Tools for Genetic Engineering and Gene Expression Control in<i>Novosphingobium aromaticivorans</i>and<i>Rhodobacter sphaeroides</i>

Hall, Ashley N.; Hall, Benjamin W.; Kinney, Kyle J.; Olsen, Gabby G.; Banta, Amy B.; Noguera, Daniel R.; Donohue, Timothy J.; Peters, Jason M.

doi:10.1101/2023.08.25.554875

Cited by 4 publications

(1 citation statement)

References 62 publications

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“…3a)). We inserted synthetic, constitutive promoters of varying strength that had been shown to function in various Proteobacteria 34,35 into Tn6677 with the goal of creating an OE gradient (Supplementary Fig. S5a and S5b).…”

Section: Targeted and Tunable Overexpression In Proteobacteriamentioning

confidence: 99%

A Targeted Genome-scale Overexpression Platform for Proteobacteria

Banta,

Myers,

Ward

et al. 2024

Preprint

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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 overexpression face key limitations. Here, we develop a facile approach for genome-scale gene overexpression in bacteria we call, "CRISPRtOE" (CRISPR transposition and OverExpression). We create a platform for comprehensive gene targeting using CRISPR-associated transposition (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 Escherichia coli, recovering known antibiotic targets and genes with unexplored roles in antibiotic function. We envision that CRISPRtOE will be a valuable overexpression tool for antibiotic mode of action, industrial strain optimization, and gene function discovery in bacteria.

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Section: Targeted and Tunable Overexpression In Proteobacteriamentioning

confidence: 99%

A Targeted Genome-scale Overexpression Platform for Proteobacteria

Banta,

Myers,

Ward

et al. 2024

Preprint

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Modular, inducible, and titratable expression systems forEscherichia coliandAcinetobacter baumannii

Bacon,

Tran,

Nadig

et al. 2024

Preprint

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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 bacteriumEscherichia coliK-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 usedtrcpromoter. We show that PabstBRis 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 theE. colienvelope stress σ factor, RpoE, and deploy the reporter inE. coliandA. baumannii, finding thatA. baumanniidoes 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 ofE. coliandA. baumannii.ImportanceAcinetobacter baumanniiis 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 inA. 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 ofAcinetobacterbiology.

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Physiological Roles of anAcinetobacter-specific σ Factor

Bacon,

Myers,

Iruegas-López

et al. 2024

Preprint

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The Gram-negative pathogenAcinetobacter baumanniiis considered an “urgent threat” to human health due to its propensity to become antibiotic resistant. Understanding the distinct regulatory paradigms used byA. baumanniito mitigate cellular stresses may uncover new therapeutic targets. Many γ-proteobacteria use the extracytoplasmic function (ECF) σ factor, RpoE, to invoke envelope homeostasis networks in response to stress.Acinetobacterspecies contain the poorly characterized ECF “SigAb;” however, it is unclear if SigAb has the same physiological role as RpoE. Here, we show that SigAb is a metal stress-responsive ECF that appears unique toAcinetobacterspecies and distinct from RpoE. We combine promoter mutagenesis, motif scanning, and ChIP-seq to define the direct SigAb regulon, which consists ofsigAbitself, the stringent response mediator,relA, and the uncharacterized small RNA, “sabS.” However, RNA-seq of strains overexpressing SigAb revealed a large, indirect regulon containing hundreds of genes. Metal resistance genes are key elements of the indirect regulon, as CRISPRi knockdown ofsigAborsabSresulted in increased copper sensitivity and excess copper induced SigAb-dependent transcription. Further, we found that two uncharacterized genes in thesigAboperon, “aabA” and “aabB”, have anti-SigAb activity. Finally, employing a targeted Tn-seq approach that uses CRISPR-associated transposons, we show thatsigAb,aabA, andaabBare important for fitness even during optimal growth conditions. Our work reveals new physiological roles for SigAb and SabS, provides a novel approach for assessing gene fitness, and highlights the distinct regulatory architecture ofA. baumannii.ImportanceAcinetobacter baumanniiis a hospital-acquired pathogen, and many strains are resistant to multiple antibiotics. Understanding howA. baumanniisenses and responds to stress may uncover novel routes to treat infections. Here, we examine how theAcinetobacter-specific transcription factor, SigAb, mitigates stress. We find that SigAb directly regulates only a small number of genes, but indirectly controls hundreds of genes that have substantial impacts on cell physiology. We show that SigAb is required for maximal growth, even during optimal conditions, and is acutely required during growth in the presence of elevated copper. Given that copper toxicity plays roles in pathogenesis and on copper-containing surfaces in hospitals, we speculate that SigAb function may be important in clinically-relevant contexts.

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Tools for Genetic Engineering and Gene Expression Control inNovosphingobium aromaticivoransandRhodobacter sphaeroides

Cited by 4 publications

References 62 publications

A Targeted Genome-scale Overexpression Platform for Proteobacteria

A Targeted Genome-scale Overexpression Platform for Proteobacteria

Modular, inducible, and titratable expression systems forEscherichia coliandAcinetobacter baumannii

Physiological Roles of anAcinetobacter-specific σ Factor

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