Mecanismos moleculares envolvidos na capacidade de tolerância ao estresse ácido de Rhizobium freireie

Abstract

The pH is the main controller of microbial diversity of soil. Rhizobium freirei is a remarkable bean endosymbiont and recognized by its ability to tolerate environmental stresses, such as acidic pH. The aim of this study was to analyze changes in gene expression at transcriptional and translational level of R. freirei grown in acidic pH. Bacteria was grown in TY medium until exponential phase in two treatments: pH 6,8 and pH 4,8. The protein extraction was performed and the extract was separated by two-dimensional electrophoresis. High-resolution images of the profiles were analyzed and the spots that showed statistical difference in the relative volume (%vol) between treatments were selected for identification by mass spectrometry (MS) and in silico identification. Differential expression of R. freirei was assessed by analysis of gshB, gstA and ropB genes after 0, 30 and 60 min of exposure in acidic pH. According to the results, the changing of central metabolic pathways seems to be the main mechanism of tolerance to acid stress in R. freirei, due to the increased expression of proteins involved in oxidative pathways (Zwf, aldolase KDPG and PckA) related to the Tricarboxilic Acid cicle (TCA). The increased expression of these proteins suggests higher carbohydrate consumption and NADH formation, reinforced by elevated expression of NuoC subunit of respiratory chain Complex 1, that converts NADH into NAD+ coupled to proton transport to periplasm and consequent decreases the cytosolic acidification. The Complex 1 of the respiratory chain is the main superoxide former; moreover the higher expression of the antioxidative enzymes AhpC and GstA at translational level, and the higher expression of gstA at transcriptional level, strongly suggests an oxidative stress induced by acid pH condition. On the other hand, the decreased AccC protein expression supports the trend of the central metabolism to acid-consuming reactions, in contrast to fatty acids biosynthesis pathways, where AccC is essential. Down-regulation of the β subunit of ATPase Complex can be related to regulation of cytosolic acidity by reduction of proton entry through ATPase Complex. Such broad range of pathways involved in the response to acid conditions suggests an adaptive response of multigenic character mainly involving cytoplasmic proteins, and reinforces the importance of the central metabolism for R. freirei ability to tolerate acidic pH conditions. In contrast to studies focused in few genes or specific mechanisms of tolerance, our results shows by the first time that the central metabolism appears to be the main mechanism of acid tolerance in R. freirei and suggests a correlation between acid and oxidative stress responses.