Análisis funcional de proteínas de respuesta a estrés de tipo sHSP en la bacteria endosimbiótica Rhizobium leguminosarum

Abstract

The Rhizobium-legume symbiosis is the result of a complex molecular network that involves the transformation of the free-living bacteria into the symbiotic forms (bacteroids) that are able to fix nitrogen inside the plant nodules. Comparative proteomics of bacteroids induced by Rhizobium leguminosarum bv. viciae UPM791 (Rlv UPM791) in pea (Pisum sativum) and lentil (Lens culinaris) plants identified about 100 rhizobial proteins that exhibited a host-dependent expression. Among them, a group of stress related proteins, small heat shock proteins (sHSP), were identified that might be related with the bacterial responses against the different environment, including several stressors, that the legume host provides. The functional role of two proteins, sHsp252, overexpressed in pea, and sHsp851, over-expressed in lens, has been studied in order to understand their role in the adaptation of Rlv UPM791 to the stress conditions in the nodules. Phylogenetic analysis showed that protein 252 and protein 851 are included in Class B and A of bacterial sHSPs, respectively, according to the previous classification defined in Bradyrhizobium japonicum. An Fnrtype promoter upstream of Rlvb_00252 gene, encoding 252 protein, and characterized by highly conserved palindromic sequence, called anaerobox, was identified. Furthermore, the expression of the 252 protein in microaerobic conditions under the control of the Rlv UPM791 FnrN transcriptional regulator was also demonstrated. In addition, heterologous expression in E. coli was observed by Fnr, the transcriptional regulator required for the expression of genes involved in anaerobic respiratory pathways in this bacteria. Plasmid-based tools were developed using a taurine inducible promoter to control the expression of these sHSP variants incorporating a C-terminal affinity StrepTag. Results showed that the over-expression of proteins 252 and 851 protects the viability of Rlv UPM791, grown under free-living conditions and exposed to 3 mM hydrogen peroxide stress at 15 and 30 min of incubation times, although the highest level of protection was given by protein 252. NCR (Nodule Cisteyne-Rich) peptides were also tested as stress agents, but in this case the over-expression of proteins 252 and 851 did not improve the viability of the bacteria for the concentrations and times tested. Other new molecular tools have been also generated to perform affinity chromatography studies that will identified the proteins that interact with sHSPs 252 and 851. These experiments will give an outstanding information to a better understanding of the role of sHSPs in the symbiosis.