Effect of the plant flavonoid luteolin on Ensifer meliloti 3001 phenotypic responses

The establishment of a successful symbiosis between the nitrogen-fixing bacterium Ensifer meliloti and compatible host legumes (Medicago spp.) depends on a complex molecular signal exchange. The early stage of signaling involves the release from plant roots of the flavonoid luteolin, which in turn induces the expression of rhizobia nodulation (nod) genes required for root infection and nodule development. To date, the bacterial response to the luteolin perception has been characterized in detail as far as gene expression is concerned. Nevertheless, despite this molecular information, a global view on E. meliloti phenotypes affected by the plant signal luteolin is still lacking. Therefore, an extensive phenotypic investigation of luteolin effect on the nitrogen-fixing E. meliloti 3001 has been performed. A thousand different growth conditions, including sensitivity to osmolites, pH stresses, antibiotics and toxic compounds, were tested by the application of the high-throughput Phenotype MicroArray (PM) technology, as well as by several dedicated assays to evaluate growth stimulation, motility, biofilm formation, N-acyl homoserine lactones and Indole-3- acetic acid (IAA) production. Results revealed that the plant signal luteolin affected a wide spectrum of E. meliloti 3001 phenotypes. E. meliloti 3001 displayed an enhanced resistance phenotype in the presence of luteolin toward a broad set of chemicals including several antibiotics, toxic ions, respiration inhibitors, membrane damagers, DNA intercalants and other potential antimicrobial agents. Moreover, the presence of luteolin significantly reduced overall AHLs production, as well as the lag phase in relation to the starting cellular density, the motility and biofilm formation under nutrient-limited growth conditions. An effect on E. meliloti indole-3-acetic acid (IAA) production was also detected in vitro as a response to luteolin. Overall, these findings suggest that the plant signal luteolin triggers a broad response in E. meliloti 3001, which was shown to be dependent on nutritional conditions sensed by the bacterium, pointing out a wide role in modifying rhizobial phenotypes, possibly in relation to plant root association and then symbiotic interaction.