In Vivo Sampling of Intracellular Heterogeneity of Pseudomonas putida Enables Multiobjective Optimization of Genetic Devices

The inner physicochemicalheterogeneity of bacterial cells generatesthree-dimensional (3D)-dependent variations of resources for effectiveexpression of given chromosomally located genes. This fact has beenexploited for adjusting the most favorable parameters for implantinga complex device for optogenetic control of biofilm formation in thesoil bacterium Pseudomonas putida.To this end, a DNA segment encoding a superactive variant of the Caulobacter crescendus diguanylate cyclase PleD expressedunder the control of the cyanobacterial light-responsive CcaSR systemwas placed in a mini-Tn5 transposon vector and randomly inserted throughthe chromosome of wild-type and biofilm-deficient variants of P. putida lacking the wsp gene cluster.This operation delivered a collection of clones covering a whole rangeof biofilm-building capacities and dynamic ranges in response to greenlight. Since the phenotypic output of the device depends on a largenumber of parameters (multiple promoters, RNA stability, translationalefficacy, metabolic precursors, protein folding, etc.), we argue thatrandom chromosomal insertions enable sampling the intracellular milieufor an optimal set of resources that deliver a preset phenotypic specification.Results thus support the notion that the context dependency can beexploited as a tool for multiobjective optimization, rather than afoe to be suppressed in Synthetic Biology constructs.

Automated summary

The inner physicochemical heterogeneity of bacterial cells leads to three-dimensional variations of resources, which has been exploited in adjusting parameters for optogenetic control of biofilm formation in Pseudomonas putida. A superactive variant of PleD diguanylate cyclase was inserted into the chromosome of wild-type and biofilm-deficient P. putida. Random chromosomal insertions enable sampling the intracellular milieu for optimal resources that deliver a preset phenotypic specification.