EFFECT OF NITROSATIVE STRESS UNDER MICROAEROBIC CONDITIONS IN Pseudomonas extremaustralis REVEALED BY TRANSCRIPTOME ANALYSIS

LÓPEZ, N.I.; SOLAR VENERO, E.C.; TRIBELLI, P.M.

Congreso; LVI SAIB Meeting ?XV SAMIGE Meeting; 2020

Pseudomonas possesses a variety of energy-generation metabolisms spanning from aerobic to anaerobic. Aerobic respiration produces reactive oxygen species (ROS), whereas anaerobic respiration, using nitrate as an electron acceptor, can generate reactive nitrogen species (RNS). ROS and RNS provoke oxidative or nitrosative stress, respectively. RNS include nitric oxide (NO) and its derivatives peroxynitrite (ONOO− ), nitrosothiols (from reaction with thiol groups), and nitrotyrosine (from the nitration of tyrosine by NO, ONOO− or NO2 − ). Pseudomonas extremaustralis is an Antarctic bacterium, capable of growing at low temperatures and under low oxygen conditions. The denitrification process in P. extremaustralis is incomplete due to the absence of nir genes that encode the enzymes responsible for catalyzing the reduction of NO2 to NO in denitrification. Considering the importance that RNS from anaerobic respiration, combined with those that may arise from reactions in the environment may have, we set out to evaluate the effect of nitrosative stress in this bacterium. We analyzed the response to nitrosative stress at low O2 tensions using S-nitrosoglutation (GSNO), a NO donor compound. NO plays several roles in bacterial metabolism, either as an intermediary in denitrification processes, acting as a signaling molecule at low concentrations, or leading to bacterial stress at high concentrations. To analyze the response to GSNO at low O2 tensions, transcriptomic data were obtained by RNA sequencing (RNA-Seq), comparing 24-hour cultures of P. extremaustralis under microaerobic conditions with or without exposure to 100 µM GSNO for 1 h. The concentration and exposure time were selected based on growth and survival analysis. Transcriptomic analysis showed differential expression of genes corresponding to various cellular processes. Among the most relevant ones, we found genes related to transcription and translation processes, amino acid transport and biosynthesis, and carbon and iron metabolism. Several stress resistance genes were also overexpressed in presence of GSNO, such as those encoding an iron-dependent superoxide dismutase, an alkyl hydroperoxide reductase, a thioredoxin, and a glutathione S-transferases-type enzyme. Results also allowed us to determine that exposure to GSNO differentially affected the expression of genes whose protein products are targets of nitrosylation (metalloproteins and proteins with free cysteine and/or tyrosine residues). Regarding carbon metabolism, exposure to GSNO resulted in the activation of inositol catabolism, a little-studied pathway that may be related to survival in interactions of bacteria with plants and animals. Additionally, iron uptake mechanisms, such as pyoverdine synthesis and iron transporter genes, were activated in presence of GSNO, probably as a response to the need to replace damaged proteins containing this metal.