Engineering the Soil Bacterium Pseudomonas synxantha 2–79 into a Ratiometric Bioreporter for Phosphorus Limitation

Abstract

Microbial bioreporters hold promise for addressing challenges in medical and environmental applications. However, the difficulty in ensuring their stable persistence and function within the target environment remains a challenge. One strategy is to integrate information about the host strain and target environment into the design-build-test cycle of the bioreporter itself. Here, we present a case study for such an environmentally motivated design process by engineering the wheat commensal bacterium Pseudomonas synxantha 2–79 into a ratiometric bioreporter for phosphorus limitation. Comparative analysis showed that an exogenous P-responsive promoter outperformed its native counterparts. This reporter can selectively sense and report phosphorus limitation at plant-relevant concentrations of 25–100 μM without cross-activation from carbon or nitrogen limitation or high cell densities. Its performance is robust over a field-relevant pH range (5.8–8), and it responds only to inorganic phosphorus, even in the presence of common soil organic P. Finally, we used fluorescein-calibrated flow cytometry to assess whether the reporter’s performance in shaken liquid culture predicts its performance in soil, finding that although the reporter is still functional at the bulk level, its variability in performance increases when grown in a soil slurry as compared to planktonic culture, with a fraction of the population not expressing the reporter proteins. Together, our environmentally aware design process provides an example of how laboratory bioengineering efforts can generate microbes with a greater promise to function reliably in their applied contexts