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

Abstract

The inner physicochemical heterogeneity of bacterial cells generates three-dimensional (3D)-dependent variations of resources for effective expression of given chromosomally located genes. This fact has been exploited for adjusting the most favorable parameters for implanting a complex device for optogenetic control of biofilm formation in the soil bacterium Pseudomonas putida. To this end, a DNA segment encoding a superactive variant of the Caulobacter crescendus diguanylate cyclase PleD expressed under the control of the cyanobacterial light-responsive CcaSR system was placed in a mini-Tn5 transposon vector and randomly inserted through the 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 range of biofilm-building capacities and dynamic ranges in response to green light. Since the phenotypic output of the device depends on a large number of parameters (multiple promoters, RNA stability, translational efficacy, metabolic precursors, protein folding, etc.), we argue that random chromosomal insertions enable sampling the intracellular milieu for an optimal set of resources that deliver a preset phenotypic specification. Results thus support the notion that the context dependency can be exploited as a tool for multiobjective optimization, rather than a foe to be suppressed in Synthetic Biology constructs.

Keywords: CcaSR system; PleD; Pseudomonas; biofilm; interoperability; transposon.

Figure 1

Intracellular heterogeneity of the P. putida cytoplasm and its consequences. Resources for transcription and translation cannot be homogeneous due to differences in DNA folding and unequal 3D distribution of the gene expression hardware. Diverse physicochemical properties of each intracellular address and uneven localization of polymerases, transcription factors, ribosomes, and metabolites along with variable DNA supercoiling conform a landscape that affects the final performance of the implemented device depending on the spot of insertion. (A) Top: fluorescence microscopy picture of the distribution of RNA polymerase (green) and ribosomes (red), reproduced with permission from reference (15) (Copyright 2019 John Wiley and Sons). Bottom: Green dots on the bottom picture mark localization of pWW0 plasmid of P. putida mt-2, reproduced from reference (12) (Copyright 2017 American Chemical Society). (B) Recreation of the uneven distribution of cellular components inside cells. (C) Insertions of a genetic device in different chromosomal locations are exposed each to different concentrations of resources, giving as a result a diverse molecular landscape for transcription, translation, and protein activity.

Figure 2

Organization of the OPT·FILM device. The genetic construct leveraged for this work (not to scale) is composed of a module for sensing green light (encoding sensor CcaS, PCB enzymes Ho1 and PcyA, and transcriptional regulator CcaR) adjacent to the phenotypic module, which, in this case, corresponds to the cdGMP and the biofilm-promoting PleD* enzyme. Genes are transcribed as separate units (except ho1 and pcyA that form a two-gene operon) punctuated by regulatory regions (promoters and Shine–Dalgarno sequences).

Figure 3

Benchmarking the OPT·FILM device in P. putida. (A) pGPD plasmid stems from the previously described pGreenL by replacing the GFP reporter sequence by the pleD* gene, which encodes a superactive diguanylate cyclase for triggering a biofilm formation phenotype. (B) P. putida KT2440 cells bearing or not pGPD plasmid were tested for biofilm formation upon exposure to green light. Cells carrying the plasmid displayed a clear light-inducible biofilm formation as quantified with the crystal violet assay. (C) Congo Red/Coomassie-stained colonies of the same strains exposed or not to green light. (D) Visual inspection of cell growth on the walls of the same cultures.

Figure 4

Genetic strategy for random sampling of chromosomal sites of P. putida with mini-Tn5 [OPT·FILM]. The OPT·FILM cassette bearing the light-inducible device for biofilm formation (top left) was cloned inside the Km^R mini-Tn5 transposon vector borne by delivery plasmid pBAMD1.2 (sketched bottom left), flanked by the MEI and MEII regions (ME: mosaic ends for transposase recognition). Upon conjugal mobilization of the resulting construct toward P. putida (whether the wild-type strain or its wsp derivative), the device becomes randomly inserted through the chromosome of the target bacterium (right). This results in placing the OPT·FILM segment in a variety of chromosomal sites, themselves located at different locations of the intracellular 3D space.

Figure 5

Biofilm formation by P. putida KT2440 clones inserted with mini-Tn5 [OPT·FILM] at diverse chromosomal locations. The biofilm index for every clone of a transposon library sample is shown. This index is calculated by dividing the absorbance measurement for biofilm at 595 nm after crystal violet staining by the absorbance at 600 nm of cultures at the beginning of the protocol. Biofilm index is a quantitative value for biofilm formation. X-fold biofilm index represents the biofilm index of a culture exposed to light divided by the biofilm index of the same culture kept in darkness. Differences in the behavior of each variant (named after the well in the plate where they were growing) can be noticed by comparing biofilm formation in plates exposed or not to green light. The plots display the results of three biological replicates. (B) Same data represented as X-fold induction. For example, strain A4 of this group of clones looks like a good case of optimized performance (located by PP_5368, Supporting Information Table S2).

Figure 6

Biofilm formation by P. putida KT2440 wsp clones inserted with mini-Tn5 [OPT·FILM]. Similar experiment to that shown in Figure 5 but carried with P. putida KT2440 wsp as transposon recipient. (A) Biofilm index of three biological replicates. (B) X-fold induction. This library showed a higher variability of biofilm-forming phenotypes (see text for Discussion).