REGULATION OF THE SUBPOLAR FLAGELLUM SYNTHESIS IN Bradyrhizobium diazoefficiens

DARDIS, CAROLINA; MENGUCCI, FLORENCIA; ALTHABEGOITI M.J; PARISI, G; LODEIRO A.R; QUELAS, J. I; MONGIARDINI, E. J

Córdoba

Congreso; XI Congreso Argentino de Microbiología General; 2015

Sociedad Argentina de Microbiología

Bradyrhizobium diazoefficiens is an a-proteobacterium with high agronomic importance due to itsability to fix atmospheric nitrogen in symbiosis with soybean. Our previous studies showed that ahyper-motile strain of B. diazoefficiens is able to compete for nodulation and produce higher soybeangrain yields than the wild type in certain conditions. This observation led us to study different aspectsof bacterial motility, including the regulation of the synthesis of its dual flagellar systems. The flagellarsynthesis is an energy-expensive process, which occurs in several steps that are tightly controlled bymaster regulators. These proteins trigger an ordered transcription cascade which means that theexpression of one gene at a given level requires the transcription of another gene at a higher level.Applying bioinformatic tools, we identified putative regulators of B. diazoefficiens USDA 110 involvedin the synthesis of the subpolar flagellum using Caulobacter crescentus as a model. We found thefollowing candidates: ctrA (master regulator), flbD (class II regulator), fliX (trans-activation factor offlbD), flbT (class III regulator) and flaF (trans-activation factor of flbT). To confirm the role of thesegenes, mutations by insertion or deletions of each one were raised. However, initial attempts to obtainctrA mutants were unsuccessful probably because the absence of CtrA causes deleterious phenotype(4).In this work, we constructed and partially characterized flbD and fliX mutants in B. diazoefficiensUSDA 110. Both mutants were obtained by double-crossing over. For the flbD, we used the counterselection method in order to avoid polar effects, while the fliX mutant was obtained by insertion of anantibiotic cassette inside the ORF. All these mutants were checked by PCR and sequencing.When we began the mutant characterization, we noticed that none of these were able to produceextracellular subpolar flagellins, although the lateral flagellins were present in both of them. In addition,we observed that swimming in soft-agar plates was reduced in each mutant compared to the wild-type.These new findings suggest that both genes are involved in the regulation of the subpolar flagellumsynthesis in B. diazoefficiens independently of the regulation of lateral flagella. In this sense, themodel of regulation seems to be similar to C. crescentus but not to Salmonella, as was thought on thefirst studies. From now on, we will continue working to determine which genes are under the control ofthese two proteins in order to construct a hierarchy model for the regulation of the synthesis of thisflagellum.