Engineering topics

Hom, ed., Erik F. Y.

Publications and source records attributed to Hom, ed., Erik F. Y..

4 records

DOE OSTI2023

Comparison of phage-derived recombinases for genetic manipulation of Pseudomonas species

ABSTRACT Several strains in the Pseudomonas genus are categorized as plant growth-promoting rhizobacteria (PGPR). Although several of these strains are strong candidates for applications as biofertilizers or biopesticides, genome editing approaches are generally limited and require further development. Editing genomes in PGPR could enable more robust agricultural applications, persistence, and biosafety measures. In this study, we investigate the use of five phage-encoded recombinases to develop a recombineering workflow in three PGPR strains: Pseudomonas protegens Pf-5, Pseudomonas protegens CHA0, and Pseudomonas putida KT2440. Using point mutations in the rpoB gene, we reach maximum recombineering efficiencies of 1.5 × 10 −4 , 3 × 10 −4 , and 5 × 10 −5 , respectively, in these strains using λ-Red Beta recombinase from Escherichia coli . We further examine recombineering efficiencies across these strains as a function of selected mutation, editing template concentration, and phosphorothiolate bond protection. This work validates the use of these tools across several environmentally and biotechnologically relevant strains to expand the possibilities of genetic manipulation in the Pseudomonas genus. IMPORTANCE The Pseudomonas genus contains many members currently being investigated for applications in biodegradation, biopesticides, biocontrol, and synthetic biology. Though several strains have been identified with beneficial properties, chromosomal manipulations to further improve these strains for commercial applications have been limited due to the lack of efficient genetic tools that have been tested across this genus. Here, we test the recombineering efficiencies of five phage-derived recombinases across three biotechnologically relevant Pseudomonas strains: P. putida KT2440, P. protegens Pf-5, and P. protegens CHA0. These results demonstrate a method to generate targeted mutations quickly and efficiently across these strains, ideally introducing a method that can be implemented across the Pseudomonas genus and a strategy that may be applied to develop analogous systems in other nonmodel bacteria.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Terpenes modulate bacterial and fungal growth and sorghum rhizobiome communities

ABSTRACT Terpenes are among the oldest and largest class of plant-specialized bioproducts that are known to affect plant development, adaptation, and biological interactions. While their biosynthesis, evolution, and function in aboveground interactions with insects and individual microbial species are well studied, how different terpenes impact plant microbiomes belowground is much less understood. Here we designed an experiment to assess how belowground exogenous applications of monoterpenes (1,8-cineole and linalool) and a sesquiterpene (nerolidol) delivered through an artificial root system impacted its belowground bacterial and fungal microbiome. We found that the terpene applications had significant and variable impacts on bacterial and fungal communities, depending on terpene class and concentration; however, these impacts were localized to the artificial root system and the fungal rhizosphere. We complemented this experiment with pure culture bioassays on responsive bacteria and fungi isolated from the sorghum rhizobiome. Overall, higher concentrations (200 µM) of nerolidol were inhibitory to Ferrovibrium and tested Firmicutes. While fungal isolates of Penicillium and Periconia were also more inhibited by higher concentrations (200 µM) of nerolidol, Clonostachys was enhanced at this higher level and together with Humicola was inhibited by the lower concentration tested (100 µM). On the other hand, 1,8-cineole had an inhibitory effect on Orbilia at both tested concentrations but had a promotive effect at 100 µM on Penicillium and Periconia . Similarly, linalool at 100 µM had significant growth promotion in Mortierella , but an inhibitory effect for Orbilia . Together, these results highlight the variable direct effects of terpenes on single microbial isolates and demonstrate the complexity of microbe-terpene interactions in the rhizobiome. Importance Terpenes represent one of the largest and oldest classes of plant-specialized metabolism, but their role in the belowground microbiome is poorly understood. Here, we used a “rhizobox” mesocosm experimental set-up to supply different concentrations and classes of terpenes into the soil compartment with growing sorghum for 1 month to assess how these terpenes affect sorghum bacterial and fungal rhizobiome communities. Changes in bacterial and fungal communities between treatments belowground were characterized, followed by bioassays screening on bacterial and fungal isolates from the sorghum rhizosphere against terpenes to validate direct microbial responses. We found that microbial growth stimulatory and inhibitory effects were localized, terpene specific, dose dependent, and transient in time. This work paves the way for engineering terpene metabolisms in plant microbiomes for improved sustainable agriculture and bioenergy crop production.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Terabase-Scale Coassembly of a Tropical Soil Microbiome

Petabases of reads are being produced by environmental metagenome sequencing. An essential step in analyzing these data is metagenome assembly, the computational reconstruction of genome sequences from microbial communities. “Coassembly” of metagenomic sequence data, in which multiple samples are assembled together, enables more complete detection of microbial genomes in an environment than “multiassembly,” in which samples are assembled individually.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2022

Diversity and Composition of Methanotroph Communities in Caves

Recent observations have shown the atmospheric greenhouse gas methane (CH 4 ) is consumed by microorganisms (methanotrophs) in caves at rates comparable to CH 4 oxidation in surface soils. Caves are abundant in karst landscapes that comprise 14% of Earth’s land surface area, and therefore may represent a potentially important, but overlooked, CH 4 sink.

59 BASIC BIOLOGICAL SCIENCES↗