Publications by authors named "Adina Howe"

38 Publications

Identification and Evolution of Cas9 tracrRNAs.

CRISPR J 2021 Jun;4(3):438-447

Department of Molecular Engineering, Corteva AgriscienceJohnston, TM, Johnston, Iowa, USA.

Clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas)9 transactivating CRISPR RNAs (tracrRNAs) form distinct structures essential for target recognition and cleavage and dictate exchangeability between orthologous proteins. As noncoding RNAs that are often apart from the CRISPR array, their identification can be arduous. In this article, a new bioinformatic method for the detection of Cas9 tracrRNAs is presented. The approach utilizes a covariance model based on both sequence homology and predicted secondary structure to locate tracrRNAs. This method predicts a tracrRNA for 98% of CRISPR-Cas9 systems identified by us. To ensure accuracy, we also benchmark our approach against biochemically vetted tracrRNAs finding false-positive and false-negative rates of 5.5% and 7.1%, respectively. Finally, the association between Cas9 amino acid sequence-based phylogeny and tracrRNA secondary structure is evaluated, revealing strong evidence that secondary structure is evolutionarily conserved among Cas9 lineages. Altogether, our findings provide insight into Cas9 tracrRNA evolution and efforts to characterize the tracrRNA of Cas9 systems.
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http://dx.doi.org/10.1089/crispr.2020.0093DOI Listing
June 2021

Antibiotic resistance gene dissipation in soil microcosms amended with antibiotics and swine manure.

J Environ Qual 2021 May 13. Epub 2021 May 13.

Dep. of Agricultural and Biosystems Engineering, IA State Univ., Elings Hall, 605 Bissell Rd., Ames, IA, 50011, USA.

The use of antibiotics in animal agriculture has exacerbated the presence of both antibiotic resistance genes (ARGs) and residual antibiotics excreted in animal manure. Field application of this manure is a common practice because its nutrient rich material can benefit crop growth. However, this practice can also introduce antibiotics and ARGs into nonagricultural settings. The integration of prairie buffer strips within and at the edge of crop fields is a potential management solution to reduce concentrations of ARGs commonly transported via water runoff and infiltration. An incubation experiment was conducted to investigate the fate of ARGs in directly manured crop field soils and the surrounding affected prairie strip soils. Row crop and prairie strip soils sampled from three sites received either an antibiotic spike and swine manure addition or a control water addition. The concentrations of select ARGs were then monitored over a 72-d period. Although soil vegetation and site location were not observed to influence ARG dissipation, the select genes did display different half-lives from one another. For example, tetM demonstrated the fastest dissipation of the genes quantified (average half-life, 5.18 d). Conversely, sul1 did not conform to the first-order linear regression kinetics used to describe the other investigated genes and was highly abundant in control prairie strip soils. The quantified half-lives of these select ARGs are comparable to previous studies and can inform monitoring and mitigative efforts aimed at reducing the spread of ARGs in the environment.
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http://dx.doi.org/10.1002/jeq2.20240DOI Listing
May 2021

Land-Use System and Forest Floor Explain Prokaryotic Metacommunity Structuring and Spatial Turnover in Amazonian Forest-to-Pasture Conversion Areas.

Front Microbiol 2021 21;12:657508. Epub 2021 Apr 21.

National Agrobiology Research Center, Embrapa Agrobiologia, Seropédica, Brazil.

Advancing extensive cattle production is a major threat to biodiversity conservation in Amazonia. The dominant vegetation cover has a drastic impact on soil microbial communities, affecting their composition, structure, and ecological services. Herein, we explored relationships between land-use, soil types, and forest floor compartments on the prokaryotic metacommunity structuring in Western Amazonia. Soil samples were taken in sites under high anthropogenic pressure and distributed along a ±800 km gradient. Additionally, the litter and a root layer, characteristic of the forest environment, were sampled. DNA was extracted, and metacommunity composition and structure were assessed through 16S rRNA gene sequencing. Prokaryotic metacommunities in the bulk soil were strongly affected by pH, base and aluminum saturation, Ca + Mg concentration, the sum of bases, and silt percentage, due to land-use management and natural differences among the soil types. Higher alpha, beta, and gamma diversities were observed in sites with higher soil pH and fertility, such as pasture soils or fertile soils of the state of Acre. When taking litter and root layer communities into account, the beta diversity was significantly higher in the forest floor than in pasture bulk soil for all study regions. Our results show that the forest floor's prokaryotic metacommunity performs a spatial turnover hitherto underestimated to the regional scale of diversity.
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http://dx.doi.org/10.3389/fmicb.2021.657508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8097146PMC
April 2021

Seasonal variations in export of antibiotic resistance genes and bacteria in runoff from an agricultural watershed in Iowa.

Sci Total Environ 2020 Oct 16;738:140224. Epub 2020 Jun 16.

Department of Agricultural and Biosystems Engineering, Iowa State University, Ames, IA, United States.

Seasonal variations of antimicrobial resistance (AMR) indicators in runoff water can help improve our understanding of AMR sources and transport within an agricultural watershed. This study aimed to monitor multiple areas throughout the Black Hawk Lake (BHL) watershed (5324 ha) in central Iowa during 2017 and 2018 that consists of both swine and cattle feeding operations as well as known areas with manure application. The measured indicators included plate counts for fecal indicator bacteria (FIB) E. coli, Enterococcus, antibiotic resistant fecal indicator bacteria (ARBs) tylosin resistant Enterococcus, tetracycline resistant Enterococcus, and antibiotic resistance genes (ARGs): ermB, ermF (macrolide), tetA, tetM, tetO, tetW (tetracycline), sul1, sul2 (sulfonamide), aadA2 (aminoglycoside), vgaA, and vgaB (pleuromutilin). Both the plate count and the ARG analyses showed seasonal trends. Plate counts were significantly greater during the growing season, while the ARGs were greater in the pre-planting and post-harvest seasons (Wilcoxon Rank-Sum Test p < 0.05). The ermB gene concentration was significantly correlated (p < 0.05) with E. coli and Enterococcus concentrations in 2017, suggesting a potential use of this ARG as an indicator of environmental AMR and human health risk. Flow rate was not a significant contributor to annual variations in bacteria and AMR indicators. Based on observed seasonal patterns, we concluded that manure application was the likely contributor to elevated ARG indicators observed in the BHL watershed, while the driver of elevated ARB indictors in the growing season can only be speculated. Understanding AMR export patterns in agricultural watersheds provides public health officials knowledge of seasonal periods of higher AMR load to recreational waters.
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http://dx.doi.org/10.1016/j.scitotenv.2020.140224DOI Listing
October 2020

An Improved Approach to Identify Bacterial Pathogens to Human in Environmental Metagenome.

J Microbiol Biotechnol 2020 Sep;30(9):1335-1342

Department of Civil and Environmental Engineering, Yonsei University, Seoul 03722, Republic of Korea.

The identification of bacterial pathogens to humans is critical for environmental microbial risk assessment. However, current methods for identifying pathogens in environmental samples are limited in their ability to detect highly diverse bacterial communities and accurately differentiate pathogens from commensal bacteria. In the present study, we suggest an improved approach using a combination of identification results obtained from multiple databases, including the multilocus sequence typing (MLST) database, virulence factor database (VFDB), and pathosystems resource integration center (PATRIC) databases to resolve current challenges. By integrating the identification results from multiple databases, potential bacterial pathogens in metagenomes were identified and classified into eight different groups. Based on the distribution of genes in each group, we proposed an equation to calculate the metagenomic pathogen identification index (MPII) of each metagenome based on the weighted abundance of identified sequences in each database. We found that the accuracy of pathogen identification was improved by using combinations of multiple databases compared to that of individual databases. When the approach was applied to environmental metagenomes, metagenomes associated with activated sludge were estimated with higher MPII than other environments (, drinking water, ocean water, ocean sediment, and freshwater sediment). The calculated MPII values were statistically distinguishable among different environments (<0.05). These results demonstrate that the suggested approach allows more for more accurate identification of the pathogens associated with metagenomes.
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http://dx.doi.org/10.4014/jmb.2005.05033DOI Listing
September 2020

Toward Antibiotic Stewardship: Route of Antibiotic Administration Impacts the Microbiota and Resistance Gene Diversity in Swine Feces.

Front Vet Sci 2020 19;7:255. Epub 2020 May 19.

Food Safety and Enteric Pathogens Research Unit, ARS-USDA National Animal Disease Center, Ames, IA, United States.

Oral antibiotics are a critical tool for fighting bacterial infections, yet their use can have negative consequences, such as the disturbance of healthy gut bacterial communities and the dissemination of antibiotic residues in feces. Altering antibiotic administration route may limit negative impacts on intestinal microbiota and reduce selective pressure for antimicrobial resistance genes (ARG) persistence and mobility. Thus, a study was performed in pigs to evaluate route of therapeutic oxytetracycline (oxytet) administration, an antibiotic commonly used in the U.S. swine industry, on intestinal microbial diversity and ARG abundance. Given that oral antibiotics would be in direct contact with intestinal bacteria, we hypothesized that oral administration would cause a major shift in intestinal bacterial community structure when compared to injected antibiotic. We further postulated that the impact would extend to the diversity and abundance of ARG in swine feces. At approximately 3 weeks-of-age, piglets were separated into three groups ( = 21-22 per group) with two groups receiving oxytet (one via injection and the second via feed) and a third non-medicated group. Oxytet levels in the plasma indicated injected antibiotic resulted in a spike 1 day after administration, which decreased over time, though oxytet was still detected in plasma 14 days after injection. Conversely, in-feed oxytet delivery resulted in lower but less variable oxytet levels in circulation and high concentrations in feces. Similar trends were observed in microbial community changes regardless of route of oxytet administration; however, the impact on the microbial community was more pronounced at all time points and in all samples with in-feed administration. Fecal ARG abundance was increased with in-feed administration over injected, with genes for tetracycline and aminoglycoside resistance enriched specifically in the feces of the in-feed group. Sequencing of plasmid-enriched samples revealed multiple genetic contexts for the resistance genes detected and highlighted the potential role of small plasmids in the movement of antibiotic resistance genes. The findings are informative for disease management in food animals, but also manure management and antibiotic therapy in human medicine for improved antibiotic stewardship.
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http://dx.doi.org/10.3389/fvets.2020.00255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249142PMC
May 2020

Improved detection of mcyA genes and their phylogenetic origins in harmful algal blooms.

Water Res 2020 Jun 19;176:115730. Epub 2020 Mar 19.

Agricultural and Biosystems Engineering, Iowa State University, Ames, IA, United States. Electronic address:

Microcystins, a group of cyanotoxins produced by cyanobacterial strains, have become a significant microbial hazard to human and animal health due to increases in the frequency and intensity of cyanobacterial harmful algal blooms (CyanoHABs). Many studies have explored the correlation between microcystin concentrations and abundances of toxin-producing genes (e.g., mcyA genes) measured using quantitative PCR, and discrepancies between toxin concentrations and gene abundances are often observed. In this study, the results show that these discrepancies are at least partially due to primer sets that do not capture the phylogenetic diversity of naturally present toxin-producers. We designed three novel primer gene probes based on known mcyA genes to improve the detection and quantification of these genes in environmental samples. These primers were shown to improve the identification of mcyA genes compared to previously published primers in freshwater metagenomes, cyanobacterial isolates, and lake water samples. Unlike previously published primers, our primer sets could selectively amplify and resolve Microcystis, Anabaena, and Planktothrix mcyA genes. In lake water samples, abundance estimations of mcyA genes were found to correlate strongly with microcystin concentrations. Based on our results, these primers offer significant improvements over previously published probes to accurately identify and quantify mcyA genes in the environment. There is an increasing need to develop models based on microbial information and environmental factors to predict CyanoHABs, and improved primers will play an important role in aiding monitoring efforts to collect reliable and consistent data on toxicity risks.
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http://dx.doi.org/10.1016/j.watres.2020.115730DOI Listing
June 2020

Catchment-scale export of antibiotic resistance genes and bacteria from an agricultural watershed in central Iowa.

PLoS One 2020 10;15(1):e0227136. Epub 2020 Jan 10.

Department of Agricultural and Biosystems Engineering, Iowa State University, Ames, Iowa, United States of America.

Antibiotics are administered to livestock in animal feeding operations (AFOs) for the control, prevention, and treatment of disease. Manure from antibiotic treated livestock contains unmetabolized antibiotics that provide selective pressure on bacteria, facilitating the expression of anti-microbial resistance (AMR). Manure application on row crops is an agronomic practice used by growers to meet crop nutrient needs; however, it can be a source of AMR to the soil and water environment. This study in central Iowa aims to directly compare AMR indicators in outlet runoff from two adjacent (221 to 229 ha) manured and non-manured catchments (manure comparison), and among three catchments (600 to 804 ha) with manure influence, no known manure application (control), and urban influences (mixed land use comparison). Monitored AMR indicators included antibiotic resistance genes (ARGs) ermB, ermF (macrolide), tetA, tetM, tetO, tetW (tetracycline), sul1, sul2 (sulfonamide), aadA2 (aminoglycoside), vgaA, and vgaB (pleuromutilin), and tylosin and tetracycline resistant enterococci bacteria. Results of the manure comparison showed significantly higher (p<0.05) tetracycline and tylosin resistant bacteria from the catchment with manure application in 2017, but no differences in 2018, possibly due to changes in antibiotic use resulting from the Veterinary Feed Directive. Moreover, the ARG analysis indicated a larger diversity of ARGs at the manure amended catchment. The mixed land use comparison showed the manure amended catchment had significantly higher (p<0.05) tetracycline resistant bacteria in 2017 and significantly higher tylosin resistant bacteria in 2017 and 2018 than the urban influenced catchment. The urban influenced catchment had significantly higher ermB concentrations in both sampling years, however the manure applied catchment runoff consisted of higher relative abundance of total ARGs. Additionally, both catchments showed higher AMR indicators compared to the control catchment. This study identifies four ARGs that might be specific to AMR as a result of agricultural sources (tetM, tetW, sul1, sul2) and optimal for use in watershed scale monitoring studies for tracking resistance in the environment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0227136PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953785PMC
April 2020

A meta-analysis of global fungal distribution reveals climate-driven patterns.

Nat Commun 2019 11 13;10(1):5142. Epub 2019 Nov 13.

Laboratory of Environmental Microbiology, Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, 14220, Praha 4, Czech Republic.

The evolutionary and environmental factors that shape fungal biogeography are incompletely understood. Here, we assemble a large dataset consisting of previously generated mycobiome data linked to specific geographical locations across the world. We use this dataset to describe the distribution of fungal taxa and to look for correlations with different environmental factors such as climate, soil and vegetation variables. Our meta-study identifies climate as an important driver of different aspects of fungal biogeography, including the global distribution of common fungi as well as the composition and diversity of fungal communities. In our analysis, fungal diversity is concentrated at high latitudes, in contrast with the opposite pattern previously shown for plants and other organisms. Mycorrhizal fungi appear to have narrower climatic tolerances than pathogenic fungi. We speculate that climate change could affect ecosystem functioning because of the narrow climatic tolerances of key fungal taxa.
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http://dx.doi.org/10.1038/s41467-019-13164-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6853883PMC
November 2019

Investigating the dispersal of antibiotic resistance associated genes from manure application to soil and drainage waters in simulated agricultural farmland systems.

PLoS One 2019 17;14(9):e0222470. Epub 2019 Sep 17.

Bioinformatics and Computational Biology Department, Iowa State University, Ames, Iowa, United States of America.

Manure from animals that have been treated with antibiotics is often used to fertilize agricultural soils and its application has previously been shown to enrich for genes associated with antibiotic resistance in agroecosystems. To investigate the magnitude of this effect, we designed a column experiment simulating manure-treated agricultural soil that utilizes artificial subsurface drainage to determine the duration and extent which this type of manure fertilization impacts the set of genes associated with antibiotic resistance in drainage water. We classified ARGs in manure-treated drainage effluent water by its source of origin. Overall, we found that 61% and 7% of the total abundance of ARGs found in drainage water samples could be attributed to manure enrichment and manure addition, respectively. Among these ARGs, we identified 75 genes unique to manure that persisted in both soil and drainage water throughout a drainage season typical of the Upper Midwestern United States. While most of these genes gradually decreased in abundance over time, the IS6100-associated tet(33) gene accrued. These results demonstrate the influence of manure applications on the composition of the resistome observed in agricultural drainage water and highlight the importance of anthropogenic ARGs in the environment.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0222470PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748443PMC
March 2020

Strategies for Building Computing Skills To Support Microbiome Analysis: a Five-Year Perspective from the EDAMAME Workshop.

mSystems 2019 Aug 20;4(4). Epub 2019 Aug 20.

Department of Agricultural and Biosystems Engineering, Iowa State University, Ames, Iowa, USA.

Here, we report our educational approach and learner evaluations of the first 5 years of the Explorations in Data Analysis for Metagenomic Advances in Microbial Ecology (EDAMAME) workshop, held annually at Michigan State University's Kellogg Biological Station from 2014 to 2018. We hope this information will be useful for others who want to organize computing-intensive workshops and will encourage quantitative skill development among microbiologists. High-throughput sequencing and related statistical and bioinformatic analyses have become routine in microbiology in the past decade, but there are few formal training opportunities to develop these skills. A weeklong workshop can offer sufficient time for novices to become introduced to best computing practices and common workflows in sequence analysis. We report our experiences in executing such a workshop targeted to professional learners (graduate students, postdoctoral scientists, faculty, and research staff).
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http://dx.doi.org/10.1128/mSystems.00297-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6702294PMC
August 2019

Characterizing the soil microbiome and quantifying antibiotic resistance gene dynamics in agricultural soil following swine CAFO manure application.

PLoS One 2019 19;14(8):e0220770. Epub 2019 Aug 19.

Department of Biology, Grinnell College, Grinnell, Iowa, United States of America.

As agriculture industrializes, concentrated animal feeding operations (CAFOs) are becoming more common. Feces from CAFOs is often used as fertilizer on fields. However, little is known about the effects manure has on the soil microbiome, which is an important aspect of soil health and fertility. In addition, due to the subtherapeutic levels of antibiotics necessary to keep the animals healthy, CAFO manure has elevated levels of antibiotic resistant bacteria. Using 16s rRNA high-throughput sequencing and qPCR, this study sought to determine the impact of swine CAFO manure application on both the soil microbiome and abundance of select antibiotic resistance genes (ARGs) and mobile element genes (erm(B), erm(C), sul1, str(B), intI1, IncW repA) in agricultural soil over the fall and spring seasons. We found the manure community to be distinct from the soil community, with a majority of bacteria belonging to Bacteroidetes and Firmicutes. The soil samples had more diverse communities dominated by Acidobacteria, Actinobacteria, Proteobacteria, Verrucomicrobia, and unclassified bacteria. We observed significant differences in the soil microbiome between all time points, except between the spring samples. However, by tracking manure associated taxa, we found the addition of the manure microbiome to be a minor driver of the shift. Of the measured genes, manure application only significantly increased the abundance of erm(B) and erm(C) which remained elevated in the spring. These results suggest bacteria in the manure do not survive well in soil and that ARG dynamics in soil following manure application vary by resistance gene.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0220770PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6699696PMC
March 2020

Complexities in understanding antimicrobial resistance across domesticated animal, human, and environmental systems.

Ann N Y Acad Sci 2019 04;1441(1):17-30

Department of Veterinary Preventive Medicine, Ohio State University, Columbus, Ohio.

Antimicrobial resistance (AMR) is a significant threat to both human and animal health. The spread of AMR bacteria and genes across systems can occur through a myriad of pathways, both related and unrelated to agriculture, including via wastewater, soils, manure applications, direct exchange between humans and animals, and food exposure. Tracing origins and drivers of AMR bacteria and genes is challenging due to the array of contexts and the complexity of interactions overlapping health practice, microbiology, genetics, applied science and engineering, as well as social and human factors. Critically assessing the diverse and sometimes contradictory AMR literature is a valuable step in identifying tractable mitigation options to stem AMR spread. In this article we review research on the nonfoodborne spread of AMR, with a focus on domesticated animals and the environment and possible exposures to humans. Attention is especially placed on delineating possible sources and causes of AMR bacterial phenotypes, including underpinning the genetics important to human and animal health.
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http://dx.doi.org/10.1111/nyas.14036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850694PMC
April 2019

RefSoil+: a Reference Database for Genes and Traits of Soil Plasmids.

mSystems 2019 Jan-Feb;4(1). Epub 2019 Feb 26.

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA.

Plasmids harbor transferable genes that contribute to the functional repertoire of microbial communities, yet their contributions to metagenomes are often overlooked. Environmental plasmids have the potential to spread antibiotic resistance to clinical microbial strains. In soils, high microbiome diversity and high variability in plasmid characteristics present a challenge for studying plasmids. To improve the understanding of soil plasmids, we present RefSoil+, a database containing plasmid sequences from 922 soil microorganisms. Soil plasmids were larger than other described plasmids, which is a trait associated with plasmid mobility. There was a weak relationship between chromosome size and plasmid size and no relationship between chromosome size and plasmid number, suggesting that these genomic traits are independent in soil. We used RefSoil+ to inform the distributions of antibiotic resistance genes among soil microorganisms compared to those among nonsoil microorganisms. Soil-associated plasmids, but not chromosomes, had fewer antibiotic resistance genes than other microorganisms. These data suggest that soils may offer limited opportunity for plasmid-mediated transfer of described antibiotic resistance genes. RefSoil+ can serve as a reference for the diversity, composition, and host associations of plasmid-borne functional genes in soil, a utility that will be enhanced as the database expands. Our study improves the understanding of soil plasmids and provides a resource for assessing the dynamics of the genes that they carry, especially genes conferring antibiotic resistances. Soil-associated plasmids have the potential to transfer antibiotic resistance genes from environmental to clinical microbial strains, which is a public health concern. A specific resource is needed to aggregate the knowledge of soil plasmid characteristics so that the content, host associations, and dynamics of antibiotic resistance genes can be assessed and then tracked between the environment and the clinic. Here, we present RefSoil+, a database of soil-associated plasmids. RefSoil+ presents a contemporary snapshot of antibiotic resistance genes in soil that can serve as a reference as novel plasmids and transferred antibiotic resistances are discovered. Our study broadens our understanding of plasmids in soil and provides a community resource of important plasmid-associated genes, including antibiotic resistance genes.
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http://dx.doi.org/10.1128/mSystems.00349-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392096PMC
February 2019

The principles of tomorrow's university.

F1000Res 2018 11;7:1926. Epub 2018 Dec 11.

School of Information Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, USA.

In the 21st Century, research is increasingly data- and computation-driven. Researchers, funders, and the larger community today emphasize the traits of openness and reproducibility. In March 2017, 13 mostly early-career research leaders who are building their careers around these traits came together with ten university leaders (presidents, vice presidents, and vice provosts), representatives from four funding agencies, and eleven organizers and other stakeholders in an NIH- and NSF-funded one-day, invitation-only workshop titled "Imagining Tomorrow's University." Workshop attendees were charged with launching a new dialog around open research - the current status, opportunities for advancement, and challenges that limit sharing. The workshop examined how the internet-enabled research world has changed, and how universities need to change to adapt commensurately, aiming to understand how universities can and should make themselves competitive and attract the best students, staff, and faculty in this new world. During the workshop, the participants re-imagined scholarship, education, and institutions for an open, networked era, to uncover new opportunities for universities to create value and serve society. They expressed the results of these deliberations as a set of 22 principles of tomorrow's university across six areas: credit and attribution, communities, outreach and engagement, education, preservation and reproducibility, and technologies. Activities that follow on from workshop results take one of three forms. First, since the workshop, a number of workshop authors have further developed and published their white papers to make their reflections and recommendations more concrete. These authors are also conducting efforts to implement these ideas, and to make changes in the university system.  Second, we plan to organise a follow-up workshop that focuses on how these principles could be implemented. Third, we believe that the outcomes of this workshop support and are connected with recent theoretical work on the position and future of open knowledge institutions.
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http://dx.doi.org/10.12688/f1000research.17425.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338243PMC
September 2019

Temporal Dynamics of Bacterial Communities in Soil and Leachate Water After Swine Manure Application.

Front Microbiol 2018 21;9:3197. Epub 2018 Dec 21.

Agricultural and Biosystems Engineering, Iowa State University, Ames, IA, United States.

Application of swine manure to agricultural land allows recycling of plant nutrients, but excess nitrate, phosphorus and fecal bacteria impact surface and drainage water quality. While agronomic and water quality impacts are well studied, little is known about the impact of swine manure slurry on soil microbial communities. We applied swine manure to intact soil columns collected from plots maintained under chisel plow or no-till with corn and soybean rotation. Targeted 16S-rRNA gene sequencing was used to characterize and to identify shifts in bacterial communities in soil over 108 days after swine manure application. In addition, six simulated rainfalls were applied during this time. Drainage water from the columns and surface soil were sampled, and DNA was extracted and sequenced. Unique DNA sequences (OTU) associated with 12 orders of bacteria were responsible for the majority of OTUs stimulated by manure application. were most prevalent, followed by , , , and . While the majority of the 12 orders decreased after day 59, relative abundances of genes associated with and in soil increased. Bacterial orders which were stimulated by manure application in soil had varied responses in drainage waters over the course of the experiment. We also identified a "manure-specific core" of five genera who comprised 13% of the manure community and were not significantly abundant in non-manured control soils. Of these five genera, was the only genus which did not return to pre-manure relative abundance in soil by day 108. Our results show that enrichment responses after manure amendment could result from displacement of native soil bacteria by manure-borne bacteria during the application process or growth of native bacteria using manure-derived available nutrients.
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http://dx.doi.org/10.3389/fmicb.2018.03197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309816PMC
December 2018

Metagenomic Insights into the Degradation of Resistant Starch by Human Gut Microbiota.

Appl Environ Microbiol 2018 12 15;84(23). Epub 2018 Nov 15.

Center for Microbial Ecology, Michigan State University, East Lansing, Michigan, USA

Several studies monitoring alterations in the community structure upon resistant starch (RS) interventions are available, although comprehensive function-based analyses are lacking. Recently, a multiomics approach based on 16S rRNA gene sequencing, metaproteomics, and metabolomics on fecal samples from individuals subjected to high and low doses of type 2 RS (RS2; 48 g and 3 g/2,500 kcal, respectively, daily for 2 weeks) in a crossover intervention experiment was performed. In the present study, we did pathway-based metagenomic analyses on samples from a subset of individuals ( = 12) from that study to obtain additional detailed insights into the functional structure at high resolution during RS2 intervention. A mechanistic framework based on obtained results is proposed where primary degradation was governed by , with as a major taxon involved, providing fermentation substrates and increased acetate concentrations for the growth of various major butyrate producers exhibiting the enzyme butyryl-coenzyme A (CoA):acetate CoA-transferase. H-scavenging sulfite reducers and acetogens concurrently increased. Individual responses of gut microbiota were noted, where seven of the 12 participants displayed all features of the outlined pattern, whereas four individuals showed mixed behavior and one subject was unresponsive. Intervention order did not affect the outcome, emphasizing a constant substrate supply for maintaining specific functional communities. Manipulation of gut microbiota is increasingly recognized as a promising approach to reduce various noncommunicable diseases, such as obesity and type 2 diabetes. Specific dietary supplements, including resistant starches (RS), are often a focus, yet comprehensive insights into functional responses of microbiota are largely lacking. Furthermore, unresponsiveness in certain individuals is poorly understood. Our data indicate that distinct parts of microbiota work jointly to degrade RS and successively form health-promoting fermentation end products. It highlights the need to consider both primary degraders and specific more-downstream-acting bacterial groups in order to achieve desired intervention outcomes. The gained insights will assist the design of personalized treatment strategies based on an individual's microbiota.
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http://dx.doi.org/10.1128/AEM.01562-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6238065PMC
December 2018

Primer set 2.0 for highly parallel qPCR array targeting antibiotic resistance genes and mobile genetic elements.

FEMS Microbiol Ecol 2018 09;94(9)

Department of Civil and Environmental Engineering, Michigan State University, East Lansing, Michigan 48824, USA.

The high-throughput antibiotic resistance gene (ARG) qPCR array, initially published in 2012, is increasingly used to quantify resistance and mobile determinants in environmental matrices. Continued utility of the array; however, necessitates improvements such as removing or redesigning questionable primer sets, updating targeted genes and coverage of available sequences. Towards this goal, a new primer design tool (EcoFunPrimer) was used to aid in identification of conserved regions of diverse genes. The total number of assays used for diverse genes was reduced from 91 old primer sets to 52 new primer sets, with only a 10% loss in sequence coverage. While the old and new array both contain 384 primer sets, a reduction in old primer sets permitted 147 additional ARGs and mobile genetic elements to be targeted. Results of validating the updated array with a mock community of strains resulted in over 98% of tested instances incurring true positive/negative calls. Common queries related to sensitivity, quantification and conventional data analysis (e.g. Ct cutoff value, and estimated genomic copies without standard curves) were also explored. A combined list of new and previously used primer sets is provided with a recommended set based on redesign of primer sets and results of validation.
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http://dx.doi.org/10.1093/femsec/fiy130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7250373PMC
September 2018

Practical implications of erythromycin resistance gene diversity on surveillance and monitoring of resistance.

FEMS Microbiol Ecol 2018 04;94(4)

Department of Agricultural and Biosystems Engineering, Iowa State University, 1201 Sukup Hall, Ames, IA 50011, USA.

Use of antibiotics in human and animal medicine has applied selective pressure for the global dissemination of antibiotic-resistant bacteria. Therefore, it is of interest to develop strategies to mitigate the continued amplification and transmission of resistance genes in environmental reservoirs such as farms, hospitals and watersheds. However, the efficacy of mitigation strategies is difficult to evaluate because it is unclear which resistance genes are important to monitor, and which primers to use to detect those genes. Here, we evaluated the diversity of one type of macrolide antibiotic resistance gene (erm) in one type of environment (manure) to determine which primers would be most informative to use in a mitigation study of that environment. We analyzed all known erm genes and assessed the ability of previously published erm primers to detect the diversity. The results showed that all known erm resistance genes group into 66 clusters, and 25 of these clusters (40%) can be targeted with primers found in the literature. These primers can target 74%-85% of the erm gene diversity in the manures analyzed.
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http://dx.doi.org/10.1093/femsec/fiy006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5939627PMC
April 2018

Feed in summer, rest in winter: microbial carbon utilization in forest topsoil.

Microbiome 2017 09 18;5(1):122. Epub 2017 Sep 18.

Institute of Microbiology of the CAS, Vídeňská 1083, 14220, Praha 4, Czech Republic.

Background: Evergreen coniferous forests contain high stocks of organic matter. Significant carbon transformations occur in litter and soil of these ecosystems, making them important for the global carbon cycle. Due to seasonal allocation of photosynthates to roots, carbon availability changes seasonally in the topsoil. The aim of this paper was to describe the seasonal differences in C source utilization and the involvement of various members of soil microbiome in this process.

Results: Here, we show that microorganisms in topsoil encode a diverse set of carbohydrate-active enzymes, including glycoside hydrolases and auxiliary enzymes. While the transcription of genes encoding enzymes degrading reserve compounds, such as starch or trehalose, was high in soil in winter, summer was characterized by high transcription of ligninolytic and cellulolytic enzymes produced mainly by fungi. Fungi strongly dominated the transcription in litter and an equal contribution of bacteria and fungi was found in soil. The turnover of fungal biomass appeared to be faster in summer than in winter, due to high activity of enzymes targeting its degradation, indicating fast growth in both litter and soil. In each enzyme family, hundreds to thousands of genes were typically transcribed simultaneously.

Conclusions: Seasonal differences in the transcription of glycoside hydrolases and auxiliary enzyme genes are more pronounced in soil than in litter. Our results suggest that mainly fungi are involved in decomposition of recalcitrant biopolymers in summer, while bacteria replace them in this role in winter. Transcripts of genes encoding enzymes targeting plant biomass biopolymers, reserve compounds and fungal cell walls were especially abundant in the coniferous forest topsoil.
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http://dx.doi.org/10.1186/s40168-017-0340-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5604414PMC
September 2017

The In-Feed Antibiotic Carbadox Induces Phage Gene Transcription in the Swine Gut Microbiome.

mBio 2017 08 8;8(4). Epub 2017 Aug 8.

National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa, USA

Carbadox is a quinoxaline-di--oxide antibiotic fed to over 40% of young pigs in the United States that has been shown to induce phage DNA transduction ; however, the effects of carbadox on swine microbiome functions are poorly understood. We investigated the longitudinal effects of carbadox on swine gut microbial gene expression (fecal metatranscriptome) and phage population dynamics (fecal dsDNA viromes). Microbial metagenome, transcriptome, and virome sequences were annotated for taxonomic inference and gene function by using FIGfam (isofunctional homolog sequences) and SEED subsystems databases. When the beta diversities of microbial FIGfam annotations were compared, the control and carbadox communities were distinct 2 days after carbadox introduction. This effect was driven by carbadox-associated lower expression of FIGfams ( = 66) related to microbial respiration, carbohydrate utilization, and RNA metabolism ( < 0.1), suggesting bacteriostatic or bactericidal effects within certain populations. Interestingly, carbadox treatment caused greater expression of FIGfams related to all stages of the phage lytic cycle 2 days following the introduction of carbadox ( ≤0.07), suggesting the carbadox-mediated induction of prophages and phage DNA recombination. These effects were diminished by 7 days of continuous carbadox in the feed, suggesting an acute impact. Additionally, the viromes included a few genes that encoded resistance to tetracycline, aminoglycoside, and beta-lactam antibiotics but these did not change in frequency over time or with treatment. The results show decreased bacterial growth and metabolism, prophage induction, and potential transduction of bacterial fitness genes in swine gut bacterial communities as a result of carbadox administration. FDA regulations on agricultural antibiotic use have focused on antibiotics that are important for human medicine. Carbadox is an antibiotic not used in humans but frequently used on U.S. pig farms. It is important to study possible side effects of carbadox use because it has been shown to promote bacterial evolution, which could indirectly impact antibiotic resistance in bacteria of clinical importance. Interestingly, the present study shows greater prophage gene expression in feces from carbadox-fed animals than in feces from nonmedicated animals 2 days after the initiation of in-feed carbadox treatment. Importantly, the phage genetic material isolated in this study contained genes that could provide resistance to antibiotics that are important in human medicine, indicating that human-relevant antibiotic resistance genes are mobile between bacteria via phages. This study highlights the collateral effects of antibiotics and demonstrates the need to consider diverse antibiotic effects whenever antibiotics are being used or new regulations are considered.
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http://dx.doi.org/10.1128/mBio.00709-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5550749PMC
August 2017

Imagining tomorrow's university in an era of open science.

F1000Res 2017 31;6:405. Epub 2017 Mar 31.

Department of Agricultural and Biosystems Engineering, Iowa State University, Ames, IA, 50011, USA.

As part of a recent workshop entitled "Imagining Tomorrow's University", we were asked to visualize the future of universities as research becomes increasingly data- and computation-driven, and identify a set of principles characterizing pertinent opportunities and obstacles presented by this shift. In order to establish a holistic view, we take a multilevel approach and examine the impact of open science on individual scholars and how this impacts as well as on the university as a whole. At the university level, open science presents a double-edged sword: when well executed, open science can accelerate the rate of scientific inquiry across the institution and beyond; however, haphazard or half-hearted efforts are likely to squander valuable resources, diminish university productivity and prestige, and potentially do more harm than good. We present our perspective on the role of open science at the university.
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http://dx.doi.org/10.12688/f1000research.11232.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437947PMC
March 2017

Allelic Variation in Outer Membrane Protein A and Its Influence on Attachment of to Corn Stover.

Front Microbiol 2017 3;8:708. Epub 2017 May 3.

Interdepartmental Microbiology Program, Iowa State UniversityAmes, IA, USA.

Understanding the genetic factors that govern microbe-sediment interactions in aquatic environments is important for water quality management and reduction of waterborne disease outbreaks. Although chemical properties of bacteria have been identified that contribute to initiation of attachment, the outer membrane proteins that contribute to these chemical properties still remain unclear. In this study we explored the attachment of 78 environmental isolates to corn stover, a representative agricultural residue. Outer membrane proteome analysis led to the observation of amino acid variations, some of which had not been previously described, in outer membrane protein A (OmpA) at 10 distinct locations, including each of the four extracellular loops, three of the eight transmembrane segments, the proline-rich linker and the dimerization domain. Some of the polymorphisms within loops 1, 2, and 3 were found to significantly co-occur. Grouping of sequences according to the outer loop polymorphisms revealed five distinct patterns that each occur in at least 5% of our isolates. The two most common patterns, I and II, are encoded by 33.3 and 20.5% of these isolates and differ at each of the four loops. Statistically significant differences in attachment to corn stover were observed among isolates expressing different versions of OmpA and when different versions of OmpA were expressed in the same genetic background. Most notable was the increased corn stover attachment associated with a loop 3 sequence of SNFDGKN relative to the standard SNVYGKN sequence. These results provide further insight into the allelic variation of OmpA and implicate OmpA in contributing to attachment to corn stover.
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http://dx.doi.org/10.3389/fmicb.2017.00708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5413513PMC
May 2017

Identification of the Core Set of Carbon-Associated Genes in a Bioenergy Grassland Soil.

PLoS One 2016 17;11(11):e0166578. Epub 2016 Nov 17.

Department of Ecology and Evolutionary Biology, Iowa State University, Ames, IA, 50011, United States of America.

Despite the central role of soil microbial communities in global carbon (C) cycling, little is known about soil microbial community structure and even less about their metabolic pathways. Efforts to characterize soil communities often focus on identifying differences in gene content across environmental gradients, but an alternative question is what genes are similar in soils. These genes may indicate critical species or potential functions that are required in all soils. Here we identified the "core" set of C cycling sequences widely present in multiple soil metagenomes from a fertilized prairie (FP). Of 226,887 sequences associated with known enzymes involved in the synthesis, metabolism, and transport of carbohydrates, 843 were identified to be consistently prevalent across four replicate soil metagenomes. This core metagenome was functionally and taxonomically diverse, representing five enzyme classes and 99 enzyme families within the CAZy database. Though it only comprised 0.4% of all CAZy-associated genes identified in FP metagenomes, the core was found to be comprised of functions similar to those within cumulative soils. The FP CAZy-associated core sequences were present in multiple publicly available soil metagenomes and most similar to soils sharing geographic proximity. In soil ecosystems, where high diversity remains a key challenge for metagenomic investigations, these core genes represent a subset of critical functions necessary for carbohydrate metabolism, which can be targeted to evaluate important C fluxes in these and other similar soils.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0166578PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5113961PMC
June 2017

Methodologies for probing the metatranscriptome of grassland soil.

J Microbiol Methods 2016 12 25;131:122-129. Epub 2016 Oct 25.

Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, United States; Department of Agricultural and Biosystems Engineering, Iowa State University, Ames, Iowa, United States.

Metatranscriptomics provides an opportunity to identify active microbes and expressed genes in complex soil communities in response to particular conditions. Currently, there are a limited number of soil metatranscriptome studies to provide guidance for using this approach in this challenging matrix. Hence, we evaluated the technical challenges of applying soil metatranscriptomics to a highly diverse, low activity natural system. We used a non-targeted rRNA removal approach, duplex nuclease specific (DSN) normalization, to generate a metatranscriptomic library from field collected soil supporting a perennial grass, Miscanthus x giganteus (a biofuel crop), and evaluated its ability to provide insight into its active community members and their expressed protein-coding genes. We also evaluated various bioinformatics approaches for analyzing our soil metatranscriptome, including annotation of unassembled transcripts, de novo assembly, and aligning reads to known genomes. Further, we evaluated various databases for their ability to provide annotations for our metatranscriptome. Overall, our results emphasize that low activity, highly genetically diverse and relatively stable microbiomes, like soil, requires very deep sequencing to sample the transcriptome beyond the common core functions. We identified several key areas that metatranscriptomic analyses will benefit from including increased rRNA removal, assembly of short read transcripts, and more relevant reference bases while providing a priority set of expressed genes for functional assessment.
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http://dx.doi.org/10.1016/j.mimet.2016.10.018DOI Listing
December 2016

Identification of Soil Microbes Capable of Utilizing Cellobiosan.

PLoS One 2016 12;11(2):e0149336. Epub 2016 Feb 12.

Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa, United States of America.

Approximately 100 million tons of anhydrosugars, such as levoglucosan and cellobiosan, are produced through biomass burning every year. These sugars are also produced through fast pyrolysis, the controlled thermal depolymerization of biomass. While the microbial pathways associated with levoglucosan utilization have been characterized, there is little known about cellobiosan utilization. Here we describe the isolation and characterization of six cellobiosan-utilizing microbes from soil samples. Each of these organisms is capable of using both cellobiosan and levoglucosan as sole carbon source, though both minimal and rich media cellobiosan supported significantly higher biomass production than levoglucosan. Ribosomal sequencing was used to identify the closest reported match for these organisms: Sphingobacterium multivorum, Acinetobacter oleivorans JC3-1, Enterobacter sp SJZ-6, and Microbacterium sps FXJ8.207 and 203 and a fungal species Cryptococcus sp. The commercially-acquired Enterobacter cloacae DSM 16657 showed growth on levoglucosan and cellobiosan, supporting our isolate identification. Analysis of an existing database of 16S rRNA amplicons from Iowa soil samples confirmed the representation of our five bacterial isolates and four previously-reported levoglucosan-utilizing bacterial isolates in other soil samples and provided insight into their population distributions. Phylogenetic analysis of the 16S rRNA and 18S rRNA of strains previously reported to utilize levoglucosan and our newfound isolates showed that the organisms isolated in this study are distinct from previously described anhydrosugar-utilizing microbial species.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0149336PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4752346PMC
July 2016

The khmer software package: enabling efficient nucleotide sequence analysis.

F1000Res 2015 25;4:900. Epub 2015 Sep 25.

Addgene, Cambridge, MA, USA.

The khmer package is a freely available software library for working efficiently with fixed length DNA words, or k-mers. khmer provides implementations of a probabilistic k-mer counting data structure, a compressible De Bruijn graph representation, De Bruijn graph partitioning, and digital normalization. khmer is implemented in C++ and Python, and is freely available under the BSD license at  https://github.com/dib-lab/khmer/.
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http://dx.doi.org/10.12688/f1000research.6924.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608353PMC
November 2015

Divergent responses of viral and bacterial communities in the gut microbiome to dietary disturbances in mice.

ISME J 2016 May 16;10(5):1217-27. Epub 2015 Oct 16.

Department of Medicine, University of Chicago, Chicago, IL, USA.

To improve our understanding of the stability of mammalian intestinal communities, we characterized the responses of both bacterial and viral communities in murine fecal samples to dietary changes between high- and low-fat (LF) diets. Targeted DNA extraction methods for bacteria, virus-like particles and induced prophages were used to generate bacterial and viral metagenomes as well as 16S ribosomal RNA amplicons. Gut microbiome communities from two cohorts of C57BL/6 mice were characterized in a 6-week diet perturbation study in response to high fiber, LF and high-refined sugar, milkfat (MF) diets. The resulting metagenomes from induced bacterial prophages and extracellular viruses showed significant overlap, supporting a largely temperate viral lifestyle within these gut microbiomes. The resistance of baseline communities to dietary disturbances was evaluated, and we observed contrasting responses of baseline LF and MF bacterial and viral communities. In contrast to baseline LF viral communities and bacterial communities in both diet treatments, baseline MF viral communities were sensitive to dietary disturbances as reflected in their non-recovery during the washout period. The contrasting responses of bacterial and viral communities suggest that these communities can respond to perturbations independently of each other and highlight the potentially unique role of viruses in gut health.
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http://dx.doi.org/10.1038/ismej.2015.183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5029215PMC
May 2016

Microbial activity in forest soil reflects the changes in ecosystem properties between summer and winter.

Environ Microbiol 2016 Jan 14;18(1):288-301. Epub 2015 Oct 14.

Laboratory of Environmental Microbiology, Institute of Microbiology of the ASCR, v.v.i., Vídeňská 1083, Praha 4, 14220, Czech Republic.

Understanding the ecology of coniferous forests is very important because these environments represent globally largest carbon sinks. Metatranscriptomics, microbial community and enzyme analyses were combined to describe the detailed role of microbial taxa in the functioning of the Picea abies-dominated coniferous forest soil in two contrasting seasons. These seasons were the summer, representing the peak of plant photosynthetic activity, and late winter, after an extended period with no photosynthate input. The results show that microbial communities were characterized by a high activity of fungi especially in litter where their contribution to microbial transcription was over 50%. Differences in abundance between summer and winter were recorded for 26-33% of bacterial genera and < 15% of fungal genera, but the transcript profiles of fungi, archaea and most bacterial phyla were significantly different among seasons. Further, the seasonal differences were larger in soil than in litter. Most importantly, fungal contribution to total microbial transcription in soil decreased from 33% in summer to 16% in winter. In particular, the activity of the abundant ectomycorrhizal fungi was reduced in winter, which indicates that plant photosynthetic production was likely one of the major drivers of changes in the functioning of microbial communities in this coniferous forest.
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http://dx.doi.org/10.1111/1462-2920.13026DOI Listing
January 2016