Biology subjects

Owens, T. K.

Publications and source records attributed to Owens, T. K..

7 recordsLinked to original sources

bioRxiv2025

Novel origin of replication for environmentally isolated Pantoea strain enables expression of heterologous proteins, pathways and products

Plasmids isolated or characterized from environmental samples serve as a resource that can be used to develop genetic tools for characterizing recently isolated or less-studied microbes. In this report, we leveraged sequences from a previously characterized groundwater plasmidome and developed a screen to identify novel plasmid origins. Putative origin sequences were used to construct a barcoded plasmid library, which contained both known and newly predicted origins. This library was tested against a panel of representative bacterial strains and led to the identification of 3 novel origins that putatively replicate in gram-negative bacteria not previously associated with these origin sequences. We empirically validated one of the newly identified origins, 6911, to be functional in both the model bacterial strain, Escherichia coli BW25113, as well as in Pantoea sp. MT58, a fast growing and metal tolerant, environmentally important bacterium from the widespread Pantoea genus. We confirmed that a plasmid bearing origin 6911 as the sole origin could replicate and had a copy number of 9 ({+/-} 2) in Pantoea sp. MT58. We successfully used a plasmid based on the new origin to express the reporter protein GFP, and two non-native metabolite pathways for the natural product, indigoidine and the terpenoid compound, isoprenol. By pairing functional novel origins of replication to non-model organisms this pipeline can expand the tool kit for genetic manipulations of both model and less-studied bacteria. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=56 SRC="FIGDIR/small/653210v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@45689corg.highwire.dtl.DTLVardef@d6daeeorg.highwire.dtl.DTLVardef@1bf4d06org.highwire.dtl.DTLVardef@59ccfd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract figure.C_FLOATNO We developed a host-origin pair identification pipeline by constructing a plasmid library containing both literature-sourced and computationally predicted origins of replication. By conjugating this library into non-model microbes, we identified functional origins in diverse bacteria, and could leverage these findings to develop genetically tractable hosts for microbial engineering. C_FIG

microbiology↗

bioRxiv2024

Improving the annotation of amino acid biosynthesis pathways: GapMind 2024

We previously described GapMind, an automated web-based tool for annotating amino acid biosynthesis pathways in bacterial and archaeal genomes. We used GapMind to identify gaps in biosynthetic pathways and systematically used comparative genomics and high-throughput genetics to identify candidate genes to fill these gaps. We confirmed the activity of ten of the proposed enzymes by using cross-species complementation assays. Highlights include a novel route to glycine, two families that can replace phosphoserine phosphatase, an alternative N-succinyl-L,L-diaminopimelate desuccinylase, an alternative N-acetylornithine deacetylase, and a bifunctional MetB/MetC. We updated GapMind to include these additional enzymes. Across 208 prokaryotes that have high-quality genomes and can grow in minimal media, the average number of unexplained missing steps or gaps in amino acid biosynthesis dropped from 1.4 per genome to 0.7 per genome. The majority of remaining gaps involve the gain or loss of phosphate groups.

microbiology↗

bioRxiv2024

Multi-laboratory Study Establishes Reproducible Methods for Plant-Microbiome Research in Fabricated Ecosystems

Inter-laboratory replicability is crucial yet challenging in microbiome research. Leveraging microbiomes to promote soil health and plant growth requires understanding underlying molecular mechanisms using reproducible experimental systems. In a global collaborative effort involving five laboratories, we aimed to help advance reproducibility in microbiome studies by testing our ability to replicate synthetic community assembly experiments. Our study compared fabricated ecosystems constructed using two different synthetic bacterial communities, the model grass Brachypodium distachyon, and sterile EcoFAB 2.0 devices. All participating laboratories observed consistent inoculum-dependent changes in plant phenotype, root exudate composition, and final bacterial community structure where Paraburkholderia sp. OAS925 could dramatically shift microbiome composition. Comparative genomics and exudate utilization linked the pH-dependent colonization ability of Paraburkholderia, which was further confirmed with motility assays. The study provides detailed protocols, benchmarking datasets, and best practices to help advance replicable science and inform future multi-laboratory reproducibility studies.

microbiology↗

bioRxiv2023

Impact of inoculation practices on microbiota assembly and community stability in a fabricated ecosystem

Studying plant-microbe-soil interactions is challenging due to their high complexity and variability in natural ecosystems. While fabricated ecosystems provide opportunities to recapitulate aspects of these systems in reduced complexity and controlled environments, inoculation can be a significant source of variation. To tackle this, we evaluated how different bacteria inoculation practices and plant harvesting time points affect the reproducibility of a microbial synthetic community (SynCom) in association with the model grass Brachypodium distachyon. We tested three microbial inoculation practices: seed inoculation, transplant inoculation, and seedling inoculation; and two harvesting points: early (14-day-old plants) and late (21 days post-inoculation). We grew our plants and bacterial strains in sterile devices (EcoFABs) and characterized the microbial community from root, rhizosphere, and sand using 16S ribosomal RNA gene sequencing. The results showed that inoculation practices significantly affected the rhizosphere microbial community only when harvesting at an early time point but not at the late stage. As the SynCom showed a persistent association with B. distachyon at 21 days post-inoculation regardless of inoculation practices, we assessed the reproducibility of each inoculation method and found that transplant inoculation showed the highest reproducibility. Moreover, plant biomass was not adversely affected by transplant inoculation treatment. We concluded that bacteria inoculation while transplanting coupled with a later harvesting time point gives the most reproducible microbial community in the EcoFAB-B. distachyon-SynCom fabricated ecosystem and recommend this method as a standardized protocol for use with fabricated ecosystem experimental systems.

plant biology↗

bioRxiv2020

Targeted Genome Editing of Bacteria Within Microbial Communities

Knowledge of microbial gene functions comes from manipulating the DNA of individual species in isolation from their natural communities. While this approach to microbial genetics has been foundational, its requirement for culturable microorganisms has left the majority of microbes and their interactions genetically unexplored. Here we describe a generalizable methodology for editing the genomes of specific organisms within a complex microbial community. First, we identified genetically tractable bacteria within a community using a new approach, Environmental Transformation Sequencing (ET-Seq), in which non-targeted transposon integrations were mapped and quantified following community delivery. ET-Seq was repeated with multiple delivery strategies for both a nine-member synthetic bacterial community and a ~200-member microbial bioremediation community. We achieved insertions in 10 species not previously isolated and identified natural competence for foreign DNA integration that depends on the presence of the community. Second, we developed and used DNA-editing All-in-one RNA-guided CRISPR-Cas Transposase (DART) systems for targeted DNA insertion into organisms identified as tractable by ET-Seq, enabling organism- and locus-specific genetic manipulation within the community context. These results demonstrate a strategy for targeted genome editing of specific organisms within microbial communities, establishing a new paradigm for microbial manipulation relevant to research and applications in human, environmental, and industrial microbiomes.

microbiology↗

bioRxiv2019

Selective carbon sources influence the end-products of microbial nitrate respiration

Respiratory and catabolic pathways are differentially distributed in 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.

microbiology↗

bioRxiv2018

Dub-seq: dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits

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 greatly increases the throughput of genome-wide overexpression assays. In Dub-seq, a shotgun expression library is cloned between dual random DNA barcodes and the precise breakpoints of DNA fragments are associated to the barcode sequences prior to performing assays. To assess the fitness of individual strains carrying these plasmids, we use DNA barcode sequencing (BarSeq), which is amenable to large-scale sample multiplexing. As a demonstration of this approach, we constructed a Dub-seq library with total Escherichia coli genomic DNA, performed 155 genome-wide fitness assays in 52 experimental conditions, and identified 813 genes with high-confidence overexpression phenotypes across 4,151 genes assayed. 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 such as transposon site sequencing or CRISPRi and will facilitate rapid and systematic functional characterization of microbial genomes.\n\nImportanceMeasuring the phenotypic consequences of overexpressing genes is a classic genetic approach for understanding protein function; for identifying drug targets, antibiotic and metal resistance mechanisms; and for optimizing strains for metabolic engineering. In microorganisms, these gain-of-function assays are typically done using laborious protocols with individually archived strains or in low-throughput following qualitative selection for a phenotype of interest, such as antibiotic resistance. However, many microbial genes are poorly characterized and the importance of a given gene may only be apparent under certain conditions. Therefore, more scalable approaches for gain-of-function assays are needed. Here, we present Dual Barcoded Shotgun Expression Library Sequencing (Dub-seq), a strategy that couples systematic gene overexpression with DNA barcode sequencing for large-scale interrogation of gene fitness under many experimental conditions at low cost. Dub-seq can be applied to many microorganisms and is a valuable new tool for large-scale gene function characterization.

microbiology↗