ROLE OF THE SIGMA FACTOR AlgT AND ITS ANTI-SIGMA MucA IN THE ANAEROBIC METABOLISM AND SENSITIVITY TO ACIDIFIED NITRITE

MARTINO, ROMÁN A.; LOPEZ, NI; HEDEMANN, GABRIELA; ZIMMERMAN, SOFÍA; MOYANO, ALEJANDRO J.; LOPEZ, VERONICA; TRIBELLI, PAULA; SMANIA, ANDREA M.

MODALIDAD VIRTUAL

Congreso; LVII Reunión Anual de Sociedad Argentina de Investigaciones Bioquímicas y Biología Molecular (SAIB) y el XVI Congreso Anual de la Asociación Civil de Microbiología General (SAMIGE); 2021

SAIB & SAMIGE

Pseudomonas aeruginosa is an opportunistic pathogen that chronically infects the airways of cystic fibrosis (CF) patients. Mucoid, mucA mutant, phenotype marks the onset of chronic infection and constitutes a sign of poor prognosis. The mucA gene is an anti-σ factor that negatively regulates alginate production by sequestration of AlgT, an alternative σ factor responsible for the transcription of the alginate biosynthetic operon. The most frequent mutation responsible for mucoid conversion is a deletion of a G residue within a homopolymeric track of five Gs (G5426), also known as mucA22 allele, causing the truncation of MucA C-terminal periplasmic domain. Mutations in mucA gene can also affect major determinants for bacterial persistence such as quorum-sensing (QS) signals, flagellum biosynthesis or survival under anaerobic and osmotic stress conditions. In this sense, a growing body of evidence has shown that mucoid mucA22 is highly sensitive to acidified nitrite (A-NO2-). However, this outcome has yet to be understood. To better understand the functional interactions between AlgT and its antagonist regulator MucA, we constructed a set of mutants containing different combinations of mucA and algT composition, namely, ΔalgT, ΔalgTΔmucA, ΔalgTmucA22, and mucA22. By measuring cell viability as well as nitrogen gas and NO2- levels, we confirmed that the mucA22 strain was highly sensitive to NO2- under anaerobic conditions. Moreover, we determined that it was impaired in the production of acyl-homoserine lactones (AHLs). However, both phenotypes were restored to wild-type levels upon algT deletion indicating that they might be the consequence of the σ factor deregulation. Based on these observations, we hypothesize that RhlR and Dnr, key regulators that control the QS system and the denitrification pathways in P. aeruginosa, respectively, were involved in the regulatory pathways leading to mucA22 ANO2- sensibility. Thus, we further constructed rhlR and dnr mutants by CRISPR-Cas9 and confirmed they were as sensible as mucA22 to A-NO2- under anaerobic conditions. Using transposon mini-Tn7 vectors, we are currently attempting to introduce the rhlR and dnr genes in the chromosome of the mucA22 strain in order to address if their expression reverse sensibility of mucA22 to A-NO2-. Our work shed light on the complex regulatory pathways connecting mucoid conversion, quorum sensing, and anaerobic growth, providing potential targets for future therapeutic strategies to control chronic P. aeruginosa.