DNA sequence data generally fails, making taxonomic characterization difficult. Here, we solve this problem with a novel iterative method, based on the expectation maximization algorithm, that reconstructs full-length small subunit gene sequences and provides estimates of relative taxon abundances. We apply the method to natural and simulated microbial communities, and correctly recover community structure from known and previously unreported rRNA gene sequences. An implementation of the method is freely available at https://github.com/csmiller/EMIRGE.

Gene Transfer from Bacteria and Archaea Facilitated Evolution of an Extremophilic Eukaryote

Hot, Toxic Eukaryote Unusually, the single-celled eukaryote red alga, Galdieria sulphuraria, can thrive in hot, acidic springs. This organism is endowed with extraordinary metabolic talents and can consume a variety of strange carbohydrates, as well as turn on photosynthesis when the food runs out. Schönknecht et al. (p. 1207; see the Perspective by Rocha) discerned from phylogenetic analysis of its genome that during its evolution, G. sulphuraria appears to have commandeered at least 75 bacterial and archaeal genes by horizontal gene transfer and then applied gene expansion to boost its metabolic repertoire. A mosaic of genes acquired from various phyla enable a red alga to grow abundantly in hot, acidic, and toxic niches. [Also see Perspective by Rocha] Some microbial eukaryotes, such as the extremophilic red alga Galdieria sulphuraria, live in hot, toxic metal-rich, acidic environments. To elucidate the underlying molecular mechanisms of adaptation, we sequenced the 13.7-megabase genome of G. sulphuraria. This alga shows an enormous metabolic flexibility, growing either photoautotrophically or heterotrophically on more than 50 carbon sources. Environmental adaptation seems to have been facilitated by horizontal gene transfer from various bacteria and archaea, often followed by gene family expansion. At least 5% of protein-coding genes of G. sulphuraria were probably acquired horizontally. These proteins are involved in ecologically important processes ranging from heavy-metal detoxification to glycerol uptake and metabolism. Thus, our findings show that a pan-domain gene pool has facilitated environmental adaptation in this unicellular eukaryote.

Genome-Directed Isolation of the Key Nitrogen Fixer Leptospirillum ferrodiazotrophum sp. nov. from an Acidophilic Microbial Community

ABSTRACT Analysis of assembled random shotgun sequence data from a low-diversity, subsurface acid mine drainage (AMD) biofilm revealed a single nif operon. This was found on a genome fragment belonging to a member of Leptospirillum group III, a lineage in the Nitrospirae phylum with no cultivated representatives. Based on the prediction that this organism is solely responsible for nitrogen fixation in the community, we pursued a selective isolation strategy to obtain the organism in pure culture. An AMD biofilm sample naturally abundant in Leptospirillum group III cells was homogenized, filtered, and serially diluted into a nitrogen-free liquid medium. The resulting culture in the terminal dilution grew autotrophically to a maximum cell density of ∼106 cells/ml, oxidizing ferrous iron as the sole energy source. 16S rRNA-internal transcribed spacer region clone library analysis confirmed that the isolate is a member of Leptospirillum group III and that the culture is axenic. We propose the name Leptospirillum ferrodiazotrophum sp. nov. for this iron-oxidizing, free-living diazotroph. This study highlights how environmental sequence data can provide insights for culturing previously uncultured microorganisms.

Asgard archaea illuminate the origin of eukaryotic cellular complexity

The origin and cellular complexity of eukaryotes represent a major enigma in biology. Current data support scenarios in which an archaeal host cell and an alphaproteobacterial (mitochondrial) endosymbiont merged together, resulting in the first eukaryotic cell. The host cell is related to Lokiarchaeota, an archaeal phylum with many eukaryotic features. The emergence of the structural complexity that characterizes eukaryotic cells remains unclear. Here we describe the ‘Asgard’ superphylum, a group of uncultivated archaea that, as well as Lokiarchaeota, includes Thor-, Odin- and Heimdallarchaeota. Asgard archaea affiliate with eukaryotes in phylogenomic analyses, and their genomes are enriched for proteins formerly considered specific to eukaryotes. Notably, thorarchaeal genomes encode several homologues of eukaryotic membrane-trafficking machinery components, including Sec23/24 and TRAPP domains. Furthermore, we identify thorarchaeal proteins with similar features to eukaryotic coat proteins involved in vesicle biogenesis. Our results expand the known repertoire of ‘eukaryote-specific’ proteins in Archaea, indicating that the archaeal host cell already contained many key components that govern eukaryotic cellular complexity.

Lineages of Acidophilic Archaea Revealed by Community Genomic Analysis

Novel, low-abundance microbial species can be easily overlooked in standard polymerase chain reaction (PCR)–based surveys. We used community genomic data obtained without PCR or cultivation to reconstruct DNA fragments bearing unusual 16S ribosomal RNA (rRNA) and protein-coding genes from organisms belonging to novel archaeal lineages. The organisms are minor components of all biofilms growing in pH 0.5 to 1.5 solutions within the Richmond Mine, California. Probes specific for 16S rRNA showed that the fraction less than 0.45 micrometers in diameter is dominated by these organisms. Transmission electron microscope images revealed that the cells are pleomorphic with unusual folded membrane protrusions and have apparent volumes of <0.006 cubic micrometer.

Community Genomic and Proteomic Analyses of Chemoautotrophic Iron-Oxidizing “Leptospirillum rubarum” (Group II) and “Leptospirillum ferrodiazotrophum” (Group III) Bacteria in Acid Mine Drainage Biofilms

ABSTRACT We analyzed near-complete population (composite) genomic sequences for coexisting acidophilic iron-oxidizing Leptospirillum group II and III bacteria (phylum Nitrospirae) and an extrachromosomal plasmid from a Richmond Mine, Iron Mountain, CA, acid mine drainage biofilm. Community proteomic analysis of the genomically characterized sample and two other biofilms identified 64.6% and 44.9% of the predicted proteins of Leptospirillum groups II and III, respectively, and 20% of the predicted plasmid proteins. The bacteria share 92% 16S rRNA gene sequence identity and >60% of their genes, including integrated plasmid-like regions. The extrachromosomal plasmid carries conjugation genes with detectable sequence similarity to genes in the integrated conjugative plasmid, but only those on the extrachromosomal element were identified by proteomics. Both bacterial groups have genes for community-essential functions, including carbon fixation and biosynthesis of vitamins, fatty acids, and biopolymers (including cellulose); proteomic analyses reveal these activities. Both Leptospirillum types have multiple pathways for osmotic protection. Although both are motile, signal transduction and methyl-accepting chemotaxis proteins are more abundant in Leptospirillum group III, consistent with its distribution in gradients within biofilms. Interestingly, Leptospirillum group II uses a methyl-dependent and Leptospirillum group III a methyl-independent response pathway. Although only Leptospirillum group III can fix nitrogen, these proteins were not identified by proteomics. The abundances of core proteins are similar in all communities, but the abundance levels of unique and shared proteins of unknown function vary. Some proteins unique to one organism were highly expressed and may be key to the functional and ecological differentiation of Leptospirillum groups II and III.