TYR9 OF THE MICROCIN J25 MOLECULE IS INVOLVED IN THE OVERPRODUCTION OF SUPEROXIDE

CHALÓN, MIRIAN C.; BELLOMIO, AUGUSTO; MORERO, ROBERTO D.; FARÍAS, RICARDO N.; VINCENT, PAULA A.

Mar del Plata

Congreso; XLIII Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología molecular.; 2007

Sociedad Argentina de Investigaciones en Bioquímica y Biología molecular.

Microcin J25 (MccJ25) have two intracellular targets: the RNA polymerase and the respiratory chain. The last one is mediated by an increment of superoxide. Previously, we showed that different parts of the molecule are responsible for interaction with each target. Recent studies suggested that tyr, trp, cys and gly residues are involved in the reactive oxygen species production. MccJ25 contain six glycines and two tyrosines (Y9 and Y20). First, we chose Y9 and Y20 for mutation. PA232 harboring a MccJ25-resistant RNAP and transformed with a plasmid carrying the fhuA gene (pGC01) was inhibited by MccJ25 and MccJ25(Y20F) under aerobic but not in anaerobic condition. The MccJ25 and MccJ25(Y20F) also inhibited the PA232 (pGC01) when the anaerobic culture was changed to aerobic one. On the contrary, MccJ25(Y9F) did not inhibit this strain either in aerobic culture or when the anaerobic culture was changed to aerobic condition. The MIC of MccJ25 and MccJ25(Y20F) were 2 and 10 µM, respectively, whereas for MccJ25(Y9F) was > 1 mM. In opposite to MccJ25 and MccJ25 (Y20F), MccJ25(Y9F) was unable to inhibit the cell respiration and to increase superoxide generation in vitro. The three peptides inhibited nearly 50% of the in vivo RNA synthesis in AB1133 (pGC01). These results define nature and position of the amino acid in the MccJ25 molecule involved in the superoxide overproduction.