@article{pmid30944879,

title = {Curated BLASŦ for Genomes},

author = {M N Price and A P Arkin},

year  = {2019},

date = {2019-01-01},

journal = {mSystems},

volume = {4},

number = {2},

abstract = {Curated BLAST for Genomes finds candidate genes for a process or an enzymatic activity within a genome of interest. In contrast to annotation tools, which usually predict a single activity for each protein, Curated BLAST asks if any of the proteins in the genome are similar to characterized proteins that are relevant. Given a query such as an enzyme's name or an EC number, Curated BLAST searches the curated descriptions of over 100,000 characterized proteins, and it compares the relevant characterized proteins to the predicted proteins in the genome of interest. In case of errors in the gene models, Curated BLAST also searches the six-frame translation of the genome. Curated BLAST is available at http://papers.genomics.lbl.gov/curated. IMPORTANCE Given a microbe's genome sequence, we often want to predict what capabilities the organism has, such as which nutrients it requires or which energy sources it can use. Or, we know the organism has a capability and we want to find the genes involved. Scientists often use automated gene annotations to find relevant genes, but automated annotations are often vague or incorrect. Curated BLAST finds candidate genes for a capability without relying on automated annotations. First, Curated BLAST finds proteins (usually from other organisms) whose functions have been studied experimentally and whose curated descriptions match a query. Then, it searches the genome of interest for similar proteins and returns a list of candidates. Curated BLAST is fast and often finds relevant genes that are missed by automated annotation.},

keywords = {arkin lab},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30943208,

title = {Correction: Filling gaps in bacterial amino acid biosynthesis pathways with high-throughput genetics},

author = {M N Price and G M Zane and J V Kuehl and R A Melnyk and J D Wall and A M Deutschbauer and A P Arkin},

year  = {2019},

date = {2019-01-01},

journal = {PLoS Genet.},

volume = {15},

number = {4},

pages = {e1008106},

abstract = {[This corrects the article DOI: 10.1371/journal.pgen.1007147.].},

keywords = {arkin lab, biodesign},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30894433,

title = {Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides},

year  = {2019},

date = {2019-01-01},

journal = {mSphere},

volume = {4},

number = {2},

abstract = {Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast Rhodosporidium toruloides is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While R. toruloides has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as Yarrowia lipolytica and Saccharomyces cerevisiae Significant advancements in the past few years have bolstered R. toruloides' engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9-based gene disruption in R. toruloides Transforming a Cas9 expression cassette harboring nourseothricin resistance and selecting transformants on this antibiotic resulted in strains of R. toruloides exhibiting successful targeted disruption of the native URA3 gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, CAR2, with much greater success; editing efficiencies of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation. Together, our results provide a framework for applying CRISPR-Cas9 to R. toruloides that will facilitate rapid and high-throughput genome engineering in this industrially relevant organism.IMPORTANCE Microbial biofuel and bioproduct platforms provide access to clean and renewable carbon sources that are more sustainable and environmentally friendly than petroleum-based carbon sources. Furthermore, they can serve as useful conduits for the synthesis of advanced molecules that are difficult to produce through strictly chemical means. R. toruloides has emerged as a promising potential host for converting renewable lignocellulosic material into valuable fuels and chemicals. However, engineering efforts to improve the yeast's production capabilities have been impeded by a lack of advanced tools for genome engineering. While this is rapidly changing, one key tool remains unexplored in R. toruloides: CRISPR-Cas9. The results outlined here demonstrate for the first time how effective multiplexed CRISPR-Cas9 gene disruption provides a framework for other researchers to utilize this revolutionary genome-editing tool effectively in R. toruloides.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30788501,

title = {A versatile platform strain for high-fidelity multiplex genome editing},

author = {R G Egbert and H S Rishi and B A Adler and D M McCormick and E Toro and R T Gill and A P Arkin},

year  = {2019},

date = {2019-01-01},

journal = {Nucleic Acids Res.},

volume = {47},

number = {6},

pages = {3244--3256},

abstract = {Precision genome editing accelerates the discovery of the genetic determinants of phenotype and the engineering of novel behaviors in organisms. Advances in DNA synthesis and recombineering have enabled high-throughput engineering of genetic circuits and biosynthetic pathways via directed mutagenesis of bacterial chromosomes. However, the highest recombination efficiencies have to date been reported in persistent mutator strains, which suffer from reduced genomic fidelity. The absence of inducible transcriptional regulators in these strains also prevents concurrent control of genome engineering tools and engineered functions. Here, we introduce a new recombineering platform strain, BioDesignER, which incorporates (i) a refactored λ-Red recombination system that reduces toxicity and accelerates multi-cycle recombination, (ii) genetic modifications that boost recombination efficiency, and (iii) four independent inducible regulators to control engineered functions. These modifications resulted in single-cycle recombineering efficiencies of up to 25% with a 7-fold increase in recombineering fidelity compared to the widely used recombineering strain EcNR2. To facilitate genome engineering in BioDesignER, we have curated eight context--neutral genomic loci, termed Safe Sites, for stable gene expression and consistent recombination efficiency. BioDesignER is a platform to develop and optimize engineered cellular functions and can serve as a model to implement comparable recombination and regulatory systems in other bacteria.},

keywords = {arkin lab, biodesign},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30746495,

title = {Oxidative Pathways of Đeoxyribose and Đeoxyribonate Catabolism},

author = {M N Price and J Ray and A T Iavarone and H K Carlson and E M Ryan and R R Malmstrom and A P Arkin and A M Deutschbauer},

year  = {2019},

date = {2019-01-01},

journal = {mSystems},

volume = {4},

number = {1},

abstract = {Using genome-wide mutant fitness assays in diverse bacteria, we identified novel oxidative pathways for the catabolism of 2-deoxy-d-ribose and 2-deoxy-d-ribonate. We propose that deoxyribose is oxidized to deoxyribonate, oxidized to ketodeoxyribonate, and cleaved to acetyl coenzyme A (acetyl-CoA) and glyceryl-CoA. We have genetic evidence for this pathway in three genera of bacteria, and we confirmed the oxidation of deoxyribose to ketodeoxyribonate in vitro. In Pseudomonas simiae, the expression of enzymes in the pathway is induced by deoxyribose or deoxyribonate, while in Paraburkholderia bryophila and in Burkholderia phytofirmans, the pathway proceeds in parallel with the known deoxyribose 5-phosphate aldolase pathway. We identified another oxidative pathway for the catabolism of deoxyribonate, with acyl-CoA intermediates, in Klebsiella michiganensis. Of these four bacteria, only P. simiae relies entirely on an oxidative pathway to consume deoxyribose. The deoxyribose dehydrogenase of P. simiae is either nonspecific or evolved recently, as this enzyme is very similar to a novel vanillin dehydrogenase from Pseudomonas putida that we identified. So, we propose that these oxidative pathways evolved primarily to consume deoxyribonate, which is a waste product of metabolism. IMPORTANCE Deoxyribose is one of the building blocks of DNA and is released when cells die and their DNA degrades. We identified a bacterium that can grow with deoxyribose as its sole source of carbon even though its genome does not contain any of the known genes for breaking down deoxyribose. By growing many mutants of this bacterium together on deoxyribose and using DNA sequencing to measure the change in the mutants' abundance, we identified multiple protein-coding genes that are required for growth on deoxyribose. Based on the similarity of these proteins to enzymes of known function, we propose a 6-step pathway in which deoxyribose is oxidized and then cleaved. Diverse bacteria use a portion of this pathway to break down a related compound, deoxyribonate, which is a waste product of metabolism. Our study illustrates the utility of large-scale bacterial genetics to identify previously unknown metabolic pathways.},

keywords = {arkin lab, biodesign},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30659179,

title = {Đual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits in bacteria},

author = {V K Mutalik and P S Novichkov and M N Price and T K Owens and M Callaghan and S Carim and A M Deutschbauer and A P Arkin},

year  = {2019},

date = {2019-01-01},

journal = {Nat Commun},

volume = {10},

number = {1},

pages = {308},

abstract = {A major challenge in genomics is the knowledge gap between sequence and its encoded function. Gain-of-function methods based on gene overexpression are attractive avenues for phenotype-based functional screens, but are not easily applied in high-throughput across many experimental conditions. Here, we present Dual Barcoded Shotgun Expression Library Sequencing (Dub-seq), a method that uses random DNA barcodes to greatly increase experimental throughput. As a demonstration of this approach, we construct a Dub-seq library with Escherichia coli genomic DNA, performed 155 genome-wide fitness assays in 52 experimental conditions, and identified overexpression phenotypes for 813 genes. We show that Dub-seq data is reproducible, accurately recapitulates known biology, and identifies hundreds of novel gain-of-function phenotypes for E. coli genes, a subset of which we verified with assays of individual strains. Dub-seq provides complementary information to loss-of-function approaches and will facilitate rapid and systematic functional characterization of microbial genomes.},

keywords = {arkin lab, biodesign},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30635343,

title = {Ŧransposon insertional mutagenesis in Saccharomyces uvarum reveals trans-acting effects influencing species-dependent essential genes},

author = {M R Sanchez and C Payen and F Cheong and B T Hovde and S Bissonnette and A P Arkin and J M Skerker and R B Brem and A A Caudy and M J Dunham},

year  = {2019},

date = {2019-01-01},

journal = {Genome Res.},

volume = {29},

number = {3},

pages = {396--406},

abstract = {To understand how complex genetic networks perform and regulate diverse cellular processes, the function of each individual component must be defined. Comprehensive phenotypic studies of mutant alleles have been successful in model organisms in determining what processes depend on the normal function of a gene. These results are often ported to newly sequenced genomes by using sequence homology. However, sequence similarity does not always mean identical function or phenotype, suggesting that new methods are required to functionally annotate newly sequenced species. We have implemented comparative analysis by high-throughput experimental testing of gene dispensability in Saccharomyces uvarum, a sister species of Saccharomyces cerevisiae. We created haploid and heterozygous diploid Tn7 insertional mutagenesis libraries in S. uvarum to identify species-dependent essential genes, with the goal of detecting genes with divergent functions and/or different genetic interactions. Comprehensive gene dispensability comparisons with S. cerevisiae predicted diverged dispensability at 12% of conserved orthologs, and validation experiments confirmed 22 differentially essential genes. Despite their differences in essentiality, these genes were capable of cross-species complementation, demonstrating that trans-acting factors that are background-dependent contribute to differential gene essentiality. This study shows that direct experimental testing of gene disruption phenotypes across species can inform comparative genomic analyses and improve gene annotations. Our method can be widely applied in microorganisms to further our understanding of genome evolution.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30633905,

title = {CRISPR-Cas9 Circular Permutants as Programmable Scaffolds for Genome Modification},

year  = {2019},

date = {2019-01-01},

journal = {Cell},

volume = {176},

number = {1-2},

pages = {254--267},

abstract = {The ability to engineer natural proteins is pivotal to a future, pragmatic biology. CRISPR proteins have revolutionized genome modification, yet the CRISPR-Cas9 scaffold is not ideal for fusions or activation by cellular triggers. Here, we show that a topological rearrangement of Cas9 using circular permutation provides an advanced platform for RNA-guided genome modification and protection. Through systematic interrogation, we find that protein termini can be positioned adjacent to bound DNA, offering a straightforward mechanism for strategically fusing functional domains. Additionally, circular permutation enabled protease-sensing Cas9s (ProCas9s), a unique class of single-molecule effectors possessing programmable inputs and outputs. ProCas9s can sense a wide range of proteases, and we demonstrate that ProCas9 can orchestrate a cellular response to pathogen-associated protease activity. Together, these results provide a toolkit of safer and more efficient genome-modifying enzymes and molecular recorders for the advancement of precision genome engineering in research, agriculture, and biomedicine.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30523276,

title = {Ŧhe selective pressures on the microbial community in a metal-contaminated aquifer},

author = {H K Carlson and M N Price and M Callaghan and A Aaring and R Chakraborty and H Liu and J V Kuehl and A P Arkin and A M Deutschbauer},

year  = {2019},

date = {2019-01-01},

journal = {ISME J},

volume = {13},

number = {4},

pages = {937--949},

abstract = {In many environments, toxic compounds restrict which microorganisms persist. However, in complex mixtures of inhibitory compounds, it is challenging to determine which specific compounds cause changes in abundance and prevent some microorganisms from growing. We focused on a contaminated aquifer in Oak Ridge, Tennessee, USA that has large gradients of pH and widely varying concentrations of uranium, nitrate, and many other inorganic ions. In the most contaminated wells, the microbial community is enriched in the Rhodanobacter genus. Rhodanobacter abundance is positively correlated with low pH and high concentrations of uranium and 13 other ions and we sought to determine which of these ions are selective pressures that favor the growth of Rhodanobacter over other taxa. Of these ions, low pH and high UO22+, Mn2+, Al3+, Cd2+, Zn2+, Co2+, and Ni2+ are both (a) selectively inhibitory of a Pseudomonas isolate from an uncontaminated well vs. a Rhodanobacter isolate from a contaminated well, and (b) reach toxic concentrations (for the Pseudomonas isolate) in the Rhodanobacter-dominated wells. We used mixtures of ions to simulate the groundwater conditions in the most contaminated wells and verified that few isolates aside from Rhodanobacter can tolerate these eight ions. These results clarify which ions are likely causal factors that impact the microbial community at this field site and are not merely correlated with taxonomic shifts. Furthermore, our general high-throughput approach can be applied to other environments, isolates, and conditions to systematically help identify selective pressures on microbial communities.},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30540439,

title = {Đesigning Spatially Đistributed Gene Regulatory Networks Ŧo Elicit Contrasting Patterns},

author = {M Tei and M L Perkins and J Hsia and M Arcak and A P Arkin},

year  = {2019},

date = {2019-01-01},

volume = {8},

number = {1},

pages = {119--126},

abstract = {Pattern formation and differential interactions are important for microbial consortia to divide labor and perform complex functions. To obtain further insight into such interactions, we present a computational method for simulating physically separated microbial colonies, each implementing different gene regulatory networks. We validate our theory by experimentally demonstrating control over gene expression patterns in a diffusion-mediated lateral inhibition circuit. We highlight the importance of spatial arrangement as a control knob for modulating system behavior. Our systematic approach provides a foundation for future applications that require understanding and engineering of multistrain microbial communities for sophisticated, synergistic functions.},

keywords = {arkin lab, biodesign},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30289197,

title = {Iron- and aluminium-induced depletion of molybdenum in acidic environments impedes the nitrogen cycle},

author = {X Ge and B J Vaccaro and M P Thorgersen and F L Poole and E L Majumder and G M Zane and K B De Le?n and W A Lancaster and J W Moon and C J Paradis and F von Netzer and D A Stahl and P D Adams and A P Arkin and J D Wall and T C Hazen and M W W Adams},

year  = {2019},

date = {2019-01-01},

journal = {Environ. Microbiol.},

volume = {21},

number = {1},

pages = {152--163},

abstract = {Anthropogenic nitrate contamination is a serious problem in many natural environments. Nitrate removal by microbial action is dependent on the metal molybdenum (Mo), which is required by nitrate reductase for denitrification and dissimilatory nitrate reduction to ammonium. The soluble form of Mo, molybdate (MoO42- ), is incorporated into and adsorbed by iron (Fe) and aluminium (Al) (oxy) hydroxide minerals. Herein we used Oak Ridge Reservation (ORR) as a model nitrate-contaminated acidic environment to investigate whether the formation of Fe- and Al-precipitates could impede microbial nitrate removal by depleting Mo. We demonstrate that Fe and Al mineral formation that occurs as the pH of acidic synthetic groundwater is increased, decreases soluble Mo to low picomolar concentrations, a process proposed to mimic environmental diffusion of acidic contaminated groundwater. Analysis of ORR sediments revealed recalcitrant Mo in the contaminated core that co-occurred with Fe and Al, consistent with Mo scavenging by Fe/Al precipitates. Nitrate removal by ORR isolate Pseudomonas fluorescens N2A2 is virtually abolished by Fe/Al precipitate-induced Mo depletion. The depletion of naturally occurring Mo in nitrate- and Fe/Al-contaminated acidic environments like ORR or acid mine drainage sites has the potential to impede microbial-based nitrate reduction thereby extending the duration of nitrate in the environment.},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{pmid29656383,

title = {Improved Method for Estimating Reaction Rates Đuring Push-Pull Ŧests},

author = {C J Paradis and E R Dixon and L M Lui and A P Arkin and J C Parker and J D Istok and E Perfect and L D McKay and T C Hazen},

year  = {2019},

date = {2019-01-01},

journal = {Ground Water},

volume = {57},

number = {2},

pages = {292--302},

abstract = {The breakthrough curve obtained from a single-well push-pull test can be adjusted to account for dilution of the injection fluid in the aquifer fluid. The dilution-adjusted breakthrough curve can be analyzed to estimate the reaction rate of a solute. The conventional dilution-adjusted method assumes that the ratios of the concentrations of the nonreactive and reactive solutes in the injection fluid vs. the aquifer fluid are equal. If this assumption is invalid, the conventional method will generate inaccurate breakthrough curves and may lead to erroneous conclusions regarding the reactivity of a solute. In this study, a new method that generates a dilution-adjusted breakthrough curve was theoretically developed to account for any possible combination of nonreactive and reactive solute concentrations in the injection and aquifer fluids. The newly developed method was applied to a field-based data set and was shown to generate more accurate dilution-adjusted breakthrough curves. The improved dilution-adjusted method presented here is simple, makes no assumptions regarding the concentrations of the nonreactive and reactive solutes in the injection and aquifer fluids, and easily allows for estimating reaction rates during push-pull tests.},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{carlson_2019,

title = {The selective pressures on the microbial community in a metal-contaminated aquifer.},

author = {Hans K Carlson and Morgan N Price and Mark Callaghan and Alex Aaring and Romy Chakraborty and Hualan Liu and Jennifer V Kuehl and Adam P Arkin and Adam M Deutschbauer},

url = {http://www.nature.com/articles/s41396-018-0328-1},

doi = {10.1038/s41396-018-0328-1},

issn = {1751-7362},

year  = {2019},

date = {2019-01-01},

urldate = {2021-05-25},

journal = {The ISME Journal},

volume = {13},

number = {4},

pages = {937-949},

abstract = {In many environments, toxic compounds restrict which microorganisms persist. However, in complex mixtures of inhibitory compounds, it is challenging to determine which specific compounds cause changes in abundance and prevent some microorganisms from growing. We focused on a contaminated aquifer in Oak Ridge, Tennessee, USA that has large gradients of pH and widely varying concentrations of uranium, nitrate, and many other inorganic ions. In the most contaminated wells, the microbial community is enriched in the Rhodanobacter genus. Rhodanobacter abundance is positively correlated with low pH and high concentrations of uranium and 13 other ions and we sought to determine which of these ions are selective pressures that favor the growth of Rhodanobacter over other taxa. Of these ions, low pH and high UO22+, Mn2+, Al3+, Cd2+, Zn2+, Co2+, and Ni2+ are both (a) selectively inhibitory of a Pseudomonas isolate from an uncontaminated well vs. a Rhodanobacter isolate from a contaminated well, and (b) reach toxic concentrations (for the Pseudomonas isolate) in the Rhodanobacter-dominated wells. We used mixtures of ions to simulate the groundwater conditions in the most contaminated wells and verified that few isolates aside from Rhodanobacter can tolerate these eight ions. These results clarify which ions are likely causal factors that impact the microbial community at this field site and are not merely correlated with taxonomic shifts. Furthermore, our general high-throughput approach can be applied to other environments, isolates, and conditions to systematically help identify selective pressures on microbial communities.},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{ge_2019,

title = {Iron- and aluminium-induced depletion of molybdenum in acidic environments impedes the nitrogen cycle.},

author = {Xiaoxuan Ge and Brian J Vaccaro and Michael P Thorgersen and Farris L Poole and Erica L Majumder and Grant M Zane and Kara B De León and Andrew W Lancaster and Ji Won Moon and Charles J Paradis and Frederick von Netzer and David A Stahl and Paul D Adams and Adam P Arkin and Judy D Wall and Terry C Hazen and Michael W W Adams},

url = {http://dx.doi.org/10.1111/1462-2920.14435},

doi = {10.1111/1462-2920.14435},

year  = {2019},

date = {2019-01-01},

urldate = {2021-05-25},

journal = {Environmental Microbiology},

volume = {21},

number = {1},

pages = {152-163},

abstract = {Anthropogenic nitrate contamination is a serious problem in many natural environments. Nitrate removal by microbial action is dependent on the metal molybdenum (Mo), which is required by nitrate reductase for denitrification and dissimilatory nitrate reduction to ammonium. The soluble form of Mo, molybdate (MoO42- ), is incorporated into and adsorbed by iron (Fe) and aluminium (Al) (oxy) hydroxide minerals. Herein we used Oak Ridge Reservation (ORR) as a model nitrate-contaminated acidic environment to investigate whether the formation of Fe- and Al-precipitates could impede microbial nitrate removal by depleting Mo. We demonstrate that Fe and Al mineral formation that occurs as the pH of acidic synthetic groundwater is increased, decreases soluble Mo to low picomolar concentrations, a process proposed to mimic environmental diffusion of acidic contaminated groundwater. Analysis of ORR sediments revealed recalcitrant Mo in the contaminated core that co-occurred with Fe and Al, consistent with Mo scavenging by Fe/Al precipitates. Nitrate removal by ORR isolate Pseudomonas fluorescens N2A2 is virtually abolished by Fe/Al precipitate-induced Mo depletion. The depletion of naturally occurring Mo in nitrate- and Fe/Al-contaminated acidic environments like ORR or acid mine drainage sites has the potential to impede microbial-based nitrate reduction thereby extending the duration of nitrate in the environment. copyright 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{paradis_2019,

title = {Improved Method for Estimating Reaction Rates During Push-Pull Tests.},

author = {Charles J Paradis and Emma R Dixon and Lauren M Lui and Adam P Arkin and Jack C Parker and Jonathan D Istok and Edmund Perfect and Larry D {McKay} and Terry C Hazen},

url = {http://dx.doi.org/10.1111/gwat.12770},

doi = {10.1111/gwat.12770},

year  = {2019},

date = {2019-01-01},

urldate = {2021-05-25},

journal = {Ground Water},

volume = {57},

number = {2},

pages = {292-302},

abstract = {The breakthrough curve obtained from a single-well push-pull test can be adjusted to account for dilution of the injection fluid in the aquifer fluid. The dilution-adjusted breakthrough curve can be analyzed to estimate the reaction rate of a solute. The conventional dilution-adjusted method assumes that the ratios of the concentrations of the nonreactive and reactive solutes in the injection fluid vs. the aquifer fluid are equal. If this assumption is invalid, the conventional method will generate inaccurate breakthrough curves and may lead to erroneous conclusions regarding the reactivity of a solute. In this study, a new method that generates a dilution-adjusted breakthrough curve was theoretically developed to account for any possible combination of nonreactive and reactive solute concentrations in the injection and aquifer fluids. The newly developed method was applied to a field-based data set and was shown to generate more accurate dilution-adjusted breakthrough curves. The improved dilution-adjusted method presented here is simple, makes no assumptions regarding the concentrations of the nonreactive and reactive solutes in the injection and aquifer fluids, and easily allows for estimating reaction rates during push-pull tests. copyright 2018 The Authors. Groundwater published by Wiley Periodicals, Inc. on behalf of National Ground Water Association.},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{smith_2018,

title = {Impact of hydrologic boundaries on microbial planktonic and biofilm communities in shallow terrestrial subsurface environments.},

author = {H J Smith and A J Zelaya and K B De León and R Chakraborty and D A Elias and T C Hazen and A P Arkin and A B Cunningham and M W Fields},

url = {http://dx.doi.org/10.1093/femsec/fiy191},

doi = {10.1093/femsec/fiy191},

year  = {2018},

date = {2018-12-01},

urldate = {2021-05-25},

journal = {FEMS Microbiology Ecology},

volume = {94},

number = {12},

abstract = {Subsurface environments contain a large proportion of planetary microbial biomass and harbor diverse communities responsible for mediating biogeochemical cycles important to groundwater used by human society for consumption, irrigation, agriculture and industry. Within the saturated zone, capillary fringe and vadose zones, microorganisms can reside in two distinct phases (planktonic or biofilm), and significant differences in community composition, structure and activity between free-living and attached communities are commonly accepted. However, largely due to sampling constraints and the challenges of working with solid substrata, the contribution of each phase to subsurface processes is largely unresolved. Here, we synthesize current information on the diversity and activity of shallow freshwater subsurface habitats, discuss the challenges associated with sampling planktonic and biofilm communities across spatial, temporal and geological gradients, and discuss how biofilms may be constrained within shallow terrestrial subsurface aquifers. We suggest that merging traditional activity measurements and sequencing/-omics technologies with hydrological parameters important to sediment biofilm assembly and stability will help delineate key system parameters. Ultimately, integration will enhance our understanding of shallow subsurface ecophysiology in terms of bulk-flow through porous media and distinguish the respective activities of sessile microbial communities from more transient planktonic communities to ecosystem service and maintenance.},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{price_2018a,

title = {Mutant phenotypes for thousands of bacterial genes of unknown function.},

author = {Morgan N Price and Kelly M Wetmore and Jordan R Waters and Mark Callaghan and Jayashree Ray and Hualan Liu and Jennifer V Kuehl and Ryan A Melnyk and Jacob S Lamson and Yumi Suh and Hans K Carlson and Zuelma Esquivel and Harini Sadeeshkumar and Romy Chakraborty and Grant M Zane and Benjamin E Rubin and Judy D Wall and Axel Visel and James Bristow and Matthew J Blow and Adam P Arkin and Adam M Deutschbauer},

url = {http://www.nature.com/articles/s41586-018-0124-0},

doi = {10.1038/s41586-018-0124-0},

issn = {0028-0836},

year  = {2018},

date = {2018-05-16},

urldate = {2021-05-25},

journal = {Nature},

volume = {557},

number = {7706},

pages = {503-509},

abstract = {One-third of all protein-coding genes from bacterial genomes cannot be annotated with a function. Here, to investigate the functions of these genes, we present genome-wide mutant fitness data from 32 diverse bacteria across dozens of growth conditions. We identified mutant phenotypes for 11,779 protein-coding genes that had not been annotated with a specific function. Many genes could be associated with a specific condition because the gene affected fitness only in that condition, or with another gene in the same bacterium because they had similar mutant phenotypes. Of the poorly annotated genes, 2,316 had associations that have high confidence because they are conserved in other bacteria. By combining these conserved associations with comparative genomics, we identified putative DNA repair proteins; in addition, we propose specific functions for poorly annotated enzymes and transporters and for uncharacterized protein families. Our study demonstrates the scalability of microbial genetics and its utility for improving gene annotations.},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{christensen_2018,

title = {Use of in-field bioreactors demonstrate groundwater filtration influences planktonic bacterial community assembly, but not biofilm composition.},

author = {Geoff A Christensen and {JiWon} Moon and Allison M Veach and Jennifer J Mosher and Ann M Wymore and Joy D van Nostrand and Jizhong Zhou and Terry C Hazen and Adam P Arkin and Dwayne A Elias},

url = {http://dx.doi.org/10.1371/journal.pone.0194663},

doi = {10.1371/journal.pone.0194663},

year  = {2018},

date = {2018-03-20},

urldate = {2021-05-25},

journal = {Plos One},

volume = {13},

number = {3},

pages = {e0194663},

abstract = {Using in-field bioreactors, we investigated the influence of exogenous microorganisms in groundwater planktonic and biofilm microbial communities as part of the Integrated Field Research Challenge (IFRC). After an acclimation period with source groundwater, bioreactors received either filtered (0.22 μM filter) or unfiltered well groundwater in triplicate and communities were tracked routinely for 23 days after filtration was initiated. To address geochemical influences, the planktonic phase was assayed periodically for protein, organic acids, physico-/geochemical measurements and bacterial community (via 16S rRNA gene sequencing), while biofilms (i.e. microbial growth on sediment coupons) were targeted for bacterial community composition at the completion of the experiment (23 d). Based on Bray-Curtis distance, planktonic bacterial community composition varied temporally and between treatments (filtered, unfiltered bioreactors). Notably, filtration led to an increase in the dominant genus, Zoogloea relative abundance over time within the planktonic community, while remaining relatively constant when unfiltered. At day 23, biofilm communities were more taxonomically and phylogenetically diverse and substantially different from planktonic bacterial communities; however, the biofilm bacterial communities were similar regardless of filtration. These results suggest that although planktonic communities were sensitive to groundwater filtration, bacterial biofilm communities were stable and resistant to filtration. Bioreactors are useful tools in addressing questions pertaining to microbial community assembly and succession. These data provide a first step in understanding how an extrinsic factor, such as a groundwater inoculation and flux of microbial colonizers, impact how microbial communities assemble in environmental systems.},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{he_2018,

title = {Microbial functional gene diversity predicts groundwater contamination and ecosystem functioning.},

author = {Zhili He and Ping Zhang and Linwei Wu and Andrea M Rocha and Qichao Tu and Zhou Shi and Bo Wu and Yujia Qin and Jianjun Wang and Qingyun Yan and Daniel Curtis and Daliang Ning and Joy D Van Nostrand and Liyou Wu and Yunfeng Yang and Dwayne A Elias and David B Watson and Michael W W Adams and Matthew W Fields and Eric J Alm and Terry C Hazen and Paul D Adams and Adam P Arkin and Jizhong Zhou},

url = {http://dx.doi.org/10.1128/mBio.02435-17},

doi = {10.1128/mBio.02435-17},

year  = {2018},

date = {2018-02-20},

urldate = {2021-05-25},

journal = {mBio},

volume = {9},

number = {1},

abstract = {Contamination from anthropogenic activities has significantly impacted Earth's biosphere. However, knowledge about how environmental contamination affects the biodiversity of groundwater microbiomes and ecosystem functioning remains very limited. Here, we used a comprehensive functional gene array to analyze groundwater microbiomes from 69 wells at the Oak Ridge Field Research Center (Oak Ridge, TN), representing a wide pH range and uranium, nitrate, and other contaminants. We hypothesized that the functional diversity of groundwater microbiomes would decrease as environmental contamination (e.g., uranium or nitrate) increased or at low or high pH, while some specific populations capable of utilizing or resistant to those contaminants would increase, and thus, such key microbial functional genes and/or populations could be used to predict groundwater contamination and ecosystem functioning. Our results indicated that functional richness/diversity decreased as uranium (but not nitrate) increased in groundwater. In addition, about 5.9% of specific key functional populations targeted by a comprehensive functional gene array (GeoChip 5) increased significantly (P textless 0.05) as uranium or nitrate increased, and their changes could be used to successfully predict uranium and nitrate contamination and ecosystem functioning. This study indicates great potential for using microbial functional genes to predict environmental contamination and ecosystem functioning.IMPORTANCE Disentangling the relationships between biodiversity and ecosystem functioning is an important but poorly understood topic in ecology. Predicting ecosystem functioning on the basis of biodiversity is even more difficult, particularly with microbial biomarkers. As an exploratory effort, this study used key microbial functional genes as biomarkers to provide predictive understanding of environmental contamination and ecosystem functioning. The results indicated that the overall functional gene richness/diversity decreased as uranium increased in groundwater, while specific key microbial guilds increased significantly as uranium or nitrate increased. These key microbial functional genes could be used to successfully predict environmental contamination and ecosystem functioning. This study represents a significant advance in using functional gene markers to predict the spatial distribution of environmental contaminants and ecosystem functioning toward predictive microbial ecology, which is an ultimate goal of microbial ecology. Copyright copyright 2018 He et al.},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{liu_2018,

title = {Magic pools: parallel assessment of transposon delivery vectors in bacteria.},

author = {Hualan Liu and Morgan N Price and Robert Jordan Waters and Jayashree Ray and Hans K Carlson and Jacob S Lamson and Romy Chakraborty and Adam P Arkin and Adam M Deutschbauer},

url = {http://dx.doi.org/10.1128/mSystems.00143-17},

doi = {10.1128/mSystems.00143-17},

year  = {2018},

date = {2018-02-01},

urldate = {2021-05-25},

journal = {mSystems},

volume = {3},

number = {1},

abstract = {Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes. However, the development of a suitable TnSeq strategy for a given bacterium can be costly and time-consuming. To meet this challenge, we describe a part-based strategy for constructing libraries of hundreds of transposon delivery vectors, which we term "magic pools." Within a magic pool, each transposon vector has a different combination of upstream sequences (promoters and ribosome binding sites) and antibiotic resistance markers as well as a random DNA barcode sequence, which allows the tracking of each vector during mutagenesis experiments. To identify an efficient vector for a given bacterium, we mutagenize it with a magic pool and sequence the resulting insertions; we then use this efficient vector to generate a large mutant library. We used the magic pool strategy to construct transposon mutant libraries in five genera of bacteria, including three genera of the phylum Bacteroidetes. IMPORTANCE Molecular genetics is indispensable for interrogating the physiology of bacteria. However, the development of a functional genetic system for any given bacterium can be time-consuming. Here, we present a streamlined approach for identifying an effective transposon mutagenesis system for a new bacterium. Our strategy first involves the construction of hundreds of different transposon vector variants, which we term a "magic pool." The efficacy of each vector in a magic pool is monitored in parallel using a unique DNA barcode that is introduced into each vector design. Using archived DNA "parts," we next reassemble an effective vector for making a whole-genome transposon mutant library that is suitable for large-scale interrogation of gene function using competitive growth assays. Here, we demonstrate the utility of the magic pool system to make mutant libraries in five genera of bacteria.},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}