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1.  Gut bacteria are rarely shared by co-hospitalized premature infants, regardless of necrotizing enterocolitis development 
eLife  null;4:e05477.
Premature infants are highly vulnerable to aberrant gastrointestinal tract colonization, a process that may lead to diseases like necrotizing enterocolitis. Thus, spread of potential pathogens among hospitalized infants is of great concern. Here, we reconstructed hundreds of high-quality genomes of microorganisms that colonized co-hospitalized premature infants, assessed their metabolic potential, and tracked them over time to evaluate bacterial strain dispersal among infants. We compared microbial communities in infants who did and did not develop necrotizing enterocolitis. Surprisingly, while potentially pathogenic bacteria of the same species colonized many infants, our genome-resolved analysis revealed that strains colonizing each baby were typically distinct. In particular, no strain was common to all infants who developed necrotizing enterocolitis. The paucity of shared gut colonizers suggests the existence of significant barriers to the spread of bacteria among infants. Importantly, we demonstrate that strain-resolved comprehensive community analysis can be accomplished on potentially medically relevant time scales.
DOI: http://dx.doi.org/10.7554/eLife.05477.001
eLife digest
The spread of potentially harmful bacteria is a major source of disease in patients staying in hospitals. Premature babies—born before 37 weeks of pregnancy—can be particularly vulnerable to these infections because their organs may not yet be fully developed. Also, young babies do not have the fully established populations of beneficial microbes that help to protect us from dangerous bacteria.
Necrotizing enterocolitis—a life-threatening disease that can cause portions of the bowel to die—is mostly seen in extremely premature babies. Although it is not known what causes this serious condition, research has suggested that a contagious microbe may be responsible.
The development of methods that can sequence DNA from whole communities of microbes, known as metagenomics, allows researchers to identify the presence of individual strains of bacteria within these communities. This makes it possible to compare and contrast the strains of bacteria present in both diseased and healthy individuals, to help identify the bacteria responsible for a disease.
Here, Raveh-Sadka et al. used a metagenomics approach to study the communities of microbes present in premature babies in a hospital unit during an outbreak of necrotizing enterocolitis. The study found that very few bacterial strains were present in more than one baby, suggesting that bacterial strains are not readily transferred between the babies while they are in the hospital. Furthermore, Raveh-Sadka et al. reveal that no single bacterial strain was shared among all the babies who developed necrotizing enterocolitis.
These findings indicate that necrotizing enterocolitis is not caused by a single strain of bacterium. Instead, if bacteria do contribute to the disease, it maybe that it is caused by a variety of potentially harmful bacteria colonizing the gut at the cost of beneficial bacteria. In future, better understanding of the barriers that limit the transfer of bacteria between premature babies could help inform efforts to reduce the spread of infections between patients in hospitals.
DOI: http://dx.doi.org/10.7554/eLife.05477.002
doi:10.7554/eLife.05477
PMCID: PMC4384745  PMID: 25735037
Microbiota; necrotizing enterocolitis; gut microbial colonization; nosocomial infections; preterm infants; metagenomics; other
2.  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.
DOI: http://dx.doi.org/10.7554/eLife.01102.001
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.
DOI: http://dx.doi.org/10.7554/eLife.01102.002
doi:10.7554/eLife.01102
PMCID: PMC3787301  PMID: 24137540
Cyanobacteria; Melainabacteria; photosynthesis; nitrogen fixation; human gut; subsurface; Human; Other
3.  Fungi Contribute Critical but Spatially Varying Roles in Nitrogen and Carbon Cycling in Acid Mine Drainage 
The ecosystem roles of fungi have been extensively studied by targeting one organism and/or biological process at a time, but the full metabolic potential of fungi has rarely been captured in an environmental context. We hypothesized that fungal genome sequences could be assembled directly from the environment using metagenomics and that transcriptomics and proteomics could simultaneously reveal metabolic differentiation across habitats. We reconstructed the near-complete 27 Mbp genome of a filamentous fungus, Acidomyces richmondensis, and evaluated transcript and protein expression in floating and streamer biofilms from an acid mine drainage (AMD) system. A. richmondensis transcripts involved in denitrification and in the degradation of complex carbon sources (including cellulose) were up-regulated in floating biofilms, whereas central carbon metabolism and stress-related transcripts were significantly up-regulated in streamer biofilms. These findings suggest that the biofilm niches are distinguished by distinct carbon and nitrogen resource utilization, oxygen availability, and environmental challenges. An isolated A. richmondensis strain from this environment was used to validate the metagenomics-derived genome and confirm nitrous oxide production at pH 1. Overall, our analyses defined mechanisms of fungal adaptation and identified a functional shift related to different roles in carbon and nitrogen turnover for the same species of fungi growing in closely located but distinct biofilm niches.
doi:10.3389/fmicb.2016.00238
PMCID: PMC4776211  PMID: 26973616
fungi; metagenomics; transcriptomics; proteomics; carbon; nitrogen; biofilm
4.  Genome-Resolved Metagenomic Analysis Reveals Roles for Candidate Phyla and Other Microbial Community Members in Biogeochemical Transformations in Oil Reservoirs 
mBio  2016;7(1):e01669-15.
ABSTRACT
Oil reservoirs are major sites of methane production and carbon turnover, processes with significant impacts on energy resources and global biogeochemical cycles. We applied a cultivation-independent genomic approach to define microbial community membership and predict roles for specific organisms in biogeochemical transformations in Alaska North Slope oil fields. Produced water samples were collected from six locations between 1,128 m (24 to 27°C) and 2,743 m (80 to 83°C) below the surface. Microbial community complexity decreased with increasing temperature, and the potential to degrade hydrocarbon compounds was most prevalent in the lower-temperature reservoirs. Sulfate availability, rather than sulfate reduction potential, seems to be the limiting factor for sulfide production in some of the reservoirs under investigation. Most microorganisms in the intermediate- and higher-temperature samples were related to previously studied methanogenic and nonmethanogenic archaea and thermophilic bacteria, but one candidate phylum bacterium, a member of the Acetothermia (OP1), was present in Kuparuk sample K3. The greatest numbers of candidate phyla were recovered from the mesothermic reservoir samples SB1 and SB2. We reconstructed a nearly complete genome for an organism from the candidate phylum Parcubacteria (OD1) that was abundant in sample SB1. Consistent with prior findings for members of this lineage, the OD1 genome is small, and metabolic predictions support an obligately anaerobic, fermentation-based lifestyle. At moderate abundance in samples SB1 and SB2 were members of bacteria from other candidate phyla, including Microgenomates (OP11), Atribacteria (OP9), candidate phyla TA06 and WS6, and Marinimicrobia (SAR406). The results presented here elucidate potential roles of organisms in oil reservoir biological processes.
IMPORTANCE
The activities of microorganisms in oil reservoirs impact petroleum resource quality and the global carbon cycle. We show that bacteria belonging to candidate phyla are present in some oil reservoirs and provide the first insights into their potential roles in biogeochemical processes based on several nearly complete genomes.
doi:10.1128/mBio.01669-15
PMCID: PMC4725000  PMID: 26787827
5.  Strain-resolved microbial community proteomics reveals simultaneous aerobic and anaerobic function during gastrointestinal tract colonization of a preterm infant 
While there has been growing interest in the gut microbiome in recent years, it remains unclear whether closely related species and strains have similar or distinct functional roles and if organisms capable of both aerobic and anaerobic growth do so simultaneously. To investigate these questions, we implemented a high-throughput mass spectrometry-based proteomics approach to identify proteins in fecal samples collected on days of life 13–21 from an infant born at 28 weeks gestation. No prior studies have coupled strain-resolved community metagenomics to proteomics for such a purpose. Sequences were manually curated to resolve the genomes of two strains of Citrobacter that were present during the later stage of colonization. Proteome extracts from fecal samples were processed via a nano-2D-LC-MS/MS and peptides were identified based on information predicted from the genome sequences for the dominant organisms, Serratia and the two Citrobacter strains. These organisms are facultative anaerobes, and proteomic information indicates the utilization of both aerobic and anaerobic metabolisms throughout the time series. This may indicate growth in distinct niches within the gastrointestinal tract. We uncovered differences in the physiology of coexisting Citrobacter strains, including differences in motility and chemotaxis functions. Additionally, for both Citrobacter strains we resolved a community-essential role in vitamin metabolism and a predominant role in propionate production. Finally, in this case study we detected differences between genome abundance and activity levels for the dominant populations. This underlines the value in layering proteomic information over genetic potential.
doi:10.3389/fmicb.2015.00654
PMCID: PMC4487087  PMID: 26191049
colonization; infant gut; metaproteomics; microbiome; microbial ecology; physiology
6.  CRISPR Immunity Drives Rapid Phage Genome Evolution in Streptococcus thermophilus 
mBio  2015;6(2):e00262-15.
ABSTRACT
Many bacteria rely on CRISPR-Cas systems to provide adaptive immunity against phages, predation by which can shape the ecology and functioning of microbial communities. To characterize the impact of CRISPR immunization on phage genome evolution, we performed long-term bacterium-phage (Streptococcus thermophilus-phage 2972) coevolution experiments. We found that in this species, CRISPR immunity drives fixation of single nucleotide polymorphisms that accumulate exclusively in phage genome regions targeted by CRISPR. Mutation rates in phage genomes highly exceed those of the host. The presence of multiple phages increased phage persistence by enabling recombination-based formation of chimeric phage genomes in which sequences heavily targeted by CRISPR were replaced. Collectively, our results establish CRISPR-Cas adaptive immunity as a key driver of phage genome evolution under the conditions studied and highlight the importance of multiple coexisting phages for persistence in natural systems.
IMPORTANCE
Phages remain an enigmatic part of the biosphere. As predators, they challenge the survival of host bacteria and archaea and set off an “arms race” involving host immunization countered by phage mutation. The CRISPR-Cas system is adaptive: by capturing fragments of a phage genome upon exposure, the host is positioned to counteract future infections. To investigate this process, we initiated massive deep-sequencing experiments with a host and infective phage and tracked the coevolution of both populations over hundreds of days. In the present study, we found that CRISPR immunity drives the accumulation of phage genome rearrangements (which enable longer phage survival) and escape mutations, establishing CRISPR as one of the fundamental drivers of phage evolution.
doi:10.1128/mBio.00262-15
PMCID: PMC4453577  PMID: 25900652
7.  Small Genomes and Sparse Metabolisms of Sediment-Associated Bacteria from Four Candidate Phyla 
mBio  2013;4(5):e00708-13.
ABSTRACT
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.
IMPORTANCE
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.
doi:10.1128/mBio.00708-13
PMCID: PMC3812714  PMID: 24149512
8.  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.
doi:10.1128/AEM.01938-12
PMCID: PMC3497393  PMID: 23001646
9.  Metabolites Associated with Adaptation of Microorganisms to an Acidophilic, Metal-Rich Environment Identified by Stable-Isotope-Enabled Metabolomics 
mBio  2013;4(2):e00484-12.
ABSTRACT
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.
IMPORTANCE
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.
doi:10.1128/mBio.00484-12
PMCID: PMC3604775  PMID: 23481603
10.  Metabolome-Proteome Differentiation Coupled to Microbial Divergence 
mBio  2010;1(5):e00246-10.
Tandem high-throughput proteomics and metabolomics were employed to functionally characterize natural microbial biofilm communities. Distinct molecular signatures exist for each analyzed sample. Deconvolution of the high-resolution molecular data demonstrates that identified proteins and detected metabolites exhibit organism-specific correlation patterns. These patterns are reflective of the functional differentiation of two bacterial species that share the same genus and that co-occur in the sampled microbial communities. Our analyses indicate that the two species have similar niche breadths and are not in strong competition with one another.
IMPORTANCE
Natural microbial assemblages represent dynamic consortia that exhibit extensive complexity at all levels. In the present study, we demonstrate that correlations between protein and metabolite abundances allow the deconvolution of complex molecular data sets into shared and organism-specific contingents. We demonstrate that evolutionary divergence is associated with the restructuring of cellular metabolic networks, which in turn allows bacterial species to occupy distinct ecological niches. The apparent lack of interspecific competition may explain the extensive population-level genetic heterogeneity observed extensively within microbial communities. The reported findings have broad implications for the in-depth investigation of the ecology and evolution of distinct microbial community members and for leveraging the solution of cryptic metabolic processes in the future.
doi:10.1128/mBio.00246-10
PMCID: PMC2962434  PMID: 20978538
11.  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.
doi:10.1111/j.1574-6976.2008.00144.x
PMCID: PMC2704941  PMID: 19054116
community genomics; CRISPR; genetic heterogeneity; metagenomics; population genomics; virus–host dynamics
12.  Analysis of five complete genome sequences for members of the class Peribacteria in the recently recognized Peregrinibacteria bacterial phylum 
PeerJ  2016;4:e1607.
Five closely related populations of bacteria from the Candidate Phylum (CP) Peregrinibacteria, part of the bacterial Candidate Phyla Radiation (CPR), were sampled from filtered groundwater obtained from an aquifer adjacent to the Colorado River near the town of Rifle, CO, USA. Here, we present the first complete genome sequences for organisms from this phylum. These bacteria have small genomes and, unlike most organisms from other lineages in the CPR, have the capacity for nucleotide synthesis. They invest significantly in biosynthesis of cell wall and cell envelope components, including peptidoglycan, isoprenoids via the mevalonate pathway, and a variety of amino sugars including perosamine and rhamnose. The genomes encode an intriguing set of large extracellular proteins, some of which are very cysteine-rich and may function in attachment, possibly to other cells. Strain variation in these proteins is an important source of genotypic variety. Overall, the cell envelope features, combined with the lack of biosynthesis capacities for many required cofactors, fatty acids, and most amino acids point to a symbiotic lifestyle. Phylogenetic analyses indicate that these bacteria likely represent a new class within the Peregrinibacteria phylum, although they ultimately may be recognized as members of a separate phylum. We propose the provisional taxonomic assignment as ‘Candidatus Peribacter riflensis’, Genus Peribacter, Family Peribacteraceae, Order Peribacterales, Class Peribacteria in the phylum Peregrinibacteria.
doi:10.7717/peerj.1607
PMCID: PMC4736985  PMID: 26844018
Peregrinibacteria; Candidate phyla radiation; Metagenomics; Complete genomes; Strain variation
13.  Elevated temperature alters proteomic responses of individual organisms within a biofilm community 
The ISME Journal  2014;9(1):180-194.
Microbial communities that underpin global biogeochemical cycles will likely be influenced by elevated temperature associated with environmental change. Here, we test an approach to measure how elevated temperature impacts the physiology of individual microbial groups in a community context, using a model microbial-based ecosystem. The study is the first application of tandem mass tag (TMT)-based proteomics to a microbial community. We accurately, precisely and reproducibly quantified thousands of proteins in biofilms growing at 40, 43 and 46 °C. Elevated temperature led to upregulation of proteins involved in amino-acid metabolism at the level of individual organisms and the entire community. Proteins from related organisms differed in their relative abundance and functional responses to temperature. Elevated temperature repressed carbon fixation proteins from two Leptospirillum genotypes, whereas carbon fixation proteins were significantly upregulated at higher temperature by a third member of this genus. Leptospirillum group III bacteria may have been subject to viral stress at elevated temperature, which could lead to greater carbon turnover in the microbial food web through the release of viral lysate. Overall, these findings highlight the utility of proteomics-enabled community-based physiology studies, and provide a methodological framework for possible extension to additional mixed culture and environmental sample analyses.
doi:10.1038/ismej.2014.113
PMCID: PMC4274423  PMID: 25050524
14.  A portable cryo-plunger for on-site intact cryogenic microscopy sample preparation in natural environments 
Microscopy research and technique  2011;75(6):829-836.
We present a modern, light portable device specifically designed for environmental samples for cryo-electron microscopy by on-site cryo-plunging. The power of cryogenic EM comes from preparation of artifact-free samples. However, in many studies the samples must be collected at remote field locations, and the time involved in transporting samples back to the laboratory for cryogenic preservation can lead to severe degradation artifacts. Thus, going back to the basics, we developed a simple mechanical device that is light and easy to transport on foot yet effective. With the system design presented here we are able to obtain cryo-samples of microbes and microbial communities not possible to culture, in their near-intact environmental conditions as well as in routine laboratory work, and in real time. This methodology thus enables us to bring the power of cryo-TEM to microbial ecology.
doi:10.1002/jemt.22001
PMCID: PMC4677670  PMID: 22213355
Cryo-electron microscopy; Cryo-plunging; Environmental microbial communities; Archaea; Extremophiles
15.  Development of an Enhanced Metaproteomic Approach for Deepening the Microbiome Characterization of the Human Infant Gut 
Journal of Proteome Research  2014;14(1):133-141.
The establishment of early life microbiota in the human infant gut is highly variable and plays a crucial role in host nutrient availability/uptake and maturation of immunity. Although high-performance mass spectrometry (MS)-based metaproteomics is a powerful method for the functional characterization of complex microbial communities, the acquisition of comprehensive metaproteomic information in human fecal samples is inhibited by the presence of abundant human proteins. To alleviate this restriction, we have designed a novel metaproteomic strategy based on double filtering (DF) the raw samples, a method that fractionates microbial from human cells to enhance microbial protein identification and characterization in complex fecal samples from healthy premature infants. This method dramatically improved the overall depth of infant gut proteome measurement, with an increase in the number of identified low-abundance proteins and a greater than 2-fold improvement in microbial protein identification and quantification. This enhancement of proteome measurement depth enabled a more extensive microbiome comparison between infants by not only increasing the confidence of identified microbial functional categories but also revealing previously undetected categories.
doi:10.1021/pr500936p
PMCID: PMC4286196  PMID: 25350865
Metaproteome; human infant gut; shotgun proteomics; double filtering
16.  Metabolic interdependencies between phylogenetically novel fermenters and respiratory organisms in an unconfined aquifer 
The ISME Journal  2014;8(7):1452-1463.
Fermentation-based metabolism is an important ecosystem function often associated with environments rich in organic carbon, such as wetlands, sewage sludge and the mammalian gut. The diversity of microorganisms and pathways involved in carbon and hydrogen cycling in sediments and aquifers and the impacts of these processes on other biogeochemical cycles remain poorly understood. Here we used metagenomics and proteomics to characterize microbial communities sampled from an aquifer adjacent to the Colorado River at Rifle, CO, USA, and document interlinked microbial roles in geochemical cycling. The organic carbon content in the aquifer was elevated via acetate amendment of the groundwater occurring over 2 successive years. Samples were collected at three time points, with the objective of extensive genome recovery to enable metabolic reconstruction of the community. Fermentative community members include organisms from a new phylum, Melainabacteria, most closely related to Cyanobacteria, phylogenetically novel members of the Chloroflexi and Bacteroidales, as well as candidate phyla genomes (OD1, BD1-5, SR1, WWE3, ACD58, TM6, PER and OP11). These organisms have the capacity to produce hydrogen, acetate, formate, ethanol, butyrate and lactate, activities supported by proteomic data. The diversity and expression of hydrogenases suggests the importance of hydrogen metabolism in the subsurface. Our proteogenomic data further indicate the consumption of fermentation intermediates by Proteobacteria can be coupled to nitrate, sulfate and iron reduction. Thus, fermentation carried out by previously unknown members of sediment microbial communities may be an important driver of nitrogen, hydrogen, sulfur, carbon and iron cycling.
doi:10.1038/ismej.2013.249
PMCID: PMC4069391  PMID: 24621521
metagenomics; proteomics; candidate phylum; hydrogenase; fermentation; sulfate reduction; microbial diversity
17.  Correlative microscopy for phylogenetic and ultrastructural characterization of microbial communities 
Transmission electron microscopy (TEM) can provide ultrastructural information for cells in microbial community samples and phylogenetic information can be recovered via molecular surveys. Here we report an approach to link these datasets by coupling fluorescence in situ hybridization (FISH) with either conventional biological or cryogenic TEM. The method could revolutionize understanding of the organization and functioning of microbial communities in natural systems.
doi:10.1111/j.1758-2229.2011.00275.x
PMCID: PMC4444221  PMID: 23757227
Correlative microscopy; CARD-FISH; microbial communities; acid mine drainage; cryo-electron microscopy; conventional electron microscopy; thin sections; Archaea
18.  Seasonal fluctuations in ionic concentrations drive microbial succession in a hypersaline lake community 
The ISME Journal  2013;8(5):979-990.
Microbial community succession was examined over a two-year period using spatially and temporally coordinated water chemistry measurements, metagenomic sequencing, phylogenetic binning and de novo metagenomic assembly in the extreme hypersaline habitat of Lake Tyrrell, Victoria, Australia. Relative abundances of Haloquadratum-related sequences were positively correlated with co-varying concentrations of potassium, magnesium and sulfate, but not sodium, chloride or calcium ions, while relative abundances of Halorubrum, Haloarcula, Halonotius, Halobaculum and Salinibacter-related sequences correlated negatively with Haloquadratum and these same ionic factors. Nanohaloarchaea and Halorhabdus-related sequence abundances were inversely correlated with each other, but not other taxonomic groups. These data, along with predicted gene functions from nearly-complete assembled population metagenomes, suggest different ecological phenotypes for Nanohaloarchaea and Halorhabdus-related strains versus other community members. Nucleotide percent G+C compositions were consistently lower in community metagenomic reads from summer versus winter samples. The same seasonal G+C trends were observed within taxonomically binned read subsets from each of seven different genus-level archaeal groups. Relative seasonal abundances were also linked to percent G+C for assembled population genomes. Together, these data suggest that extreme ionic conditions may exert selective pressure on archaeal populations at the level of genomic nucleotide composition, thus contributing to seasonal successional processes. Despite the unavailability of cultured representatives for most of the organisms identified in this study, effective coordination of physical and biological measurements has enabled discovery and quantification of unexpected taxon-specific, environmentally mediated factors influencing microbial community structure.
doi:10.1038/ismej.2013.221
PMCID: PMC3996697  PMID: 24335829
archaea; halophile; hypersaline; metagenome; microbial succession
19.  De Novo Sequences of Haloquadratum walsbyi from Lake Tyrrell, Australia, Reveal a Variable Genomic Landscape 
Archaea  2015;2015:875784.
Hypersaline systems near salt saturation levels represent an extreme environment, in which organisms grow and survive near the limits of life. One of the abundant members of the microbial communities in hypersaline systems is the square archaeon, Haloquadratum walsbyi. Utilizing a short-read metagenome from Lake Tyrrell, a hypersaline ecosystem in Victoria, Australia, we performed a comparative genomic analysis of H. walsbyi to better understand the extent of variation between strains/subspecies. Results revealed that previously isolated strains/subspecies do not fully describe the complete repertoire of the genomic landscape present in H. walsbyi. Rearrangements, insertions, and deletions were observed for the Lake Tyrrell derived Haloquadratum genomes and were supported by environmental de novo sequences, including shifts in the dominant genomic landscape of the two most abundant strains. Analysis pertaining to halomucins indicated that homologs for this large protein are not a feature common for all species of Haloquadratum. Further, we analyzed ATP-binding cassette transporters (ABC-type transporters) for evidence of niche partitioning between different strains/subspecies. We were able to identify unique and variable transporter subunits from all five genomes analyzed and the de novo environmental sequences, suggesting that differences in nutrient and carbon source acquisition may play a role in maintaining distinct strains/subspecies.
doi:10.1155/2015/875784
PMCID: PMC4330952  PMID: 25709557
20.  Diverse and divergent protein post-translational modifications in two growth stages of a natural microbial community 
Nature Communications  2014;5:4405.
Detailed characterization of post-translational modifications (PTMs) of proteins in microbial communities remains a significant challenge. Here we directly identify and quantify a broad range of PTMs (hydroxylation, methylation, citrullination, acetylation, phosphorylation, methylthiolation, S-nitrosylation and nitration) in a natural microbial community from an acid mine drainage site. Approximately 29% of the identified proteins of the dominant Leptospirillum group II bacteria are modified, and 43% of modified proteins carry multiple PTM types. Most PTM events, except S-nitrosylations, have low fractional occupancy. Notably, PTM events are detected on Cas proteins involved in antiviral defense, an aspect of Cas biochemistry not considered previously. Further, Cas PTM profiles from Leptospirillum group II differ in early versus mature biofilms. PTM patterns are divergent on orthologues of two closely related, but ecologically differentiated, Leptospirillum group II bacteria. Our results highlight the prevalence and dynamics of PTMs of proteins, with potential significance for ecological adaptation and microbial evolution.
Characterizing post-translational modifications of proteins in microbial communities is challenging. Here, the authors identify and quantify a great number and diversity of such modifications in two growth stages of a natural microbial biofilm.
doi:10.1038/ncomms5405
PMCID: PMC4279252  PMID: 25059763
21.  Comparison of environmental and isolate Sulfobacillus genomes reveals diverse carbon, sulfur, nitrogen, and hydrogen metabolisms 
BMC Genomics  2014;15:1107.
Background
Bacteria of the genus Sulfobacillus are found worldwide as members of microbial communities that accelerate sulfide mineral dissolution in acid mine drainage environments (AMD), acid-rock drainage environments (ARD), as well as in industrial bioleaching operations. Despite their frequent identification in these environments, their role in biogeochemical cycling is poorly understood.
Results
Here we report draft genomes of five species of the Sulfobacillus genus (AMDSBA1-5) reconstructed by cultivation-independent sequencing of biofilms sampled from the Richmond Mine (Iron Mountain, CA). Three of these species (AMDSBA2, AMDSBA3, and AMDSBA4) have no cultured representatives while AMDSBA1 is a strain of S. benefaciens, and AMDSBA5 a strain of S. thermosulfidooxidans. We analyzed the diversity of energy conservation and central carbon metabolisms for these genomes and previously published Sulfobacillus genomes. Pathways of sulfur oxidation vary considerably across the genus, including the number and type of subunits of putative heterodisulfide reductase complexes likely involved in sulfur oxidation. The number and type of nickel-iron hydrogenase proteins varied across the genus, as does the presence of different central carbon pathways. Only the AMDSBA3 genome encodes a dissimilatory nitrate reducatase and only the AMDSBA5 and S. thermosulfidooxidans genomes encode assimilatory nitrate reductases. Within the genus, AMDSBA4 is unusual in that its electron transport chain includes a cytochrome bc type complex, a unique cytochrome c oxidase, and two distinct succinate dehydrogenase complexes.
Conclusions
Overall, the results significantly expand our understanding of carbon, sulfur, nitrogen, and hydrogen metabolism within the Sulfobacillus genus.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1107) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-1107
PMCID: PMC4378227  PMID: 25511286
22.  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.
doi:10.3389/fmicb.2014.00231
PMCID: PMC4032931  PMID: 24904545
continuous culture; enrichment; chemostat; Roseobacter; Maritimibacter; stop codon
23.  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.
doi:10.1128/AEM.01946-13
PMCID: PMC3811486  PMID: 23995931
24.  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.
doi:10.1038/ismej.2012.148
PMCID: PMC3603403  PMID: 23190730
autotroph; metagenomics; proteomics; sediment; subsurface; syntrophy
25.  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.
doi:10.1038/ismej.2012.103
PMCID: PMC3554402  PMID: 23038172
iron-reducing bacteria; biomineralization; correlative microscopy; cryo-transmission electron microscopy; STXM; X-ray absorption spectroscopy

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