Análisis funcional de proteínas de respuesta a estrés expresadas diferencialmente en bacterias endosimbióticas en asociación con distintos hospedadores

In the Rhizobium-legume symbiosis, sophisticated plant- and bacterial-dependent mechanisms lead to the establishment of the symbiotic form of bacteria (bacteroid) that fixes nitrogen within root nodules. Comparative proteomics between bacteroids induced by Rhizobium leguminosarum bv. viciae UPM791 (Rlv UPM791) in pea and lentil nodules allowed to identify a list of over 100 rhizobial proteins expressed with a host-dependent variation. One of the most relevant group of proteins potentially involved in the host adaptation were the small Heat Shock Proteins (sHsp), chaperones that stabilize unfolded proteins produced in the cell under stress conditions. In order to analyse the functional role of these proteins in the differential symbiosis adaptation, one sHsp over-expressed in pea (protein 252), and other over-expressed in lens (protein 851) were chosen for study. Mutants affected on each of the proteins showed a moderate decrease of symbiotic performance in pea plants. The structural modelization of both proteins was consistent with the information available about the structures, including their potential ability to form oligomers, wich is crucial for their potential chaperone activity. The regulation of the expression of these sHsps has been studied by the construction of promoter-lacZ fusions, performing assays with different stressors agents such as temperature, low oxygen, ethanol, high salinity, and hypochlorite. The results revealed a microaerobiosis-dependent expression of 252’s promoter. In contrast, relevant induction was not shown in the 851’s promoter. Symbiotic induction was also analysed by measuring ß-galactosidase activity in pea and lens bacteroids. Finally, plasmid-based tools to study the function of these proteins were developed by incorporating a taurine-dependent inducible promoter and a C-terminal affinity tag to the corresponding genes. The funtionality of the inducible promoter present in these plasmids was confirmed in free living cells and in bacteroids. Furthermore, the protein 851 was detected by inmunoblotting in free living strains induced by taurine, as well as in bacteroids