Biology subjects

Herbert, R. A.

Publications and source records attributed to Herbert, R. A..

5 recordsLinked to original sources

bioRxiv2025

BarTn7: Optimizing Bacterial Lineage Tracking at Sub-Species Resolution for Population Dynamics in Ecological and Evolutionary Studies

Communities of bacteria undergo population bottlenecks which are crucial to their population, ecological, and evolutionary dynamics. However, conventional amplicon sequencing cannot distinguish such demographic shifts between very closely related lineages. Here we describe BarTn7, an optimized method for bacterial lineage tracking through chromosomal integration of phenotypically neutral DNA barcodes with transposon Tn7. By combining conventional conjugative plasmids into a single vector and leveraging a parts-based strategy to optimize delivery to different recipients, BarTn7 increases barcoding efficiency and enables the systematic application of this tool to diverse bacteria. We tested BarTn7 in multiple bacterial species and confirmed a lack of lineage-specific growth effects. We then used BarTn7 to measure the colonization of Panicum virgatum roots under differing phosphate concentrations. Lineage tracking enabled discrimination between unique colonization events and the proliferation of existing bacteria during root colonization, the ratio of which differed between three plant-associated bacterial species. The strategy further allowed the measurement of phosphate-dependent ingress rates of the root endosphere by Paraburkholderia phytofirmans PsJN. We then demonstrated the effectiveness of BarTn7 to detect adaptive mutation(s) and facilitate identification of mutant lineages. Additionally, we demonstrated that BarTn7 is more accurate than conventional 16S rRNA gene sequencing at measuring community composition of a 5-member synthetic bacterial community (SynCom) and compares favorably to shotgun metagenomics. Our results illustrate the utility of BarTn7 as a simple, cost-efficient, and broadly applicable method of measuring bacterial population dynamics at sub-species resolution.

microbiology↗

bioRxiv2021

Genomics Characterization of an Engineered Corynebacterium glutamicum in Bioreactor Cultivation under Ionic Liquid Stress

Corynebacterium glutamicum is an ideal microbial chassis for the production of valuable bioproducts including amino acids and next-generation biofuels. Here we resequence engineered isopentenol (IP) producing C. glutamicum BRC-JBEI 1.1.2 strain and assess differential transcriptional profiles using RNA sequencing under industrially relevant conditions including scale transition and compare the presence vs. absence of an ionic liquid, cholinium lysinate ([Ch][Lys]). Analysis of the scale transition from shake flask to bioreactor with transcriptomics identified a distinct pattern of metabolic and regulatory responses needed for growth in this industrial format. These differential changes in gene expression corroborate altered accumulation of organic acids and bioproducts, including succinate, acetate, and acetoin that occur when cells are grown in the presence of 50mM [Ch][Lys] in the stirred-tank reactor. This new genome assembly and differential expression analysis of cells grown in a stirred tank bioreactor clarify the cell response of a C. glutamicum strain engineered to produce IP.

systems biology↗

bioRxiv2021

High Throughput Fitness Profiling Reveals Loss Of GacS-GacA Regulation Improves Indigoidine Production In Pseudomonas putida

Pseudomonas putida KT2440 is an emerging industrial microbe amenable for use with renewable carbon streams including aromatics such as para-coumarate (pCA). We examined this microbe under common stirred-tank bioreactor parameters with quantitative fitness assays using a pooled transposon library containing nearly all (4,778) non-essential genes. Assessing differential fitness values by monitoring changes in mutant strain abundance over time identified 31 genes with improved fitness in multiple bioreactor-relevant parameters. Twenty-one genes from this subset were reconstructed, including GacA, a signaling protein, TtgB, an ABC transporter, and PP_0063, a lipid A acyltransferase. Twelve deletion strains with roles in varying cellular functions were evaluated for conversion of pCA, to a heterologous bioproduct, indigoidine. Several mutants, such as the {Delta}gacA strain improved both fitness in a bioreactor and showed an 8-fold improvement in indigoidine production (4.5 g/L, 0.29 g/g pCA, 23% MTY) from pCA as the carbon source.

systems biology↗

bioRxiv2020

Iron Supplementation Eliminates Antagonistic Interactions Between Root Associated Bacteria

The rhizosphere microbiome (rhizobiome) plays a critical role in plant health and development. However the processes by which the constituent microbes interact to form and maintain a community are not well understood. To investigate these molecular processes, we examined pairwise interactions between 11 different microbial isolates under selected nutrient-rich and nutrient-limited conditions. We observed that when grown with media supplemented with 56 mM glucose, 2 microbial isolates were able to inhibit the growth of 6 out of 11 other microbes tested. The interaction between microbes persisted even after the antagonistic microbe was removed, upon exposure to spent media. To probe the genetic basis for these antagonistic interactions, we used a barcoded transposon library in a proxy bacterium, Pseudomonas putida, to identify genes which showed enhanced sensitivity to the antagonistic factor(s) secreted by Acinetobacter sp. 02. Iron metabolism-related gene clusters in P. putida were implicated by this systems-level analysis. The supplementation of iron prevented the antagonistic interaction in the original microbial pair supporting the hypothesis that iron limitation drives antagonistic microbial interactions between rhizobionts. We conclude that rhizobiome community composition is influenced by competition for limiting nutrients with implications for growth and development of the plant.

microbiology↗

bioRxiv2020

Genome-scale metabolic rewiring to achieve predictable titers rates and yield of a non-native product at scale

Achieving high titer rates and yields (TRY) remains a bottleneck in the production of heterologous products through microbial systems, requiring elaborate engineering and many iterations. Reliable scaling of engineered strains is also rarely addressed in the first designs of the engineered strains. Both high TRY and scale are challenging metrics to achieve due to the inherent trade-off between cellular use of carbon towards growth vs. target metabolite production. We hypothesized that being able to strongly couple product formation with growth may lead to improvements across both metrics. In this study, we use elementary mode analysis to predict metabolic reactions that could be targeted to couple the production of indigoidine, a sustainable pigment, with the growth of the chosen host, Pseudomonas putida KT2440. We then filtered the set of 16 predicted reactions using -omics data. We implemented a total of 14 gene knockdowns using a CRISPRi method optimized for P. putida and show that the resulting engineered P. putida strain could achieve high TRY. The engineered pairing of product formation with carbon use also shifted production from stationary to exponential phase and the high TRY phenotype was maintained across scale. In one design cycle, we constructed an engineered P. putida strain that demonstrates close to 50% maximum theoretical yield (0.33 g indigoidine/g glucose consumed), reaching 25.6 g/L indigoidine and a rate of 0.22g/l/h in exponential phase. These desirable phenotypes were maintained from batch to fed-batch cultivation mode, and from 100ml shake flasks to 250 mL ambr(R) and 2 L bioreactors.

bioengineering↗