Relationship between the triggering of the stringent response and the expression of the Type 3 Secretion System in Bradyrhizobium diazoefficiens

COLLA, DELFINA; CASTELLANI, LUCAS G.; TORRES TEJERIZO, GONZALO A.; PÉREZ GIMÉNEZ, JULIETA

Mendoza

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2022

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular (SAIB)

Bradyrhizobium diazoefficiens USDA 110 is a Gram-negative soil bacterium which fixes nitrogen in symbiosis with soybean plants (Glycine max) in so-called root nodules. In return, the plant provides photo-assimilated products. In B. diazoefficiens, the Type 3 Secretion System (T3SS) is inducible by genistein, a soybean flavonoid that activates the transcriptional regulator TtsI. TtsI regulates the expression of the T3SS, including the structural gene rhcJ, and the effector proteins involved in the suppression of the host's defenses. Also, the T3SS induction is affected by the second messenger guanosine tetraphosphate and guanosine pentaphosphate variations (collectively referred as (p)ppGpp). In response to starvation, bacteria trigger the Stringent Response (SR) and (p)ppGpp is synthesized. A B. diazoefficiens rsh mutant, incapable of triggering the SR, establishes a defective symbiosis with soybean. In addition, this mutant induces far less the expression of the T3SS regulator ttsI and the rhcJ structural gene in the presence of genistein compared to the induction of the WT and also, does not suppress host’s defenses.In order to find determinants linking SR and T3SS, we performed a comparative exoproteomic analyses. Extracellular proteins were obtained from WT and rsh mutant (MUT) strains, induced with genistein (G) or without induction (WI). These proteins were separated and identified by Orbitrap and the spectra were analyzed with DIA-NN software. We obtained about 1000 proteins per sample (WT G, WT WI, MUT G and MUT WI), from which different comparisons allow us to obtain differentially expressed proteins. As an example, in the WT G vs MUT G comparison, we found that the T3SS’s effector protein NopE1 was overexpressed, indicating that T3SS is upregulated in the WT.As a connection between SR and T3SS was described, we hypothesized if the nutritional stress that triggers SR might also trigger the expression of T3SS. For this purpose, we measured the transcriptional levels of the ttsI and rhcJ genes of the WT and rsh mutant subjected to nutritional stress by qRT-PCR. We incubated the WT and the rsh mutant with genistein, without induction as a control condition and with a C and N free solution, condition where the (p)ppGpp is accumulated in the WT. The transcript levels of genes ttsI and rhcJ were upregulated in the WT under a nutritional stress, in comparison with the rsh mutant. Even more, the expression of T3SS genes under stress condition was higher than the expression in presence of genistein. Under nutritional starvation, the symbiotic interaction is stimulated. Our results confirmed that in B. diazoefficiens USDA 110 there is a link between the T3SS expression and the nutrient starvation condition, and that the expression of T3SS is triggered by the SR, but further work will help to understand the complete pathways involved during this activation.