Microorganisms play a vital role in shaping the soil environment and enhancing plant growth by interacting with plant root systems. Because of the vast diversity of cell types involved, combined with dynamic and spatial heterogeneity, identifying the causal contribution of a defined factor, such as a microbial exopolysaccharide (EPS), remains elusive. Synthetic approaches that enable orthogonal control of microbial pathways are a promising means to dissect such complexity. Here we report the implementation of a synthetic, light-activated, transcriptional control platform using the blue-light responsive DNA binding protein EL222 in the nitrogen fixing soil bacterium Sinorhizobium meliloti. By fine-tuning the system, we successfully achieved optical control of an EPS production pathway without significant basal expression under noninducing (dark) conditions. Optical control of EPS recapitulated important behaviors such as a mucoid plate phenotype and formation of structured biofilms, enabling spatial control of biofilm structures in S. meliloti. The successful implementation of optically controlled gene expression in S. meliloti enables systematic investigation of how genotype and microenvironmental factors together shape phenotype in situ.

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AbstractMicroorganisms play a vital role in shaping the soil environment and enhancing plant growth by interacting with plant root systems. Due to the vast diversity of cell types involved, combined with dynamic and spatial heterogeneity, identifying the causal contribution of a defined factor, such as a microbial exopolysaccharide (EPS), remains elusive. Synthetic approaches that enable orthogonal control of microbial pathways are a promising means to dissect such complexity. Here we report the implementation of a synthetic, light-activated, transcriptional control platform in the nitrogen fixing soil bacterium Sinorhizobium meliloti. By fine tuning the system, we successfully achieved optical control of an EPS production pathway without significant basal expression under non-inducing (dark) conditions. Optical control of EPS recapitulated important behaviors such as a mucoid plate phenotype and formation of structured biofilms, enabling spatial control of biofilm structures in S. meliloti. The successful implementation of optically controlled gene expression in S. meliloti enables systematic investigation of how genotype and microenvironmental factors together shape phenotype in situ.SignificanceMicroorganisms are key players in sustaining the soil environment and plant growth. Symbiotic associations of soil microbes and plants provide a major source of nitrogen in agricultural systems, prevent water contamination from synthetic fertilizer application, and support crop growth in marginal soils. However, measuring the impact of microbial gene products on beneficial function remains a major challenge. This work provides a critical step toward addressing this challenge by implementing external gene regulation in a well characterized nitrogen fixing soil bacterium. We show that light exposure enables spatial and temporal control of the extracellular polysaccharide production functionality essential for symbiosis. Remote control of genes enables the benefits of candidate microorganisms to be systematically measured and enhanced within complex natural settings.

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Generating robust, predictable perturbations in cellular protein levels will advance our understanding of protein function and enable control of physiological outcomes in biotechnology applications. Previous studies have shown that controlling RNA transcription achieves perturbations in protein levels over a timescale of several hours. Here, we demonstrate the potential for harnessing the protein degradation machinery to achieve robust, rapid control of a specific protein level in the yeast Saccharomyces cerevisiae. Using a light-driven protein degradation machinery and red fluorescent proteins as reporters, we show that under constant transcriptional induction, repeated triangular fluctuations in protein levels can be generated by controlling the protein degradation rate. Consistent with previous results using transcriptional control, we observed a continuous decrease in the magnitude of fluctuations as the modulation frequency increased, indicating low-pass filtering of input perturbation. However, compared to hour-scale fluctuations observed using transcriptional control, modulating the protein degradation rate enabled five to ten minute-scale fluctuations. Our study demonstrates the potential for repeated control of protein levels by controlling protein degradation rate, at timescales much shorter than that achieved by transcriptional control.