Daniel P. Smith

ASRP: the Arabidopsis Small RNA Project Database.

Eukaryotes produce functionally diverse classes of small RNAs (20–25 nt). These include microRNAs (miRNAs), which act as regulatory factors during growth and development, and short-interfering RNAs (siRNAs), which function in several epigenetic and post-transcriptional silencing systems. The Arabidopsis Small RNA Project (ASRP) seeks to characterize and functionally analyze the major classes of endogenous small RNAs in plants. The ASRP database provides a repository for sequences of small RNAs cloned from various Arabidopsis genotypes and tissues. Version 3.0 of the database contains 1920 unique sequences, with tools to assist in miRNA and siRNA identification and analysis. The comprehensive database is publicly available through a web interface at http://asrp.cgrb.oregonstate.edu.

Exercise prevents weight gain and alters the gut microbiota in a mouse model of high fat diet-induced obesity.

Background Diet-induced obesity (DIO) is a significant health concern which has been linked to structural and functional changes in the gut microbiota. Exercise (Ex) is effective in preventing obesity, but whether Ex alters the gut microbiota during development with high fat (HF) feeding is unknown. Objective Determine the effects of voluntary Ex on the gastrointestinal microbiota in LF-fed mice and in HF-DIO. Methods Male C57BL/6 littermates (5 weeks) were distributed equally into 4 groups: low fat (LF) sedentary (Sed) LF/Sed, LF/Ex, HF/Sed and HF/Ex. Mice were individually housed and LF/Ex and HF/Ex cages were equipped with a wheel and odometer to record Ex. Fecal samples were collected at baseline, 6 weeks and 12 weeks and used for bacterial DNA isolation. DNA was subjected both to quantitative PCR using primers specific to the 16S rRNA encoding genes for Bacteroidetes and Firmicutes and to sequencing for lower taxonomic identification using the Illumina MiSeq platform. Data were analyzed using a one or two-way ANOVA or Pearson correlation. Results HF diet resulted in significantly greater body weight and adiposity as well as decreased glucose tolerance that were prevented by voluntary Ex (p<0.05). Visualization of Unifrac distance data with principal coordinates analysis indicated clustering by both diet and Ex at week 12. Sequencing demonstrated Ex-induced changes in the percentage of major bacterial phyla at 12 weeks. A correlation between total Ex distance and the ΔCt Bacteroidetes: ΔCt Firmicutes ratio from qPCR demonstrated a significant inverse correlation (r2 = 0.35, p = 0.043). Conclusion Ex induces a unique shift in the gut microbiota that is different from dietary effects. Microbiota changes may play a role in Ex prevention of HF-DIO.

Energy Starved Candidatus Pelagibacter Ubique Substitutes Light-Mediated ATP Production for Endogenous Carbon Respiration

Previous studies have demonstrated that Candidatus Pelagibacter ubique, a member of the SAR11 clade, constitutively expresses proteorhodopsin (PR) proteins that can function as light-dependent proton pumps. However, exposure to light did not significantly improve the growth rate or final cell densities of SAR11 isolates in a wide range of conditions. Thus, the ecophysiological role of PR in SAR11 remained unresolved. We investigated a range of cellular properties and here show that light causes dramatic changes in physiology and gene expression in Cand. P. ubique cells that are starved for carbon, but provides little or no advantage during active growth on organic carbon substrates. During logarithmic growth there was no difference in oxygen consumption by cells in light versus dark. Energy starved cells respired endogenous carbon in the dark, becoming spheres that approached the minimum predicted size for cells, and produced abundant pili. In the light, energy starved cells maintained size, ATP content, and higher substrate transport rates, and differentially expressed nearly 10% of their genome. These findings show that PR is a vital adaptation that supports Cand. P. ubique metabolism during carbon starvation, a condition that is likely to occur in the extreme conditions of ocean environments.

Environmental proteomics of microbial plankton in a highly productive coastal upwelling system

Metaproteomics is one of a suite of new approaches providing insights into the activities of microorganisms in natural environments. Proteins, the final products of gene expression, indicate cellular priorities, taking into account both transcriptional and posttranscriptional control mechanisms that control adaptive responses. Here, we report the proteomic composition of the < 1.2 μm fraction of a microbial community from Oregon coast summer surface waters, detected with two-dimensional liquid chromatography coupled with electrospray tandem mass spectrometry. Spectra corresponding to proteins involved in protein folding and biosynthesis, transport, and viral capsid structure were the most frequently detected. A total of 36% of all the detected proteins were best matches to the SAR11 clade, and other abundant coastal microbial clades were also well represented, including the Roseobacter clade (17%), oligotrophic marine gammaproteobacteria group (6%), OM43 clade (1%). Viral origins were attributed to 2.5% of proteins. In contrast to oligotrophic waters, phosphate transporters were not highly detected in this nutrient-rich system. However, transporters for amino acids, taurine, polyamines and glutamine synthetase were among the most highly detected proteins, supporting predictions that carbon and nitrogen are more limiting than phosphate in this environment. Intriguingly, one of the highly detected proteins was methanol dehydrogenase originating from the OM43 clade, providing further support for recent reports that the metabolism of one-carbon compounds by these streamlined methylotrophs might be an important feature of coastal ocean biogeochemistry.

One Carbon Metabolism in SAR11 Pelagic Marine Bacteria

The SAR11 Alphaproteobacteria are the most abundant heterotrophs in the oceans and are believed to play a major role in mineralizing marine dissolved organic carbon. Their genomes are among the smallest known for free-living heterotrophic cells, raising questions about how they successfully utilize complex organic matter with a limited metabolic repertoire. Here we show that conserved genes in SAR11 subgroup Ia (Candidatus Pelagibacter ubique) genomes encode pathways for the oxidation of a variety of one-carbon compounds and methyl functional groups from methylated compounds. These pathways were predicted to produce energy by tetrahydrofolate (THF)-mediated oxidation, but not to support the net assimilation of biomass from C1 compounds. Measurements of cellular ATP content and the oxidation of 14C-labeled compounds to 14CO2 indicated that methanol, formaldehyde, methylamine, and methyl groups from glycine betaine (GBT), trimethylamine (TMA), trimethylamine N-oxide (TMAO), and dimethylsulfoniopropionate (DMSP) were oxidized by axenic cultures of the SAR11 strain Ca. P. ubique HTCC1062. Analyses of metagenomic data showed that genes for C1 metabolism occur at a high frequency in natural SAR11 populations. In short term incubations, natural communities of Sargasso Sea microbial plankton expressed a potential for the oxidation of 14C-labeled formate, formaldehyde, methanol and TMAO that was similar to cultured SAR11 cells and, like cultured SAR11 cells, incorporated a much larger percentage of pyruvate and glucose (27–35%) than of C1 compounds (2–6%) into biomass. Collectively, these genomic, cellular and environmental data show a surprising capacity for demethylation and C1 oxidation in SAR11 cultures and in natural microbial communities dominated by SAR11, and support the conclusion that C1 oxidation might be a significant conduit by which dissolved organic carbon is recycled to CO2 in the upper ocean.

Membership and Behavior of Ultra-Low-Diversity Pathogen Communities Present in the Gut of Humans during Prolonged Critical Illness

ABSTRACT We analyzed the 16S rRNA amplicon composition in fecal samples of selected patients during their prolonged stay in an intensive care unit (ICU) and observed the emergence of ultra-low-diversity communities (1 to 4 bacterial taxa) in 30% of the patients. Bacteria associated with the genera Enterococcus and Staphylococcus and the family Enterobacteriaceae comprised the majority of these communities. The composition of cultured species from stool samples correlated to the 16S rRNA analysis and additionally revealed the emergence of Candida albicans and Candida glabrata in ~75% of cases. Four of 14 ICU patients harbored 2-member pathogen communities consisting of one Candida taxon and one bacterial taxon. Bacterial members displayed a high degree of resistance to multiple antibiotics. The virulence potential of the 2-member communities was examined in C. elegans during nutrient deprivation and exposure to opioids in order to mimic local conditions in the gut during critical illness. Under conditions of nutrient deprivation, the bacterial members attenuated the virulence of fungal members, leading to a “commensal lifestyle.” However, exposure to opioids led to a breakdown in this commensalism in 2 of the ultra-low-diversity communities. Application of a novel antivirulence agent (phosphate-polyethylene glycol [Pi-PEG]) that creates local phosphate abundance prevented opioid-induced virulence among these pathogen communities, thus rescuing the commensal lifestyle. To conclude, the gut microflora in critically ill patients can consist of ultra-low-diversity communities of multidrug-resistant pathogenic microbes. Local environmental conditions in gut may direct pathogen communities to adapt to either a commensal style or a pathogenic style. IMPORTANCE During critical illness, the normal gut microbiota becomes disrupted in response to host physiologic stress and antibiotic treatment. Here we demonstrate that the community structure of the gut microbiota during prolonged critical illness is dramatically changed such that in many cases only two-member pathogen communities remain. Most of these ultra-low-membership communities display low virulence when grouped together (i.e., a commensal lifestyle); individually, however, they can express highly harmful behaviors (i.e., a pathogenic lifestyle). The commensal lifestyle of the whole community can be shifted to a pathogenic one in response to host factors such as opioids that are released during physiologic stress and critical illness. This shift can be prevented by using compounds such as Pi-PEG15-20 that interrupt bacterial virulence expression. Taking the data together, this report characterizes the plasticity seen with respect to the choice between a commensal lifestyle and a pathogenic lifestyle among ultra-low-diversity pathogen communities that predominate in the gut during critical illness and offers novel strategies for prevention of sepsis. During critical illness, the normal gut microbiota becomes disrupted in response to host physiologic stress and antibiotic treatment. Here we demonstrate that the community structure of the gut microbiota during prolonged critical illness is dramatically changed such that in many cases only two-member pathogen communities remain. Most of these ultra-low-membership communities display low virulence when grouped together (i.e., a commensal lifestyle); individually, however, they can express highly harmful behaviors (i.e., a pathogenic lifestyle). The commensal lifestyle of the whole community can be shifted to a pathogenic one in response to host factors such as opioids that are released during physiologic stress and critical illness. This shift can be prevented by using compounds such as Pi-PEG15-20 that interrupt bacterial virulence expression. Taking the data together, this report characterizes the plasticity seen with respect to the choice between a commensal lifestyle and a pathogenic lifestyle among ultra-low-diversity pathogen communities that predominate in the gut during critical illness and offers novel strategies for prevention of sepsis.

The presence of the glycolysis operon in SAR11 genomes is positively correlated with ocean productivity

Bacteria in the SAR11 clade are highly abundant in marine surface waters, but currently little is known about the carbon compounds that support these large heterotrophic populations. To better understand the carbon requirements of these organisms, we conducted a multiphasic exploration of carbohydrate utilization among SAR11 isolates from the Northeast Pacific Ocean and the Sargasso Sea. A comparison of three SAR11 genomes showed they all lacked a recognizable PTS system, the oxidative portion of the pentose phosphate shunt (zwf-, pgl-), genes for the Embden-Meyerhoff-Parnas (pfk-, pyk-) and Entner-Doudoroff (eda-) pathways of glycolysis. Strain HTCC7211, isolated from an ocean gyre, was missing other glycolysis genes as well. Growth assays, radioisotopes, metagenomics and microarrays were used to test the hypothesis that these isolates might be limited in their abilities to transport and oxidize exogenous carbohydrates. Galactose, fucose, rhamnose, arabinose, ribose and mannose could not serve as carbon sources for the isolates tested. However, differences in glucose utilization were detected between coastal and ocean gyre isolates, with the coastal isolates capable of transporting, incorporating and oxidizing glucose while the open ocean isolate could not. Subsequent microarray analysis of a coastal isolate suggested that an operon encoding a variant of the Entner-Doudoroff pathway is likely responsible for the observed differences in glucose utilization. Metagenomic analysis indicated this operon is more commonly found in coastal environments and is positively correlated with chlorophyll a concentrations. Our results indicated that glycolysis is a variable metabolic property of SAR11 metabolism and suggest that glycolytic SAR11 are more common in productive marine environments.

Beyond the genome: community-level analysis of the microbial world

The development of culture-independent strategies to study microbial diversity and function has led to a revolution in microbial ecology, enabling us to address fundamental questions about the distribution of microbes and their influence on Earth’s biogeochemical cycles. This article discusses some of the progress that scientists have made with the use of so-called “omic” techniques (metagenomics, metatranscriptomics, and metaproteomics) and the limitations and major challenges these approaches are currently facing. These ‘omic methods have been used to describe the taxonomic structure of microbial communities in different environments and to discover new genes and enzymes of industrial and medical interest. However, microbial community structure varies in different spatial and temporal scales and none of the ‘omic techniques are individually able to elucidate the complex aspects of microbial communities and ecosystems. In this article we highlight the importance of a spatiotemporal sampling design, together with a multilevel ‘omic approach and a community analysis strategy (association networks and modeling) to examine and predict interacting microbial communities and their impact on the environment.

Forensic analysis of the microbiome of phones and shoes

BackgroundMicrobial interaction between human-associated objects and the environments we inhabit may have forensic implications, and the extent to which microbes are shared between individuals inhabiting the same space may be relevant to human health and disease transmission. In this study, two participants sampled the front and back of their cell phones, four different locations on the soles of their shoes, and the floor beneath them every waking hour over a 2-day period. A further 89 participants took individual samples of their shoes and phones at three different scientific conferences.ResultsSamples taken from different surface types maintained significantly different microbial community structures. The impact of the floor microbial community on that of the shoe environments was strong and immediate, as evidenced by Procrustes analysis of shoe replicates and significant correlation between shoe and floor samples taken at the same time point. Supervised learning was highly effective at determining which participant had taken a given shoe or phone sample, and a Bayesian method was able to determine which participant had taken each shoe sample based entirely on its similarity to the floor samples. Both shoe and phone samples taken by conference participants clustered into distinct groups based on location, though much more so when an unweighted distance metric was used, suggesting sharing of low-abundance microbial taxa between individuals inhabiting the same space.ConclusionsCorrelations between microbial community sources and sinks allow for inference of the interactions between humans and their environment.

Saliva from Obese Individuals Suppresses the Release of Aroma Compounds from Wine

Background Recent evidence suggests that a lower extent of the retronasal aroma release correspond to a higher amount of ad libitum food intake. This has been regarded as one of the bases of behavioral choices towards food consumption in obese people. In this pilot study we investigated the hypothesis that saliva from obese individuals could be responsible for an alteration of the retro-nasal aroma release. We tested this hypothesis in vitro, by comparing the release of volatiles from a liquid food matrix (wine) after its interaction with saliva from 28 obese (O) and 28 normal-weight (N) individuals. Methods and Findings Amplicon sequencing of the 16S rRNA V4 region indicated that Firmicutes and Actinobacteria were more abundant in O, while Proteobacteria and Fusobacteria dominated in N. Streptococcaceae were significantly more abundant in the O subjects and constituted 34% and 19% on average of the saliva microbiota of O and N subjects, respectively. The Total Antioxidant Capacity was higher in O vs N saliva samples. A model mouth system was used to test whether the in-mouth wine aroma release differs after the interaction with O or N saliva. In O samples, a 18% to 60% significant decrease in the mean concentration of wine volatiles was detected as a result of interaction with saliva, compared with N. This suppression was linked to biochemical differences in O and N saliva composition, which include protein content. Conclusion Microbiological and biochemical differences were found in O vs N saliva samples. An impaired retronasal aroma release from white wine was detected in vitro and linked to compositional differences between saliva from obese and normal-weight subjects. Additional in vivo investigations on diverse food matrices could contribute to understanding whether a lower olfactory stimulation due to saliva composition can be a co-factor in the development/maintenance of obesity.