ABSTRACT
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.
Subject(s)
Biofilms/growth & development , Optogenetics/methods , Polysaccharides, Bacterial/biosynthesis , Signal Transduction/radiation effects , Sinorhizobium meliloti/genetics , Sinorhizobium meliloti/metabolism , Bacterial Proteins/metabolism , Binding Sites , Gene Expression/radiation effects , Gene Expression Regulation, Bacterial/radiation effects , Light , Plant Roots/microbiology , Ribosomes/metabolism , Soil Microbiology , Sphingomonadaceae/metabolism , Symbiosis/genetics , Transcription Factors/metabolismABSTRACT
In Sinorhizobium meliloti, catabolite repression is influenced by a noncanonical nitrogen-type phosphotransferase system (PTS(Ntr)). In this PTS(Ntr), the protein HPr is phosphorylated on histidine-22 by the enzyme EI(Ntr) and the flux of phosphate through this residue onto downstream proteins leads to an increase in succinate-mediated catabolite repression (SMCR). In order to explore the molecular determinants of HPr phosphorylation by EI(Ntr), both proteins were purified and the activity of EI(Ntr) was measured. Experimentally determined kinetic parameters of EI(Ntr) activity were significantly slower than those determined for the carbohydrate-type EI in Escherichia coli. Enzymatic assays showed that glutamine, a signal of nitrogen availability in many Gram-negative bacteria, strongly inhibits EI(Ntr). Binding experiments using the isolated GAF domain of EI(Ntr) (EIGAF) showed that it is the domain responsible for detection of glutamine. EI(Ntr) activity was not affected by α-ketoglutarate, and no binding between the EIGAF and α-ketoglutarate could be detected. These data suggest that in S. melilloti, EI(Ntr) phosphorylation of HPr is regulated by signals from both carbon metabolism (phosphoenolpyruvate) and nitrogen metabolism (glutamine).
