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© 2020 The Authors. Published under the terms of the CC BY 4.0 license Mutually exclusive expression of the var multigene family is key to immune evasion and pathogenesis in Plasmodium falciparum, but few factors have been shown to play a direct role. We adapted a CRISPR-based proteomics approach to identify novel factors associated with var genes in their natural chromatin context. Catalytically inactive Cas9 (“dCas9”) was targeted to var gene regulatory elements, immunoprecipitated, and analyzed with mass spectrometry. Known and novel factors were enriched including structural proteins, DNA helicases, and chromatin remodelers. Functional characterization of PfISWI, an evolutionarily divergent putative chromatin remodeler enriched at the var gene promoter, revealed a role in transcriptional activation. Proteomics of PfISWI identified several proteins enriched at the var gene promoter such as acetyl-CoA synthetase, a putative MORC protein, and an ApiAP2 transcription factor. These findings validate the CRISPR/dCas9 proteomics method and define a new var gene-associated chromatin complex. This study establishes a tool for targeted chromatin purification of unaltered genomic loci and identifies novel chromatin-associated factors potentially involved in transcriptional control and/or chromatin organization of virulence genes in the human malaria parasite.

Study designProspective cross-sectional survey.ObjectiveTo determine the perspectives of parents of patients undergoing posterior instrumented fusion for adolescent idiopathic scoliosis (AIS) regarding simultaneous surgery and trainee participation.Summary of background dataSimultaneous ("at the same time") surgery is under scrutiny by the public, government, payers, and the medical community. The objective of this study is to determine the perspectives of parents of patients undergoing posterior instrumented fusion for adolescent idiopathic scoliosis. Our goal is to inform the national conversation on this subject with real patient and family voices.MethodsA survey was prospectively administered to 31 consecutive parents of patients undergoing posterior instrumented fusion for adolescent idiopathic scoliosis at a large academic medical center. "Overlapping" was defined as simultaneity during "noncritical" parts of an operation. "Concurrent" was defined as simultaneity that includes "critical" part(s) of an operation. Participants were asked to provide levels of agreement with overlapping and concurrent surgery and anesthesia, as well as with trainee involvement.ResultsOn average, respondents "strongly agree" with the need to be informed about overlapping or concurrent surgery. They "disagree" with both overlapping and concurrent scheduling, and "disagree" with trainees operating without direct supervision, even for "noncritical" parts. Informing parents about the presence of a back-up surgeon or research demonstrating safety of simultaneous surgery did not make them agreeable to simultaneous scheduling.ConclusionParents have a strong desire to be informed of simultaneous spinal surgery and anesthesia as part of consent on behalf of their children. Their disagreement with simultaneous surgery, as well as with trainees operating without direct supervision, suggests discordance with current guidelines and practice and should inform the national conversation moving for

Infection with the unicellular eukaryotic pathogen Plasmodium falciparum causes malaria, a mosquito-borne disease affecting more than 200 million and killing 400,000 people each year. Underlying the asexual replication within human red blood cells is a tight regulatory network of gene expression and protein synthesis.

© 2020 The Authors. Published under the terms of the CC BY 4.0 license Mutually exclusive expression of the var multigene family is key to immune evasion and pathogenesis in Plasmodium falciparum, but few factors have been shown to play a direct role. We adapted a CRISPR-based proteomics approach to identify novel factors associated with var genes in their natural chromatin context. Catalytically inactive Cas9 (“dCas9”) was targeted to var gene regulatory elements, immunoprecipitated, and analyzed with mass spectrometry. Known and novel factors were enriched including structural proteins, DNA helicases, and chromatin remodelers. Functional characterization of PfISWI, an evolutionarily divergent putative chromatin remodeler enriched at the var gene promoter, revealed a role in transcriptional activation. Proteomics of PfISWI identified several proteins enriched at the var gene promoter such as acetyl-CoA synthetase, a putative MORC protein, and an ApiAP2 transcription factor. These findings validate the CRISPR/dCas9 proteomics method and define a new var gene-associated chromatin complex. This study establishes a tool for targeted chromatin purification of unaltered genomic loci and identifies novel chromatin-associated factors potentially involved in transcriptional control and/or chromatin organization of virulence genes in the human malaria parasite.

BackgroundOsteochondral injuries of the elbow are limiting and affect the ability of pediatric and adolescent athletes to participate in sports.PurposeTo report short- and midterm outcomes on athletes undergoing microfracture or fragment fixation of osteochondral elbow lesions and evaluate the effects thereof on sporting activity.Study designCase series; Level of evidence, 4.MethodsThis was a retrospective study analyzing patients who underwent surgical treatment via microfracture or fragment fixation for osteochondral elbow lesions. Patients were treated at a single institution by a single surgeon between 2012 and 2019. Diagnosis was confirmed with magnetic resonance imaging, and patients were indicated for surgery after having persistent symptoms despite trialing rest, immobilization, and/or activity restriction for at least 3 months. Demographic data including sports of choice were collected preoperatively. Imaging and intraoperative findings were documented, and any complications were noted. Range of motion (ROM) was compared pre- to postoperatively. Return-to-sport evaluation included the ability to play the preoperative sport of choice.ResultsIn total, 23 patients (25 elbows) were included with a mean follow-up of 23.5 months (range, 6-60.3 months) and a mean age of 13.8 years. Of 25 lesions, 20 (80%) were on the athlete's dominant side. There was significant improvement from pre- to postoperative ROM, including extension (mean ± SD, 6.4° ± 5.3° to 0.04° ± 0.2°; P < .00001), flexion (129.2° ± 10.6° to 138.6° ± 4.4°; P = .00013), and arc of ROM (122.6° ± 13.2° to 138.6° ± 4.4°; P < .00001). Mean lesion size was 81.9 ± 59.3 mm2 (range, 15-225 mm2). All elbows demonstrated radiographic healing postoperatively. Mean time to release to sport was 4.48 ± 1.38 months (range, 2.5-8 months). Six (26.1%) patients changed or stopped their preoperative sporting activity, including 2 of 4 gymnasts and 4 of 11 baseball players.ConclusionArthroscopic technique with lesi

BackgroundOsteochondral injuries of the elbow are limiting and affect the ability of pediatric and adolescent athletes to participate in sports.PurposeTo report short- and midterm outcomes on athletes undergoing microfracture or fragment fixation of osteochondral elbow lesions and evaluate the effects thereof on sporting activity.Study designCase series; Level of evidence, 4.MethodsThis was a retrospective study analyzing patients who underwent surgical treatment via microfracture or fragment fixation for osteochondral elbow lesions. Patients were treated at a single institution by a single surgeon between 2012 and 2019. Diagnosis was confirmed with magnetic resonance imaging, and patients were indicated for surgery after having persistent symptoms despite trialing rest, immobilization, and/or activity restriction for at least 3 months. Demographic data including sports of choice were collected preoperatively. Imaging and intraoperative findings were documented, and any complications were noted. Range of motion (ROM) was compared pre- to postoperatively. Return-to-sport evaluation included the ability to play the preoperative sport of choice.ResultsIn total, 23 patients (25 elbows) were included with a mean follow-up of 23.5 months (range, 6-60.3 months) and a mean age of 13.8 years. Of 25 lesions, 20 (80%) were on the athlete's dominant side. There was significant improvement from pre- to postoperative ROM, including extension (mean ± SD, 6.4° ± 5.3° to 0.04° ± 0.2°; P < .00001), flexion (129.2° ± 10.6° to 138.6° ± 4.4°; P = .00013), and arc of ROM (122.6° ± 13.2° to 138.6° ± 4.4°; P < .00001). Mean lesion size was 81.9 ± 59.3 mm2 (range, 15-225 mm2). All elbows demonstrated radiographic healing postoperatively. Mean time to release to sport was 4.48 ± 1.38 months (range, 2.5-8 months). Six (26.1%) patients changed or stopped their preoperative sporting activity, including 2 of 4 gymnasts and 4 of 11 baseball players.ConclusionArthroscopic technique with lesi

Malaria pathogenesis results from the asexual replication of Plasmodium falciparum within human red blood cells, which relies on a precisely timed cascade of gene expression over a 48-h life cycle. Although substantial post-transcriptional regulation of this hardwired program has been observed, it remains unclear how these processes are mediated on a transcriptome-wide level. To this end, we identified mRNA modifications in the P. falciparum transcriptome and performed a comprehensive characterization of N6-methyladenosine (m⁶A) over the course of blood-stage development. Using mass spectrometry and m⁶A RNA sequencing, we demonstrate that m6A is highly developmentally regulated, exceeding m⁶A levels known in any other eukaryote. We characterize a distinct m⁶A writer complex and show that knockdown of the putative m⁶A methyltransferase, PfMT-A70, by CRISPR interference leads to increased levels of transcripts that normally contain m6A. In accordance, we find an inverse correlation between m6A methylation and mRNA stability or translational efficiency. We further identify two putative m6A-binding YTH proteins that are likely to be involved in the regulation of these processes across the parasite’s life cycle. Our data demonstrate unique features of an extensive m⁶A mRNA methylation programme in malaria parasites and reveal its crucial role in dynamically fine-tuning the transcriptional cascade of a unicellular eukaryote.

Heterochromatin plays a central role in the process of immune evasion, pathogenesis, and transmission of the malaria parasite Plasmodium falciparum during blood stage infection. Here, we use ChIP sequencing to demonstrate that sporozoites from mosquito salivary glands expand heterochromatin at subtelomeric regions to silence blood-stage-specific genes. Our data also revealed that heterochromatin enrichment is predictive of the transcription status of clonally variant genes members that mediate cytoadhesion in blood stage parasites. A specific member (here called NF54varsporo) of the var gene family remains euchromatic, and the resultant PfEMP1 (NF54_SpzPfEMP1) is expressed at the sporozoite surface. NF54_SpzPfEMP1-specific antibodies efficiently block hepatocyte infection in a strain-specific manner. Furthermore, human volunteers immunized with infective sporozoites developed antibodies against NF54_SpzPfEMP1. Overall, we show that the epigenetic signature of var genes is reset in mosquito stages. Moreover, the identification of a strain-specific sporozoite PfEMP1 is highly relevant for vaccine design based on sporozoites.

Heterochromatin plays a central role in the process of immune evasion, pathogenesis, and transmission of the malaria parasite Plasmodium falciparum during blood stage infection. Here, we use ChIP sequencing to demonstrate that sporozoites from mosquito salivary glands expand heterochromatin at subtelomeric regions to silence blood-stage-specific genes. Our data also revealed that heterochromatin enrichment is predictive of the transcription status of clonally variant genes members that mediate cytoadhesion in blood stage parasites. A specific member (here called NF54varsporo) of the var gene family remains euchromatic, and the resultant PfEMP1 (NF54_SpzPfEMP1) is expressed at the sporozoite surface. NF54_SpzPfEMP1-specific antibodies efficiently block hepatocyte infection in a strain-specific manner. Furthermore, human volunteers immunized with infective sporozoites developed antibodies against NF54_SpzPfEMP1. Overall, we show that the epigenetic signature of var genes is reset in mosquito stages. Moreover, the identification of a strain-specific sporozoite PfEMP1 is highly relevant for vaccine design based on sporozoites.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Civil and Environmental Engineering, 2017., Cataloged from PDF version of thesis., Includes bibliographical references., Microorganisms support all life in the oceans and are fundamental to maintaining a habitable biosphere on Earth. However an understanding of their taxonomic and functional distributions across space and time are just beginning to emerge and numerous niches within the marine environment are still awaiting exploration. The motivation for this thesis is to improve our understanding of distributions of microbes and their metabolic potential at Station ALOHA, a long-term study site representative of the North Pacific Subtropical Gyre (NPSG). We observed changes in diversity and community composition at Station ALOHA across time, ocean depth and on plastic debris, a new anthropogenically derived niche in the NPSG. Despite surface waters only experiencing mild seasonal variation in the abiotic environment, using near monthly picoplankton samples collected across a 2-year period at 25m depth, we observed that microbial community composition correlated with solar irradiance, thereby demonstrating seasonal trends. Ocean surface microbes are known to differ fundamentally from those found in the ocean's interior, yet the nature of the transitions from shallow to deep surface water communities is not well understood. Using a high resolution depth series across twelve time points, we observed that microbial communities partitioned into four groups that consisted of all samples above the deep chlorophyll maximum (DCM), 125m samples below the DCM, all 200 m samples and all 500, 770 and 1000m samples. Our data also revealed a sharp discontinuity in genomic traits including GC%, genome size and proteome elemental composition spanning the DCM, suggesting that nitrogen limitation was key to shaping this sharp genomic transition zone across disparate clades. In contrast, we observed that plastic debris in the NPSG forms a habitat for complex microbial assemblages that have organisms, lifestyles and metabolic pathways that are distinct and potentially less nutrient limited than picoplank, by Jessica A. Bryant., Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Civil and Environmental Engineering, 2017., Cataloged from PDF version of thesis., Includes bibliographical references., Microorganisms support all life in the oceans and are fundamental to maintaining a habitable biosphere on Earth. However an understanding of their taxonomic and functional distributions across space and time are just beginning to emerge and numerous niches within the marine environment are still awaiting exploration. The motivation for this thesis is to improve our understanding of distributions of microbes and their metabolic potential at Station ALOHA, a long-term study site representative of the North Pacific Subtropical Gyre (NPSG). We observed changes in diversity and community composition at Station ALOHA across time, ocean depth and on plastic debris, a new anthropogenically derived niche in the NPSG. Despite surface waters only experiencing mild seasonal variation in the abiotic environment, using near monthly picoplankton samples collected across a 2-year period at 25m depth, we observed that microbial community composition correlated with solar irradiance, thereby demonstrating seasonal trends. Ocean surface microbes are known to differ fundamentally from those found in the ocean's interior, yet the nature of the transitions from shallow to deep surface water communities is not well understood. Using a high resolution depth series across twelve time points, we observed that microbial communities partitioned into four groups that consisted of all samples above the deep chlorophyll maximum (DCM), 125m samples below the DCM, all 200 m samples and all 500, 770 and 1000m samples. Our data also revealed a sharp discontinuity in genomic traits including GC%, genome size and proteome elemental composition spanning the DCM, suggesting that nitrogen limitation was key to shaping this sharp genomic transition zone across disparate clades. In contrast, we observed that plastic debris in the NPSG forms a habitat for complex microbial assemblages that have organisms, lifestyles and metabolic pathways that are distinct and potentially less nutrient limited than picoplank, by Jessica A. Bryant., Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Civil and Environmental Engineering, 2017., Cataloged from PDF version of thesis., Includes bibliographical references., Microorganisms support all life in the oceans and are fundamental to maintaining a habitable biosphere on Earth. However an understanding of their taxonomic and functional distributions across space and time are just beginning to emerge and numerous niches within the marine environment are still awaiting exploration. The motivation for this thesis is to improve our understanding of distributions of microbes and their metabolic potential at Station ALOHA, a long-term study site representative of the North Pacific Subtropical Gyre (NPSG). We observed changes in diversity and community composition at Station ALOHA across time, ocean depth and on plastic debris, a new anthropogenically derived niche in the NPSG. Despite surface waters only experiencing mild seasonal variation in the abiotic environment, using near monthly picoplankton samples collected across a 2-year period at 25m depth, we observed that microbial community composition correlated with solar irradiance, thereby demonstrating seasonal trends. Ocean surface microbes are known to differ fundamentally from those found in the ocean's interior, yet the nature of the transitions from shallow to deep surface water communities is not well understood. Using a high resolution depth series across twelve time points, we observed that microbial communities partitioned into four groups that consisted of all samples above the deep chlorophyll maximum (DCM), 125m samples below the DCM, all 200 m samples and all 500, 770 and 1000m samples. Our data also revealed a sharp discontinuity in genomic traits including GC%, genome size and proteome elemental composition spanning the DCM, suggesting that nitrogen limitation was key to shaping this sharp genomic transition zone across disparate clades. In contrast, we observed that plastic debris in the NPSG forms a habitat for complex microbial assemblages that have organisms, lifestyles and metabolic pathways that are distinct and potentially less nutrient limited than picoplank, by Jessica A. Bryant., Ph. D.

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 8 (2017): 1786, doi:10.3389/fmicb.2017.01786., Semi-labile dissolved organic matter (DOM) accumulates in surface waters of the oligotrophic ocean gyres and turns over on seasonal to annual timescales. This reservoir of DOM represents an important source of carbon, energy, and nutrients to marine microbial communities but the identity of the microorganisms and the biochemical pathways underlying the cycling of DOM remain largely uncharacterized. In this study we describe bacteria isolated from the North Pacific Subtropical Gyre (NPSG) near Hawaii that are able to degrade phosphonates associated with high molecular weight dissolved organic matter (HMWDOM), which represents a large fraction of semi-labile DOM. We amended dilution-to-extinction cultures with HMWDOM collected from NPSG surface waters and with purified HMWDOM enriched with polysaccharides bearing alkylphosphonate esters. The HMWDOM-amended cultures were enriched in Roseobacter isolates closely related to Sulfitobacter and close relatives of hydrocarbon-degrading bacteria of the Oceanospirillaceae family, many of which encoded phosphonate degradation pathways. Sulfitobacter cultures encoding C-P lyase were able to catabolize methylphosphonate and 2-hydroxyethylphosphonate, as well as the esters of these phosphonates found in native HMWDOM polysaccharides to acquire phosphorus while producing methane and ethylene, respectively. Conversely, growth of these isolates on HMWDOM polysaccharides as carbon source did not support robust increases in cell yields, suggesting that the constituent carbohydrates in HMWDOM were not readily available to these individual isolates. We postulate that the complete remineralization of HMWDOM polysaccharides requires more complex microbial inter-species interactions. The degradation of phosphonate esters and other common substitutions in marine polysaccharides may be key steps in the turnover of marine DOM., Financial support for this work was provided by the National Science Foundation Center for Microbial Oceanography: Research and Education (award #EF0424599 to DK and ED), the National Science Foundation HOT program (OCE-1260164 to M. J. Church and DK), the Gordon and Betty Moore Foundation (grants #492.01 and #3777 to ED, #3298 to DR, and #3794 to DK), and the Simons Foundation (award ID 329108 to DK, DR, and ED). Additional support was provided by the Agouron Institute through a fellowship to OS.

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 8 (2017): 1786, doi:10.3389/fmicb.2017.01786., Semi-labile dissolved organic matter (DOM) accumulates in surface waters of the oligotrophic ocean gyres and turns over on seasonal to annual timescales. This reservoir of DOM represents an important source of carbon, energy, and nutrients to marine microbial communities but the identity of the microorganisms and the biochemical pathways underlying the cycling of DOM remain largely uncharacterized. In this study we describe bacteria isolated from the North Pacific Subtropical Gyre (NPSG) near Hawaii that are able to degrade phosphonates associated with high molecular weight dissolved organic matter (HMWDOM), which represents a large fraction of semi-labile DOM. We amended dilution-to-extinction cultures with HMWDOM collected from NPSG surface waters and with purified HMWDOM enriched with polysaccharides bearing alkylphosphonate esters. The HMWDOM-amended cultures were enriched in Roseobacter isolates closely related to Sulfitobacter and close relatives of hydrocarbon-degrading bacteria of the Oceanospirillaceae family, many of which encoded phosphonate degradation pathways. Sulfitobacter cultures encoding C-P lyase were able to catabolize methylphosphonate and 2-hydroxyethylphosphonate, as well as the esters of these phosphonates found in native HMWDOM polysaccharides to acquire phosphorus while producing methane and ethylene, respectively. Conversely, growth of these isolates on HMWDOM polysaccharides as carbon source did not support robust increases in cell yields, suggesting that the constituent carbohydrates in HMWDOM were not readily available to these individual isolates. We postulate that the complete remineralization of HMWDOM polysaccharides requires more complex microbial inter-species interactions. The degradation of phosphonate esters and other common substitutions in marine polysaccharides may be key steps in the turnover of marine DOM., Financial support for this work was provided by the National Science Foundation Center for Microbial Oceanography: Research and Education (award #EF0424599 to DK and ED), the National Science Foundation HOT program (OCE-1260164 to M. J. Church and DK), the Gordon and Betty Moore Foundation (grants #492.01 and #3777 to ED, #3298 to DR, and #3794 to DK), and the Simons Foundation (award ID 329108 to DK, DR, and ED). Additional support was provided by the Agouron Institute through a fellowship to OS.

The core properties of microbial genomes, including GC content and genome size, are known to vary widely among different bacteria and archaea1,2. Several hypotheses have been proposed to explain this genomic variability, but the fundamental drivers that shape bacterial and archaeal genomic properties remain uncertain3,4,5,6,7. Here, we report the existence of a sharp genomic transition zone below the photic zone, where bacterial and archaeal genomes and proteomes undergo a community-wide punctuated shift. Across a narrow range of increasing depth of just tens of metres, diverse microbial clades trend towards larger genome size, higher genomic GC content, and proteins with higher nitrogen but lower carbon content. These community-wide changes in genome features appear to be driven by gradients in the surrounding environmental energy and nutrient fields. Collectively, our data support hypotheses invoking nutrient limitation as a central driver in the evolution of core bacterial and archaeal genomic and proteomic properties.

The core properties of microbial genomes, including GC content and genome size, are known to vary widely among different bacteria and archaea1,2. Several hypotheses have been proposed to explain this genomic variability, but the fundamental drivers that shape bacterial and archaeal genomic properties remain uncertain3,4,5,6,7. Here, we report the existence of a sharp genomic transition zone below the photic zone, where bacterial and archaeal genomes and proteomes undergo a community-wide punctuated shift. Across a narrow range of increasing depth of just tens of metres, diverse microbial clades trend towards larger genome size, higher genomic GC content, and proteins with higher nitrogen but lower carbon content. These community-wide changes in genome features appear to be driven by gradients in the surrounding environmental energy and nutrient fields. Collectively, our data support hypotheses invoking nutrient limitation as a central driver in the evolution of core bacterial and archaeal genomic and proteomic properties.

Few microbial time-series studies have been conducted in open ocean habitats having low seasonal variability such as the North Pacific Subtropical Gyre (NPSG), where surface waters experience comparatively mild seasonal variation. To better describe microbial seasonal variability in this habitat, we analyzed rRNA amplicon and shotgun metagenomic data over two years at the Hawaii Ocean Time-series Station ALOHA. We postulated that this relatively stable habitat might reveal different environmental factors that influence planktonic microbial community diversity than those previously observed in more seasonally dynamic habitats. Unexpectedly, the data showed that microbial diversity at 25 m was positively correlated with average wind speed 3 to 10 days prior to sampling. In addition, microbial community composition at 25 m exhibited significant correlations with solar irradiance. Many bacterial groups whose relative abundances varied with solar radiation corresponded to taxa known to exhibit strong seasonality in other oceanic regions. Network co-correlation analysis of 25 m communities showed seasonal transitions in composition, and distinct successional cohorts of co-occurring phylogenetic groups. Similar network analyses of metagenomic data also indicated distinct seasonality in genes originating from cyanophage, and several bacterial clades including SAR116 and SAR324. At 500 m, microbial community diversity and composition did not vary significantly with any measured environmental parameters. The minimal seasonal variability in the NPSG facilitated detection of more subtle environmental influences, such as episodic wind variation, on surface water microbial diversity. Community composition in NPSG surface waters varied in response to solar irradiance, but less dramatically than reported in other ocean provinces., Gordon and Betty Moore Foundation (Grant 492.01), Gordon and Betty Moore Foundation (Grant 3777), United States. Environmental Protection Agency (STAR Fellowship)

Oxygen minimum zones (OMZs) are natural physical features of the world's oceans. They create steep physiochemical gradients in the water column, which most notably include a dramatic draw down in oxygen concentrations over small vertical distances (<100 m). Microbial communities within OMZs play central roles in ocean and global biogeochemical cycles, yet we still lack a fundamental understanding of how microbial biodiversity is distributed across OMZs. Here, we used metagenomic sequencing to investigate microbial diversity across a vertical gradient in the water column during three seasons in the Eastern Tropical South Pacific (ETSP) OMZ. Based on analysis of small subunit ribosomal RNA (SSU rRNA) gene fragments, we found that both taxonomic and phylogenetic diversity declined steeply along the transition from oxygen-rich surface water to the permanent OMZ. We observed similar declines in the diversity of protein-coding gene categories, suggesting a decrease in functional (trait) diversity with depth. Metrics of functional and trait dispersion indicated that microbial communities are phylogenetically and functionally more overdispersed in oxic waters, but clustered within the OMZ. These dispersion patterns suggest that community assembly drivers (e.g., competition, environmental filtering) vary strikingly across the oxygen gradient. To understand the generality of our findings, we compared OMZ results to two marine depth gradients in subtropical oligotrophic sites and found that the oligotrophic sites did not display similar patterns, likely reflecting unique features found in the OMZ. Finally, we discuss how our results may relate to niche theory, diversity–energy relationships and stress gradients., Agouron Institute, Gordon and Betty Moore Foundation, National Science Foundation (U.S.). Science and Technology Center (Award EF0424599)

Understanding patterns in the distribution and abundance of functional traits across a landscape is of fundamental importance to ecology. Mapping these distributions is particularly challenging for species-rich groups with sparse trait measurement coverage, such as flowering plants, insects, and microorganisms. Here, we use likelihood-based character reconstruction to infer and analyze the spatial distribution of unmeasured traits. We apply this framework to a microbial dataset comprised of 11,732 ketosynthase alpha gene sequences extracted from 144 soil samples from three continents to document the spatial distribution of putative microbial polyketide antibiotic production. Antibiotic production is a key competitive strategy for soil microbial survival and performance. Additionally, novel antibiotic discovery is highly relevant to human health, making natural antibiotic production by soil microorganisms a major target for bioprospecting. Our comparison of trait-based biogeographical patterns to patterns based on taxonomy and phylogeny is relevant to our basic understanding of microbial biogeography as well as the pressing need for new antibiotics.

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of John Wiley & Sons for personal use, not for redistribution. The definitive version was published in Environmental Microbiology 16 (2014): 2815-2830, doi:10.1111/1462-2920.12254., A considerable fraction of the Earth's organic carbon exists in dissolved form in seawater. To investigate the roles of planktonic marine microbes in the biogeochemical cycling of this dissolved organic matter (DOM), we performed controlled seawater incubation experiments and followed the responses of an oligotrophic surface water microbial assemblage to perturbations with DOM derived from an axenic culture of Prochlorococcus, or high-molecular weight DOM concentrated from nearby surface waters. The rapid transcriptional responses of both Prochlorococcus and Pelagibacter populations suggested the utilization of organic nitrogen compounds common to both DOM treatments. Along with these responses, both populations demonstrated decreases in gene transcripts associated with nitrogen stress, including those involved in ammonium acquisition. In contrast, responses from low abundance organisms of the NOR5/OM60 gammaproteobacteria were observed later in the experiment, and included elevated levels of gene transcripts associated with polysaccharide uptake and oxidation. In total, these results suggest that numerically dominant oligotrophic microbes rapidly acquire nitrogen from commonly available organic sources, and also point to an important role for carbohydrates found within the DOM pool for sustaining the less abundant microorganisms in these oligotrophic systems., This work was supported by a National Science Foundation Science and Technology Center Award EF0424599 (E.F.D and D.M.K.), grants to D.M.K., D.J.R and E.F.D from the Gordon and Betty Moore Foundation, a gift from the Agouron Institute (to E.F.D.) and a fellowship (202180) to A.K.S. from the Canadian Institutes of Health Research (CIHR).

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of John Wiley & Sons for personal use, not for redistribution. The definitive version was published in Environmental Microbiology 16 (2014): 2815-2830, doi:10.1111/1462-2920.12254., A considerable fraction of the Earth's organic carbon exists in dissolved form in seawater. To investigate the roles of planktonic marine microbes in the biogeochemical cycling of this dissolved organic matter (DOM), we performed controlled seawater incubation experiments and followed the responses of an oligotrophic surface water microbial assemblage to perturbations with DOM derived from an axenic culture of Prochlorococcus, or high-molecular weight DOM concentrated from nearby surface waters. The rapid transcriptional responses of both Prochlorococcus and Pelagibacter populations suggested the utilization of organic nitrogen compounds common to both DOM treatments. Along with these responses, both populations demonstrated decreases in gene transcripts associated with nitrogen stress, including those involved in ammonium acquisition. In contrast, responses from low abundance organisms of the NOR5/OM60 gammaproteobacteria were observed later in the experiment, and included elevated levels of gene transcripts associated with polysaccharide uptake and oxidation. In total, these results suggest that numerically dominant oligotrophic microbes rapidly acquire nitrogen from commonly available organic sources, and also point to an important role for carbohydrates found within the DOM pool for sustaining the less abundant microorganisms in these oligotrophic systems., This work was supported by a National Science Foundation Science and Technology Center Award EF0424599 (E.F.D and D.M.K.), grants to D.M.K., D.J.R and E.F.D from the Gordon and Betty Moore Foundation, a gift from the Agouron Institute (to E.F.D.) and a fellowship (202180) to A.K.S. from the Canadian Institutes of Health Research (CIHR).

Gametogenesis is a complex process that results in a highly differentiated gamete capable of transmitting genetic and epigenetic information to the next generation. In the cases of mammalian spermatogenesis and yeast sporulation, an extreme post-meiotic compaction of the genome is key to gamete function. While genome compaction in sperm is reliant upon a histone-to-protamine transition, yeast spores accomplish compaction with a full complement of histones. Although the mechanisms behind such striking chromatin dynamics are largely unknown, several histone variants and post-translational modifications, especially acetylation of histone H4, have been implicated in these processes. The following studies elucidate the roles of two proteins, BRD4 and the linker histone (Hho1), in chromatin compaction during mouse spermatogenesis and yeast sporulation, respectively. In the post-meiotic phase of mouse spermatogenesis, BRD4 forms a ring structure around the haploid nucleus at the cytoskeletal base of the developing acrosome, which has been implicated in nuclear compaction and shaping. Chromatin immunoprecipitation followed by mass spectrometry and sequencing in post-meiotic cells revealed that BRD4, a bromodomain-containing protein, binds to acetylated histone H4 and is enriched in intergenic regions of the genome where histone replacement by protamines is most predominant in mature sperm. Thus, BRD4 may provide a structural link between the contractile force of acrosome formation and the removal of acetylated histones from the genome. In contrast to sperm, spores must use a histone-based chromatin compaction mechanism. During sporulation, Hho1 plays a dual role: transcriptional repression and nuclear compaction. Hho1 is depleted during meiosis and enriched in post-meiotic spore chromatin. Moreover, Hho1 shows a high genome-wide binding correlation with Ume6, the master repressor of meiotic genes. Meiotic depletion of both of these proteins may lead to the activation of key

Background Combined metagenomic and metatranscriptomic datasets make it possible to study the molecular evolution of diverse microbial species recovered from their native habitats. The link between gene expression level and sequence conservation was examined using shotgun pyrosequencing of microbial community DNA and RNA from diverse marine environments, and from forest soil. Results Across all samples, expressed genes with transcripts in the RNA sample were significantly more conserved than non-expressed gene sets relative to best matches in reference databases. This discrepancy, observed for many diverse individual genomes and across entire communities, coincided with a shift in amino acid usage between these gene fractions. Expressed genes trended toward GC-enriched amino acids, consistent with a hypothesis of higher levels of functional constraint in this gene pool. Highly expressed genes were significantly more likely to fall within an orthologous gene set shared between closely related taxa (core genes). However, non-core genes, when expressed above the level of detection, were, on average, significantly more highly expressed than core genes based on transcript abundance normalized to gene abundance. Finally, expressed genes showed broad similarities in function across samples, being relatively enriched in genes of energy metabolism and underrepresented by genes of cell growth. Conclusions These patterns support the hypothesis, predicated on studies of model organisms, that gene expression level is a primary correlate of evolutionary rate across diverse microbial taxa from natural environments. Despite their complexity, meta-omic datasets can reveal broad evolutionary patterns across taxonomically, functionally, and environmentally diverse communities., Gordon and Betty Moore Foundation, Agouron Institute, National Science Foundation (U.S.), Center for Microbial Oceanography: Research and Education

Background Combined metagenomic and metatranscriptomic datasets make it possible to study the molecular evolution of diverse microbial species recovered from their native habitats. The link between gene expression level and sequence conservation was examined using shotgun pyrosequencing of microbial community DNA and RNA from diverse marine environments, and from forest soil. Results Across all samples, expressed genes with transcripts in the RNA sample were significantly more conserved than non-expressed gene sets relative to best matches in reference databases. This discrepancy, observed for many diverse individual genomes and across entire communities, coincided with a shift in amino acid usage between these gene fractions. Expressed genes trended toward GC-enriched amino acids, consistent with a hypothesis of higher levels of functional constraint in this gene pool. Highly expressed genes were significantly more likely to fall within an orthologous gene set shared between closely related taxa (core genes). However, non-core genes, when expressed above the level of detection, were, on average, significantly more highly expressed than core genes based on transcript abundance normalized to gene abundance. Finally, expressed genes showed broad similarities in function across samples, being relatively enriched in genes of energy metabolism and underrepresented by genes of cell growth. Conclusions These patterns support the hypothesis, predicated on studies of model organisms, that gene expression level is a primary correlate of evolutionary rate across diverse microbial taxa from natural environments. Despite their complexity, meta-omic datasets can reveal broad evolutionary patterns across taxonomically, functionally, and environmentally diverse communities., Gordon and Betty Moore Foundation, Agouron Institute, National Science Foundation (U.S.), Center for Microbial Oceanography: Research and Education