21Sixty bacterial strains were encountered by random amplification of polymorphic DNA 22 (RAPD) and repetitive extragenic palindromic (REP) typing in a series of 306 Lactococcus 23 lactis isolates collected during the manufacturing and ripening stages of five traditional, 24 starter-free cheeses made from raw milk. Among the 60 strains, 17 were shown to produce 25 bacteriocin-like compounds in both solid and liquid media. At a genotypic level, 16 of the 26 strains were identified by molecular methods as belonging to L. lactis subsp. lactis and one 27 to L. lactis subsp. cremoris. Among the L. lactis subsp. lactis strains, phenotypic and 28 genetic data determined that eleven produced either nisin A (nine strains) or nisin Z (two 29 strains), and that five produced lactococcin 972. Variable levels of the two bacteriocins 30 were produced by the different strains. In addition, nisin was shown to be produced in 31 inexpensive, dairy-and meat-based media, which will allow the practical application of its 32 producing strains in industrial processes. Specific PCR and nucleotide and deduced amino 33 acid sequence analysis identified as a lactococcin G-like bacteriocin the inhibitor produced 34 by the single L. lactis subsp. cremoris isolate. Beyond the use of bacteriocins as functional 35 ingredients for the biopreservation of foods, the newly identified bacteriocin-producing L. 36 lactis strains from traditional cheeses may also be useful for designing starter cultures with 37 protective properties and/or adjunct cultures for accelerating cheese ripening. 38 39

ABSTRACTLactococcin 972 (Lcn972) is a nonlantibiotic bacteriocin that inhibits cell wall biosynthesis by binding to lipid II. In this work, two mutants resistant to Lcn972,Lactococcus lactisD1 and D1-20, with high (>320 arbitrary units [AU]/ml) and low (80 AU/ml) susceptibilities, respectively, have been isolated. Resistance to Lcn972 did not impose a burden to growth under laboratory conditions, nor did it substantially alter the physicochemical properties of the cell surface. However, the peptidoglycan of the mutants featured a higher content of muropeptides with tripeptide side chains than the wild-type strain, linking for the first time peptidoglycan remodelling to bacteriocin resistance. Moreover,L. lactislacking a functionald,d-carboxypeptidase DacA (i.e., with a high content of pentapeptide side chain muropeptides) was shown to be more susceptible to Lcn972. Cross-resistance to lysozyme and nisin and enhanced susceptibility to penicillin G and bacitracin was also observed. Intriguingly, the Lcn972-resistant mutants were not infected by the lytic phage c2 and less efficiently infected by phage sk1. Lack of c2 infectivity was linked to a 22.6-kbp chromosomal deletion encompassing the phage receptor protein genepip. The deletion also included maltose metabolic genes and the two-component system (TCS) F. However, a clear correlation between these genes and resistance to Lcn972 could not be clearly established, pointing to the presence of as-yet-unidentified mutations that account for Lcn972 resistance.

The non-starter microbiota of Cheddar cheese mostly comprises mesophilic lactobacilli, such as Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, and Lactobacillus plantarum. These bacteria are recognized for their potential to improve Cheddar cheese flavor when used as adjunct cultures. In this study, three strains of L. paracasei (DPC2071, DPC4206, and DPC4536) were evaluated for their contribution to the enhancement and diversification of flavor in short-aged Cheddar cheese. The strains were selected based on their previously determined genomic diversity, variability in proteolytic enzyme activities and metabolic capability in cheese model systems. The addition of adjunct cultures did not affect the gross composition or levels of lipolysis of the cheeses. The levels of free amino acids (FAA) in cheeses showed a significant increase after 28 days of ripening. However, the concentrations of individual amino acids in the cheeses did not significantly differ except for some amino acids (aspartic acid, threonine, serine, and tryptophan) at Day 14. Volatile profile analysis revealed that the main compounds that differentiated the cheeses were of lipid origin, such as long chain aldehydes, acids, ketones, and lactones. This study demonstrated that the adjunct L. paracasei strains contributed to the development and diversification of compounds related to flavor in short-aged Cheddar cheeses.

pBL1 is a Lactococcus lactis theta-replicating 10.9-kbp plasmid that encodes the synthetic machinery of the bacteriocin Lcn972. In this work, the transcriptomes of exponentially growing L. lactis strains with and without pBL1 were compared. A discrete response was observed, with a total of 10 genes showing significantly changed expression. Upregulation of the lactococcal oligopeptide uptake (opp) system was observed, which was likely linked to a higher nitrogen demand required for Lcn972 biosynthesis. Strikingly, celB, coding for the membrane porter IIC of the cellobiose phosphoenolpyruvate-dependent phosphotransferase system (PTS), and the upstream gene llmg0186 were downregulated. Growth profiles for L. lactis strains MG1363, MG1363/pBL1, and MG1363 ⌬celB grown in chemically defined medium (CDM) containing cellobiose confirmed slower growth of MG1363/pBL1 and MG1363 ⌬celB, while no differences were observed with growth on glucose. The presence of pBL1 shifted the fermentation products toward a mixed acid profile and promoted substantial changes in intracellular pool sizes for glycolytic intermediates in cells growing on cellobiose as determined by highpressure liquid chromatography (HPLC) and nuclear magnetic resonance (NMR). Overall, these data support the genetic evidence of a constriction in cellobiose uptake. Notably, several cell wall precursors accumulated, while other UDP-activated sugar pools were lower, which could reflect rerouting of precursors toward the production of structural or storage polysaccharides. Moreover, cells growing slowly on cellobiose and those lacking celB were more tolerant to Lcn972 than cellobiose-adapted cells. Thus, downregulation of celB could help to build up a response against the antimicrobial activity of Lcn972, enhancing self-immunity of the producer cells.