Antimicrobial activity and structure-function relationship of different chimerical bacteriocins

ACUÑA, LEONARDO; MORERO, ROBERTO D.; GALVÁN, EMILCE; SESMA, FERNANDO; BELLOMIO, AUGUSTO

Salta

Congreso; 3rd Latin American Protein Society Meeting; 2010

  Bacteriocins are ribosomally-synthesized antimicrobial peptides produced by Gram (-) and (+) bacteria with antibacterial spectra generally limited to phylogenetically related microbial strains. We carried out different transcriptional fusions between structural genes of enterocin CRL35 (a bacteriocin produced by Enterococcus mundtii CRL35) and colicin V (a microcin produced by Escherichia coli) in order to obtain recombinant broad spectrum chimerical bacteriocins, namely MunA-CvaC and CvaC-MunA. The engineered genes were cloned into several E. coli expression vectors: i) pT7, pUHE22, pET28b (carring or not a His tag); ii) pET43.1a to produce a NusA-MunA-CvaC fusion protein with factor Xa cleavage site-encoding sequence and pRSETa (carrying a His-tag and a factor Xa cleavage site-encoding sequence). All constructions were expressed in riphampycin-treated E. coli Rosetta Gami2 [DE3] (pLysS) after IPTG induction. The antimicrobial activity of the different cellular extracts and partially purified bacteriocin preparations were assayed against Gram (-) and Gram (+) indicator strains which are sensitive, immune and resistant to the native parental bacteriocins. All products of MunACvaC expression resulted in biologically-active fusion proteins active on both Gram (+) and Gram (-) bacteria except NusA-MunA-CvaC wich had no activity against Gram (+) bacteria. On the other hand CvaC-MunA showed activity only against Gram (+) bacteria.     The results suggest that the antimicrobial activity of CvaC requires a free C-terminus of the microcin in the chimerical molecule, while MunA-dependent activity of the hybrid bacteriocins are preserved regardless the position of the enterocin domain in the chimera.