*3.5. Amino Acids Critical for the Anti-Gram-Negative Activity of Lacticaseicin 30*

Different variants of lacticaseicin 30 were generated by site-directed mutagenesis using the pT7-6his-030 plasmid. These substitutions were performed in regions expected to play a key role in the folding of the bacteriocin. Importantly, these amino acid substitutions were introduced in the middle of each predicted α-helix in order to induce a conformational change. The antibacterial activity of lacticaseicin 30 and its variants was measured against the four selected Gram-negative bacteria (Table 2). Overall, amino acid substitutions led to a decrease in the antibacterial activity, except for E32G, T33P and D57G peptides; these MIC values remained unchanged and similar to those of lacticaseicin 30 (Table 2). Furthermore, when Glu6, Tyr78 and Tyr93 are mutated, they induce a net decrease of activity, suggesting a role in the anti-Gram-negative activity. Moreover, the MIC values obtained for lacticaseicin 30 and its truncated forms suggested that the Gram-negative activity (*E. coli*, *Salmonella*, *Proteus* and *Pseudomonas*) require the presence of helix 1. Helix 2 and its acidic residue E32 appeared to not be a determinant for the anti-Gram-negative activity evaluated in this study. Helix 3 and its aromatic residue T52 seems to be weakly required for the anti-Gram-negative activity. Helix 4 and its aromatic residue Y78 is, however, important for the anti-Gram-negative activity. Remarkably, helix 5 is important for activity against *Escherichia coli* and *Proteus* but is less important against *Salmonella* and *Pseudomonas* (Tables 1 and 2).


**Table 2.** Minimum inhibitory concentrations (μg/mL) of lacticaseicin 30 and its variant peptides.
