The Role of Two Linear β-Glucans Activated by c-di-GMP in Rhizobium etli CFN42

Bacterial exopolysaccharides (EPS) have been implicated in a variety of functions that assist in bacterial survival, colonization, and host-microbe interactions. Among them, bacterial linear β-glucans are polysaccharides formed by D-glucose units linked by β-glycosidic bonds, which include curdlan, cellulose, and the new described Mixed Linkage β-Glucan (MLG). Bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a universal bacterial second messenger that usually promote EPS production. Here, we report Rhizobium etli as the first bacterium capable of producing cellulose and MLG. Significant amounts of these two β-glucans are not produced under free-living laboratory conditions, but their production is triggered upon elevation of intracellular c-di-GMP levels, both contributing to Congo red (CR⁺) and Calcofluor (CF⁺) phenotypes. Cellulose turned out to be more relevant for free-living phenotypes promoting flocculation and biofilm formation under high c-di-GMP conditions. None of these two EPS are essential for attachment to roots of Phaseolus vulgaris, neither for nodulation nor for symbiotic nitrogen fixation. However, both β-glucans separately contribute to the fitness of interaction between R. etli and its host. Overproduction of these β-glucans, particularly cellulose, appears detrimental for symbiosis. This indicates that their activation by c-di-GMP must be strictly regulated in time and space and should be controlled by different, yet unknown, regulatory pathways.