Dataset Information

Caged Cumate Enables Proximity-Dependent Control Over Gene Expression.

ABSTRACT: Cell-cell interactions underlie diverse physiological processes yet remain challenging to examine with conventional imaging tools. Here we report a novel strategy to illuminate cell proximity using transcriptional activators. We repurposed cumate, a small molecule inducer of gene expression, by caging its key carboxylate group with a nitrile. Nitrilase-expressing activator cells released the cage, liberating cumate for consumption by reporter cells. Reporter cells comprising a cumate-responsive switch expressed a target gene when in close proximity to the activator cells. Overall, this strategy provides a versatile platform to image and potentially manipulate cellular interactions over time.

SUBMITTER: Love AC

PROVIDER: S-EPMC9870035 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Caged Cumate Enables Proximity-Dependent Control Over Gene Expression.

Love Anna C AC Tran Sabrina H SH Prescher Jennifer A JA

Chembiochem : a European journal of chemical biology 20210608 14

Cell-cell interactions underlie diverse physiological processes yet remain challenging to examine with conventional imaging tools. Here we report a novel strategy to illuminate cell proximity using transcriptional activators. We repurposed cumate, a small molecule inducer of gene expression, by caging its key carboxylate group with a nitrile. Nitrilase-expressing activator cells released the cage, liberating cumate for consumption by reporter cells. Reporter cells comprising a cumate-responsive ...[more]

PMID: 34031982

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Project description:Inducible gene expression systems are powerful genetic tools to study bacterial physiology, probing essential and toxic gene functions, gene dosage effects, and overexpression phenotypes. For the opportunistic human pathogen Pseudomonas aeruginosa, dedicated inducible gene expression systems are scarce. In the current study, we developed a minimal synthetic 4-isopropylbenzoic acid (cumate)-inducible promoter, called PQJ, that is tunable over several orders of magnitude. This was achieved by combining semirandomized housekeeping promoter libraries and control elements from the Pseudomonas putida strain F1 cym/cmt system with powerful fluorescence-activated cell sorting (FACS) to select functionally optimized variants. Using flow cytometry and live-cell fluorescence microscopy, we demonstrate that PQJ responds rapidly and homogenously to the inducer cumate in a graded manner at the single-cell level. PQJ and cumate are orthogonal to the frequently used isopropyl β-d-thiogalactopyranoside (IPTG)-regulated lacIq-Ptac expression system. The modular design of the cumate-inducible expression cassette together with the FACS-based enrichment strategy presented here facilitates portability, thus serving as a blueprint for the development of tailored gene expression systems for a wide range of bacteria. IMPORTANCE Reverse genetics is a powerful approach to study bacterial physiology and behavior by relying on well-developed genetic tools, such as inducible promoters. For the human pathogen Pseudomonas aeruginosa, well-characterized inducible promoters are scarce. In the current work, we used a synthetic biology-based approach to develop a cumate-inducible promoter for P. aeruginosa, termed PQJ, that shows excellent induction properties at the single-cell level. This genetic tool provides the means for qualitative and quantitative gene function studies describing P. aeruginosa's physiology and virulence in vitro and in vivo. Because this synthetic approach to constructing species-specific inducible promoters is portable, it can serve as a blueprint for similar tailored gene expression systems in bacteria largely lacking such tools, including, for example, representatives of the human microbiota.

Dataset Information

Caged Cumate Enables Proximity-Dependent Control Over Gene Expression.

Publications

Caged Cumate Enables Proximity-Dependent Control Over Gene Expression.

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