Search for interaction partners of ECF sigma factor SigX in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a gammaproteobacteria that usually lives in water and soil, but is capable of infecting phylogenetically distant organisms. Its versatility reflects a sophisticated genic regulation machinery, which includes sigma factors, subunits of RNA polymerase that recognize promotors of target genes and are usually downregulated by anti-sigma proteins. Among the extracytoplasmic function sigma factors (ECF) there is SigX, which regulates the fatty acid synthesis and the expression of genes related to c-di-GMP metabolism, and for which, until now, no stability regulator has been found. One of the proteins induced in conditions in which SigX presents a lower concentration is PA14_41730, a Rimk-like protein. The objective of this work is to analyze the relationship between SigX and the proteins coded by the operon PA14_41690, PA14_41710 and PA14_41730, besides looking for other proteins that might regulate SigX function. Therefore, this work will be split in two phases: the first will consist in coexpressing said proteins and verify their interaction with SigX through copurification of E. coli extracts via affinity chromatography. On the second phase, His-FLAG-SigX will be overexpressed in P. aeruginosa, and both pulldown and mass spectrometry techniques will be used to isolate and identify proteins that may interact with this sigma factor and display a role in its regulation.