Title : Assignment of sigma factors of RNA polymerase to promoters by in vivo and in vitro systems in Rhodococcus sp.
Many Rhodococcus strains are able to transform and metabolize a wide range of toxic organic compounds, which contaminate various environments. Thanks to their diverse metabolic activities, rhodococci are promising microorganisms for biotechnological applications in biodegradations, bioremediations, and enzymatic biotransformations. Rhodococcus strains are distinguished by their cell wall which contains mycolic acids. These compounds are also present in the cell wall of other bacteria of the Mycolata group, such as Corynebacterium and Mycobacterium species. Many Rhodococcus strains were classified as extremophiles, which are able to cope with various types of stresses, e.g. desiccation, heat, cold and osmotic stress. In most cases, severe stresses cause decrease in biodegradation efficiency. In contrast, stresses have in some cases positive effects on production of the desired compounds (e.g. desiccation stress on the production of triacylglycerols, osmotic stress on the synthesis of fatty acids). Most stress responses, and particularly their mechanisms, have been described in rhodococci in much less detail than their enzymatic machinery and their use for biotechnological processes. Global, genome-wide studies, which can describe complex stress response mechanisms and impact of stresses on the biotechnological processes, are still rare in rhodococci. We developed in vitro and in vivo assays to examine the connection between alternative sigma factors of RNA polymerase and genes activated by various stress conditions. These assays are based on the procedures designed for the related species, Corynebacterium glutamicum. We showed that the R. erythropolis CCM2595 genes frmB1 and frmB2 which encode S-formylglutathione hydrolases (named corynomycolyl transferases in C. glutamicum) are controlled by SigD, just like the homologous genes cmt1 and cmt2 in C. glutamicum. The new protocol of the in vivo and in vitro assays will enable us to classify promoters of Rhodococcus stress genes according to their connection to sigma factors and to assign the genes to the corresponding sigma regulons. The complex stress responses could thus be analyzed in terms of regulation of gene expression by sigma factors.