@article{lui_2020,

title = {A method for achieving complete microbial genomes and better quality bins from metagenomics data},

author = {Lauren M Lui and Torben N Nielsen and Adam P Arkin},

url = {http://biorxiv.org/lookup/doi/10.1101/2020.03.05.979740},

doi = {10.1101/2020.03.05.979740},

year  = {2020},

date = {2020-03-06},

urldate = {2021-05-25},

journal = {BioRxiv},

abstract = {Metagenomics facilitates the study of the genetic information from uncultured microbes and complex microbial communities. Assembling complete microbial genomes (i.e., circular with no misassemblies) from metagenomics data is difficult because most samples have high organismal complexity and strain diversity. Only 63 circularized bacterial and archaeal genomes have been assembled from metagenomics data despite the thousands of datasets that are available. Circularized genomes are important for (1) building a reference collection as scaffolds for future assemblies, (2) providing complete gene content of a genome, (3) confirming little or no contamination of a genome, (4) studying the genomic context and synteny of genes, and (5) linking protein coding genes to ribosomal RNA genes to aid metabolic inference in 16S rRNA gene sequencing studies. We developed a method to achieve circularized genomes using iterative assembly, binning, and read mapping. In addition, this method exposes potential misassemblies from k-mer based assemblies. We chose species of the Candidate Phyla Radiation (CPR) to focus our initial efforts because they have small genomes and are only known to have one copy of ribosomal RNA genes. We present 34 circular CPR genomes, one circular Margulisbacteria genome, and two circular megaphage genomes from 19 public and published datasets. We demonstrate findings that would likely be difficult without circularizing genomes, including that ribosomal genes are likely not operonic in the majority of CPR, and that some CPR harbor diverged forms of RNase P RNA.},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{ning2020quantitativeb,

title = {A quantitative framework reveals the ecological drivers of grassland soil microbial community assembly in response to warming},

author = {Daliang Ning, Mengting Yuan, Linwei Wu, Ya Zhan, Xue Guo, Xishu Zhou , Yunfeng Yang, Adam P. Arkin, Mary K. Firestone, Jizhong Zhou},

year  = {2020},

date = {2020-02-25},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{Mutalik2020,

title = {High-throughput mapping of the phage resistance landscape in E. coli},

author = {Vivek K. Mutalik, Benjamin A. Adler, Harneet S. Rishi, Denish Piya, Crystal Zhong,Britt Koskella, Richard Calendar, Pavel Novichkov, Morgan N. Price, Adam M. Deutschbauer, Adam P. Arkin},

year  = {2020},

date = {2020-02-16},

keywords = {arkin lab, biodesign},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32817099,

title = {Effects of Genetic and Physiological Đivergence on the Evolution of a Sulfate-Reducing Bacterium under Conditions of Elevated Ŧemperature},

author = {M L Kempher and X Tao and R Song and B Wu and D A Stahl and J D Wall and A P Arkin and A Zhou and J Zhou},

year  = {2020},

date = {2020-01-01},

journal = {mBio},

volume = {11},

number = {4},

abstract = {Adaptation via natural selection is an important driver of evolution, and repeatable adaptations of replicate populations, under conditions of a constant environment, have been extensively reported. However, isolated groups of populations in nature tend to harbor both genetic and physiological divergence due to multiple selective pressures that they have encountered. How this divergence affects adaptation of these populations to a new common environment remains unclear. To determine the impact of prior genetic and physiological divergence in shaping adaptive evolution to accommodate a new common environment, an experimental evolution study with the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH) was conducted. Two groups of replicate populations with genetic and physiological divergence, derived from a previous evolution study, were propagated in an elevated-temperature environment for 1,000 generations. Ancestor populations without prior experimental evolution were also propagated in the same environment as a control. After 1,000 generations, all the populations had increased growth rates and all but one had greater fitness in the new environment than the ancestor population. Moreover, improvements in growth rate were moderately affected by the divergence in the starting populations, while changes in fitness were not significantly affected. The mutations acquired at the gene level in each group of populations were quite different, indicating that the observed phenotypic changes were achieved by evolutionary responses that differed between the groups. Overall, our work demonstrated that the initial differences in fitness between the starting populations were eliminated by adaptation and that phenotypic convergence was achieved by acquisition of mutations in different genes.IMPORTANCE Improving our understanding of how previous adaptation influences evolution has been a long-standing goal in evolutionary biology. Natural selection tends to drive populations to find similar adaptive solutions for the same selective conditions. However, variations in historical environments can lead to both physiological and genetic divergence that can make evolution unpredictable. Here, we assessed the influence of divergence on the evolution of a model sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, in response to elevated temperature and found a significant effect at the genetic but not the phenotypic level. Understanding how these influences drive evolution will allow us to better predict how bacteria will adapt to various ecological constraints.},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32252814,

title = {Small and mighty: adaptation of superphylum Patescibacteria to groundwater environment drives their genome simplicity},

author = {R Tian and D Ning and Z He and P Zhang and S J Spencer and S Gao and W Shi and L Wu and Y Zhang and Y Yang and B G Adams and A M Rocha and B L Detienne and K A Lowe and D C Joyner and D M Klingeman and A P Arkin and M W Fields and T C Hazen and D A Stahl and E J Alm and J Zhou},

year  = {2020},

date = {2020-01-01},

journal = {Microbiome},

volume = {8},

number = {1},

pages = {51},

abstract = {The newly defined superphylum Patescibacteria such as Parcubacteria (OD1) and Microgenomates (OP11) has been found to be prevalent in groundwater, sediment, lake, and other aquifer environments. Recently increasing attention has been paid to this diverse superphylum including > 20 candidate phyla (a large part of the candidate phylum radiation, CPR) because it refreshed our view of the tree of life. However, adaptive traits contributing to its prevalence are still not well known. Here, we investigated the genomic features and metabolic pathways of Patescibacteria in groundwater through genome-resolved metagenomics analysis of > 600 Gbp sequence data. We observed that, while the members of Patescibacteria have reduced genomes (~ 1 Mbp) exclusively, functions essential to growth and reproduction such as genetic information processing were retained. Surprisingly, they have sharply reduced redundant and nonessential functions, including specific metabolic activities and stress response systems. The Patescibacteria have ultra-small cells and simplified membrane structures, including flagellar assembly, transporters, and two-component systems. Despite the lack of CRISPR viral defense, the bacteria may evade predation through deletion of common membrane phage receptors and other alternative strategies, which may explain the low representation of prophage proteins in their genomes and lack of CRISPR. By establishing the linkages between bacterial features and the groundwater environmental conditions, our results provide important insights into the functions and evolution of this CPR group. We found that Patescibacteria has streamlined many functions while acquiring advantages such as avoiding phage invasion, to adapt to the groundwater environment. The unique features of small genome size, ultra-small cell size, and lacking CRISPR of this large lineage are bringing new understandings on life of Bacteria. Our results provide important insights into the mechanisms for adaptation of the superphylum in the groundwater environments, and demonstrate a case where less is more, and small is mighty.},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{Berliner2020,

title = {Towards a Biomanufactory on Mars},

author = {Aaron Berliner and Jacob M Hilzinger and Anthony J Abel and Matthew McNulty and George Makrygiorgos and Nils J H Averesch and Soumyajit Sen Gupta and Alexander Benvenuti and Daniel Caddell and Stefano Cestellos-Blanco},

doi = {doi: 10.20944/preprints202012.0714.v1},

year  = {2020},

date = {2020-01-01},

journal = {preprints.org},

publisher = {Preprints},

keywords = {cubes},

pubstate = {published},

tppubtype = {article}

}

@article{Abel2020,

title = {Systems-informed genome mining for electroautotrophic microbial production},

author = {Anthony J Abel and Jacob M Hilzinger and Adam P Arkin and Douglas S Clark},

year  = {2020},

date = {2020-01-01},

journal = {bioRxiv},

publisher = {Cold Spring Harbor Laboratory},

keywords = {cubes},

pubstate = {published},

tppubtype = {article}

}

@article{pmid32372050,

title = {Selective carbon sources influence the end products of microbial nitrate respiration},

author = {H K Carlson and L M Lui and M N Price and A E Kazakov and A V Carr and J V Kuehl and T K Owens and T Nielsen and A P Arkin and A M Deutschbauer},

year  = {2020},

date = {2020-00-01},

journal = {ISME J},

volume = {14},

number = {8},

pages = {2034--2045},

abstract = {Respiratory and catabolic genes are differentially distributed across microbial genomes. Thus, specific carbon sources may favor different respiratory processes. We profiled the influence of 94 carbon sources on the end products of nitrate respiration in microbial enrichment cultures from diverse terrestrial environments. We found that some carbon sources consistently favor dissimilatory nitrate reduction to ammonium (DNRA/nitrate ammonification) while other carbon sources favor nitrite accumulation or denitrification. For an enrichment culture from aquatic sediment, we sequenced the genomes of the most abundant strains, matched these genomes to 16S rDNA exact sequence variants (ESVs), and used 16S rDNA amplicon sequencing to track the differential enrichment of functionally distinct ESVs on different carbon sources. We found that changes in the abundances of strains with different genetic potentials for nitrite accumulation, DNRA or denitrification were correlated with the nitrite or ammonium concentrations in the enrichment cultures recovered on different carbon sources. Specifically, we found that either L-sorbose or D-cellobiose enriched for a Klebsiella nitrite accumulator, other sugars enriched for an Escherichia nitrate ammonifier, and citrate or formate enriched for a Pseudomonas denitrifier and a Sulfurospirillum nitrate ammonifier. Our results add important nuance to the current paradigm that higher concentrations of carbon will always favor DNRA over denitrification or nitrite accumulation, and we propose that, in some cases, carbon composition can be as important as carbon concentration in determining nitrate respiratory end products. Furthermore, our approach can be extended to other environments and metabolisms to characterize how selective parameters influence microbial community composition, gene content, and function.},

keywords = {arkin lab, enigma},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31848309,

title = {Genomewide and Enzymatic Analysis Reveals Efficient d-Galacturonic Acid Metabolism in the Basidiomycete Yeast Rhodosporidium toruloides},

author = {R J Protzko and C A Hach and S T Coradetti and M A Hackhofer and S Magosch and N Thieme and G M Geiselman and A P Arkin and J M Skerker and J E Dueber and J P Benz},

year  = {2019},

date = {2019-12-01},

journal = {mSystems},

volume = {4},

number = {6},

abstract = {Biorefining of renewable feedstocks is one of the most promising routes to replace fossil-based products. Since many common fermentation hosts, such as Saccharomyces cerevisiae, are naturally unable to convert many component plant cell wall polysaccharides, the identification of organisms with broad catabolism capabilities represents an opportunity to expand the range of substrates used in fermentation biorefinery approaches. The red basidiomycete yeast Rhodosporidium toruloides is a promising and robust host for lipid- and terpene-derived chemicals. Previous studies demonstrated assimilation of a range of substrates, from C5/C6 sugars to aromatic molecules similar to lignin monomers. In the current study, we analyzed the potential of R. toruloides to assimilate d-galacturonic acid, a major sugar in many pectin-rich agricultural waste streams, including sugar beet pulp and citrus peels. d-Galacturonic acid is not a preferred substrate for many fungi, but its metabolism was found to be on par with those of d-glucose and d-xylose in R. toruloides A genomewide analysis by combined transcriptome sequencing (RNA-seq) and RB-TDNA-seq revealed those genes with high relevance for fitness on d-galacturonic acid. While R. toruloides was found to utilize the nonphosphorylative catabolic pathway known from ascomycetes, the maximal velocities of several enzymes exceeded those previously reported. In addition, an efficient downstream glycerol catabolism and a novel transcription factor were found to be important for d-galacturonic acid utilization. These results set the basis for use of R. toruloides as a potential host for pectin-rich waste conversions and demonstrate its suitability as a model for metabolic studies with basidiomycetes.IMPORTANCE The switch from the traditional fossil-based industry to a green and sustainable bioeconomy demands the complete utilization of renewable feedstocks. Many currently used bioconversion hosts are unable to utilize major components of plant biomass, warranting the identification of microorganisms with broader catabolic capacity and characterization of their unique biochemical pathways. d-Galacturonic acid is a plant component of bioconversion interest and is the major backbone sugar of pectin, a plant cell wall polysaccharide abundant in soft and young plant tissues. The red basidiomycete and oleaginous yeast Rhodosporidium toruloides has been previously shown to utilize a range of sugars and aromatic molecules. Using state-of-the-art functional genomic methods and physiological and biochemical assays, we elucidated the molecular basis underlying the efficient metabolism of d-galacturonic acid. This study identified an efficient pathway for uronic acid conversion to guide future engineering efforts and represents the first detailed metabolic analysis of pectin metabolism in a basidiomycete fungus.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31387058,

title = {Ħigh spatiotemporal variability of bacterial diversity over short time scales with unique hydrochemical associations within a shallow aquifer},

author = {A J Zelaya and A E Parker and K L Bailey and P Zhang and J Van Nostrand and D Ning and D A Elias and J Zhou and T C Hazen and A P Arkin and M W Fields},

year  = {2019},

date = {2019-11-01},

journal = {Water Res.},

volume = {164},

pages = {114917},

abstract = {Understanding microbial community structure and function within the subsurface is critical to assessing overall quality and maintenance of groundwater; however, the factors that determine microbial community assembly, structure, and function in groundwater systems and their impact on water quality remains poorly understood. In this study, three shallow wells (FW301, FW303, FW305) in a non-contaminated shallow aquifer in the ENIGMA-Oak Ridge Field Research Center (Oak Ridge, TN) were sampled approximately 3 times a week over a period of three months to measure changes in groundwater geochemistry and microbial diversity. It was expected that the sampled microbial diversity from two historic field wells (FW301, FW303) would be relatively stable, while diversity from a newer well (FW305) would be less stable over time. The wells displayed some degree of hydrochemical variability over time unique to each well, with FW303 being overall the most stable well and FW301 being the most dynamic based upon dissolved oxygen, conductivity, and nitrate. Community analysis via ss-rRNA paired-end sequencing and distribution-based clustering revealed higher OTU richness, diversity, and variability in groundwater communities of FW301 than the other two wells for diversity binned over all time points. Microbial community composition of a given well was on average > 50% dissimilar to any other well at a given time (days), yet, functional gene diversity as measured with GeoChip remained relatively constant. Similarities in community structure across wells were observed with respect to the presence of 20 shared bacterial groups in all samples in all wells, although at varying levels over the tested time period. Similarity percentage (SIMPER) analysis revealed that variability in FW301 was largely attributed to low abundance, highly-transient populations, while variability in the most hydrochemically stable well (FW303) was due to fluctuations in more highly abundant and frequently present taxa. Additionally, the youngest well FW305 showed a dramatic shift in community composition towards the end of the sampling period that was not observed in the other wells, suggesting possible succession events over time. Time-series analysis using vector auto-regressive models and Granger causality showed unique relationships between richness and geochemistry over time in each well. These results indicate temporally dynamic microbial communities over short time scales, with day-to-day population shifts in local community structure influenced by available source community diversity and local groundwater hydrochemistry.},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{zelaya_2019,

title = {High spatiotemporal variability of bacterial diversity over short time scales with unique hydrochemical associations within a shallow aquifer.},

author = {Anna J Zelaya and Albert E Parker and Kathryn L Bailey and Ping Zhang and Joy Van Nostrand and Daliang Ning and Dwayne A Elias and Jizhong Zhou and Terry C Hazen and Adam P Arkin and Matthew W Fields},

url = {http://dx.doi.org/10.1016/j.watres.2019.114917},

doi = {10.1016/j.watres.2019.114917},

year  = {2019},

date = {2019-11-01},

urldate = {2021-05-25},

journal = {Water Research},

volume = {164},

pages = {114917},

abstract = {Understanding microbial community structure and function within the subsurface is critical to assessing overall quality and maintenance of groundwater; however, the factors that determine microbial community assembly, structure, and function in groundwater systems and their impact on water quality remains poorly understood. In this study, three shallow wells (FW301, FW303, FW305) in a non-contaminated shallow aquifer in the ENIGMA-Oak Ridge Field Research Center (Oak Ridge, TN) were sampled approximately 3 times a week over a period of three months to measure changes in groundwater geochemistry and microbial diversity. It was expected that the sampled microbial diversity from two historic field wells (FW301, FW303) would be relatively stable, while diversity from a newer well (FW305) would be less stable over time. The wells displayed some degree of hydrochemical variability over time unique to each well, with FW303 being overall the most stable well and FW301 being the most dynamic based upon dissolved oxygen, conductivity, and nitrate. Community analysis via ss-rRNA paired-end sequencing and distribution-based clustering revealed higher OTU richness, diversity, and variability in groundwater communities of FW301 than the other two wells for diversity binned over all time points. Microbial community composition of a given well was on average textgreater 50% dissimilar to any other well at a given time (days), yet, functional gene diversity as measured with GeoChip remained relatively constant. Similarities in community structure across wells were observed with respect to the presence of 20 shared bacterial groups in all samples in all wells, although at varying levels over the tested time period. Similarity percentage (SIMPER) analysis revealed that variability in FW301 was largely attributed to low abundance, highly-transient populations, while variability in the most hydrochemically stable well (FW303) was due to fluctuations in more highly abundant and frequently present taxa. Additionally, the youngest well FW305 showed a dramatic shift in community composition towards the end of the sampling period that was not observed in the other wells, suggesting possible succession events over time. Time-series analysis using vector auto-regressive models and Granger causality showed unique relationships between richness and geochemistry over time in each well. These results indicate temporally dynamic microbial communities over short time scales, with day-to-day population shifts in local community structure influenced by available source community diversity and local groundwater hydrochemistry. Copyright copyright 2019 Elsevier Ltd. All rights reserved.},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{thorgersen_2019,

title = {Nitrate-Utilizing Microorganisms Resistant to Multiple Metals from the Heavily Contaminated Oak Ridge Reservation.},

author = {Michael P Thorgersen and Xiaoxuan Ge and Farris L Poole and Morgan N Price and Adam P Arkin and Michael W W Adams},

url = {http://dx.doi.org/10.1128/AEM.00896-19},

doi = {10.1128/AEM.00896-19},

year  = {2019},

date = {2019-09-01},

urldate = {2021-05-25},

journal = {Applied and Environmental Microbiology},

volume = {85},

number = {17},

abstract = {Contamination of environments with nitrate generated by industrial processes and the use of nitrogen-containing fertilizers is a growing problem worldwide. While nitrate can be removed from contaminated areas by microbial denitrification, nitrate frequently occurs with other contaminants, such as heavy metals, that have the potential to impede the process. Here, nitrate-reducing microorganisms were enriched and isolated from both groundwater and sediments at the Oak Ridge Reservation (ORR) using concentrations of nitrate and metals (Al, Mn, Fe, Co, Ni, Cu, Cd, and U) similar to those observed in a contaminated environment at ORR. Seven new metal-resistant, nitrate-reducing strains were characterized, and their distribution across both noncontaminated and contaminated areas at ORR was examined. While the seven strains have various pH ranges for growth, carbon source preferences, and degrees of resistance to individual and combinations of metals, all were able to reduce nitrate at similar rates both in the presence and absence of the mixture of metals found in the contaminated ORR environment. Four strains were identified in groundwater samples at different ORR locations by exact 16S RNA sequence variant analysis, and all four were found in both noncontaminated and contaminated areas. By using environmentally relevant metal concentrations, we successfully isolated multiple organisms from both ORR noncontaminated and contaminated environments that are capable of reducing nitrate in the presence of extreme mixed-metal contamination.IMPORTANCE Nitrate contamination is a global issue that affects groundwater quality. In some cases, cocontamination of groundwater with nitrate and mixtures of heavy metals could decrease microbially mediated nitrate removal, thereby increasing the duration of nitrate contamination. Here, we used metal and nitrate concentrations that are present in a contaminated site at the Oak Ridge Reservation to isolate seven metal-resistant strains. All were able to reduce nitrate in the presence of high concentrations of a mixture of heavy metals. Four of seven strains were located in pristine as well as contaminated sites at the Oak Ridge Reservation. Further study of these nitrate-reducing strains will uncover mechanisms of resistance to multiple metals that will increase our understanding of the effect of nitrate and metal contamination on groundwater microbial communities. Copyright copyright 2019 American Society for Microbiology.},

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{price_2019b,

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

author = {Morgan N Price and Grant M Zane and Jennifer V Kuehl and Ryan A Melnyk and Judy D Wall and Adam M Deutschbauer and Adam P Arkin},

url = {http://dx.doi.org/10.1371/journal.pgen.1008106},

doi = {10.1371/journal.pgen.1008106},

year  = {2019},

date = {2019-04-03},

urldate = {2021-05-25},

journal = {PLoS Genetics},

volume = {15},

number = {4},

pages = {e1008106},

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

keywords = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{price_2019a,

title = {Curated BLAST for genomes.},

author = {Morgan N Price and Adam P Arkin},

url = {http://dx.doi.org/10.1128/mSystems.00072-19},

doi = {10.1128/mSystems.00072-19},

year  = {2019},

date = {2019-04-01},

urldate = {2021-05-25},

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 = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{price_2019,

title = {Oxidative pathways of deoxyribose and deoxyribonate catabolism.},

author = {Morgan N Price and Jayashree Ray and Anthony T Iavarone and Hans K Carlson and Elizabeth M Ryan and Rex R Malmstrom and Adam P Arkin and Adam M Deutschbauer},

url = {http://dx.doi.org/10.1128/mSystems.00297-18},

doi = {10.1128/mSystems.00297-18},

year  = {2019},

date = {2019-02-05},

urldate = {2021-05-25},

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 = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{mutalik_2019,

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

author = {Vivek K Mutalik and Pavel S Novichkov and Morgan N Price and Trenton K Owens and Mark Callaghan and Sean Carim and Adam M Deutschbauer and Adam P Arkin},

url = {http://www.nature.com/articles/s41467-018-08177-8},

doi = {10.1038/s41467-018-08177-8},

issn = {2041-1723},

year  = {2019},

date = {2019-01-18},

urldate = {2021-05-25},

journal = {Nature Communications},

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 = {enigma},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31397669,

title = {Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a},

author = {G J Knott and B F Cress and J J Liu and B W Thornton and R J Lew and B Al-Shayeb and D J Rosenberg and M Hammel and B A Adler and M J Lobba and M Xu and A P Arkin and C Fellmann and J A Doudna},

year  = {2019},

date = {2019-01-01},

journal = {Elife},

volume = {8},

abstract = {CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the L. bacterium Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The Ac. species Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31253673,

title = {Nitrate-Utilizing Microorganisms Resistant to Multiple Metals from the Ħeavily Contaminated Oak Ridge Reservation},

author = {M P Thorgersen and X Ge and F L Poole and M N Price and A P Arkin and M W W Adams},

year  = {2019},

date = {2019-01-01},

volume = {85},

number = {17},

abstract = {Contamination of environments with nitrate generated by industrial processes and the use of nitrogen-containing fertilizers is a growing problem worldwide. While nitrate can be removed from contaminated areas by microbial denitrification, nitrate frequently occurs with other contaminants, such as heavy metals, that have the potential to impede the process. Here, nitrate-reducing microorganisms were enriched and isolated from both groundwater and sediments at the Oak Ridge Reservation (ORR) using concentrations of nitrate and metals (Al, Mn, Fe, Co, Ni, Cu, Cd, and U) similar to those observed in a contaminated environment at ORR. Seven new metal-resistant, nitrate-reducing strains were characterized, and their distribution across both noncontaminated and contaminated areas at ORR was examined. While the seven strains have various pH ranges for growth, carbon source preferences, and degrees of resistance to individual and combinations of metals, all were able to reduce nitrate at similar rates both in the presence and absence of the mixture of metals found in the contaminated ORR environment. Four strains were identified in groundwater samples at different ORR locations by exact 16S RNA sequence variant analysis, and all four were found in both noncontaminated and contaminated areas. By using environmentally relevant metal concentrations, we successfully isolated multiple organisms from both ORR noncontaminated and contaminated environments that are capable of reducing nitrate in the presence of extreme mixed-metal contamination.IMPORTANCE Nitrate contamination is a global issue that affects groundwater quality. In some cases, cocontamination of groundwater with nitrate and mixtures of heavy metals could decrease microbially mediated nitrate removal, thereby increasing the duration of nitrate contamination. Here, we used metal and nitrate concentrations that are present in a contaminated site at the Oak Ridge Reservation to isolate seven metal-resistant strains. All were able to reduce nitrate in the presence of high concentrations of a mixture of heavy metals. Four of seven strains were located in pristine as well as contaminated sites at the Oak Ridge Reservation. Further study of these nitrate-reducing strains will uncover mechanisms of resistance to multiple metals that will increase our understanding of the effect of nitrate and metal contamination on groundwater microbial communities.},

keywords = {arkin lab, enigma},

pubstate = {published},

tppubtype = {article}

}

@article{pmid31064836,

title = {Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in Pseudomonas putida Lysine Metabolism},

author = {M G Thompson and J M Blake-Hedges and P Cruz-Morales and J F Barajas and S C Curran and C B Eiben and N C Harris and V T Benites and J W Gin and W A Sharpless and F F Twigg and W Skyrud and R N Krishna and J H Pereira and E E K Baidoo and C J Petzold and P D Adams and A P Arkin and A M Deutschbauer and J D Keasling},

year  = {2019},

date = {2019-01-01},

journal = {mBio},

volume = {10},

number = {3},

abstract = {Despite intensive study for 50 years, the biochemical and genetic links between lysine metabolism and central metabolism in Pseudomonas putida remain unresolved. To establish these biochemical links, we leveraged random barcode transposon sequencing (RB-TnSeq), a genome-wide assay measuring the fitness of thousands of genes in parallel, to identify multiple novel enzymes in both l- and d-lysine metabolism. We first describe three pathway enzymes that catabolize l-2-aminoadipate (l-2AA) to 2-ketoglutarate (2KG), connecting d-lysine to the TCA cycle. One of these enzymes, P. putida 5260 (PP_5260), contains a DUF1338 domain, representing a family with no previously described biological function. Our work also identified the recently described coenzyme A (CoA)-independent route of l-lysine degradation that results in metabolization to succinate. We expanded on previous findings by demonstrating that glutarate hydroxylase CsiD is promiscuous in its 2-oxoacid selectivity. Proteomics of selected pathway enzymes revealed that expression of catabolic genes is highly sensitive to the presence of particular pathway metabolites, implying intensive local and global regulation. This work demonstrated the utility of RB-TnSeq for discovering novel metabolic pathways in even well-studied bacteria, as well as its utility a powerful tool for validating previous research.IMPORTANCEP. putida lysine metabolism can produce multiple commodity chemicals, conferring great biotechnological value. Despite much research, the connection of lysine catabolism to central metabolism in P. putida remained undefined. Here, we used random barcode transposon sequencing to fill the gaps of lysine metabolism in P. putida We describe a route of 2-oxoadipate (2OA) catabolism, which utilizes DUF1338-containing protein P. putida 5260 (PP_5260) in bacteria. Despite its prevalence in many domains of life, DUF1338-containing proteins have had no known biochemical function. We demonstrate that PP_5260 is a metalloenzyme which catalyzes an unusual route of decarboxylation of 2OA to d-2-hydroxyglutarate (d-2HG). Our screen also identified a recently described novel glutarate metabolic pathway. We validate previous results and expand the understanding of glutarate hydroxylase CsiD by showing that can it use either 2OA or 2KG as a cosubstrate. Our work demonstrated that biological novelty can be rapidly identified using unbiased experimental genetics and that RB-TnSeq can be used to rapidly validate previous results.},

keywords = {arkin lab, biodesign},

pubstate = {published},

tppubtype = {article}

}

@article{pmid30967567,

title = {Iterative screening methodology enables isolation of strains with improved properties for a FACS-based screen and increased L-ĐOPA production},

author = {J Savitskaya and R J Protzko and F Z Li and A P Arkin and J E Dueber},

year  = {2019},

date = {2019-01-01},

journal = {Sci Rep},

volume = {9},

number = {1},

pages = {5815},

abstract = {Optimizing microbial hosts for the large-scale production of valuable metabolites often requires multiple mutations and modifications to the host's genome. We describe a three-round screen for increased L-DOPA production in S. cerevisiae using FACS enrichment of an enzyme-coupled biosensor for L-DOPA. Multiple rounds of screening were enabled by a single build of a barcoded in vitro transposon-mediated disruption library. New background strains for screening were built for each iteration using results from previous iterations. The same in vitro transposon-mediated disruption library was integrated by homologous recombination into new background strains in each round of screening. Compared with creating new transposon insertions in each round, this method takes less time and saves the cost of additional sequencing to characterize transposon insertion sites. In the first two rounds of screening, we identified deletions that improved biosensor compartmentalization and, consequently, improved our ability to screen for L-DOPA production. In a final round, we discovered that deletion of heme oxygenase (HMX1) increases total heme concentration and increases L-DOPA production, using dopamine measurement as a proxy. We further demonstrated that deleting HMX1 may represent a general strategy for P450 function improvement by improving activity of a second P450 enzyme, BM3, which performs a distinct reaction.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}