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; MONGIARDINO, ELÍAS JAVIER; ITURRALDE, ESTEBAN TOMÁS; PÉREZ-GIMÉNEZ, JULIETA; LÓPEZ-GARCÍA, SILVINA L

Online

Congreso; V Congreso Argentino de Microbiología Agrícola y Ambiental (V CAMAyA); 2021

División de Microbiología Agrícola y Ambiental (DiMAyA)

Bacteria respond to sudden nutritional starvation through the stringent response (SR) producing thesecond messenger guanosine tetra/pentaphosphate ((p)ppGpp) that regulates bacterial metabolismwith the purpose to survive in the new environment. Studies done with Bradyrhizobium diazoefficiensUSDA 110 (soybean symbiont) showed that the bacteria were more infective and developed morenodules 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 inN-sufficiency. This suggests that the improvements of the symbiotic parameters are due to theexpression of different genes regulated by the amount of (p)ppGpp. This hypothesis is supported bythe fact that B. diazoefficiens LP5065, a mutant in rsh gene whose product regulates the synthesisof (p)ppGpp, was less competitive to nodulate soybean and formed smaller nodules that weredeficient in N2 fixation.In order to obtain a global overview of the SR in B. diazoefficiens a comparison proteomic experimentbetween the wild type (WT) and mutant (MUT) was done, through a nanoHPLC coupled to massspectrometry (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 theUntreated condition (U).On one hand, the stress effect was shown when we compared the WT T vs WT U. In the WT Tcondition we found different metabolic pathways down regulated such as the biosynthesis of aminoacids and cofactors, carbon metabolism and the ABC transporters. In turn, were up regulated thebiosynthesis of fatty acids and purine metabolism. On the other hand, the mutation effect has beenexposed 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, carbonmetabolism, glyoxylate cycle, purine metabolism among other pathways.In Caulobacter crescentus, a direct relationship between (p)ppGpp levels and the cell cycle controlregulator, CtrA, was found. Surprisingly, in the comparison of WT T vs U and WT U vs MUT U, wefound that CtrA expression did not change. This regulator also acts as a master regulator in thecontrol 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 theexpression 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 overthe expression of CtrA and the regulation of subpolar flagellar system expression in B. diazoefficiens.