Optogenetics inSinorhizobium melilotienables spatial control of exopolysaccharide production and biofilm structure

Abstract

<jats:title>Abstract</jats:title><jats:p>Microorganisms 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 <jats:italic>Sinorhizobium meliloti.</jats:italic> 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 <jats:italic>S. meliloti.</jats:italic> The successful implementation of optically controlled gene expression in <jats:italic>S. meliloti</jats:italic> enables systematic investigation of how genotype and microenvironmental factors together shape phenotype <jats:italic>in situ</jats:italic>.</jats:p><jats:sec><jats:title>Significance</jats:title><jats:p>Microorganisms 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.</jats:p></jats:sec>

Optogenetic Tools for Control of Public Goods in Saccharomyces cerevisiae

“…To extend the techniques described in this article to mixed-kingdom communities, optogenetic systems developed for bacteria ( 73 , 74 ) could be utilized. Indeed, in Sinorhizobium meliloti , a nitrogen-fixing soil bacterium, the blue light-sensitive transcription factor EL222 was recently used to control production of the public good exopolysaccharide, enabling manipulation of biofilm formation ( 41 ). Hybrid optochemical approaches also hold promise for repurposing existing inducible systems, as a recent study showed that photocaged isopropyl-β- d -thiogalactopyranoside (IPTG) could be used to control coculture interactions in the bacterium Corynebacterium glutamicum ( 39 ).…”

“…Light is a powerful actuator, as it is inexpensive, easily controlled in time and space, and S. cerevisiae contains no known native photoreceptors ( 34 ). Light can be rapidly added and removed from cell cultures or spatially targeted ( 35 – 38 ), meaning it can be used to study how regulation of microbial interactions determines microbial community development ( 39 – 41 ). We report here the development of an optogenetic tool that allows the expression of a specific metabolic enzyme of interest to be put under light control in S. cerevisiae .…”

Optogenetic tools for public goods control in Saccharomyces cerevisiae

“…To extend the techniques described in this article to mixed-kingdom communities, optogenetic systems developed for bacteria 80,81 could be utilized. Indeed in Sinorhizobium meliloti , a nitrogen fixing soil bacterium, the blue-light sensitive transcription factor EL222 was recently used to control production of the public good exopolysaccharide enabling manipulation of biofilm formation 41 . Hybrid optochemical approaches also hold promise for repurposing existing inducible systems, as a recent study showed that photocaged IPTG could be used to control coculture interactions in the bacterium Corynebacterium glutamicum 39 .…”

“…Light is a powerful actuator as it is inexpensive, easily controlled in time and space, and S. cerevisiae contains no known native photoreceptors 34 . Light can be rapidly added and removed from cell cultures or spatially targeted 35– 38 , meaning it can be used to study how regulation of microbial interactions determines microbial community development 39–41 . We report here the development of an optogenetic tool that allows the expression of a specific metabolic enzyme of interest to be put under light control in S. cerevisiae .…”