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1.  Recoding of the stop codon UGA to glycine by a BD1-5/SN-2 bacterium and niche partitioning between Alpha- and Gammaproteobacteria in a tidal sediment microbial community naturally selected in a laboratory chemostat 
Sandy coastal sediments are global hotspots for microbial mineralization of organic matter and denitrification. These sediments are characterized by advective porewater flow, tidal cycling and an active and complex microbial community. Metagenomic sequencing of microbial communities sampled from such sediments showed that potential sulfur oxidizing Gammaproteobacteria and members of the enigmatic BD1-5/SN-2 candidate phylum were abundant in situ (>10% and ~2% respectively). By mimicking the dynamic oxic/anoxic environmental conditions of the sediment in a laboratory chemostat, a simplified microbial community was selected from the more complex inoculum. Metagenomics, proteomics and fluorescence in situ hybridization showed that this simplified community contained both a potential sulfur oxidizing Gammaproteobacteria (at 24 ± 2% abundance) and a member of the BD1-5/SN-2 candidate phylum (at 7 ± 6% abundance). Despite the abundant supply of organic substrates to the chemostat, proteomic analysis suggested that the selected gammaproteobacterium grew partially autotrophically and performed hydrogen/formate oxidation. The enrichment of a member of the BD1-5/SN-2 candidate phylum enabled, for the first time, direct microscopic observation by fluorescent in situ hybridization and the experimental validation of the previously predicted translation of the stop codon UGA into glycine.
PMCID: PMC4032931  PMID: 24904545
continuous culture; enrichment; chemostat; Roseobacter; Maritimibacter; stop codon
2.  New Approaches Indicate Constant Viral Diversity despite Shifts in Assemblage Structure in an Australian Hypersaline Lake 
Applied and Environmental Microbiology  2013;79(21):6755-6764.
It is widely stated that viruses represent the most significant source of biodiversity on Earth, yet characterizing the diversity of viral assemblages in natural systems remains difficult. Viral diversity studies are challenging because viruses lack universally present, phylogenetically informative genes. Here, we developed an approach to estimate viral diversity using a series of functional and novel conserved genes. This approach provides direct estimates of viral assemblage diversity while retaining resolution at the level of individual viral populations in a natural system. We characterized viral assemblages in eight samples from hypersaline Lake Tyrrell (LT), Victoria, Australia, using 39,636 viral contigs. We defined viral operational taxonomic units (OTUs) in two ways. First, we used genes with three different functional predictions that were abundantly represented in the data set. Second, we clustered proteins of unknown function based on sequence similarity, and we chose genes represented by three clusters with numerous members to define OTUs. In combination, diversity metrics indicated between 412 and 735 sampled populations, and the number of populations remained relatively constant across samples. We determined the relative representation of each viral OTU in each sample and found that viral assemblage structures correlate with salinity and solution chemistry. LT viral assemblages were near-replicates from the same site sampled a few days apart but differed significantly on other spatial and temporal scales. The OTU definition approach proposed here paves the way for metagenomics-based analyses of viral assemblages using ecological models previously applied to bacteria and archaea.
PMCID: PMC3811486  PMID: 23995931
3.  The dynamic genetic repertoire of microbial communities 
Fems Microbiology Reviews  2008;33(1):109-132.
Community genomic data have revealed multiple levels of variation between and within microbial consortia. This variation includes large-scale differences in gene content between ecosystems as well as within-population sequence heterogeneity. In the present review, we focus specifically on how fine-scale variation within microbial and viral populations is apparent from community genomic data. A major unresolved question is how much of the observed variation is due to neutral vs. adaptive processes. Limited experimental data hint that some of this fine-scale variation may be in part functionally relevant, whereas sequence-based and modeling analyses suggest that much of it may be neutral. While methods for interpreting population genomic data are still in their infancy, we discuss current interpretations of existing datasets in the light of evolutionary processes and models. Finally, we highlight the importance of virus–host dynamics in generating and shaping within-population diversity.
PMCID: PMC2704941  PMID: 19054116
community genomics; CRISPR; genetic heterogeneity; metagenomics; population genomics; virus–host dynamics
4.  Biostimulation induces syntrophic interactions that impact C, S and N cycling in a sediment microbial community 
The ISME Journal  2012;7(4):800-816.
Stimulation of subsurface microorganisms to induce reductive immobilization of metals is a promising approach for bioremediation, yet the overall microbial community response is typically poorly understood. Here we used proteogenomics to test the hypothesis that excess input of acetate activates complex community functioning and syntrophic interactions among autotrophs and heterotrophs. A flow-through sediment column was incubated in a groundwater well of an acetate-amended aquifer and recovered during microbial sulfate reduction. De novo reconstruction of community sequences yielded near-complete genomes of Desulfobacter (Deltaproteobacteria), Sulfurovum- and Sulfurimonas-like Epsilonproteobacteria and Bacteroidetes. Partial genomes were obtained for Clostridiales (Firmicutes) and Desulfuromonadales-like Deltaproteobacteria. The majority of proteins identified by mass spectrometry corresponded to Desulfobacter-like species, and demonstrate the role of this organism in sulfate reduction (Dsr and APS), nitrogen fixation and acetate oxidation to CO2 during amendment. Results indicate less abundant Desulfuromonadales, and possibly Bacteroidetes, also actively contributed to CO2 production via the tricarboxylic acid (TCA) cycle. Proteomic data indicate that sulfide was partially re-oxidized by Epsilonproteobacteria through nitrate-dependent sulfide oxidation (using Nap, Nir, Nos, SQR and Sox), with CO2 fixed using the reverse TCA cycle. We infer that high acetate concentrations, aimed at stimulating anaerobic heterotrophy, led to the co-enrichment of, and carbon fixation in Epsilonproteobacteria. Results give an insight into ecosystem behavior following addition of simple organic carbon to the subsurface, and demonstrate a range of biological processes and community interactions were stimulated.
PMCID: PMC3603403  PMID: 23190730
autotroph; metagenomics; proteomics; sediment; subsurface; syntrophy
5.  Iron-reducing bacteria accumulate ferric oxyhydroxide nanoparticle aggregates that may support planktonic growth 
The ISME Journal  2012;7(2):338-350.
Iron-reducing bacteria (FeRB) play key roles in anaerobic metal and carbon cycling and carry out biogeochemical transformations that can be harnessed for environmental bioremediation. A subset of FeRB require direct contact with Fe(III)-bearing minerals for dissimilatory growth, yet these bacteria must move between mineral particles. Furthermore, they proliferate in planktonic consortia during biostimulation experiments. Thus, a key question is how such organisms can sustain growth under these conditions. Here we characterized planktonic microbial communities sampled from an aquifer in Rifle, Colorado, USA, close to the peak of iron reduction following in situ acetate amendment. Samples were cryo-plunged on site and subsequently examined using correlated two- and three-dimensional cryogenic transmission electron microscopy (cryo-TEM) and scanning transmission X-ray microscopy (STXM). The outer membranes of most cells were decorated with aggregates up to 150 nm in diameter composed of ∼3 nm wide amorphous, Fe-rich nanoparticles. Fluorescent in situ hybridization of lineage-specific probes applied to rRNA of cells subsequently imaged via cryo-TEM identified Geobacter spp., a well-studied group of FeRB. STXM results at the Fe L2,3 absorption edges indicate that nanoparticle aggregates contain a variable mixture of Fe(II)–Fe(III), and are generally enriched in Fe(III). Geobacter bemidjiensis cultivated anaerobically in the laboratory on acetate and hydrous ferric oxyhydroxides also accumulated mixed-valence nanoparticle aggregates. In field-collected samples, FeRB with a wide variety of morphologies were associated with nano-aggregates, indicating that cell surface Fe(III) accumulation may be a general mechanism by which FeRB can grow while in planktonic suspension.
PMCID: PMC3554402  PMID: 23038172
iron-reducing bacteria; biomineralization; correlative microscopy; cryo-transmission electron microscopy; STXM; X-ray absorption spectroscopy
6.  Architecture and Gene Repertoire of the Flexible Genome of the Extreme Acidophile Acidithiobacillus caldus 
PLoS ONE  2013;8(11):e78237.
Acidithiobacillus caldus is a sulfur oxidizing extreme acidophile and the only known mesothermophile within the Acidithiobacillales. As such, it is one of the preferred microbes for mineral bioprocessing at moderately high temperatures. In this study, we explore the genomic diversity of A. caldus strains using a combination of bioinformatic and experimental techniques, thus contributing first insights into the elucidation of the species pangenome.
Principal Findings
Comparative sequence analysis of A. caldus ATCC 51756 and SM-1 indicate that, despite sharing a conserved and highly syntenic genomic core, both strains have unique gene complements encompassing nearly 20% of their respective genomes. The differential gene complement of each strain is distributed between the chromosomal compartment, one megaplasmid and a variable number of smaller plasmids, and is directly associated to a diverse pool of mobile genetic elements (MGE). These include integrative conjugative and mobilizable elements, genomic islands and insertion sequences. Some of the accessory functions associated to these MGEs have been linked previously to the flexible gene pool in microorganisms inhabiting completely different econiches. Yet, others had not been unambiguously mapped to the flexible gene pool prior to this report and clearly reflect strain-specific adaption to local environmental conditions.
For many years, and because of DNA instability at low pH and recurrent failure to genetically transform acidophilic bacteria, gene transfer in acidic environments was considered negligible. Findings presented herein imply that a more or less conserved pool of actively excising MGEs occurs in the A. caldus population and point to a greater frequency of gene exchange in this econiche than previously recognized. Also, the data suggest that these elements endow the species with capacities to withstand the diverse abiotic and biotic stresses of natural environments, in particular those associated with its extreme econiche.
PMCID: PMC3826726  PMID: 24250794
7.  Small Genomes and Sparse Metabolisms of Sediment-Associated Bacteria from Four Candidate Phyla 
mBio  2013;4(5):e00708-13.
Cultivation-independent surveys of microbial diversity have revealed many bacterial phyla that lack cultured representatives. These lineages, referred to as candidate phyla, have been detected across many environments. Here, we deeply sequenced microbial communities from acetate-stimulated aquifer sediment to recover the complete and essentially complete genomes of single representatives of the candidate phyla SR1, WWE3, TM7, and OD1. All four of these genomes are very small, 0.7 to 1.2 Mbp, and have large inventories of novel proteins. Additionally, all lack identifiable biosynthetic pathways for several key metabolites. The SR1 genome uses the UGA codon to encode glycine, and the same codon is very rare in the OD1 genome, suggesting that the OD1 organism could also transition to alternate coding. Interestingly, the relative abundance of the members of SR1 increased with the appearance of sulfide in groundwater, a pattern mirrored by a member of the phylum Tenericutes. All four genomes encode type IV pili, which may be involved in interorganism interaction. On the basis of these results and other recently published research, metabolic dependence on other organisms may be widely distributed across multiple bacterial candidate phyla.
Few or no genomic sequences exist for members of the numerous bacterial phyla lacking cultivated representatives, making it difficult to assess their roles in the environment. This paper presents three complete and one essentially complete genomes of members of four candidate phyla, documents consistently small genome size, and predicts metabolic capabilities on the basis of gene content. These metagenomic analyses expand our view of a lifestyle apparently common across these candidate phyla.
PMCID: PMC3812714  PMID: 24149512
8.  The human gut and groundwater harbor non-photosynthetic bacteria belonging to a new candidate phylum sibling to Cyanobacteria 
eLife  2013;2:e01102.
Cyanobacteria were responsible for the oxygenation of the ancient atmosphere; however, the evolution of this phylum is enigmatic, as relatives have not been characterized. Here we use whole genome reconstruction of human fecal and subsurface aquifer metagenomic samples to obtain complete genomes for members of a new candidate phylum sibling to Cyanobacteria, for which we propose the designation ‘Melainabacteria’. Metabolic analysis suggests that the ancestors to both lineages were non-photosynthetic, anaerobic, motile, and obligately fermentative. Cyanobacterial light sensing may have been facilitated by regulators present in the ancestor of these lineages. The subsurface organism has the capacity for nitrogen fixation using a nitrogenase distinct from that in Cyanobacteria, suggesting nitrogen fixation evolved separately in the two lineages. We hypothesize that Cyanobacteria split from Melainabacteria prior or due to the acquisition of oxygenic photosynthesis. Melainabacteria remained in anoxic zones and differentiated by niche adaptation, including for symbiosis in the mammalian gut.
eLife digest
Microbes are ubiquitous in the world and exist in complex communities called microbiomes that have colonized many environments, including the human gut. Until modern techniques for sequencing nucleic acids became available, many of the organisms found in these microbiomes could not be studied because they could not be cultured in the laboratory. However, advances in sequencing technology have made it possible to study the evolution and properties of these microbes, including their impact on human health.
Bacteria belonging to the phylum Cyanobacteria had a significant effect on the prehistoric Earth because they were the first organisms to produce gaseous oxygen as a byproduct of photosynthesis, and thus shaped the Earth’s oxygen-rich atmosphere. Early plants took up these bacteria in a symbiotic relationship, and plastids—the organelles in plant cells that perform photosynthesis and produce oxygen–are the descendants of Cyanobacteria.
Organisms evolutionarily related to Cyanobacteria have been found in the human gut and in various aquatic sources, but these bacteria have not been studied because it has not been possible to isolate or culture them. Now, Di Rienzi, Sharon et al. have used modern sequencing techniques to obtain complete genomes for some of these bacteria, which they assign to a new phylum called Melainabacteria.
By analyzing these genomes, Di Rienzi, Sharon et al. were able to make predictions about the cell structure and metabolic abilities of Melainabacteria. Like Cyanobacteria, they have two membranes surrounding the bacterial cell; unlike Cyanobacteria, however, they have flagella that propel them through liquid or across surfaces. Most interestingly, Melainabacteria are not able to perform photosynthesis, but instead produce energy through fermentation and release hydrogen gas that can be consumed by other microorganisms.
The genome of the bacteria isolated from water reveals that it has the capacity to fix nitrogen. Cyanobacteria can also fix atmospheric nitrogen, but the protein complexes used by the two phyla are not related, which suggests that nitrogen fixation evolved after the evolutionary divergence of Cyanobacteria and Melainabacteria.
By exploring previously published datasets of bacterial communities, Di Rienzi, Sharon et al. found that Melainabacteria are common in aquatic habitats. They are also prevalent in the guts of herbivorous mammals and humans with a predominantly vegetarian diet. Melainabacteria from the human gut also synthesize several B and K vitamins, which suggests that these bacteria are beneficial to their host because in addition to aiding with the digestion of plant fibers, they are also a source of vitamins.
PMCID: PMC3787301  PMID: 24137540
Cyanobacteria; Melainabacteria; photosynthesis; nitrogen fixation; human gut; subsurface; Human; Other
9.  New Group in the Leptospirillum Clade: Cultivation-Independent Community Genomics, Proteomics, and Transcriptomics of the New Species “Leptospirillum Group IV UBA BS” 
Applied and Environmental Microbiology  2013;79(17):5384-5393.
Leptospirillum spp. are widespread members of acidophilic microbial communities that catalyze ferrous iron oxidation, thereby increasing sulfide mineral dissolution rates. These bacteria play important roles in environmental acidification and are harnessed for bioleaching-based metal recovery. Known members of the Leptospirillum clade of the Nitrospira phylum are Leptospirillum ferrooxidans (group I), Leptospirillum ferriphilum and “Leptospirillum rubarum” (group II), and Leptospirillum ferrodiazotrophum (group III). In the Richmond Mine acid mine drainage (AMD) system, biofilm formation is initiated by L. rubarum; L. ferrodiazotrophum appears in later developmental stages. Here we used community metagenomic data from unusual, thick floating biofilms to identify distinguishing metabolic traits in a rare and uncultivated community member, the new species “Leptospirillum group IV UBA BS.” These biofilms typically also contain a variety of Archaea, Actinobacteria, and a few other Leptospirillum spp. The Leptospirillum group IV UBA BS species shares 98% 16S rRNA sequence identity and 70% average amino acid identity between orthologs with its closest relative, L. ferrodiazotrophum. The presence of nitrogen fixation and reverse tricarboxylic acid (TCA) cycle proteins suggest an autotrophic metabolism similar to that of L. ferrodiazotrophum, while hydrogenase proteins suggest anaerobic metabolism. Community transcriptomic and proteomic analyses demonstrate expression of a multicopper oxidase unique to this species, as well as hydrogenases and core metabolic genes. Results suggest that the Leptospirillum group IV UBA BS species might play important roles in carbon fixation, nitrogen fixation, hydrogen metabolism, and iron oxidation in some acidic environments.
PMCID: PMC3753937  PMID: 23645189
10.  Comparative genomics in acid mine drainage biofilm communities reveals metabolic and structural differentiation of co-occurring archaea 
BMC Genomics  2013;14:485.
Metal sulfide mineral dissolution during bioleaching and acid mine drainage (AMD) formation creates an environment that is inhospitable to most life. Despite dominance by a small number of bacteria, AMD microbial biofilm communities contain a notable variety of coexisting and closely related Euryarchaea, most of which have defied cultivation efforts. For this reason, we used metagenomics to analyze variation in gene content that may contribute to niche differentiation among co-occurring AMD archaea. Our analyses targeted members of the Thermoplasmatales and related archaea. These results greatly expand genomic information available for this archaeal order.
We reconstructed near-complete genomes for uncultivated, relatively low abundance organisms A-, E-, and Gplasma, members of Thermoplasmatales order, and for a novel organism, Iplasma. Genomic analyses of these organisms, as well as Ferroplasma type I and II, reveal that all are facultative aerobic heterotrophs with the ability to use many of the same carbon substrates, including methanol. Most of the genomes share genes for toxic metal resistance and surface-layer production. Only Aplasma and Eplasma have a full suite of flagellar genes whereas all but the Ferroplasma spp. have genes for pili production. Cryogenic-electron microscopy (cryo-EM) and tomography (cryo-ET) strengthen these metagenomics-based ultrastructural predictions. Notably, only Aplasma, Gplasma and the Ferroplasma spp. have predicted iron oxidation genes and Eplasma and Iplasma lack most genes for cobalamin, valine, (iso)leucine and histidine synthesis.
The Thermoplasmatales AMD archaea share a large number of metabolic capabilities. All of the uncultivated organisms studied here (A-, E-, G-, and Iplasma) are metabolically very similar to characterized Ferroplasma spp., differentiating themselves mainly in their genetic capabilities for biosynthesis, motility, and possibly iron oxidation. These results indicate that subtle, but important genomic differences, coupled with unknown differences in gene expression, distinguish these organisms enough to allow for co-existence. Overall this study reveals shared features of organisms from the Thermoplasmatales lineage and provides new insights into the functioning of AMD communities.
PMCID: PMC3750248  PMID: 23865623
Metagenomics; Acid mine drainage; Thermoplasmatales; Ferroplasma; Iron oxidation; Comparative genomics
11.  Virus-Host and CRISPR Dynamics in Archaea-Dominated Hypersaline Lake Tyrrell, Victoria, Australia 
Archaea  2013;2013:370871.
The study of natural archaeal assemblages requires community context, namely, a concurrent assessment of the dynamics of archaeal, bacterial, and viral populations. Here, we use filter size-resolved metagenomic analyses to report the dynamics of 101 archaeal and bacterial OTUs and 140 viral populations across 17 samples collected over different timescales from 2007–2010 from Australian hypersaline Lake Tyrrell (LT). All samples were dominated by Archaea (75–95%). Archaeal, bacterial, and viral populations were found to be dynamic on timescales of months to years, and different viral assemblages were present in planktonic, relative to host-associated (active and provirus) size fractions. Analyses of clustered regularly interspaced short palindromic repeat (CRISPR) regions indicate that both rare and abundant viruses were targeted, primarily by lower abundance hosts. Although very few spacers had hits to the NCBI nr database or to the 140 LT viral populations, 21% had hits to unassembled LT viral concentrate reads. This suggests local adaptation to LT-specific viruses and/or undersampling of haloviral assemblages in public databases, along with successful CRISPR-mediated maintenance of viral populations at abundances low enough to preclude genomic assembly. This is the first metagenomic report evaluating widespread archaeal dynamics at the population level on short timescales in a hypersaline system.
PMCID: PMC3703381  PMID: 23853523
12.  Heterotrophic Archaea Contribute to Carbon Cycling in Low-pH, Suboxic Biofilm Communities 
Applied and Environmental Microbiology  2012;78(23):8321-8330.
Archaea are widely distributed and yet are most often not the most abundant members of microbial communities. Here, we document a transition from Bacteria- to Archaea-dominated communities in microbial biofilms sampled from the Richmond Mine acid mine drainage (AMD) system (∼pH 1.0, ∼38°C) and in laboratory-cultivated biofilms. This transition occurs when chemoautotrophic microbial communities that develop at the air-solution interface sink to the sediment-solution interface and degrade under microaerobic and anaerobic conditions. The archaea identified in these sunken biofilms are from the class Thermoplasmata, and in some cases, the highly divergent ARMAN nanoarchaeal lineage. In several of the sunken biofilms, nanoarchaea comprise 10 to 25% of the community, based on fluorescent in situ hybridization and metagenomic analyses. Comparative community proteomic analyses show a persistence of bacterial proteins in sunken biofilms, but there is clear evidence for amino acid modifications due to acid hydrolysis. Given the low representation of bacterial cells in sunken biofilms based on microscopy, we infer that hydrolysis reflects proteins derived from lysed cells. For archaea, we detected ∼2,400 distinct proteins, including a subset involved in proteolysis and peptide uptake. Laboratory cultivation experiments using complex carbon substrates demonstrated anaerobic enrichment of Ferroplasma and Aplasma coupled to the reduction of ferric iron. These findings indicate dominance of acidophilic archaea in degrading biofilms and suggest that they play roles in anaerobic nutrient cycling at low pH.
PMCID: PMC3497393  PMID: 23001646
13.  Effect of Rainfall-Induced Soil Geochemistry Dynamics on Grassland Soil Microbial Communities 
Applied and Environmental Microbiology  2012;78(21):7587-7595.
In Mediterranean-type grassland ecosystems, the timing of rainfall events controls biogeochemical cycles, as well as the phenology and productivity of plants and animals. Here, we investigate the effect of short-term (days) soil environmental conditions on microbial community structure and composition during a natural wetting and drying cycle. Soil samples were collected from a meadow in Northern California at four time points after the first two rainfall events of the rainy season. We used 16S rRNA microarrays (PhyloChip) to track changes in bacterial and archaeal community composition. Microbial communities at time points 1 and 3 were significantly different than communities at time points 2 and 4. Based on ordination analysis, the available carbon, soil moisture, and temperature explained most of the variation in community structure. For the first time, a complementary and more comprehensive approach using linear regression and generalized logical networks were used to identify linear and nonlinear associations among environmental variables and with the relative abundance of subfamilies. Changes in soil moisture and available carbon were correlated with the relative abundance of many phyla. Only the phylum Actinobacteria showed a lineage-specific relationship to soil moisture but not to carbon or nitrogen. The results indicate that the use of a high taxonomic rank in correlations with nutritional indicators might obscure divergent subfamily-level responses to environmental parameters. An important implication of this research is that there is short-term variation in microbial community composition driven in part by rainfall fluctuation that may not be evident in long-term studies with coarser time resolution.
PMCID: PMC3485702  PMID: 22904056
14.  Quantifying Heavy Metals Sequestration by Sulfate-Reducing Bacteria in an Acid Mine Drainage-Contaminated Natural Wetland 
Bioremediation strategies that depend on bacterial sulfate reduction for heavy metals remediation harness the reactivity of these metals with biogenic aqueous sulfide. Quantitative knowledge of the degree to which specific toxic metals are partitioned into various sulfide, oxide, or other phases is important for predicting the long-term mobility of these metals under environmental conditions. Here we report the quantitative partitioning into sedimentary biogenic sulfides of a suite of metals and metalloids associated with acid mine drainage contamination of a natural estuarine wetland for over a century.
PMCID: PMC3594707  PMID: 23487496
acid mine drainage; heavy metals; metal-sulfides; wetlands; bioremediation; electron microprobe; bacterial sulfate reduction; sulfate-reducing bacteria
15.  Metabolites Associated with Adaptation of Microorganisms to an Acidophilic, Metal-Rich Environment Identified by Stable-Isotope-Enabled Metabolomics 
mBio  2013;4(2):e00484-12.
Microorganisms grow under a remarkable range of extreme conditions. Environmental transcriptomic and proteomic studies have highlighted metabolic pathways active in extremophilic communities. However, metabolites directly linked to their physiology are less well defined because metabolomics methods lag behind other omics technologies due to a wide range of experimental complexities often associated with the environmental matrix. We identified key metabolites associated with acidophilic and metal-tolerant microorganisms using stable isotope labeling coupled with untargeted, high-resolution mass spectrometry. We observed >3,500 metabolic features in biofilms growing in pH ~0.9 acid mine drainage solutions containing millimolar concentrations of iron, sulfate, zinc, copper, and arsenic. Stable isotope labeling improved chemical formula prediction by >50% for larger metabolites (>250 atomic mass units), many of which were unrepresented in metabolic databases and may represent novel compounds. Taurine and hydroxyectoine were identified and likely provide protection from osmotic stress in the biofilms. Community genomic, transcriptomic, and proteomic data implicate fungi in taurine metabolism. Leptospirillum group II bacteria decrease production of ectoine and hydroxyectoine as biofilms mature, suggesting that biofilm structure provides some resistance to high metal and proton concentrations. The combination of taurine, ectoine, and hydroxyectoine may also constitute a sulfur, nitrogen, and carbon currency in the communities.
Microbial communities are central to many critical global processes and yet remain enigmatic largely due to their complex and distributed metabolic interactions. Metabolomics has the possibility of providing mechanistic insights into the function and ecology of microbial communities. However, our limited knowledge of microbial metabolites, the difficulty of identifying metabolites from complex samples, and the inability to link metabolites directly to community members have proven to be major limitations in developing advances in systems interactions. Here, we show that combining stable-isotope-enabled metabolomics with genomics, transcriptomics, and proteomics can illuminate the ecology of microorganisms at the community scale.
PMCID: PMC3604775  PMID: 23481603
16.  Dynamic Viral Populations in Hypersaline Systems as Revealed by Metagenomic Assembly 
Applied and Environmental Microbiology  2012;78(17):6309-6320.
Viruses of the Bacteria and Archaea play important roles in microbial evolution and ecology, and yet viral dynamics in natural systems remain poorly understood. Here, we created de novo assemblies from 6.4 Gbp of metagenomic sequence from eight community viral concentrate samples, collected from 12 h to 3 years apart from hypersaline Lake Tyrrell (LT), Victoria, Australia. Through extensive manual assembly curation, we reconstructed 7 complete and 28 partial novel genomes of viruses and virus-like entities (VLEs, which could be viruses or plasmids). We tracked these 35 populations across the eight samples and found that they are generally stable on the timescale of days and transient on the timescale of years, with some exceptions. Cross-detection of the 35 LT populations in three previously described haloviral metagenomes was limited to a few genes, and most previously sequenced haloviruses were not detected in our samples, though 3 were detected upon reducing our detection threshold from 90% to 75% nucleotide identity. Similar results were obtained when we applied our methods to haloviral metagenomic data previously reported from San Diego, CA: 10 contigs that we assembled from that system exhibited a variety of detection patterns on a timescale of weeks to 1 month but were generally not detected in LT. Our results suggest that most haloviral populations have a limited or, possibly, a temporally variable global distribution. This study provides high-resolution insight into viral biogeography and dynamics and it places “snapshot” viral metagenomes, collected at a single time and location, in context.
PMCID: PMC3416638  PMID: 22773627
17.  De novo metagenomic assembly reveals abundant novel major lineage of Archaea in hypersaline microbial communities 
The ISME Journal  2011;6(1):81-93.
This study describes reconstruction of two highly unusual archaeal genomes by de novo metagenomic assembly of multiple, deeply sequenced libraries from surface waters of Lake Tyrrell (LT), a hypersaline lake in NW Victoria, Australia. Lineage-specific probes were designed using the assembled genomes to visualize these novel archaea, which were highly abundant in the 0.1–0.8 μm size fraction of lake water samples. Gene content and inferred metabolic capabilities were highly dissimilar to all previously identified hypersaline microbial species. Distinctive characteristics included unique amino acid composition, absence of Gvp gas vesicle proteins, atypical archaeal metabolic pathways and unusually small cell size (approximately 0.6 μm diameter). Multi-locus phylogenetic analyses demonstrated that these organisms belong to a new major euryarchaeal lineage, distantly related to halophilic archaea of class Halobacteria. Consistent with these findings, we propose creation of a new archaeal class, provisionally named ‘Nanohaloarchaea'. In addition to their high abundance in LT surface waters, we report the prevalence of Nanohaloarchaea in other hypersaline environments worldwide. The simultaneous discovery and genome sequencing of a novel yet ubiquitous lineage of uncultivated microorganisms demonstrates that even historically well-characterized environments can reveal unexpected diversity when analyzed by metagenomics, and advances our understanding of the ecology of hypersaline environments and the evolutionary history of the archaea.
PMCID: PMC3246234  PMID: 21716304
assembly; halophile; hypersaline; metagenome; Nanohaloarchaea
18.  X-ray structure of the fourth type of archaeal tRNA splicing endonuclease: insights into the evolution of a novel three-unit composition and a unique loop involved in broad substrate specificity 
Nucleic Acids Research  2012;40(20):10554-10566.
Cleavage of introns from precursor transfer RNAs (tRNAs) by tRNA splicing endonuclease (EndA) is essential for tRNA maturation in Archaea and Eukarya. In the past, archaeal EndAs were classified into three types (α′2, α4 and α2β2) according to subunit composition. Recently, we have identified a fourth type of archaeal EndA from an uncultivated archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2, which is deeply branched within Euryarchaea. The ARMAN-2 EndA forms an ε2 homodimer and has broad substrate specificity like the α2β2 type EndAs found in Crenarchaea and Nanoarchaea. However, the precise architecture of ARMAN-2 EndA was unknown. Here, we report the crystal structure of the ε2 homodimer of ARMAN-2 EndA. The structure reveals that the ε protomer is separated into three novel units (αN, α and βC) fused by two distinct linkers, although the overall structure of ARMAN-2 EndA is similar to those of the other three types of archaeal EndAs. Structural comparison and mutational analyses reveal that an ARMAN-2 type-specific loop (ASL) is involved in the broad substrate specificity and that K161 in the ASL functions as the RNA recognition site. These findings suggest that the broad substrate specificities of ε2 and α2β2 EndAs were separately acquired through different evolutionary processes.
PMCID: PMC3488258  PMID: 22941657
19.  Quantitative proteomic analyses of the response of acidophilic microbial communities to different pH conditions 
The ISME Journal  2011;5(7):1152-1161.
Extensive genomic characterization of multi-species acid mine drainage microbial consortia combined with laboratory cultivation has enabled the application of quantitative proteomic analyses at the community level. In this study, quantitative proteomic comparisons were used to functionally characterize laboratory-cultivated acidophilic communities sustained in pH 1.45 or 0.85 conditions. The distributions of all proteins identified for individual organisms indicated biases for either high or low pH, and suggests pH-specific niche partitioning for low abundance bacteria and archaea. Although the proteome of the dominant bacterium, Leptospirillum group II, was largely unaffected by pH treatments, analysis of functional categories indicated proteins involved in amino acid and nucleotide metabolism, as well as cell membrane/envelope biogenesis were overrepresented at high pH. Comparison of specific protein abundances indicates higher pH conditions favor Leptospirillum group III, whereas low pH conditions promote the growth of certain archaea. Thus, quantitative proteomic comparisons revealed distinct differences in community composition and metabolic function of individual organisms during different pH treatments. Proteomic analysis revealed other aspects of community function. Different numbers of phage proteins were identified across biological replicates, indicating stochastic spatial heterogeneity of phage outbreaks. Additionally, proteomic data were used to identify a previously unknown genotypic variant of Leptospirillum group II, an indication of selection for a specific Leptospirillum group II population in laboratory communities. Our results confirm the importance of pH and related geochemical factors in fine-tuning acidophilic microbial community structure and function at the species and strain level, and demonstrate the broad utility of proteomics in laboratory community studies.
PMCID: PMC3146278  PMID: 21228889
acid mine drainage; communities; genotyping; perturbation; proteomics
20.  Phage-Induced Expression of CRISPR-Associated Proteins Is Revealed by Shotgun Proteomics in Streptococcus thermophilus 
PLoS ONE  2012;7(5):e38077.
The CRISPR/Cas system, comprised of clustered regularly interspaced short palindromic repeats along with their associated (Cas) proteins, protects bacteria and archaea from viral predation and invading nucleic acids. While the mechanism of action for this acquired immunity is currently under investigation, the response of Cas protein expression to phage infection has yet to be elucidated. In this study, we employed shotgun proteomics to measure the global proteome expression in a model system for studying the CRISPR/Cas response in S. thermophilus DGCC7710 infected with phage 2972. Host and viral proteins were simultaneously measured following inoculation at two different multiplicities of infection and across various time points using two-dimensional liquid chromatography tandem mass spectrometry. Thirty-seven out of forty predicted viral proteins were detected, including all proteins of the structural virome and viral effector proteins. In total, 1,013 of 2,079 predicted S. thermophilus proteins were detected, facilitating the monitoring of host protein synthesis changes in response to virus infection. Importantly, Cas proteins from all four CRISPR loci in the S. thermophilus DGCC7710 genome were detected, including loci previously thought to be inactive. Many Cas proteins were found to be constitutively expressed, but several demonstrated increased abundance following infection, including the signature Cas9 proteins from the CRISPR1 and CRISPR3 loci, which are key players in the interference phase of the CRISPR/Cas response. Altogether, these results provide novel insights into the proteomic response of S. thermophilus, specifically CRISPR-associated proteins, upon phage 2972 infection.
PMCID: PMC3364186  PMID: 22666452
21.  Identification of Biofilm Matrix-Associated Proteins from an Acid Mine Drainage Microbial Community ▿ †  
Applied and Environmental Microbiology  2011;77(15):5230-5237.
In microbial communities, extracellular polymeric substances (EPS), also called the extracellular matrix, provide the spatial organization and structural stability during biofilm development. One of the major components of EPS is protein, but it is not clear what specific functions these proteins contribute to the extracellular matrix or to microbial physiology. To investigate this in biofilms from an extremely acidic environment, we used shotgun proteomics analyses to identify proteins associated with EPS in biofilms at two developmental stages, designated DS1 and DS2. The proteome composition of the EPS was significantly different from that of the cell fraction, with more than 80% of the cellular proteins underrepresented or undetectable in EPS. In contrast, predicted periplasmic, outer membrane, and extracellular proteins were overrepresented by 3- to 7-fold in EPS. Also, EPS proteins were more basic by ∼2 pH units on average and about half the length. When categorized by predicted function, proteins involved in motility, defense, cell envelope, and unknown functions were enriched in EPS. Chaperones, such as histone-like DNA binding protein and cold shock protein, were overrepresented in EPS. Enzymes, such as protein peptidases, disulfide-isomerases, and those associated with cell wall and polysaccharide metabolism, were also detected. Two of these enzymes, identified as β-N-acetylhexosaminidase and cellulase, were confirmed in the EPS fraction by enzymatic activity assays. Compared to the differences between EPS and cellular fractions, the relative differences in the EPS proteomes between DS1 and DS2 were smaller and consistent with expected physiological changes during biofilm development.
PMCID: PMC3147463  PMID: 21685158
22.  A Semi-Quantitative, Synteny-Based Method to Improve Functional Predictions for Hypothetical and Poorly Annotated Bacterial and Archaeal Genes 
PLoS Computational Biology  2011;7(10):e1002230.
During microbial evolution, genome rearrangement increases with increasing sequence divergence. If the relationship between synteny and sequence divergence can be modeled, gene clusters in genomes of distantly related organisms exhibiting anomalous synteny can be identified and used to infer functional conservation. We applied the phylogenetic pairwise comparison method to establish and model a strong correlation between synteny and sequence divergence in all 634 available Archaeal and Bacterial genomes from the NCBI database and four newly assembled genomes of uncultivated Archaea from an acid mine drainage (AMD) community. In parallel, we established and modeled the trend between synteny and functional relatedness in the 118 genomes available in the STRING database. By combining these models, we developed a gene functional annotation method that weights evolutionary distance to estimate the probability of functional associations of syntenous proteins between genome pairs. The method was applied to the hypothetical proteins and poorly annotated genes in newly assembled acid mine drainage Archaeal genomes to add or improve gene annotations. This is the first method to assign possible functions to poorly annotated genes through quantification of the probability of gene functional relationships based on synteny at a significant evolutionary distance, and has the potential for broad application.
Author Summary
Based on trends between gene sequence divergence and gene order divergence over time, we developed a new synteny-based method to refine functional annotation. This method uses these trends to determine the probability that any two syntenous genes (genes that are sequential in two organisms) are functionally related. Organisms that are distant relatives have few syntenous genes, but these syntenous genes have a very high probability of functional relatedness. We applied this method to newly assembled genomes of co-occurring, uncultivated acid mine drainage Archaea in order to improve their gene annotations. This application revealed important physiological differences between the co-occurring organisms in this clade, including the ability of some but not all of the Archaea to manufacture vitamin B12 and to carry out anaerobic energy metabolism. We also used this method to identify new genes possibly involved in vitamin B12 synthesis, ether lipid synthesis, molybdopterin synthesis and utilization, and microbial immunity through the CRISPR system.
PMCID: PMC3197636  PMID: 22028637
23.  A novel three-unit tRNA splicing endonuclease found in ultrasmall Archaea possesses broad substrate specificity 
Nucleic Acids Research  2011;39(22):9695-9704.
tRNA splicing endonucleases, essential enzymes found in Archaea and Eukaryotes, are involved in the processing of pre-tRNA molecules. In Archaea, three types of splicing endonuclease [homotetrameric: α4, homodimeric: α2, and heterotetrameric: (αβ)2] have been identified, each representing different substrate specificity during the tRNA intron cleavage. Here, we discovered a fourth type of archaeal tRNA splicing endonuclease (ε2) in the genome of the acidophilic archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2 and its closely related species, ARMAN-1. The enzyme consists of two duplicated catalytic units and one structural unit encoded on a single gene, representing a novel three-unit architecture. Homodimeric formation was confirmed by cross-linking assay, and site-directed mutagenesis determined that the conserved L10-pocket interaction between catalytic and structural unit is necessary for the assembly. A tRNA splicing assay reveal that ε2 endonuclease cleaves both canonical and non-canonical bulge–helix–bulge motifs, similar to that of (αβ)2 endonuclease. Unlike other ARMAN and Euryarchaeota, tRNAs found in ARMAN-2 are highly disrupted by introns at various positions, which again resemble the properties of archaeal species with (αβ)2 endonuclease. Thus, the discovery of ε2 endonuclease in an archaeon deeply branched within Euryarchaeota represents a new example of the coevolution of tRNA and their processing enzymes.
PMCID: PMC3239211  PMID: 21880595
24.  Improved genome annotation through untargeted detection of pathway-specific metabolites 
BMC Genomics  2011;12(Suppl 1):S6.
Mass spectrometry-based metabolomics analyses have the potential to complement sequence-based methods of genome annotation, but only if raw mass spectral data can be linked to specific metabolic pathways. In untargeted metabolomics, the measured mass of a detected compound is used to define the location of the compound in chemical space, but uncertainties in mass measurements lead to "degeneracies" in chemical space since multiple chemical formulae correspond to the same measured mass. We compare two methods to eliminate these degeneracies. One method relies on natural isotopic abundances, and the other relies on the use of stable-isotope labeling (SIL) to directly determine C and N atom counts. Both depend on combinatorial explorations of the "chemical space" comprised of all possible chemical formulae comprised of biologically relevant chemical elements.
Of 1532 metabolic pathways curated in the MetaCyc database, 412 contain a metabolite having a chemical formula unique to that metabolic pathway. Thus, chemical formulae alone can suffice to infer the presence of some metabolic pathways. Of 248,928 unique chemical formulae selected from the PubChem database, more than 95% had at least one degeneracy on the basis of accurate mass information alone. Consideration of natural isotopic abundance reduced degeneracy to 64%, but mainly for formulae less than 500 Da in molecular weight, and only if the error in the relative isotopic peak intensity was less than 10%. Knowledge of exact C and N atom counts as determined by SIL enabled reduced degeneracy, allowing for determination of unique chemical formula for 55% of the PubChem formulae.
To facilitate the assignment of chemical formulae to unknown mass-spectral features, profiling can be performed on cultures uniformly labeled with stable isotopes of nitrogen (15N) or carbon (13C). This makes it possible to accurately count the number of carbon and nitrogen atoms in each molecule, providing a robust means for reducing the degeneracy of chemical space and thus obtaining unique chemical formulae for features measured in untargeted metabolomics having a mass greater than 500 Da, with relative errors in measured isotopic peak intensity greater than 10%, and without the use of a chemical formula generator dependent on heuristic filtering. These chemical formulae can serve as indicators for the presence of particular metabolic pathways.
PMCID: PMC3223729  PMID: 21810208
25.  EMIRGE: reconstruction of full-length ribosomal genes from microbial community short read sequencing data 
Genome Biology  2011;12(5):R44.
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
PMCID: PMC3219967  PMID: 21595876

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