Biology subjects

Bacon, E. E.

Publications and source records attributed to Bacon, E. E..

4 recordsLinked to original sources

bioRxiv2024

Modular, inducible, and titratable expression systems for Escherichia coli and Acinetobacter baumannii

Gene expression systems that transcend species barriers are needed for cross-species analysis of gene function. In particular, expression systems that can be utilized in both model and pathogenic bacteria underpin comparative functional approaches that inform conserved and variable features of bacterial physiology. Here, we develop replicative and integrative vectors alongside a novel, IPTG-inducible promoter that can be used in the model bacterium Escherichia coli K-12 as well as strains of the antibiotic-resistant pathogen, Acinetobacter baumannii. We generate modular vectors that transfer by conjugation at high efficiency and either replicate or integrate into the genome, depending on design. Embedded in these vectors, we also developed a synthetic, IPTG-inducible promoter, PabstBR, that induces to a high level, but is less leaky than the commonly used trc promoter. We show that PabstBR is titratable at both the population and single cell level, regardless of species, highlighting the utility of our expression systems for cross-species functional studies. Finally, as a proof of principle, we use our integrating vector to develop a reporter for the E. coli envelope stress {sigma} factor, RpoE, and deploy the reporter in E. coli and A. baumannii, finding that A. baumannii does not recognize RpoE-dependent promoters unless RpoE is heterologously expressed. We envision that these vector and promoter tools will be valuable for the community of researchers that study fundamental biology of E. coli and A. baumannii. ImportanceAcinetobacter baumannii is a multidrug-resistant, hospital-acquired pathogen with the ability to cause severe infections. Understanding the unique biology of this non-model bacterium may lead to the discovery of new weaknesses that can be targeted to treat antibiotic-resistant infections. Here, we provide expression tools that can be used to study gene function in A. baumannii, including in drug-resistant clinical isolates. These tools are also compatible with the model bacterium, Escherichia coli, enabling cross-species comparisons of gene function. We anticipate that the use of these tools by the scientific community will accelerate our understanding of Acinetobacter biology.

microbiology↗

bioRxiv2024

A Targeted Genome-scale Overexpression Platform for Proteobacteria

Targeted, genome-scale gene perturbation screens using Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) and activation (CRISPRa) have revolutionized eukaryotic genetics, advancing medical, industrial, and basic research. Although CRISPRi knockdowns have been broadly applied in bacteria, options for genome-scale gene overexpression face key limitations. Here, we develop a facile approach for genome-scale overexpression in bacteria we call, "CRISPRtOE" (CRISPR transposition and OverExpression). We first create a platform for comprehensive gene targeting using CRISPR-associated transposons (CAST) and show that transposition occurs at a higher frequency in non-transcribed DNA. We then demonstrate that CRISPRtOE can upregulate gene expression in Proteobacteria with medical and industrial relevance by integrating synthetic promoters of varying strength upstream of target genes. Finally, we employ CRISPRtOE screening at the genome-scale in the model bacterium Escherichia coli and the non-model biofuel producer Zymomonas mobilis, recovering known and novel antibiotic and engineering targets. We envision that CRISPRtOE will be a valuable overexpression tool for antibiotic mode of action, industrial strain optimization, and gene function discovery in bacteria. ImportanceSystematic alteration of bacterial gene expression enables identification of genes relevant to diverse fields of study and practical applications. Although many targeted, genome-scale genetic tools exist for reducing or eliminating gene expression, there are few facile approaches for systematic gene overexpression in bacteria. Here, we develop a targeted overexpression approach for Proteobacteria of medical and industrial importance that precisely inserts strong promoters upstream of genes using CRISPR-associated transposons. We demonstrate that this approach can be used to systematically overexpress genes in both model (Escherichia coli K-12) and non-model (Zymomonas mobilis) Proteobacteria for the purpose of understanding antibiotic resistance mechanisms and improving strain resilience in biofuel production conditions, respectively.

systems biology↗

bioRxiv2023

Essential Gene Knockdowns Reveal Genetic Vulnerabilities and Antibiotic Sensitivities in Acinetobacter baumannii

The emergence of multidrug-resistant Gram-negative bacteria underscores the need to define genetic vulnerabilities that can be therapeutically exploited. The Gram-negative pathogen, Acinetobacter baumannii, is considered an urgent threat due to its propensity to evade antibiotic treatments. Essential cellular processes are the target of existing antibiotics and a likely source of new vulnerabilities. Although A. baumannii essential genes have been identified by transposon sequencing (Tn-seq), they have not been prioritized by sensitivity to knockdown or antibiotics. Here, we take a systems biology approach to comprehensively characterize A. baumannii essential genes using CRISPR interference (CRISPRi). We show that certain essential genes and pathways are acutely sensitive to knockdown, providing a set of vulnerable targets for future therapeutic investigation. Screening our CRISPRi library against last-resort antibiotics uncovered genes and pathways that modulate beta-lactam sensitivity, an unexpected link between NADH dehydrogenase activity and growth inhibition by polymyxins, and anticorrelated phenotypes that underpin synergy between polymyxins and rifamycins. Our study demonstrates the power of systematic genetic approaches to identify vulnerabilities in Gram-negative pathogens and uncovers antibiotic-essential gene interactions that better inform combination therapies. ImportanceAcinetobacter baumannii is a hospital-acquired pathogen that is resistant to many common antibiotic treatments. To combat resistant A. baumannii infections, we need to identify promising therapeutic targets and effective antibiotic combinations. In this study, we comprehensively characterize the genes and pathways that are critical for A. baumannii viability. We show that genes involved in aerobic metabolism are central to A. baumannii physiology and may represent appealing drug targets. We also find antibiotic-gene interactions that may impact the efficacy of carbapenems, rifamycins, and polymyxins, providing a new window into how these antibiotics function in mono- and combination therapies. Our studies offer a useful approach for characterizing interactions between drugs and essential genes in pathogens to inform future therapies.

systems biology↗

bioRxiv2022

Essential Gene Phenotypes Reveal Antibiotic Mechanisms and Synergies in Acinetobacter baumannii

The emergence of multidrug-resistant Gram-negative bacteria underscores the need to define genetic vulnerabilities in relevant pathogens. The Gram-negative pathogen, Acinetobacter baumannii, poses an urgent threat by evading antibiotic treatment through both intrinsic and acquired mechanisms. Antibiotics kill bacteria by targeting essential gene products, but antibiotic-essential gene interactions have not been studied systematically in A. baumannii. Here, we use CRISPR interference (CRISPRi) to comprehensively phenotype A. baumannii essential genes. We show that certain essential genes are acutely sensitive to knockdown, providing a set of promising therapeutic targets. Screening our CRISPRi library against last-resort antibiotics revealed essential pathways that modulate beta-lactam resistance, an unexpected link between NADH dehydrogenase function and polymyxin killing, and the genetic basis for synergy between polymyxins and rifamycins. Our results demonstrate the power of systematic genetic approaches to identify weaknesses in Gram-negative pathogens and uncover antibiotic mechanisms that better inform combination therapies. SignificanceAcinetobacter baumannii is a hospital-acquired pathogen that is resistant to common antibiotic treatments. A. baumannii infections, we need to identify promising therapeutic targets and effective antibiotic combinations. Here, we characterize genes critical for A. baumannii viability and their interactions with antibiotics. We find that genes involved in proton gradient formation required for oxygen-dependent energy generation are central to A. baumannii physiology and represent appealing drug targets. We show that polymyxins interact with proton gradient genes, explaining how these antibiotics inhibit growth at sub-lethal concentrations and their efficacy in combination therapies. Our studies reveal antibiotic-gene interactions in A. baumannii that can inform future therapies.

microbiology↗