Proteomic studies of the stringent response in Bradyrhizobium diazoefficiens show independence between the levels of the second messenger (p)ppGpp and the expression of master regulator CtrA

COLLA, DELFINA; ITURRALDE, ESTEBAN TOMÁS; LÓPEZ GARCÍA S.L.; MONGIARDINI, E.J.; PÉREZ GIMÉNEZ, JULIETA

LA PLATA - LA PLATA

Congreso; V Congreso de Microbiología Agrícola y Ambiental; 2021

División Agrícola y Ambiental de la Asociación Argentina de Microbiología

Bacteria respond to sudden nutritional starvation through the stringent response (SR) producing the second messenger guanosine tetra/pentaphosphate ((p)ppGpp) that regulates bacterial metabolism with the purpose to survive in the new environment. Studies done with Bradyrhizobium diazoefficiens USDA 110 (soybean symbiont) showed that the bacteria were more infective and developed more nodules when they were cultivated with low N; under this condition the SR is triggered. Besides, bacteria cultivated in low N are more competitive to nodulate in comparison with those cultivated in N-sufficiency. This suggest that the improvements of the symbiotic parameters are due to the expression of different genes regulated by the amount of (p)ppGpp. This hypothesis is supported by the fact that B. diazoefficiens LP5065, a mutant in rsh gene whose product regulates the synthesis of (p)ppGpp, was less competitive to nodulate soybean and formed smaller nodules that were deficient in N2 fixation.In order to obtain a global overview of the SR in B. diazoefficiens a comparison proteomic experiment between the wild type (WT) and mutant (MUT) was done, through a nanoHPLC coupled to mass spectrometry (MS) with Orbitrap technology. For that purpose, we chose two conditions: Treated (T) with a free solution of C and N, where (p)ppGpp levels are increased in the wild type strain, and the Untreated condition (U).On one hand, the stress effect was shown when we compared the WT T vs WT U. In the WT T condition we found different metabolic pathways down regulated such as the biosynthesis of amino acids and cofactors, carbon metabolism and the ABC transporters. In turn, were up regulated the biosynthesis of fatty acids and purine metabolism. On the other hand, the mutation effect has been exposed in the comparison of WT U vs MUT U, where in the WT second messenger levels are basal. In this comparison, we found up regulated the biosynthesis of amino acids, ABC transporters, carbon metabolism, glyoxylate cycle, purine metabolism among other pathways. In Caulobacter crescentus, a direct relationship between (p)ppGpp levels and the cell cycle control regulator, CtrA, was found. Surprisingly, in the comparison of WT T vs U and WT U vs MUT U, we found that CtrA expression did not change. This regulator also acts as a master regulator in the control of subpolar flagellar system expression in B. diazoefficiens, the constitutively polar flagellum. Consistent with our finding, the swimming ability of both strains was not altered as well as the expression of the subpolar flagellum measured as a function of extracellular flagellin expression. Due to that we could conclude that, in B. diazoefficiens, CtrA expression is independent of the (p)ppGpp levels. Even though, we need more evidence to understand the influence of the SR over the expression of CtrA and the regulation of subpolar flagellar system expression in B. diazoefficiens.