Biophysical and functional studies of Microcin V domains using suicide probes

PINTANEL, JOSE; RIOS COLOMBO, N.S.; BELLOMIO, A; CHALON, M.C.

Rosario

Congreso; LIX Reunion Anual de la Sociedad Argentina de Investigaciones en Bioquimica y Biologia Molecular (SAIB); 2023

Bacteriocins are small peptides of ribosomal synthesis, which have antimicrobial activity against phylogenetically related bacteria. Microcin V (MccV) is a linear peptide of 88 amino acids, it kills sensitive cells by acting at the level of the internal membrane through the dissipation of the membrane potential. Suicide probes consist of the fusion of a single-pass membrane protein called EtpM with a bacteriocin at the C-terminus. They have a toxic effect on the bacteria that express them. It is proposed to construct truncated versions of the EtpM-MccV suicide probes that include the different domains of the peptide, to elucidate in vivo the role of each of them in the mechanism of MccV toxicity. By using computer software and genetic engineering techniques, truncated suicide probes were constructed from the chimeric protein. Viability tests were performed to evaluate their toxicity once they were expressed in E. coli strains by adding arabinose. Fluorescence spectroscopy using DiSC3(5) probe were studied to determine changes in membrane potential. Two truncated suicide probes were constructed and through viability assays we observed that, under the conditions used, the proteins turned out to be non-toxic for the E. coli strains that expressed them. On the other hand, when subjecting the cells to the study with the fluorescent molecule, we saw that one of the constructions dissipates the membrane potential (the protein that has a transmembrane portion) but the other (which does not have this portion) does not. Our results confirmed that using suicide probes it is possible to detect changes in membrane permeability even though the effect is not sufficient to produce cell death.