Structure-Function Relationship in Microcin J25

SERGIO SOCÍAS; PAULA VINCENT; KONSTANTIN SEVERINOV; RAÚL SALOMÓN

Rosario, Argentina

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología molecular; 2006

Sociedad Argentina de Investigaciones en Bioquímica y Biología molecular

To study structure-function relationships in the peptide antibiotic MccJ25, the structural gene was subjected to site-directed mutagenesis. Antibacterial activity of the peptide mutants was tested against E. coli DH5a  and MC4100. We obtained 279 mutants, from which 218 were inactive and 61 active. From the latter, 3 have wild-type activity, 48 were less active, and 10 were more active than native MccJ25. When tested against strains hyperexpressing the MccJ25 importers FhuA and SbmA, less active mutants became as active as MccJ25, suggesting that the mutated amino acid is involved in microcin import. Using the same strains, no activity change was observed with the other mutant peptides, indicating that the mutated residue is either important for antibiotic action or is needed for MccJ25 maturation and export. In other experiments, we deleted the entire leader peptide as well as almost all of it, leaving only the three amino acids to the left of the cleavage site. In both cases, we provided a Shine-Dalgarno and an ATG codon. Anyone of these constructs led to the production of extracellular active microcin, suggesting that integrity of the leader peptide is essential for MccJ25 production.