Avaliação da atividade promotora dos genes codificadores de fatores sigma em Corynebacterium pseudotuberculosis em resposta a estresses abióticos

Abstract

Corynebacterium pseudotuberculosis (Cp) is a Gram-positive bacterium and the etiological agent of Caseous Lymphadenitis, a disease that causes great economic losses in the sheep and goat industries worldwide. This pathogen persists for a long time within the host or in the external environment, resisting adverse conditions. The transient activation of the RNA polymerase sigma factors provides the adaptation to environmental variations. A single sigma factor recognizes a specific set of promoter sequences in the bacterial genome and may activate the expression of up to hundreds of physiologically related genes, allowing the bacterial cell to persist and survive. In the present study, the differential induction of the Green Fluorescent Protein (GFP) was evaluated, using a flow cytometer and the promoters from different sigma factor-encoding sequences. For this purpose, promoters from the genes sigA, B, C, D, E, H, K, and M, in Cp, were cloned upstream the GFP-encoding sequence present in the promoterless pSM20 vector. All plasmid constructs were obtained using the strain Top10 of Escherichia coli and confirmed through digestion, PCR and DNA sequencing. Subsequently, the strain 1002 of Cp was transformed with each of the recombinant plasmids. The resulting strains were cultured until the beginning of the exponential growth phase and submitted to oxidative, osmotic and cell surface stresses. The promoter PsigA was activated under high osmolarity and cell surface stress, induced by SDS. PsigB was induced only in the presence of SDS. PsigD was activated following exposure to high osmolarity, SDS and oxidative stress, induced by hydrogen peroxide. PsigE and PsigH were activated by SDS, but deactivated under osmotic stress and exposure to lysozyme, respectively. PsigC, PsigK and PsigM were not induced by any of the stress conditions tested in this study. These activation/ deactivation profiles demonstrate the capacity of our plasmid constructs to provide relevant information on the activity of sigma factors, when Cp is submitted to various environmental conditions. Currently, additional environmental conditions are being tested, in order to achieve a better understanding of the genetic regulation in Cp. Also, the present study raises the possibility to assess intracellular sigma factor activation, using murine macrophage cells infected in vitro.