Periodic Reporting for period 1 - REDBAC (Occurrence, Regulation, and Function of Bacterial mRNA Editing Under Diverse Environments)
Reporting period: 2023-09-01 to 2026-02-28
Our project seeks to revolutionize our understanding of bacterial genetics by investigating the occurrence, regulation, and function of adenosine-to-inosine (A-to-I) mRNA editing in bacteria. Traditionally, bacteria have been viewed as haploid organisms whose genetic potential is fixed, unable to diversify their proteome through mechanisms such as alternative splicing. However, recent discoveries—including our own—suggest that bacteria might generate alternative protein isoforms by editing their messenger RNA, thereby introducing functional diversity at both the single-cell and population level.
We hypothesized that A-to-I mRNA editing modifies the protein-coding sequence post-transcriptionally, enabling the bacterial ribosome to translate one gene into multiple protein isoforms with distinct activities. This process appears to be regulated by environmental cues and may serve as a flexible adaptive strategy, allowing bacteria to rapidly respond to fluctuating conditions and stressors such as nutrient deprivation or antibiotic exposure. Through this mechanism, bacteria may introduce what is essentially “pseudo-heterozygosity,” conferring advantages in survival and competition that were previously unrecognized in these organisms.
The overall objectives of the project are threefold:
• To systematically identify and catalog mRNA editing events in diverse bacterial species and under varied environmental conditions, using both existing RNA-seq datasets and newly developed laboratory protocols.
• To elucidate the genetic and biochemical mechanisms that regulate these editing events, focusing on sequence motifs, enzyme-substrate relationships, and environmental triggers.
• To determine the functional significance of mRNA editing for bacterial physiology, including its impacts on growth, adaptation, antibiotic resistance, and pathogenicity.
By addressing these objectives, the project aims to make a foundational impact on bacterial genetics, overturning established dogma and providing new explanations for elements of bacterial adaptation that remain poorly understood. The expected significance of these findings extends beyond basic science, offering new insights into infection biology, antibiotic resistance mechanisms, and potential avenues for diagnostic and therapeutic innovation in biomedicine. The research also promises to deliver valuable tools and datasets to the wider scientific community, thereby enabling broader exploration of RNA-level regulation in bacteria.
Overall, the project sets the scene for a paradigm shift in how bacterial diversity and adaptability are conceptualized and studied, with direct implications for public health, microbiology, and biotechnology.
We hypothesized that A-to-I mRNA editing modifies the protein-coding sequence post-transcriptionally, enabling the bacterial ribosome to translate one gene into multiple protein isoforms with distinct activities. This process appears to be regulated by environmental cues and may serve as a flexible adaptive strategy, allowing bacteria to rapidly respond to fluctuating conditions and stressors such as nutrient deprivation or antibiotic exposure. Through this mechanism, bacteria may introduce what is essentially “pseudo-heterozygosity,” conferring advantages in survival and competition that were previously unrecognized in these organisms.
The overall objectives of the project are threefold:
• To systematically identify and catalog mRNA editing events in diverse bacterial species and under varied environmental conditions, using both existing RNA-seq datasets and newly developed laboratory protocols.
• To elucidate the genetic and biochemical mechanisms that regulate these editing events, focusing on sequence motifs, enzyme-substrate relationships, and environmental triggers.
• To determine the functional significance of mRNA editing for bacterial physiology, including its impacts on growth, adaptation, antibiotic resistance, and pathogenicity.
By addressing these objectives, the project aims to make a foundational impact on bacterial genetics, overturning established dogma and providing new explanations for elements of bacterial adaptation that remain poorly understood. The expected significance of these findings extends beyond basic science, offering new insights into infection biology, antibiotic resistance mechanisms, and potential avenues for diagnostic and therapeutic innovation in biomedicine. The research also promises to deliver valuable tools and datasets to the wider scientific community, thereby enabling broader exploration of RNA-level regulation in bacteria.
Overall, the project sets the scene for a paradigm shift in how bacterial diversity and adaptability are conceptualized and studied, with direct implications for public health, microbiology, and biotechnology.
In the first two years of the project, we have made the following series of discoveries:
Project 1:
• A-to-I mRNA editing is widespread and occurs in 64 gamaproteobacterial species.
• Hundreds of transcripts are edited across the examined species.
• Most editing events are predicted to affect protein sequence.
• Conserved regulatory determinants control A-to-I mRNA editing formation in bacteria – a seven-base sequence motif and RNA stem-loop structure.
• Mutating TadA, the mediating enzyme, in Acinetobacter baylyi reduces A-to-I mRNA editing activity across all sites.
• Conversely, overexpressing TadA (the editing enzyme) in Acinetobacter baylyi resulted in the editing of more than 300 transcripts, attesting to the potential of TadA expression to reshape the bacterial transcriptome and proteome.
• A-to-I mRNA editing enables bacteria to produce two endogenous protein isoforms from a single gene.
• TadA mutant with deficient editing activity does not grow at high temperatures, suggesting that RNA editing has a functional role in bacteria.
Project 2:
• A-to-I mRNA editing can alter protein function in bacteria (increases the toxicity of HokB).
• A-to-I mRNA editing constitutes a novel mechanism controlling disulfide bond formation in bacteria at the RNA level (unknown in eukaryotes as well, to our knowledge).
• A-to-I mRNA editing can induce bacterial death or early entrance to the stationary phase via HokB toxicity.
• A-to-I mRNA editing of hokB is conserved in pathogenic species and strains, supporting functional importance with possible clinical relevance.
Project 3:
• Discovered the highest number of mRNA editing events in any bacterium to date (Vibrio alginolyticus): Identified 38 A-to-I RNA editing sites in Vibrio alginolyticus, greatly expanding the known bacterial RNA editome.
• Exposed dynamic regulation of editing across growth phases: Demonstrated that editing frequencies vary in specific transcripts depending on the bacterial growth phase, supporting a mechanism of gene-specific control.
• Revealed species-specific sequence determinants of editing: Found that editing events occur within a shorter, less conserved sequence motif compared to other gammaproteobacteria, suggesting unique recognition features in V. alginolyticus.
• Showed extensive and evolutionarily conserved editing of cqsA quorum-sensing gene: Uncovered that the quorum-sensing gene cqsA is the most heavily edited (∼90% of transcripts), and that editing at this locus is conserved across multiple Vibrio species, including human pathogens.
• Established functional consequences of RNA editing in natural bacterial contexts: Demonstrated for the first time that blocking editing of endogenous cqsA alters swarming motility in a temperature-dependent manner, revealing a direct ecological and evolutionary impact.
Project 1:
• A-to-I mRNA editing is widespread and occurs in 64 gamaproteobacterial species.
• Hundreds of transcripts are edited across the examined species.
• Most editing events are predicted to affect protein sequence.
• Conserved regulatory determinants control A-to-I mRNA editing formation in bacteria – a seven-base sequence motif and RNA stem-loop structure.
• Mutating TadA, the mediating enzyme, in Acinetobacter baylyi reduces A-to-I mRNA editing activity across all sites.
• Conversely, overexpressing TadA (the editing enzyme) in Acinetobacter baylyi resulted in the editing of more than 300 transcripts, attesting to the potential of TadA expression to reshape the bacterial transcriptome and proteome.
• A-to-I mRNA editing enables bacteria to produce two endogenous protein isoforms from a single gene.
• TadA mutant with deficient editing activity does not grow at high temperatures, suggesting that RNA editing has a functional role in bacteria.
Project 2:
• A-to-I mRNA editing can alter protein function in bacteria (increases the toxicity of HokB).
• A-to-I mRNA editing constitutes a novel mechanism controlling disulfide bond formation in bacteria at the RNA level (unknown in eukaryotes as well, to our knowledge).
• A-to-I mRNA editing can induce bacterial death or early entrance to the stationary phase via HokB toxicity.
• A-to-I mRNA editing of hokB is conserved in pathogenic species and strains, supporting functional importance with possible clinical relevance.
Project 3:
• Discovered the highest number of mRNA editing events in any bacterium to date (Vibrio alginolyticus): Identified 38 A-to-I RNA editing sites in Vibrio alginolyticus, greatly expanding the known bacterial RNA editome.
• Exposed dynamic regulation of editing across growth phases: Demonstrated that editing frequencies vary in specific transcripts depending on the bacterial growth phase, supporting a mechanism of gene-specific control.
• Revealed species-specific sequence determinants of editing: Found that editing events occur within a shorter, less conserved sequence motif compared to other gammaproteobacteria, suggesting unique recognition features in V. alginolyticus.
• Showed extensive and evolutionarily conserved editing of cqsA quorum-sensing gene: Uncovered that the quorum-sensing gene cqsA is the most heavily edited (∼90% of transcripts), and that editing at this locus is conserved across multiple Vibrio species, including human pathogens.
• Established functional consequences of RNA editing in natural bacterial contexts: Demonstrated for the first time that blocking editing of endogenous cqsA alters swarming motility in a temperature-dependent manner, revealing a direct ecological and evolutionary impact.
Thus, our study of bacterial mRNA editing—invisible to DNA sequencing—may account for previously overlooked aspects (e.g. pathogenicity and antibiotic resistance) in bacteria.
Future research is focused on the following 3 aims:
1. Determine the prevalence and regulation of RNA editing in additional bacterial species.
2. Determine the function of A-to-I RNA editing in different species and on different proteins.
3. Determine the state of A-to-I RNA editing within single bacterial cells and, subsequently, whether it constitutes a DNA-independent mechanism to produce genetic diversity in clonal bacterial populations, enhancing fitness under different conditions.
Future research is focused on the following 3 aims:
1. Determine the prevalence and regulation of RNA editing in additional bacterial species.
2. Determine the function of A-to-I RNA editing in different species and on different proteins.
3. Determine the state of A-to-I RNA editing within single bacterial cells and, subsequently, whether it constitutes a DNA-independent mechanism to produce genetic diversity in clonal bacterial populations, enhancing fitness under different conditions.