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At least 793 records · Page 44

Ecophysiological and genomic analyses of a representative isolate of highly abundant Bacillus cereus strains in contaminated subsurface sediments

Abstract Bacillus cereus strain CPT56D‐587‐MTF (CPTF) was isolated from the highly contaminated Oak Ridge Reservation (ORR) subsurface. This site is contaminated with high levels of nitric acid and multiple heavy metals. Amplicon sequencing of the 16S rRNA genes (V4 region) in sediment from this area revealed an amplicon sequence variant (ASV) with 100% identity to the CPTF 16S rRNA sequence. Notably, this CPTF‐matching ASV had the highest relative abundance in this community survey, with a median relative abundance of 3.77% and comprised 20%–40% of reads in some samples. Pangenomic analysis revealed that strain CPTF has expanded genomic content compared to other B. cereus species—largely due to plasmid acquisition and expansion of transposable elements. This suggests that these features are important for rapid adaptation to native environmental stressors. We connected genotype to phenotype in the context of the unique geochemistry of the site. These analyses revealed that certain genes (e.g. nitrate reductase, heavy metal efflux pumps) that allow this strain to successfully occupy the geochemically heterogenous microniches of its native site are characteristic of the B. cereus species while others such as acid tolerance are mobile genetic element associated and are generally unique to strain CPTF.

59 BASIC BIOLOGICAL SCIENCES↗

Metagenome‐Assembled Genomes for Oligotrophic Nitrifiers From a Mountainous Gravelbed Floodplain

Riparian floodplains are important regions for biogeochemical cycling, including nitrogen. Here, we present MAGs from nitrifying microorganisms, including ammonia-oxidising archaea (AOA) and comammox bacteria from Slate River (SR) floodplain sediments (Crested Butte, CO, US). Additionally, we explore MAGs from potential nitrite-oxidising bacteria (NOB) from the Nitrospirales. AOA diversity in SR is lower than observed in other western US floodplain sediments and Nitrosotalea-like lineages such as the genus TA-20 are the dominant AOA. No ammonia-oxidising bacteria (AOB) MAGs were recovered. Microorganisms from the Palsa-1315 genus (clade B comammox) are the most abundant ammonia-oxidizers in SR floodplain sediments. Established NOB are conspicuously absent; however, we recovered MAGs from uncultured lineages of the NS-4 family (Nitrospirales) and Nitrospiraceae that we propose as putative NOB. Nitrite oxidation may be carried out by organisms sister to established Nitrospira NOB lineages based on the genomic content of uncultured Nitrospirales clades. Nitrifier MAGs recovered from SR floodplain sediments harbour genes for using alternative sources of ammonia, such as urea, cyanate, biuret, triuret and nitriles. In conclusion, the SR floodplain therefore appears to be a low ammonia flux environment that selects for oligotrophic nitrifiers.

60 APPLIED LIFE SCIENCES↗

Biomass yield improvement in switchgrass through genomic prediction of flowering time

The seasonal timing of the transition from vegetative to reproductive growth has a major impact on biomass accumulation in switchgrass. Late-flowering switchgrass cultivars produce greater biomass, a critical trait for sustainable bioenergy production. Genomic prediction (GP) may allow rapid selection of late-flowering individuals with reduced time and expense for field evaluations. To evaluate GP, two flowering time traits (heading date and anthesis date) were collected on 1532 genotypes from four breeding populations: Midwest, Gulf, Atlantic, and Hybrid. These were sequenced using genotype-by-sequencing (530,792 single-nucleotide polymorphisms). Predictive ability of single-trait and multi-trait models were evaluated by cross-validation, by prediction of a progeny trial (n = 122), and through prediction of yield performance in a parallel experiment (n = 52). Predictive ability was not improved by sharing information among breeding groups. Overall, multi-trait models provided an advantage during cross-validation, but a smaller advantage during progeny prediction. Within populations, GP resulted in lower per-cycle progress than previously reported field evaluations (3.1 vs. 5.0 day –1 cycle –1 ). However, GP cycles are potentially much faster than field evaluations. When directly predicting biomass yield, the Hybrid training population had a predictive ability of 0.54–0.63. This reinforces the strong linkage between biomass yields in swards and flowering time. Furthermore, these results highlight the value of GP for rapid yield improvement in switchgrass, particularly in a breeding program designed to share information between biomass yield trials and low-cost flowering time evaluations.

09 BIOMASS FUELS↗

Combining GWAS and population genomic analyses to characterize coevolution in a legume‐rhizobia symbiosis

Abstract The mutualism between legumes and rhizobia is clearly the product of past coevolution. However, the nature of ongoing evolution between these partners is less clear. To characterize the nature of recent coevolution between legumes and rhizobia, we used population genomic analysis to characterize selection on functionally annotated symbiosis genes as well as on symbiosis gene candidates identified through a two‐species association analysis. For the association analysis, we inoculated each of 202 accessions of the legume host Medicago truncatula with a community of 88 Sinorhizobia (Ensifer) meliloti strains. Multistrain inoculation, which better reflects the ecological reality of rhizobial selection in nature than single‐strain inoculation, allows strains to compete for nodulation opportunities and host resources and for hosts to preferentially form nodules and provide resources to some strains. We found extensive host by symbiont, that is, genotype‐by‐genotype, effects on rhizobial fitness and some annotated rhizobial genes bear signatures of recent positive selection. However, neither genes responsible for this variation nor annotated host symbiosis genes are enriched for signatures of either positive or balancing selection. This result suggests that stabilizing selection dominates selection acting on symbiotic traits and that variation in these traits is under mutation‐selection balance. Consistent with the lack of positive selection acting on host genes, we found that among‐host variation in growth was similar whether plants were grown with rhizobia or N‐fertilizer, suggesting that the symbiosis may not be a major driver of variation in plant growth in multistrain contexts.

59 BASIC BIOLOGICAL SCIENCES↗

Functional and comparative genomics reveals conserved noncoding sequences in the nitrogen–fixing clade

Nitrogen is one of the most inaccessible plant nutrients, but certain species have overcome this limitation by establishing symbiotic interactions with nitrogen-fixing bacteria in the root nodule. This root–nodule symbiosis (RNS) is restricted to species within a single clade of angiosperms, suggesting a critical, but undetermined, evolutionary event at the base of this clade. To identify putative regulatory sequences implicated in the evolution of RNS, we evaluated the genomes of 25 species capable of nodulation and identified 3091 conserved noncoding sequences (CNS) in the nitrogen-fixing clade (NFC). We show that the chromatin accessibility of 452 CNS correlates significantly with the regulation of genes responding to lipochitooligosaccharides in Medicago truncatula. These included 38 CNS in proximity to 19 known genes involved in RNS. Five such regions are upstream of MtCRE1, Cytokinin Response Element 1, required to activate a suite of downstream transcription factors necessary for nodulation in M. truncatula. Genetic complementation of an Mtcre1 mutant showed a significant decrease of nodulation in the absence of the five CNS, when they are driving the expression of a functional copy of MtCRE1. CNS identified in the NFC may harbor elements required for the regulation of genes controlling RNS in M. truncatula.

59 BASIC BIOLOGICAL SCIENCES↗

Integration of physiologically relevant photosynthetic energy flows into whole genome models of light‐driven metabolism

SUMMARY Characterizing photosynthetic productivity is necessary to understand the ecological contributions and biotechnology potential of plants, algae, and cyanobacteria. Light capture efficiency and photophysiology have long been characterized by measurements of chlorophyll fluorescence dynamics. However, these investigations typically do not consider the metabolic network downstream of light harvesting. By contrast, genome‐scale metabolic models capture species‐specific metabolic capabilities but have yet to incorporate the rapid regulation of the light harvesting apparatus. Here, we combine chlorophyll fluorescence parameters defining photosynthetic and non‐photosynthetic yield of absorbed light energy with a metabolic model of the pennate diatom Phaeodactylum tricornutum. This integration increases the model predictive accuracy regarding growth rate, intracellular oxygen production and consumption, and metabolic pathway usage. Through the quantification of excess electron transport, we uncover the sequential activation of non‐radiative energy dissipation processes, cross‐compartment electron shuttling, and non‐photochemical quenching as the rapid photoacclimation strategy in P. tricornutum. Interestingly, the photon absorption thresholds that trigger the transition between these mechanisms were consistent at low and high incident photon fluxes. We use this understanding to explore engineering strategies for rerouting cellular resources and excess light energy towards bioproducts in silico . Overall, we present a methodology for incorporating a common, informative data type into computational models of light‐driven metabolism and show its utilization within the design–build–test–learn cycle for engineering of photosynthetic organisms.

59 BASIC BIOLOGICAL SCIENCES↗

Genome‐wide association identifies a BAHD acyltransferase activity that assembles an ester of glucuronosylglycerol and phenylacetic acid

SUMMARY Genome‐wide association studies (GWAS) are an effective approach to identify new specialized metabolites and the genes involved in their biosynthesis and regulation. In this study, GWAS of Arabidopsis thaliana soluble leaf and stem metabolites identified alleles of an uncharacterized BAHD‐family acyltransferase (AT5G57840) associated with natural variation in three structurally related metabolites. These metabolites were esters of glucuronosylglycerol, with one metabolite containing phenylacetic acid as the acyl component of the ester. Knockout and overexpression of AT5G57840 in Arabidopsis and heterologous overexpression in Nicotiana benthamiana and Escherichia coli demonstrated that it is capable of utilizing phenylacetyl‐CoA as an acyl donor and glucuronosylglycerol as an acyl acceptor. We, thus, named the protein Glucuronosylglycerol Ester Synthase (GGES). Additionally, phenylacetyl glucuronosylglycerol increased in Arabidopsis CYP79A2 mutants that overproduce phenylacetic acid and was lost in knockout mutants of UDP‐sulfoquinovosyl: diacylglycerol sulfoquinovosyl transferase, an enzyme required for glucuronosylglycerol biosynthesis and associated with glycerolipid metabolism under phosphate‐starvation stress. GGES is a member of a well‐supported clade of BAHD family acyltransferases that arose by duplication and neofunctionalized during the evolution of the Brassicales within a larger clade that includes HCT as well as enzymes that synthesize other plant‐specialized metabolites. Together, this work extends our understanding of the catalytic diversity of BAHD acyltransferases and uncovers a pathway that involves contributions from both phenylalanine and lipid metabolism.

09 BIOMASS FUELS↗

The Frequency of Sex: Population Genomics Reveals Differences in Recombination and Population Structure of the Aflatoxin-Producing Fungus Aspergillus flavus

The apparent rarity of sex in many fungal species has raised questions about how much sex is needed to purge deleterious mutations and how differences in frequency of sex impact fungal evolution. We sought to determine how differences in the extent of recombination between populations of Aspergillus flavus impact the evolution of genes associated with the synthesis of aflatoxin, a notoriously potent carcinogen. We sequenced the genomes of, and quantified aflatoxin production in, 94 isolates of A. flavus sampled from seven states in eastern and central latitudinal transects of the United States. The overall population is subdivided into three genetically differentiated populations (A, B, and C) that differ greatly in their extent of recombination, diversity, and aflatoxin-producing ability. Estimates of the number of recombination events and linkage disequilibrium decay suggest relatively frequent sex only in population A. Population B is sympatric with population A but produces significantly less aflatoxin and is the only population where the inability of nonaflatoxigenic isolates to produce aflatoxin was explained by multiple gene deletions. Population expansion evident in population B suggests a recent introduction or range expansion. Population C is largely nonaflatoxigenic and restricted mainly to northern sampling locations through restricted migration and/or selection. Despite differences in the number and type of mutations in the aflatoxin gene cluster, codon optimization and site frequency differences in synonymous and nonsynonymous mutations suggest that low levels of recombination in some A. flavus populations are sufficient to purge deleterious mutations.

59 BASIC BIOLOGICAL SCIENCES↗

Draft genome sequence of Yarrowia lipolytica NRRL Y-64008, an oleaginous yeast capable of growing on lignocellulosic hydrolysates

ABSTRACT Yarrowia lipolytica is an oleaginous yeast that produces high titers of fatty acid-derived biofuels and biochemicals. It can grow on hydrophobic carbon sources and lignocellulosic hydrolysates. The genome sequence of Y. lipolytica NRRL Y-64008 is reported to aid in its development as a biotechnological chassis for producing biofuels and bioproducts.

59 BASIC BIOLOGICAL SCIENCES↗

Complete genomes of Mucilaginibacter sabulilitoris SNA2 and Mucilaginibacter sp. cycad4: microbes with the potential for plant growth promotion

Mucilaginibacter species have been isolated from various environments, often in association with plants. Here, we report the complete genomes of Mucilaginibacter sabulilitoris SNA2 and Mucilaginibacter sp. cycad4. The former is the first available for that species, and based on 16S sequence analysis, the latter strain is likely a new species.

Mucilaginibacter↗

Draft Genome Sequence of the Biofuel-Relevant Microalga Desmodesmus armatus

A draft genome of 906 scaffolds of 115.8 Mb was created for Desmodesmus armatus , a diploid, lipid- and storage carbohydrate-accumulating microalga proven relevant for large-scale, outdoor cultivation, and serves as a model alga platform for improving photosynthetic efficiency and carbon assimilation for next-generation bioenergy production.

09 BIOMASS FUELS↗

SvAnna: efficient and accurate pathogenicity prediction of coding and regulatory structural variants in long-read genome sequencing

Structural variants (SVs) are implicated in the etiology of Mendelian diseases but have been systematically underascertained owing to sequencing technology limitations. Long-read sequencing enables comprehensive detection of SVs, but approaches for prioritization of candidate SVs are needed. Structural variant Annotation and analysis (SvAnna) assesses all classes of SVs and their intersection with transcripts and regulatory sequences, relating predicted effects on gene function with clinical phenotype data. SvAnna places 87% of deleterious SVs in the top ten ranks. The interpretable prioritizations offered by SvAnna will facilitate the widespread adoption of long-read sequencing in diagnostic genomics. SvAnna is available at https://github.com/TheJacksonLaboratory/SvAnna.

59 BASIC BIOLOGICAL SCIENCES↗

Systematic and scalable genome-wide essentiality mapping to identify nonessential genes in phages

Phages are one of the key ecological drivers of microbial community dynamics, function, and evolution. Despite their importance in bacterial ecology and evolutionary processes, phage genes are poorly characterized, hampering their usage in a variety of biotechnological applications. Methods to characterize such genes, even those critical to the phage life cycle, are labor intensive and are generally phage specific. Here, we develop a systematic gene essentiality mapping method scalable to new phage–host combinations that facilitate the identification of nonessential genes. As a proof of concept, we use an arrayed genome-wide CRISPR interference (CRISPRi) assay to map gene essentiality landscape in the canonical coliphages λ and P1. Results from a single panel of CRISPRi probes largely recapitulate the essential gene roster determined from decades of genetic analysis for lambda and provide new insights into essential and nonessential loci in P1. We present evidence of how CRISPRi polarity can lead to false positive gene essentiality assignments and recommend caution towards interpreting CRISPRi data on gene essentiality when applied to less studied phages. Finally, we show that we can engineer phages by inserting DNA barcodes into newly identified inessential regions, which will empower processes of identification, quantification, and tracking of phages in diverse applications.

59 BASIC BIOLOGICAL SCIENCES↗

Comparative genomic and phenotypic characterization of invasive non-typhoidal Salmonella isolates from Siaya, Kenya

Non-typhoidal Salmonella (NTS) is a major global health concern that often causes bloodstream infections in areas of the world affected by malnutrition and comorbidities such as HIV and malaria. Developing a strategy to control the emergence and spread of highly invasive and antimicrobial resistant NTS isolates requires a comprehensive analysis of epidemiological factors and molecular pathogenesis. Here, we characterize 11 NTS isolates that caused bloodstream infections in pediatric patients in Siaya, Kenya from 2003–2010. Nine isolates were identified as S . Typhimurium sequence type 313 while the other two were S . Enteritidis. Comprehensive genotypic and phenotypic analyses were performed to compare these isolates to those previously identified in sub-Saharan Africa. We identified a S . Typhimurium isolate referred to as UGA14 that displayed novel plasmid, pseudogene and resistance features as compared to other isolates reported from Africa. Notably, UGA14 is able to ferment both lactose and sucrose due to the acquisition of insertion elements on the pKST313 plasmid. These findings show for the first time the co-evolution of plasmid-mediated lactose and sucrose metabolism along with cephalosporin resistance in NTS further elucidating the evolutionary mechanisms of invasive NTS phenotypes. These results further support the use of combined genomic and phenotypic approaches to detect and characterize atypical NTS isolates in order to advance biosurveillance efforts that inform countermeasures aimed at controlling invasive and antimicrobial resistant NTS.

59 BASIC BIOLOGICAL SCIENCES↗

Evaluating E. coli genome‐scale metabolic model accuracy with high‐throughput mutant fitness data

Abstract The Escherichia coli genome‐scale metabolic model (GEM) is an exemplar systems biology model for the simulation of cellular metabolism. Experimental validation of model predictions is essential to pinpoint uncertainty and ensure continued development of accurate models. Here, we quantified the accuracy of four subsequent E. coli GEMs using published mutant fitness data across thousands of genes and 25 different carbon sources. This evaluation demonstrated the utility of the area under a precision–recall curve relative to alternative accuracy metrics. An analysis of errors in the latest (iML1515) model identified several vitamins/cofactors that are likely available to mutants despite being absent from the experimental growth medium and highlighted isoenzyme gene‐protein‐reaction mapping as a key source of inaccurate predictions. A machine learning approach further identified metabolic fluxes through hydrogen ion exchange and specific central metabolism branch points as important determinants of model accuracy. This work outlines improved practices for the assessment of GEM accuracy with high‐throughput mutant fitness data and highlights promising areas for future model refinement in E. coli and beyond.

59 BASIC BIOLOGICAL SCIENCES↗

Metagenome-assembled genomes from Wind River Basin floodplain sediments Riverton, Wyoming site (August 2015)

Microorganisms play a key role in cycling nutrients and contaminants in the terrestrial environment depending on their genetic potential. Here we present metagenome-assembled genomes (MAGs) for the bacterial and archaeal community in floodplain sediment samples taken August 29, 2015 at a location (KB1) close to DOE Legacy Management well 855 at the Riverton, Wyoming floodplain site in the Wind River Basin (WRB). The groundwater at this site exhibits persistent U, Mo, and sulfate plumes and is one of the field sites in focus for the SLAC Groundwater Quality SFA program. Sediment samples from a deep soil pit were collected from 0 to 234 cm depth below surface at discrete depths every ~10-20 cm for microbial analyses. 13 metagenomes were sequenced through JGI and can be found under Gold sequencing project: Gs0131241. Metagenomes were assembled, binned, and refined using metawrap to generate MAGs (>50% complete and < 10% contamination based on checkM scores).This dataset includes a zip file of 2216 MAG fasta files and a csv file with quality, taxonomic classification (GTDB RS220), and metagenome accessions for MAGs. This dataset also includes a file-level metadata (flmd.csv) file that lists each file contained in the dataset with associated metadata and a data dictionary (dd.csv) file that contains column/row headers used throughout the files along with a definition, units, and data type

54 ENVIRONMENTAL SCIENCES↗

Metagenome-assembled genomes from Wind River Basin floodplain sediments Riverton, Wyoming site (June to October 2019)

Microorganisms play a key role in cycling nutrients and contaminants in the terrestrial environment depending on their genetic potential. Here we present metagenome-assembled genomes (MAGs) for the bacterial and archaeal community in floodplain sediment samples taken at three time points from June 12, 2019 to October 23,2019 at a location (PTT1) close to DOE Legacy Management well 855 at the Riverton, Wyoming floodplain site in the Wind River Basin (WRB). The groundwater at this site exhibits persistent U, Mo, and sulfate plumes and is one of the field sites in focus for the SLAC Groundwater Quality SFA program. Sediment samples were collected from 60 to 180 cm below surface every 30cm for microbial analyses through metagenomic sequencing. 15 metagenomes were sequenced through JGI and can be found under Gold sequencing project: Gs0131241. Metagenomes were assembled, binned, and refined using metawrap to generate MAGs (>50% complete and < 10% contamination based on checkM scores). This dataset includes a zip file of 780 MAG fasta files and a csv file with quality, taxonomic classification (GTDB RS220), and metagenome accessions for MAGs. This dataset also includes a file-level metadata (flmd.csv) file that lists each file contained in the dataset with associated metadata and a data dictionary (dd.csv) file that contains column/row headers used throughout the files along with a definition, units, and data type. A sample metadata file (samples.csv) that contains site information has also been included.

54 ENVIRONMENTAL SCIENCES↗