*3.3. Design of Lacticaseicin 30 Variants*

Lacticaseicin 30 variants were designed and constructed in order to locate the regions and amino acids specifically involved in the anti-Gram-negative activity, since such activity is rarely reported for LAB-bacteriocins. To this end, the DNA coding for the N-terminal region carrying the first two helices (from Met1 to Asp39), or the central and C-terminal regions carrying the last three helices (from Glu40 to His111) were cloned into the pET-32b plasmid, leading to the N-ter-lacticaseicin 30 and C-ter-lacticaseicin 30 variants, respectively (Figure 4). In addition, a shortened N-terminal region (from Met1 to Thr20) carrying only the first α-helix, designed as N-ter-H1-lacticaseicin 30 (Figure 4), was also expressed in the same system, considering nonetheless that thioredoxin (TRX) and 6his-tags located upstream of the multicloning site improve solubility of the variant peptides and their purification by the Ni-NTA chromatography.

In addition, several mutants were generated by site-directed mutagenesis, in order to identify the amino acids which, play a role in the anti-Gram-negative activity, and to understand the involvement of the predicted helical segments. To this purpose, charged (Glu, Asp) or aromatic (Tyr) amino acids located inside each α-helix were replaced by uncharged amino acids (Gly, Ser) (Figures 4 and S1). Furthermore, amino acids located in the center of each helix were substituted with proline, which is known for its role in breaking or introducing kinks in helices due to the lack of amide proton (Figures 4 and S1).

**Figure 2.** AlphaFold2 predicted structure of lacticaseicin 30 showing five helices (various shades of red) connected by coil regions (green). Five predicted H-bonds (blue) stabilize the position of the three helices.

**Figure 3.** (**A**) Circular dichroism spectra of lacticaseicin 30 at pH 7 (blue) and pH 5 (red) recorded in the absence or presence of 10 mM SDS micelles. (**B**) The secondary structure content (%) of lacticaseicin 30 at pH 7 and 5 was predicted using the BestSel Software.

**Figure 4.** Amino acid sequence and predicted three-dimensional structure of lacticaseicin 30. The predicted helical regions (helix 1 to helix 5) are underlined. Truncated forms of lacticaseicin 30 are shown by colored dotted lines (red: N-ter-Lacticaseicin 30, orange: N-ter-H1-Lacticaseicin 30 and blue: C-ter-Lacticaseicin 30). Mutated amino acids of variants are in red. Lacticaseicin 30 variants generated by directed mutagenesis are designated by a number between 1 and 10 (1: E6G, 2: T7P, 3: E32G, 4: T33P, 5: T52P, 6: D57G, 7: A74P, 8: Y78S, 9: Y93S and 10: A97P). Activities in the same range as native lacticaseicin 30 and total absence of activity are shown by + and −, respectively; \* and ":" indicate successively a gradual decrease of activity.

#### *3.4. The N-Terminal Region Is Sufficient to Exert Anti-Gram-Negative Activity*

After being produced, purified and quantified, lacticaseicin 30 and its variants, N-terlacticaseicin 30, C-ter-lacticaseicin 30 and N-ter-H1-lacticaseicin 30, were assessed for their antibacterial activity, which was determined at pH5 against four Gram-negative bacterial strains and *Listeria innocua* CIP 80.11 as Gram-positive bacterium (Table 1). Lacticaseicin 30 inhibited the growth of all target bacteria (Table 1), and particularly of *Escherichia coli* ATCC 8739 and *Proteus vulgaris* ATCC 33420, against which MIC values were the lowest (40 μg/mL), contrary to those obtained against *Pseudomonas aeruginosa* ATCC 27853 (160 μg/mL). The antibacterial activity against Gram-negative bacteria was clearly evidenced. Remarkably, the shortened variants, N-ter-lacticaseicin 30 and C-ter-lacticaseicin 30, both exhibited significant activity against the aforementioned Gram-negative target bacteria, as well as against the Gram-positive *Listeria innocua* CIP 80.11 (Table 1). The MIC values obtained with the N-ter-lacticaseicin 30 and C-ter-lacticaseicin 30 variants revealed that the N-terminal 1–39 region of lacticaseicin 30 was sufficient for the anti-Gram-negative activity against most of the targets (*P. aeruginosa* was no longer inhibited efficiently), whereas that of the truncated N-terminal peptide N-ter-H1-lacticasecin 30 was fully abolished, arguing that the first helix at the bacteriocin N-terminus is insufficient for the anti-Gram-negative activity.


**Table 1.** Minimum inhibitory concentrations (MIC) values (μg/mL) of lacticaseicin 30 and its truncated forms.
