Response to lethal UVA radiation in the Antartic bacterium Pseudomonas extremaustralis

BRITO MARIA GABRIELA; LOPEZ NANCY I; TRIBELLI PAULA M.; MONTESINOS, NAHUEL V.; PEZZONI MAGDALENA; COSTA CRISTINA S.

Congreso; V Congreso Argentino de Microbiología Agrícola y Ambiental (CAMAyA),; 2021

Asociación Argentina de Microbiología división CAMAYA

Pseudomonas extremaustralis is an Antarctic bacterium with high stress resistance, able to grow under cold conditions. It is capable to produce polyhydroxyalkanoates (PHAs) mainly as polyhydroxybutyrate (PHB) and, to a lesser extent, medium-chain length polyhydroxyalkanoates (mclPHAs). In this work main objective is to analyze the role of PHAs and cold adaptation in the survival of P. extremaustralis after lethal UVA exposure. To achieve this objective, bacteria (P. extremaustralis 14-3b and its PHA- and PHB- derivative mutant strains and the PHB complemented strain) were grown at 10 °C or 30 °C to stationary phase in LB or LBO (LB supplemented with sodium octanoate as carbon source for PHAs accumulation). After washes the bacterial suspensions adjusted to DO600nm=0.4 were divided into two 30-mL fractions, which were each placed in a glass beaker. One of these fractions was irradiated from above at a fluence rate of 20 W/m2 at the level of the free surface of the suspension, while the other fraction remained in the dark. Serial dilutions of cell suspensions exposed to UVA radiation or maintained in the dark were performed and plated on LB solid medium. Plates were immediately incubated at 30 °C in the dark to prevent light-induced DNA repair and the colonies were counted after 24?48 h. Survival was expressed as a fraction of the number of colony forming units (CFU) per mL at initial time (t0). Catalase activity was measured by H2O2 decomposition while redox state was analyzed using NADH/NAD+ determinations. Additionally, polyhydroxyalkanoates accumulation was measure using gas chromatography (GC) methodology. PHAs granule interaction with UV light was analyzed by light scattering and absorption determinations.P. extremaustralis presented higher radiation resistance under polymer accumulation conditions. This result was also observed in the derivative mutant strain PHA-, deficient for mclPHAs production. On the contrary, the PHB- derivative mutant, deficient for PHB production, showed high sensitivity to UVA exposure. Complementation of the PHB- strain restored the wild-type resistance level, indicating that the UVA-sensitive phenotype is due to the lack of PHB. All strains exhibited high sensitivity to radiation when cultured under PHAs non-accumulation conditions. A slight decrease in PHB content was observed after UVA exposure in association with increased survival. The scattering of UVA radiation by intracellular PHAs granules could also result in bacterial cell protection. In addition, cold conditions improved UVA tolerance, probably depending on PHB mobilization. Results showed that PHB accumulation is crucial in the resistance to UVA in P. extremaustralis. Mechanisms involved probably entail depolymerization and light scattering acting as a screen, both conferring protection against oxidative stress.