Enterocin CRL35 is active against Gram-negative bacteria regardless of a specific receptor when is anchored in the membrane

CHALÓN MC; BARRAZA DE; ACUÑA L; SESMA F; MORERO RD; MINAHK CJ; BELLOMIO A

Congreso; VII CONGRESO ARGENTINO DE MICROBIOLOGÍA GENERAL SAMIGE DEL BICENTENARIO; 2011

SAMIGE

Enterocin CRL35 is a pediocin-like bacteriocin (subclass IIa) produced by Enterococcus mundtiiCRL35. Subclass IIa bacteriocins act on the cell membrane of gram positive bacteria through apore formation. Apparently, the peptides bind to the cytoplasmic membrane through a veryspecific receptor, the mannose phosphotransferase system. There are two theories on themechanism of pore formation: i) they would induce a conformational changes in the receptor toform a channel that remains open or , ii) they would use the receptor simply as an anchor andthen form the pore in the membrane. In both cases, the pore formation leads to the leakage ofions, dissipation of proton motive force and release of ATP and essential metabolites. In orderto study the mechanism by which enterocin CRL35 induces the loss of membrane integrity, weled the bacteriocin to the cell membrane of E. coli, by fusing it with EtpM, a protein of the type IIsecretion system from E. coli O157: H7. Previously in our laboratory, we carried out atranscriptional fusion between structural genes of enterocin CRL35 and colicin V thus obtainingthe chimera MunA-CvaC active against Gram-positive and Gram- negative bacteria. In this workwe constructed the fusions etpM-munA and etpM-munA-cvaC under the tight control of the PBAD promoter (repressed by glucose and induced by arabinose). In addition we carried out the coexpression of EtpM-MunA and EtpM-MunA-CvaC with the enterocin CRL35 and colicin Vimmunity proteins. This result is surprising because it is shown for the very first time thatenterocin CRL35 is capable of acting on Gram-negative bacteria when it is anchored in theplasma membrane, independently of its specific receptor.