VINCENT Paula Andrea
congresos y reuniones científicas
Ile13 of the microcin J25 molecule is involved in interaction with the receptor Fhua
Carlos Paz, Córdoba
Congreso; XLIV Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología molecular.; 2008
E. coli RNA polymerase (RNAP) is the target of microcin J25 (MccJ25). MccJ25 enters cells after binding to specific membrane transporters: FhuA in the outer membrane and SbmA in the inner membrane. We studied a MccJ25 mutant carrying a substitution of Ile13 by Lys (I13K). The results showed that the I13K mutant inhibited RNAP in vitro. However, the mutant was defective in its ability to inhibit cellular growth. A possible explanation could be that the mutant is unable to reach the cytoplasm of the sensitive cells or that it has a higher affinity by YojI, a MccJ25 export pump. Following this hypothesis we tested the activity of the mutant against strains with a chromosomal deletion of yojI and hyperexpressing FhuA and SbmA. Interestingly, the mutant was almost as active as wild-type microcin only against an E. coli strain carrying a plasmid with fhuA cloned, suggesting that the lower activity of the mutant is due to a deficient transport into the cell. Moreover, in experiments in which the FhuA protein was bypassed, the mutant showed an activity similar to wild-type MccJ25. In addition, in a competition assay with colicin M, MccJ25 was able to reduce colicin uptake by FhuA, while the I13K mutant did not. Together, these results suggest that the Ile13 residue in the MccJ25 molecule is needed for uptake of the antibiotic by FhuA, but it is not required for MccJ25 inhibition of RNAP.