Abstract
<jats:title>Abstract</jats:title> <jats:p> Optogenetics uses light to provide precise, reversible, and non-invasive control over bacterial functions including gene expression with high spatiotemporal resolution. Although many optogenetic systems have been developed for <jats:italic>Escherichia coli</jats:italic> , only a limited number is available for other prokaryotes, such as pseudomonads. Here, we establish a toolbox of genomically integrated optogenetic gene cassettes for light-responsive regulation of target gene expression in <jats:italic>Pseudomonas putida</jats:italic> with UV-A, blue, green, and red light. Using transposon Tn7-mediated chromosomal integration, we implemented four optogenetic systems: the photocaged IPTG/P <jats:sub>tac</jats:sub> -LacI system, the LOV-based Dusk switch, the cyanobacteriochrome system CcaS/R, and different bacteriophytochrome-based REDusk variants. Benchmarking with the mCherry reporter demonstrated high dynamic ranges of up to ∼270-fold, low basal expression, and largely homogeneous population responses in <jats:italic>P. putida</jats:italic> . Spatial illumination further enabled patterned single- and dual-color gene expression. As a proof of concept, we applied the toolbox for light-controlled regulation of pyoverdine (PVD) biosynthesis in <jats:italic>P. putida</jats:italic> . The expression of the alternative sigma factor PfrI, which upregulates the production of the siderophore during iron-limitation, was placed under optogenetic control in a Δ <jats:italic>pfrI</jats:italic> background. The red-light responsive switches resulted in the strongest induction of PVD synthesis and enabled spatial control of siderophore-mediated microbial interactions. To demonstrate transferability, light-dependent pyoverdine production was further established in the human pathogen <jats:italic>Pseudomonas aeruginosa</jats:italic> PAO1. Together, this optogenetic plug-and-play toolbox enables non-invasive, spatiotemporal reprogramming of gene expression and cellular processes in pseudomonads and expands the available optogenetic repertoire beyond established model organisms. </jats:p>