Bordetella bronchiseptica LapD interacts with two different diguanylate cyclases

SISTI, FEDERICO; AMBROSIS, NICOLÁS MARTÍN; FERNANDEZ, JULIETA

Congreso; LIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular.; 2017

Cyclic-di-GMP (cdG) is a widely distributed second messenger in the bacterial world. It is produced by diguanylate cyclases (DGCs) and can direct many cellular processes like motility and biofilm formation. Over the last years our group has been working on phenotypes regulated by this molecule in the genus Bordetella. We have previously described that cdG regulates biofilm formation in B. bronchiseptica. This process relies on a group of proteins homologue to the Pseudomonas fluorescens Lap system: LapD, LapG and BrtA.    We speculate that there might be at least one DGC that physically interact with LapD in order to provide cdG in a specific manner to the mentioned Lap system. Using a bioinformatic methodology we have looked into every B. bronchiseptica membrane DGC and predicted that BdcG, a putative DGC, physically interacts with LapD. We have also considered that BdcA could interact with LapD because our previous results showed a strong correlation between BdcA activity and biofilm formation.     In order to advance on the knowledge of the recently found BdcG, we analyze for DGC activity using two different methodologies (heterologous expression on a P. fluorescens reporter strain and cdG direct measurement). Neither of the approaches could confirm if BdcG is an active DGC.      To figure out if mentioned DGCs wether interact with LapD, we performed Bacterial Two Hybrid experiments. Our results show that BdcA and BdcG physically interact with LapD.    Furthermore we have also generated simple and double knock-out mutants of bdcG and bdcA. We hypothesized that deletion of DGCs may impaired biofilm formation ability. Neither simple nor double mutants showed a biofilm formation defect.    Considering the mentioned results and taking in mind that B. bronchiseptica genome has 10 sequences that encode for DGCs our results suggest that other DGCs are part of the regulation network through Lap proteins.