CONICET | Buscador de Institutos y Recursos Humanos

Bradyrhizobium diazoefficiens is an α-proteobacterium with high agronomic importance due to its ability to fix atmospheric nitrogen in symbiosis with soybean. Our previous studies showed that a hyper-motile strain of B. diazoefficiens is able to compete for nodulation and produce higher soybean grain yields than the wild type in certain conditions (1). This observation led us to study different aspects of bacterial motility, including the regulation of the synthesis of its dual flagellar systems (2). The flagellar synthesis is an energy-expensive process, which occurs in several steps that are tightly controlled by master regulators. These proteins trigger an ordered transcription cascade which means that the expression of one gene at a given level requires the transcription of another gene at a higher level (3). This regulation is associated with the correct protein synthesis and their assembly into the three structures (basal body, hook and filament) which forms the entire flagellum.Applying bioinformatic tools, we identified putative regulators of B. diazoefficiens USDA 110 involved in the synthesis of the subpolar flagellum using Caulobacter crescentus as a model. We found the following candidates: ctrA (master regulator), flbD (class II regulator), fliX (trans-activation factor of flbD), flbT (class III regulator) and flaF (trans-activation factor of flbT). To confirm the role of these genes, mutations by insertion or deletions of each one were raised. However, initial attempts to obtain ctrA mutants were unsuccessful probably because the absence of CtrA causes deleterious phenotype (4).In this work, we constructed and characterized flbD, fliX, flbT and flaF mutants in B. diazoefficiens USDA 110. For mutant characterization, we checked if they were able to produce extracellular subpolar flagellins, and we noticed that they were not present in flbD, fliX and flaF mutants although we could see them in the flbT mutant.Then we try to elucidate if these mutations produced a change in the transcription levels of some of the genes that are involved in flagellar assembly. For this, we measured through real time assays and observed that flbD and fliX mutants have low levels of RNAm of genes of the rod and hook, while flbT mutant has high levels of RNAm of three flagellins, and has not influence in the regulation of the other flagellin transcript.These new findings suggest that flbD, fliX, flbT and flaF are involved in the regulation of subpolar flagellum synthesis in B. diazoefficiens independently of the regulation of lateral flagella. It seems that flbD and fliX act in a higher step than flbT and flaF, regulating the transcription of genes of the rod and hook, and without it, the flagellum cannot assemble correctly. On the other hand, flbT seems to be a negative regulator of three of the four subpolar flagellins, and flaF a positive factor and because of this, flaF mutant does not present extracellular flagellins while flbT still does it. In this sense, the model of regulation seams to be similar to C. crescentus but not to Salmonella, as was thought on the first studies (5).