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Research describes a methodology to design vectors for targeted heterologous gene expression and exporting proteins to specific sites in Lactococcus lactis cells. Results show that staphylococcal nuclease is a suitable vector reporter protein by virtue of its biochemical properties and easy detection by activity tests on solid media.

The Streptococcus pyogenes M6 cell envelope-associated proteins have C-terminal sorting signals that mediate the cell wall anchoring of heterologous proteins. The membrane-bound M6 protein is encoded by the emm6 gene in lactic acid bacteria (LAB) other than Streptococcus pyogenes such as Escherichia coli and Lactobacillus sp. M6 preprotein signals also specified the export and anchoring of M6 to the cell wall of noncolonizing strains and digestive tract colonizing strains of LAB.

The production of plantaricins S and T by Lactobacillus plantarum LPCO10 isolated from a green olive fermentation was described. L. plantarum LPCO10 produced plantaricin S during log phase and also plantaricin T with a different activity spectrum in late stationary phase. Plantaricin S exhibited a bactericidal mode of action. Both bacteriocins are only active against lactic acid bacteria. In sodium dodecyl sulfate-polyacrylamide gels, plantaricin S migrated as a 2.5-kDa peptide and plantaricin T as a lower-molecular-weight peptide.

A sequential purification of lacticin 481, a bacteriocin produced by Lactococcus lactis subsp. lactis CNRZ 481, was described. The methodology involved using ammonnium sulfate precipitation, gel filtration and reversed-phase high-pressure liquid chromatography. Amino acid composition and sequence analysis showed that the bacteriocin contained a majority of nonpolar amino acids and revealed the presence of lanthionine residues.

j.g. leblanc, m.s. garro, a. silvestroni, c. connes, j.-c. piard, f. sesma and g. savoy de giori. 2004.Consumption of soya-derived products has been hampered by the presence ofα-galactooligosaccharides (α-GOS) because mammals lack pancreaticα-galactosidase (α-Gal) which is necessary for their hydrolysis. These sugars thus reach the large intestine causing gastrointestinal disorders in sensitive individuals. The use of lactic acid bacteria (LAB) expressingα-Gal is a promising solution for the degradation ofα-GOS in soyamilk.The capacity of the LABLactobacillus fermentumCRL 722 to properly degradeα-GOS was studiedin vitrousing controlled fermentation conditions andin vivousing a rat model.Lactobacillus fermentumCRL 722 was able to grow on commercial soyamilk and completely eliminated stachyose and raffinose during fermentation because of its highα-Gal activity. Rats fed soyamilk fermented by this LAB had smaller caecums compared with rats fed unfermented soyamilk.Soyamilk fermentation byLact. fermentumCRL 722 results in the reduction ofα-GOS concentrations in soyamilk, thus eliminating possible undesirable physiological effects normally associated with its consumption.Fermentation withLact. fermentumCRL 722 could prevent gastrointestinal disorders in sensitive individuals normally associated with the consumption of soya-based products. This LAB could thus be used in the elaboration of novel fermented vegetable products which better suit the digestive capacities of consumers. [ABSTRACT FROM AUTHOR]

The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis ssp. lactis. This polypeptide contains 27 amino acids, including the unusual residues dehydrobutyrine and the thioether-bridging lanthionine and 3-methyllanthionine. Lacticin 481 belongs to a structurally distinct group of lantibiotics, which also include streptococcin A-FF22, salivaricin A and variacin. Here we report the first complete structure of this type of lantibiotic. The exact location of the thioether bridges in lacticin 481 was determined by a combination of peptide chemistry, mass spectrometry and NMR spectroscopy, showing connections between residues 9 and 14, 11 and 25, and 18 and 26.

The lacticin 481-producer (Lct+), L. lactis subsp. lactis (L. lactis) CNRZ 481 harbours 5 plasmids of 6.5, 7.5, 20, 37 and 69 kb. Novobiocin treatment of L. lactis 481 led to the appearance of lacticin 481 deficient variants which had all lost the 69 kb plasmid. Conjugal transfer of the lacticin 481 structural gene (lct) into the plasmid free strain L. lactis IL1441 yielded Lct+ transconjugants at a 10(-4) frequency, which carried a plasmid with an apparent size of 120-130 kb. Southern hybridization analyses showed that the lct gene was located on the 69 kb plasmid in L. lactis 481 and on the 120-130 kb plasmid in the transconjugants. The lct gene was in higher copy number in transconjugants than in the parental strain resulting in two-fold higher lacticin 481 production in the former strain.

The structural gene for the lactococcal lantibiotic lacticin 481 (lct) has been identified and cloned using a degenerated 20-mer DNA oligonucleotide based on the amino-terminal 7 amino acid residues of the purified protein. The transcription of the lct gene was analyzed, and its promoter was mapped. DNA sequence analysis of the lct gene revealed an open reading frame encoding a peptide of 51 amino acids. Comparison of its deduced amino acid sequence with the amino-terminal sequence and the amino acid composition of lacticin 481 indicates that the 51-residue peptide is prelacticin 481, containing a 27-residue carboxyl-terminal propeptide and a 24-residue amino-terminal leader peptide which lacks the properties of a typical signal sequence and which is significantly different from the leaders of other lantibiotics. The predicted amino acid sequence of prolacticin 481 contains 3 cysteines, 2 serines, and 2 threonines which were not detectable in amino acid analyses of mature lacticin 481. Based on these results and on characterization by two-dimensional NMR techniques, a structural model is proposed in which 2 cysteine residues are involved in lanthionine and one in beta-methyllanthionine formation, and a 4th threonine residue is dehydrated. This model predicts a molecular mass for lacticin 481 of 2,901, which is in excellent agreement with that obtained from mass spectrometry.

Plasmid profiles of 35 Lactobacillus plantarum strains isolated from different green olive fermentors were obtained. A large number of plasmids in the CCC form (from 5 to 16) were present in all the tested strains as confirmed by a second dimension electrophoresis of DNA. These plasmids, all of which remain cryptic, ranged from 2.0 to 68 kb in size. Novobiocin, sodium dodecyl sulphate and ethidium bromide were used as plasmid-curing agents but only novobiocin induced loss of extrachromosomal DNA at a high frequency in these strains.