Biology subjects

Ojeda, E.

Publications and source records attributed to Ojeda, E..

3 recordsLinked to original sources

bioRxiv2025

Evolutionary Constraints on RNA Polymerase Gene Positioning in the Genome of Fast-Growing Bacteria..

How gene order along chromosomes affects cellular homeostasis and genome evolution remains poorly understood. Bacterial chromosomes are organized along the replication origin (oriC)-terminus (ter) axis. The spatial arrengement of genes within this axis may influence cellular physiology, genome evolution, and transcriptional regulation. The catalytic core of the sole bacterial RNA polymerase (RNAP) is encoded by rpoB and rpoC within the universally conserved rplKAJL-rpoBC locus. In fast growing bacteria this locus is located near oriC potentially linking genome organization to cellular physiology. Here, we investigated the functional relevance of rplKAJL-rpoBC chromosomal positioning by relocating it within Vibrio cholerae genome. While viable, strains harboring rplKAJL-rpoBC far from oriC exhibited nutrient-dependent fitness defects. These phenotypes correlated with reduced locus copy number and RNAP abundance, without changes in its subcellular distribution. Introducing a second copy of rpIKAJL-rpoBC at a distal genomic location from oriC abolished the phenotypes, demonstrating that replication-associated gene dosage effects were the main mechanism behind the observed physiological changes. Further uncoupling rpoB and rpoC from the rplKAJL-rpoBC locus revealed that fitness costs were specifically linked to RNAP gene relocation. Our results reveal that selective pressures act to maintain RNAP-encoding genes near oriC to optimize replication-associated dosage effects, ensuring efficient RNAP production during exponential growth. We discuss the relationship between gene order and ecological strategies linked to bacterial growth. Our work underscores gene order as an underestimated but critical factor shaping cellular physiology and genome evolution. SignificanceBacterial chromosomes have a highly plastic gene content but the importance of their spatial organization and gene order is started to be appreciated as a driver of cellular physiology. Growing evidence suggests that the spatial organization of bacterial genomes plays a critical role in gene expression, replication dynamics, and fitness. In this study, we demonstrate that the conserved positioning of rpoB and rpoC, encoding the catalytic core of RNA polymerase (RNAP), near the replication origin (oriC) is essential for optimal bacterial growth. By relocating the rplKAJL-rpoBC locus in Vibrio cholerae, we show that its chromosomal position directly impacts nutrient-dependent fitness, RNAP abundance, and replication-associated gene dosage effects. These findings suggest that selective pressures maintain genes encoding RNAP near oriC to ensure efficient transcriptional output during exponential growth. More broadly, our study highlights gene order as an underappreciated factor in bacterial genome evolution, beyond its known roles in operon structure and transcriptional regulation. Given the fundamental role of RNAP, our results have implications for understanding bacterial ecological strategies, adaptation, and synthetic genome design. By integrating positional genomics with physiological studies, we provide a theoretical and experimental framework for investigating how genome architecture shapes cellular function.

microbiology↗

bioRxiv2024

Focal persistence and phylodynamics of Heartland virus in Georgia

Heartland virus (HRTV) is an emerging tick-bone virus associated with severe illness in the U.S. There are large gaps in knowledge of HRTV diversity, evolution, and transmission due to a paucity of HRTV-positive samples and genome sequences. We identified a focal site of HRTV- positive Amblyomma americanum ticks in central Georgia and developed a novel multiplex- amplicon sequencing assay to generate full HRTV genome sequences. By screening over 21,000 field-collected ticks from 2021-2023, we identified six positive pools. Five were collected from the site in central Georgia where our group first detected HRTV-positive ticks in 2019, and one from a site in western Georgia approximately 175 km away. The HRTV genome sequences from Georgia were highly related, even across this distance and over five years. Reference HRTV genome sequences from across the U.S. were also geographically clustered. Time-scaled phylogenetic analysis suggested recent spread of HRTV in the U.S., with all available sequences sharing a common ancestor within the last 300 years, and sequences from Georgia sharing a common ancestor within the last 40 years. Our observed spatial clustering of HRTV and the high degree of genetic conservation in our persistent focus suggest the importance of small spatial dynamics in HRTV transmission ecology. Author SummaryHeartland virus (HRTV) was first discovered in humans in 2009 and has since caused over 60 cases of severe and fatal disease in the United States. HRTV is transmitted by the lone star tick, Amblyomma americanum, across the Southeast, East coast, and Midwest. Little information is known about how this virus circulates and changes across time and space due to a lack of genetic data. Here, we created a new procedure to generate more genetic sequence data for HRTV and collected over 21,000 ticks to screen for HRTV across three years in Georgia. We generated 6 new HRTV sequences and compared them to existing sequences from our group in Georgia, and across the country, finding evidence of regional clustering of HRTV and highly related HRTV across time in Georgia. Our analyses additionally found that this virus was likely introduced to the U.S. in the last 300 years. Our study provides new context and information in understanding the landscape and transmission of HRTV in the U.S.

genomics↗

bioRxiv2024

Utility of Cellular Measurements of Non-Specific Endocytosis to Assess the Target-Independent Clearance of Monoclonal Antibodies

Past studies have demonstrated higher clearance for monoclonal antibodies possessing increased rates of non-specific endocytosis. However, this metric is oftentimes evaluated indirectly using biophysical techniques or cell surface binding studies that may not provide insight into the specific rates of cellular turnover. Furthermore, few examples evaluating non-specific endocytosis have been reported for a therapeutic antibody that reached clinical assessment. In the current report, we evaluated a therapeutic human immunoglobulin G2 monoclonal antibody targeted against the interleukin-4 receptor alpha chain (IL-4R) that exhibited elevated target independent clearance in previous Phase 1 and 2 studies. We confirmed high non-specific clearance of the anti-IL-4R antibody as compared to a reference antibody during pharmacokinetic assessments in wild type mice where target-mediated disposition was absent. We then developed a cell-based method capable of measuring cellular protein endocytosis and demonstrated the anti-IL-4R antibody exhibited marked non-specific uptake relative to the reference compound. Antibody homology modeling identified the anti-IL-4R antibody possessed positive charge patches whose removal via targeted mutations substantially reduced its non-specific endocytosis. We then expanded the scope of the study by evaluating a panel of consisting of both preclinical and clinical monoclonal antibodies and demonstrate those with the highest rates of non-specific uptake in vitro exhibit elevated target independent clearance, low subcutaneous bioavailability, or both. Our results support the observation that high non-specific endocytosis is a negative attribute in monoclonal antibody development and demonstrate the utility of a generic cell-based screen as a quantitative tool to measure non-specific endocytosis of protein therapeutics at the single-cell level. Highlights- Developed a novel, reproducible cellular assay to directly quantify non-specific endocytosis of therapeutic proteins. - A previous clinical candidate monoclonal antibody with rapid target-independent clearance in mice and humans possessed extensive non-specific endocytosis that was due to exposed positive charge features. - Demonstration of distinct rates of endocytosis into mammalian cells for disparate monoclonal antibodies, even those with common specificity for targets or isoelectric points. - Cell-based assay to quantify the potential impact of non-specific endocytosis on target-independent clearance and/or subcutaneous bioavailability of monoclonal antibodies.

biochemistry↗