Cytochromes bd-I and bo3 are essential for the bactericidal effect of microcin J25 on Escherichia coli cells.

Microcin J25 has two targets in sensitive bacteria, the RNA polymerase, and the respiratory chain through inhibition of cellular respiration. In this work, the effect of microcin J25 in E. coli mutants that lack the terminal oxidases cytochrome bd-I and cytochrome bo3 was analyzed. The mutant strains lacking cytochrome bo3 or cytochrome bd-I were less sensitive to the peptide. In membranes obtained from the strain that only expresses cytochrome bd-I a great ROS overproduction was observed in the presence of microcin J25. Nevertheless, the oxygen consumption was less inhibited in this strain, probably because the oxygen is partially reduced to superoxide. There was no overproduction of ROS in membranes isolated from the mutant strain that only express cytochrome bo3 and the inhibition of the cellular respiration was similar to the wild type. It is concluded that both cytochromes bd-I and bo3 are affected by the peptide. The results establish for the first time a relationship between the terminal oxygen reductases and the mechanism of action of microcin J25.

Effects of protective agents on membrane fluidity of freeze-dried Lactobacillus delbrueckii ssp. bulgaricus.

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 degrees 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.

Inhibition of enterocin CRL35 antibiotic activity by mono- and divalent ions.

AIM: The aim of this work was to study the influence of different cations on the enterocin CRL35 activity. METHODS AND RESULTS: The antilisterial activity of enterocin CRL35 was tested by performing viability curves and measuring the dissipation of the proton motive force by fluorescent methods upon the addition of Ca2+, Mg2+, Li+, K+ and Na+ chlorides. The peptide uptake by sensitive cells was studied in the different conditions as well. The addition of calcium and magnesium chlorides (0.5-2 mmol l(-1)) induced an inhibition of the peptide activity. Potassium, sodium and lithium chlorides have an inhibitory effect as well, but at different range of concentration compared with divalent cations (50-150 mmol l(-1)). Interestingly, we found a differential protection effect among monovalent ions, KCl being almost nonprotective, meanwhile LiCl shows the stronger effect and NaCl has an intermediate effect. The ion effect depends on the pH, being more efficient in acidic media. Both mono and divalent ions inhibited the ability of the peptide to dissipate the transmembrane electric potential and pH gradient. Furthermore, the peptide uptake was also inhibited. CONCLUSIONS: The enterocin CRL35 activity is strongly dependent on the pH and the nature of the salts present in the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings will allow definition of the best system in which this peptide can be applied as biopreservative.

Effect of enterocin CRL35 on Listeria monocytogenes cell membrane.

The antimicrobial peptide Enterocin CRL35, a class II bacteriocin, produces at high concentrations (8 microg ml(-1)) localized holes in the wall and cellular membrane of Listeria monocytogenes, reflected in the efflux of macromolecules such as proteins and other ultraviolet-absorbing materials. At lower concentrations (0.5 microg ml(-1)), neither ultra structural changes nor macromolecules efflux were observed, however potassium and phosphate ions were released, dissipating the proton motive force. As a result the bacteria were killed.