Anr is necessary to develop oxidative stress resistance in Pseudomonas extremaustralis

TRIBELLI, P.M.; OPPEZZO, O.J.; NIKEL, PI; LÓPEZ, N.I.

Mar del Plata

Congreso; VIII Congreso Argentino de Microbiología General Samige del Bicentenario; 2012

Pseudomonas extremaustralis is a highly stress resistant bacterial species isolated from an Antarctic environment that is able to synthesize high amounts of poly(3-hydroxybutyrate) (PHB). This nonpathogenic bacterium shares general characteristics with representative Pseudomonas species but also presents metabolic differences with P. aeruginosa such as a nitrite accumulation during microaerobic growth, high stress resistance and PHB accumulation. Anr is the global anaerobic regulator involved in low oxygen tension metabolism. The relationship between Anr and the oxidative stress resistance was investigated in P.extremaustralis. The anr mutant strain presented a lower oxidative stress resistance in agar plate assay in presence of hydrogen peroxide (H2O2). The inhibition of growth was also measured in presence of different H2O2 concentrations in microaerobic planktonic cultures. The mutant strain presented higher inhibition of growth than the wild type strain. In addition, presence of oxygen reactive species (ROS) was determined by flow cytometry in aerobic and microaerobic cultures in presence or not of H2O2. In aerobic conditions the level of ROS was similar in both strains. However, in microaerobiosis anr strain showed strong differences, since the level of ROS was significantly higher in this strain, with and without H2O2. Therefore, antioxidative defenses in microaerobic cultures with and without H2O2 were investigated. Catalase activity of Anr and reduced thiol pool, determined by flow cytometry, resulted higher in absence of hydrogen peroxide in the mutant strain indicating the display of antioxidative defense systems in response to a basal stressed state in cells. In presence of H2O2, the thiol pool drooped significantly in the wild type strain, while in the mutant strain this was not observed, suggesting an impairment in the consumption of this molecules to ROS detoxification. In silico analysis allowed the prediction of genes potentially regulated by Anr in the whole genome sequence of P. extremaustralis. Among them, genes encoding for several chaperones and cold shock proteins, the glutathione synthase, a sulfate transporter and a thiol peroxidase were detected. These results in concert suggest that Anr is crucial to maintain a correct balance in cells and its absence provokes a stressed state and the increment in the antioxidative battery. However in presence of an exogenous source of oxidative stress the mutant strain is no capable to cope with it, thus suggesting an important role of Anr in the oxidative stress resistance.