Grantee Research Project Results
2023 Progress Report: Microbial Community Models for Measuring Survival and Persistence of SynBio Microbes in Soil
EPA Grant Number: R840206Title: Microbial Community Models for Measuring Survival and Persistence of SynBio Microbes in Soil
Investigators: Farny, Natalie G
Institution: Worcester Polytechnic Institute
EPA Project Officer: Callan, Richard
Project Period: July 1, 2021 through June 30, 2024
Project Period Covered by this Report: July 1, 2022 through June 30,2023
Project Amount: $449,213
RFA: Assessment Tools for Biotechnology Products (2020) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability , Safer Chemicals
Objective:
The overarching goal of the work supported by this grant is to understand, predict, and control the relationships between soil microbial communities (SMCs) and genetically engineered microbes (GEMs). In order to better understand and predict the behavior of GEMs within a soil environment, we aim to develop scalable, high-throughput laboratory models to measure survival and persistence of GEMs in soils. These models will enable us to understand how a GEM released into an environment – for example to remediate soil contaminated with TNT – will behave. The models also permit us to test how best to control the GEM after release. We propose that GEM control could be achieved by altering the balance of members within an existing SMC.
Progress Summary:
In the previous reporting period, we described a method to extract the solubilized organic matter (solubilized extract of soil organic material, SESOM) and a subset of the soil microbial community (SMC), then used this liquid as a medium for survival assays with P. putida. We reported that we do recapitulate the survival patterns seen by colony-forming unit (CFU) assay when an extraction of the native microbiome is present, wherein our GEM population declines between days 4 and 7 of culture. We have since confirmed that the liquid SESOM extracted SMC is stable in liquid culture at room temperature over a period of 28 days. We performed 16S rDNA deep sequencing. DNA was isolated at zero, 14-day and 28-day timepoints, and 16S libraries were prepared by PCR and sequenced on the Illumina platform. These analyses of the SMC at the class level reveal remarkable stability in the representation of most bacterial classes relative to the solid soil samples. The culture maintains species that are not culturable in isolation, and in relatively similar proportions. We have done this complete analysis in two different but similar soils and show that the distinct signature of the SMC within a given soil type is maintained. PCoA analysis of weighted UniFrac for each soil type reveals separation of the soil brands by the first principal component, which accounts for over 31% of the variation within the soil types. This highly significant finding suggests that non-sterile SESOM models maintain their distinct SMC signatures throughout our 28-day liquid culture period, making them the best known liquid model of an SMC of which we are aware. This model is readily scalable and amenable to automated liquid handling and flow cytometry analysis, thus achieving our goals for development of a facile soil model for GEM risk assessment.
Despite much effort, our TNT-sensing genetic circuit was not able to detect the presence of TNT in our hands. We identified that the problem was weak promoter activation in response to TNT, which was further exacerbated by large transcriptional changes that occur when laboratory P. putida strains are switched from rich broth medium to restrictive growth conditions. We therefore will switch our focus to lead contamination, because heavy metal contaminants are an important cause of soil degradation. We are engineering a lead biosensor, based on the PbrR transcriptional regulator, which works robustly in E. coli in our hands, and is reported to function in a range of soil organisms in the literature.
We have observed that many engineered functions, including inducible gene circuits which are designed in the lab to function in LB medium, are not functional in the soil environment. We believe that a fundamental lack of understanding of global changes to transcriptional programs underlies this problem. To better understand the promoters that are active in the soil environment, we performed RNA-seq from P. putida grown to stationary phase in LB versus SESOM. Our results reveal hundreds of strongly upregulated and downregulated genes. We hypothesize that alternative sigma factor activity is responsible for the massive changes observed, and are following up on that hypothesis.
To examine the relationship between our engineered P. putida and other soil microbes, we used sterilized SESOM to co-culture the species and measure their survival and relative abundance by flow cytometry. For example, we observe that our engineered P. putida rapidly outcompetes Citrobacter freundii in liquid soil extracts, and that the growth advantage of P. putida is enhanced with increasing temperature. Similar results were obtained in competition assays with Burkholderia thailandensis. We have also developed a method to identify whether individual cells in the co-culture are living or dead which clearly separates out co-cultures into four populations, as expected. In Year 3 we will apply these methods to study relationships between the engineered P. putida and various environmental isolates, including strains of wild P. putida.
Future Activities:
With our liquid SESOM model in place and new data transcriptional regulation of P. putida in SESOM, we will make rapid progress in three key areas: 1. Utilizing the liquid SESOM model to determine the effects of lead contamination on SMC composition GEM survival and persistence
(Objectives 1.1 and 1.2). 2. Building an engineered P. putida strain with a functional lead biosensor, for testing in our solid soil and liquid SESOM models. The lead biosensor is a model for a GEM with an engineered function. 3. Completing individual competition assays between our model GEM and culturable species to identify potential biocontrol species. (Objective 2). We will continue to assess interspecies relationships between our GEM and culturable strains. We will further apply live/dead staining analysis to classify interspecies relationships. Finally, potential biocontrol strains will be added to the non-sterile SESOM with our GEM to assess modulation of GEM survival. Upcoming presentations of the research are planned at the 6th International Conference on Microbiome Engineering (Dec. 8 - 10, 2023, Berkeley, CA) and an invited seminar at Stanford University on Dec. 12, 2023.
Supplemental Keywords:
Risk assessment, bioremediation, biocontainment, soil ecology, TNT, soil, bacteria, soil microbial community (SMC), genetically engineered microbe (GEM).Relevant Websites:
Natalie Farny’s faculty website Exit Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.