The enterocin CRL35 biosynthetic gene cluster was cloned and sequenced. The sequence was revealed to be highly identical to that of the mundticin KS gene cluster (S. Kawamoto, J. Shima, R. Sato, T. Eguchi, S. Ohmomo, J. Shibato, N. Horikoshi, K. Takeshita, and T. Sameshima, Appl. Environ. Microbiol. 68: 3830-3840, 2002). Short synthetic peptides were designed based on the bacteriocin sequence and were evaluated in antimicrobial competitive assays. The peptide KYYGNGVSCNKKGCS produced an enhancement of enterocin CRL35 antimicrobial activity in a buffer system.

Aims: To evaluate the effect of protective agents upon survival of Lactobacillus delbrueckii ssp. bulgaricus during freeze‐drying and storage, and selective amino acids on cell membrane fluidity. Methods and Results: The protective effect of amino‐acids and sugars at different concentrations was studied by determining the viability of lyophilized cells after storage under air at 30°C. Survival following freeze‐drying was improved by all compounds. During storage, neither proline nor maltose had protective effects on lyophilized Lact. delbrueckii ssp. bulgaricus. Glutamate 5% and aspartate 5% showed similar protection capability during freeze‐drying (94–95%) and after storage (92–99%). Fluorescence probes (DPH and TMA–DPH) were used to study the effect of both amino acids on membrane fluidity. Polarization decreased with increasing concentrations of glutamate or aspartate. Lowest values were observed with TMA–DPH. Conclusions: Glutamate 5% and aspartate 5% allowed maintaining high viability rates during freeze‐drying and storage of Lact. delbrueckii ssp. bulgaricus because of an increase of the membrane fluidity by inserting in the interfacial region of bacterial plasma membrane. Significance and Impact of the Study: These results show the first evidence of the mechanisms underlying glutamate and aspartate as lyoprotectors.

The antimicrobial peptide Enterocin CRL35, a class II bacteriocin, produces at high concentrations (8 μg ml−1) localized holes in the wall and cellul

Objective: The aim of this study was to evaluate the interaction of several conventional antibiotics with sub-lethal concentrations of enterocin CRL35, a cationic peptide, on Listeria innocua 7. Methods: Susceptibility of L. innocua 7 cells to the combination of enterocin CRL35 and non-peptide antibiotics (cefalexin, ampicillin, ciprofloxacin, nalidixic acid, erythromycin, chloramphenicol, vancomycin and tetracycline) was assayed using the broth dilution method and killing curves. Fractional inhibitory concentration (FIC) index was calculated to assess synergy. The transmembrane electrical potential and pH gradient were determined by specific fluorescent probes. Results: We found positive interactions between the cationic peptide and three conventional antibiotics (tetracycline, erythromycin and chloramphenicol) which are excluded from the cells by efflux pumps dependent on the membrane proton gradient. Furthermore, enterocin CRL35 even at sub-lethal concentrations induced the dissipation of both components of the proton motive force (Δp), i.e. transmembrane electrical potential and pH gradient and hence the alteration of processes dependent on it. Conclusion: We hypothesize that enterocin CRL35 increases the effectiveness of these antibiotics by impairment of the bacterial active efflux systems and the consequent accumulation of these toxic compounds in the cytoplasm.