IDENTIFICATION OF PROTEINS INDUCED BY POLYCYCLIC AROMATIC HYDROCARBONS (PAHs) IN ACTINOBACTERIAL STRAINS USING ONE AND TWO-DIMENSIONAL GEL ELECTROPHORESIS

BOURGUIGNON N; ISSAC P; CRISTINA ESTEVEZ; IRAZUSTA VERÓNICA; AMOROSO M.J.; FERRERO M

Córdoba Capital

Congreso; Congreso Argentino de Microbiología General SAMIGE - 2015; 2015

Among the most abundant environmental pollutants, polycyclic aromatic hydrocarbons (PAHs) are one of the major concerns because of their persistence and toxicity. A high diversity of actinobacterial strains with the ability to degrade PAH were studied because their metabolic versatility and high resistance to environmental stress conditions. Rhodococcus sp. 20, Rhodococcus sp. 016, Rhodococcus sp. P18, Rhodococcus sp. F27 and Amycolatopsis tucumanensis AB0, isolated from contaminated marine sediment and soil, were able to growth and remove naphthalene, phenantrene or pyrene from the culture medium. Strains were cultured in minimum medium supplemented with and without PAHs. After 72 h of incubation at 30 °C and 180 rpm the pellets were harvested and disrupted by sonication or French press. Soluble cellular protein fractions were evaluated SDS-PAGE (1-DE). Analysis of 1-DE revealed variations in the protein composition showing the up-regulation of multiple proteins for the three PAH treatments compared with the uninduced control sample. A total of 23proteins were identified including propane, methane monooxygenase, toluene hydroxylase, which areinvolved in metabolism of aromatic compounds; catalases, oxidoreductases, molecular chaperone GroEL and aldehyde dehydrogenase, were expressed to control oxidative stress during thehydrocarbons catabolism. We also found proteins related to carbohydrate metabolism (aldehyde dehydrogenase, acetone carboxylase, amidohydrolase, lydantoinase), and energy production (ATPsynthase, 5-oxoprolinase). Some proteins were detected uniquely upon exposure to a specific PAH, for instance most of the induced proteins in Rhodococcus sp. 016 were detected with pyrene; whereas others were shared, like methane monooxygenase for Rhodococcus sp. F27; which indicates thatinduction triggers not only specific responses but common responses in these strains.In addition, 2-DE studies revealed that when A. tucumanensis was exposed to phenanthrene it produced an over-expression of 22 spots. The identity of spots was related with the proteins identified in 1-DE. It is important to note the repetitive presence of methane monooxygenase and toluene hydroxylase. Proteins involved in energy production (ATP synthase, RNA polymerase, GTP-binding protein), detoxifying enzymes (sugar ABC transporter, universal stress protein) and those related to active metabolism (succinyl-CoA synthetase, elongation factor Ts, acetyl-CoA synthetase, enoyl-CoA hydratase, dihydrolipoamide acetyltransferase) were also up-regulated during phenanthrene degradation. Our results have shown that the actinobacteria studied degrade PAH through monooxidation of aromatic rings. Also, they up-expressed proteins involved in defense against oxidative stress and others related with energy gain. This is the first study that addresses the composition of proteins to reveal the metabolism of phenanthrene in A. tucumanensis.