The small heat shock proteins (sHSP) are characterized by a chaperone activity to prevent irreversible protein denaturation. This study deals with the sHSP Lo18 induced by multiple stresses in Oenococcus oeni, a lactic acid bacterium. Using in situ immunocytochemistry and cellular fractionation experiments, we demonstrated the association of Lo18 with the membrane in O. oeni cells submitted to heat shock. The same result was obtained after exposure of cells to ethanol or benzyl alcohol, agents known to have an influence on membranes. For the different stresses, the protein was located on the periphery of the cell at membrane level and was also found within the cytoplasm. In order to determine if Lo18 could interact with the phospholipids, we used model membranes made of lipids extracted from O. oeni cells. Using fluorescence anisotropy of diphenylhexatriene (DPH) and generalized polarization of Laurdan, we showed that purified Lo18 interacts with these liposomes, and increases the molecular order of the lipid bilayer in these membranes when the temperature reaches 33.8 degrees C. All these data suggest that Lo18 could be involved in an adaptive response allowing the maintenance of membrane integrity during stress conditions in O. oeni cells.

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Using promoter exchange and gene knock-out strategies, two mutant strains, the so-called BBG116 and BBG125, were constructed from Bacillus subtilis wild-type strain ATCC 6633, a surfactin and mycosubtilin producer. Compared to the parental strain, both mutants overproduced constitutively mycosubtilin, while BBG125 had lost the ability to synthesize surfactin. Surprisingly, BBG125 was found to produce about 2-fold less mycosubtilin than BBG116 despite an expected higher availability of the cytoplasmic precursors and cofactors pool for biosynthesis. Further physiological characterization of BBG125 also highlighted: (i) a strong influence of temperature on mycosubtilin biosynthesis in BBG125 with a maximal productivity observed at 22°C, compared to 15 and 30°C; (ii) substantial changes in fatty acid profiles and thereby in mycosubtilin isoforms, compared to the wild-type strain; and (iii) the presence of five novel mycosubtilin isoforms. The antifungal activities of the new mix were higher than or equal to those of purified isoforms.