Pore-forming toxins (PFTs) are proteins that are secreted as soluble molecules and are inserted into membranes to form oligomeric transmembrane pores. In this paper, we report the crystal structure of Fragaceatoxin C (FraC), a PFT isolated from the sea anemone Actinia fragacea, at 1.8 Å resolution. It consists of a crown-shaped nonamer with an external diameter of about 11.0 nm and an internal diameter of approximately 5.0 nm. Cryoelectron microscopy studies of FraC in lipid bilayers reveal the pore structure that traverses the membrane. The shape and dimensions of the crystallographic oligomer are fully consistent with the membrane pore. The FraC structure provides insight into the interactions governing the assembly process and suggests the structural changes that allow for membrane insertion. We propose a nonameric pore model that spans the membrane by forming a lipid-free α-helical bundle pore.

Microcin J25 (MccJ25) uptake by Escherichia coli requires the outer membrane receptor FhuA and the inner membrane proteins TonB, ExbD, ExbB, and SbmA. MccJ25 appears to have two intracellular targets: (i) RNA polymerase (RNAP), which has been described in E. coli and Salmonella enterica serovars, and (ii) the respiratory chain, reported only in S. enterica serovars. In the current study, it is shown that the observed difference between the actions of microcin on the respiratory chain in E. coli and S. enterica is due to the relatively low microcin uptake via the chromosomally encoded FhuA. Higher expression by a plasmid-encoded FhuA allowed greater uptake of MccJ25 by E. coli strains and the consequent inhibition of oxygen consumption. The two mechanisms, inhibition of RNAP and oxygen consumption, are independent of each other. Further analysis revealed for the first time that MccJ25 stimulates the production of reactive oxygen species (O 2˙؊ ) in bacterial cells, which could be the main reason for the damage produced on the membrane respiratory chain.MccJ25 is active on gram-negative bacteria related to the producer strain, including some pathogenic strains (43,44,55). Four plasmid genes, mcjABCD, are involved in MccJ25 production: mcjA, mcjB, and mcjC code for an MccJ25 precursor and two processing enzymes required for the in vivo synthesis of the mature peptide, respectively, and mcjD encodes the McjD immunity protein (53). McjD, a homologous ABC exporter family protein, participates in MccJ25 secretion (52). Thus, immunity is mediated by active efflux of the peptide, keeping its intracellular concentration below a critical level (53). Recently, it has been demonstrated that YojI, a chromosomal protein with ABC-type exporter homology (36), is also able to export MccJ25 from the cells (14). TolC, an E. coli outer membrane protein, is necessary for MccJ25 secretion mediated by either McjD or YojI (11,14). On the other hand, the uptake of MccJ25 by E. coli is dependent on the outer membrane receptor FhuA (15, 45) and the four inner membrane proteins TonB, ExbD, ExbB, and SbmA, the first three of which constitute the Ton complex (38), while the last one acts as a transporter (46).Convincing evidence showing that RNA polymerase (RNAP) is the target for MccJ25 action in E. coli was previously provided by our laboratory. The peptide inhibits the enzyme activity by obstructing the secondary channel and consequently preventing access of the substrates to its active sites (1,12,34,57). Later, it was demonstrated that MccJ25 can bind and penetrate into the phospholipid monolayer and disrupt the electric potential of liposomes composed of phospholipids from gram-negative bacteria (5, 40). These results encouraged the study of the effect of MccJ25 on the bacterial membrane. MccJ25 was found to disrupt the membrane potential inhibiting oxygen consumption in Salmonella enterica serovar Newport (41) and S. enterica serovar Typhimurium transformed with a plasmid carrying fhuA from E. coli (55), suggesting the presence of a se...

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Graphical abstractHighlights► A chimerical gene encoding a hybrid bacteriocin–microcin was generated by PCR. ► Ent35–MccV is a hybrid bacteriocin containing enterocin CRL35 and microcin V. ► Ent35–MccV is active against Gram-positive and Gram-negative bacteria. ► Enterohemorrhagic Escherichia coli and Listeriamonocytogenes are sensitive to Ent35–MccV.

This review attempts to analyze the mechanism of action and immunity of class IIa bacteriocins. These peptides are promising alternative food preservatives and they have a great potential application in medical sciences. Class IIa bacteriocins act on the cytoplasmic membrane of Gram-positive cells dissipating the transmembrane electrical potential by forming pores. However, their toxicity and immunity mechanism remains elusive. Here we discuss the role of the mannose phosphotransferase system (man-PTS) as the receptor for class IIa bacteriocins and the influence of the membrane composition on the activity of these antimicrobial peptides. A model that is consistent with experimental results obtained by different researchers involves the non-specific binding of the bacteriocin to the negatively charged membrane of target bacteria. This step would facilitate a specific binding to the receptor protein, altering its functionality and forming an independent pore in which the bacteriocin is inserted in the membrane. An immunity protein could specifically recognize and block the pore. Bacteriocins function in bacterial ecosystems and energetic costs associated with their production are also discussed. Theoretical models based on solid experimental evidence are vital to understand bacteriocins mechanism of action and to promote new technological developments.

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