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We designed an expression and export system that enabled the targeting of a reporter protein (the staphylococcal nuclease Nuc) to specific locations in Lactococcus lactis cells, i.e., cytoplasm, cell wall, or medium. Optimization of protein secretion and of protein cell wall anchoring was performed with L. lactis cells by modifying the signals located at the N and C termini, respectively, of the reporter protein. Efficient translocation of precursor (ϳ95%) is obtained using the signal peptide from the lactococcal Usp45 protein and provided that the mature protein is fused to overall anionic amino acids at its N terminus; those residues prevented interactions of Nuc with the cell envelope. Nuc could be covalently anchored to the peptidoglycan by using the cell wall anchor motif of the Streptococcus pyogenes M6 protein. However, the anchoring step proved to not be totally efficient in L. lactis, as considerable amounts of protein remained membrane associated. Our results may suggest that the defect is due to limiting sortase in the cell. The optimized expression and export vectors also allowed secretion and cell wall anchoring of Nuc in food-fermenting and commensal strains of Lactobacillus. In all strains tested, both secreted and cell wall-anchored Nuc was enzymatically active, suggesting proper enzyme folding in the different locations. These results provide the first report of a targeting system in lactic acid bacteria in which the final location of a protein is controlled and biological activity is maintained.

Summary -Lactic acid bacteria can produce a variety of substances with antibacterial activity which are described in this article. Non-peptide antibacterial substances are distinguished from bacteriocins, which have a proteinaceous active site. Among the former, reuterin produced by Lactobacillus reuteri is a broad spectrum inhibitor active against Gram-positive and Gram-negative bacteria, yeasts, molds and protozoas. It is a glycerol derivative, l3-hydroxypropionaldehyde.Bacteriocins can be produced by most lactic acid bacteria species and their spectrum of activity is generally restricted to organisms taxonomically close to the producer. The biochemical properties of bacteriocins, their structure and nature of their genetic determinants are highly variable. This literature review discusses the similarities and differences existing in this group of substances.

Lactic acid bacteria (LAB) isolated from fish products (fresh fish, smoked and marinated fish, fish intestinal tract) were screened for bacteriocin production and immunity in conditions eliminating the effects of organic acids and hydrogen peroxide. Twenty-two isolates which were found to produce bacteriocin-like compounds were identified as Carnobacteria, Lactococci and Enterococci on the basis of morphological examination, gas production from glucose, growth temperatures, configuration of lactic acid, carbohydrates fermentation and deamination of arginine. Two Carnobacteria named V1 and V41 were selected for further studies and identified by DNA-DNA hybridization as Carnobacterium piscicola and Carnobacterium divergens, respectively. Their respective bacteriocins named piscicocin V1 and divercin V41 were heat-resistant and sensitive to various proteolytic enzymes. These bacteriocins were active against Listeria monocytogenes and exhibited a different spectrum of activity against LAB. Both bacteriocins had a bactericidal and non-bacteriolytic mode of action. Maximum production of piscicocin V1 and divercin V41 in Man Rogosa Sharpe (MRS) medium broth occurred at the beginning of the stationary phase and was higher at 20°C than at 30°C. When the cultures were maintained at pH 6.5, bacteriocin production was significantly increased.

Divercin V41 is a new bacteriocin produced by Carnobacterium divergens V41, a lactic acid bacterium isolated from fish viscera. The amino acid sequence of divercin V41 showed high homologies with pediocin PA-1 and enterocin A. Two disulphide bonds were present in the hydrophilic N-terminal domain and in the highly variable hydrophobic C-terminal domain, respectively. A DNA probe designed from the N-terminal sequence of the purified peptide was used t o locate the structural gene of divercin V41. A 6 kb chromosomal fragment containing the divercin V41 structural gene (dvnA) was cloned and sequenced. The results indicate that divercin V41 is synthesized as a pre-bacteriocin of 66 amino acids. The 23-residue N-terminal extension is cleaved off t o yield the mature 43-amino-acid divercin V41. In addition, the fragment encodes putative proteins commonly found within bacteriocin operons, including an ATPdependent transporter, two immunity-like proteins and the two components of a lantibiotic-type signal-transducing system. The genetic organization of the fragment suggested important gene rearrangements.

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To cite this version:Jc Piard, M Desmazeaud. Inhibiting factors produced by lactic acid bacteria. 1. Oxygen metabolites and catabolism end-products. Le Lait, INRA Editions, 1991, 71 (5), pp.525-541. Lait (1991) Summary -Lactic acid bacteria can produce a variety of antimicrobial compounds which may affect both the lactic acid bacteria themselves as weil as undesirable or pathogenic strains. In this first section, we describe the biosynthesis, mode of action and activity spectra of these inhibitors. Metabolites of oxygen (hydrogen peroxide and free radicals) exhibit a bacteriostatic or bactericidal activity against lactic or non-Iactic flora. When associated with the lactoperoxidase/thiocyanate system, hydrogen peroxide leads to the formation of inhibitory compounds which are bacteriostatic for lactic acid bacteria and bactericidal for Gram-negative bacteria. The antimicrobial activity of organic acids (Iactic, acetic and formic) and of pH are closely linked. It appears that the non-dissociated fraction of these acids is the major inhibitory form. Thus, acetic acid whose pKa is higher than that of lactic acid exhibits the highest inhibitory activity. The antimicrobial activities of diacetyl, acetaldehyde and of the D isomers of amino acids are also described, although their effects are slight in usual lactic fermentations. 8acteriocins are the last type of inhibitory substance produced by some lactie acid bacteria. They will be described in a second section.lactic acid bacteria / oxygen 1 acidity 1 diacetyll acetaldehyde Résumé -Facteurs inhibiteurs produits par les bactéries lactiques. 1. Métabolites de l'oxygène et produits du catabolisme. Les bactéries lactiques sont capables de produire une variété de produits inhibiteurs dont les effets peuvent se répercuter sur la flore lactique el/e-même mais aussi sur les flores indésirables ou pathogènes. Dans cette première partie, nous décrivons les méca-nismes d'apparition, les modes d'action et les spectres d'activité de ces inhibiteurs. Les métabolites de l'oxygène (le peroxyde d'hydrogène et les radicaux libres) peuvent avoir des effets biologiques de

The genome of Lactococcus lactis strain IL1403 harbors a putative pilus biogenesis cluster consisting of a sortase C gene flanked by 3 LPxTG protein encoding genes (yhgD, yhgE, and yhhB), called here pil. However, pili were not detected under standard growth conditions. Over-expression of the pil operon resulted in production and display of pili on the surface of lactococci. Functional analysis of the pilus biogenesis machinery indicated that the pilus shaft is formed by oligomers of the YhgE pilin, that the pilus cap is formed by the YhgD pilin and that YhhB is the basal pilin allowing the tethering of the pilus fibers to the cell wall. Oligomerization of pilin subunits was catalyzed by sortase C while anchoring of pili to the cell wall was mediated by sortase A. Piliated L. lactis cells exhibited an auto-aggregation phenotype in liquid cultures, which was attributed to the polymerization of major pilin, YhgE. The piliated lactococci formed thicker, more aerial biofilms compared to those produced by non-piliated bacteria. This phenotype was attributed to oligomers of YhgE. This study provides the first dissection of the pilus biogenesis machinery in a non-pathogenic Gram-positive bacterium. Analysis of natural lactococci isolates from clinical and vegetal environments showed pili production under standard growth conditions. The identification of functional pili in lactococci suggests that the changes they promote in aggregation and biofilm formation may be important for the natural lifestyle as well as for applications in which these bacteria are used.