Two 2[5H]-furanones as possible signaling molecules in Lactobacillus helveticus.

Two 2[5H]-furanones, in association with medium-chain fatty acids, were released in whey by Lactobacillus helveticus exposed to oxidative and heat stresses. This species plays an important role in cheese technology, particularly for Swiss-type cheeses and Grana cheese. Moreover, it significantly contributes to cheese ripening by means of an early autolysis and the release of enzymes during processing. Experimental evidence of the involvement of the two 2[5H]-furanones, detected by a gas chromatography-mass spectrometry/solid-phase microextraction technique, in the autolysis phenomenon has been obtained. Zymograms performed by using renaturing sodium dodecyl sulfate-polyacrylamide gels were used to detect the bioactivity of the supernatants containing the two furanones on fresh cells of the same strain. In addition to bands corresponding to known autolysins, new autolysins were detected concomitant with the exposure of Lactobacillus helveticus to the supernatants, which can be regarded as conditioned media (CM), and to a commercial furanone, 5-ethyl-3-hydroxy-4-methyl-2[5H]-furanone (HEMFi), having spectral data similar to those of the newly described 2[5H]-furanones. Morphological changes were observed when fresh cells were exposed to CM containing the two 2[5H]-furanones and HEMFi. The two furanones produced by Lactobacillus helveticus, which met a number of criteria to be included in cell-cell signaling molecules, have a presumptive molecular mass lower than those of already known 3[2H]-furanones having an autolytic activity and being produced by gram-negative bacteria. Moreover, they present a different chemical structure with respect to the furanones already identified as products of Lactococcus lactis subsp. cremoris or to those identified in some cheeses with Lactobacillus helveticus as a starter culture.

Alteration in cellular fatty acid composition as a response to salt, acid, oxidative and thermal stresses in Lactobacillus helveticus.

The fundamental question in this study is concerned with whether the increase of unsaturated fatty acids in the cell membrane is a general response of certain thermotolerant strains or species when exposed to superoptimal temperatures, and in combination with other stresses, especially oxidative stress. A strain of Lactobacillus helveticus, a species widely used as a starter in the dairy industry and able to tolerate high temperature and NaCl concentrations as well as acidic conditions, was chosen for this study. Cells of strain CNBL 1156, grown in its natural medium (i.e. milk whey), were exposed for 100 min to sublethal combinations of temperature, NaCl, H(2)O(2) and pH, modulated according to a Central Composite Design. The fatty acid composition of cell lipid extract was identified by GC/MS. Polynomial equations, able to describe the individual interactive and quadratic effects of the independent variables on cell fatty acid composition, were obtained. The results and the mathematical models relative to the individual fatty acids indirectly suggest that desaturase activation or hyperinduction play an important role in the response to heat stress. In fact, the relative proportions of oleic, linoleic and palmitic acids increased with temperature in a range between 38 and 54 degrees C. The fatty acid profiles included vernolic acid (up to 37% of total fatty acids), an epoxide of linoleic acid not previously reported in microbial cells. In particular, this epoxide was present in cells exposed to low pH in combination with high temperatures and oxidative stress. In conclusion, these results provide experimental support to the hypothesis that the increase of an oxygen-consuming desaturase system, with a consequent increase in fatty acid desaturation, is a cellular response to environmental stresses able to protect the cells of this anaerobic micro-organism from toxic oxygen species and high temperatures.

The S-layer gene of Lactobacillus helveticus CNRZ 892: cloning, sequence and heterologous expression.

Lactobacillus helveticus CNRZ 892 contains a surface layer (S-layer) composed of protein monomers of 43 kDa organized in regular arrays. The gene encoding this protein (slpH) has been cloned in Escherichia coli and sequenced. slpH consists of 440 codons and is preceded by a ribosome-binding site (RBS) and followed by a putative rho-independent terminator. Indeed, Northern analysis revealed that slpH is a monocistronic gene. The gene is preceded by a possible promotor of which the -35 and -10 hexanucleotides are separated by 17 nt. By primer extension analysis the transcription start site was mapped at 7 nt downstream of the -10 sequence while the deduced amino acid sequence of SlpH shows a leader peptide of 30 aa. The slpH gene has been amplified by PCR and the fragment, carrying the complete gene from the RBS to the stop codon, has been cloned in a lactococcal gene expression vector downstream of promoter P32. Lactococcus lactis MG1363 carrying the resulting plasmid produced and secreted an S-layer monomer with the same molecular mass as the authentic L. helveticus CNRZ 892 SlpH, as judged by SDS-PAGE. Immunoelectron microscopy revealed that SlpH was bound to the lactococcal cell walls in small clumps and accumulated in the growth medium as small sheets.