Mutations of Microcin J25 that Affect Entry of the Antibiotic Into Escherichia coli Cells

DE CRISTÓBAL, R.E; ZENOFF, A.M; FARÍAS, R.N; SOLBIATI, J.O; SALOMÓN R.A.

Bariloche, Río Negro, Argentina 17 al 21 de Noviembe de 2003

Congreso; XXXIX Reunión Nacional SAIB; 2003

Sociedad Argentina de Investigaciones Bioquímicas y Biología Molecular

Microcin J25 (MccJ25) is a 2,107 Da Cyclopeptide antibiotic of 21 amino acids produced by Escherichia coli MccJ25 can enter sensitive cells by binding to specific membrane receptors: FhuA, in the outer membrane, and TonB and SbmA, in the inner membrane. E coli RNA polymerase is the intracellular target of MccJ25. We are interested in identifying which residues within MccJ25 are critical for interaction with the target site and with the membrane receptors. MccJ25 shows a predominance of uncharged and hydrophobic residues over the charged residues Glu and His. We have mutagenized the unique His residue in MccJ25 in orde to obtain 3 different analogs in which His has been replaced by Ala, Lys or Arg. Respectively. This mutant peptides were purified by HPLC and their effect on RNA polymerase was assessed in vitro. No significant difference on RNA polymerase activity was noted as compared with the control, wild type microcin. .We also performed in vivo assays to calculate the minimal inhibitory concentration (CIM) of each one of the mutant microcins against E. coli strains where the different membrane receptors were overexpressed or not. From these experiments we found that E. coli cells are more resistant to the mutant microcins HisxAla and HisxArg than to the wild- type microcina and the Hisx Lys mutant. Additionalexperiments showed that the interaction with the inner memebrane receptor SbmA is affected in these mutants