Fluorescent labeled microcin incorporation in living cells

DUPUY, FERNANDO G.; NIKLISON CHIROU, MARÍA V.; FERNÁNDEZ DE ARCURI, BEATRIZ; BELLOMIO, AUGUSTO AND MORERO, ROBERTO D.

Montevideo, Uruguay

Congreso; XXXVI Reunión Anual de la Sociedad Argentina de Biofisica; 2007

Sociedad Argentina de Biofisica

Microcin J25 is a plasmid encoded 21 aminoacid antibiotic peptide with a distinctive lasso-structure produced by Escherichia coli and active against Escherichia coli, Salmonella enteritidis serovars and Shigela strains. It has been shown that the target in E. coli is the b subunit of the RNA polymerase. However, it’s also known that in Salmonella newport cells MccJ25 inhibits the plasmatic membrane’s respiratory enzymes, suggesting a dual mechanism of action. To study with more detail the antibiotic activity of the peptide, a fluorescent labeled microcin was sinthetizated. To achieve this, a mutant peptide containing lysine instead of isoleucine(13) was purificated, the MccJ25 I13K. This peptide was chosed because this amino acid substitution has no effect on the biological activity of the peptide. For the labelling reaction, the mutant peptide was incubated with the amine-reactive probe fluorescein isothiocyanate, which form a thiourea bond with free amine groups. A mixture of peptide and fluorescent probe 1:3 ratio (w/w) was incubated in alcaline medium at room temperature during two hours in the dark. The purification of the labeled peptide was carried out in two steps. First, the non reacted fluorescent probe was eliminated by a C-8 hydrophobic chromatography. Then, the methanolic fraction was purified by HPLC on a C18 column in order to separate the labeled peptide from the non labeled one. The purified fluorescein-labeled microcin showed antibiotic activity against MccJ25 sensible-strains similar to the native peptide. Uptake assays of the microcin derivative with several strains were performed following the maximum emission fluorescence at 520nm. A bacterial suspension was incubated with the peptide at sublethal concentrations at 37°C in a TRIS buffer medium supplemented with glucose. Aliquots were taken at different times and the residual peptide in the supernadant was determined. The sensible strains Salmonella newport and E. coli AB1133 (pGC01) and the resistant strains E. coli SBG 231 y E. coli AB 259 (pTUC 200) were used. The independent effect of 100 mM 2,4 dinitrophenol, 200 mM vanadate or heat plus azide on the derivative peptide uptake were studied. The results indicates that MccJ25 incorporation is an active mechanism ATP driven in the different strains tested, and the incorporation is seen both in sensitive and resistant strains.