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.

The temperate bacteriophage TP712 was unable to plaque on Lactococcus lactis DftsH lacking the membrane protease FtsH and complementation in trans restored the WT phenotype. Absence of ftsH did not hinder phage adsorption, phage DNA delivery or activation of the lytic cycle. Thin sections revealed that TP712 virions appeared to be correctly assembled inside the DftsH host, but were not released. These virions were infective, demonstrating that a functional host FtsH is required by TP712 to proceed effectively with lysis of the host.Large-scale dairy fermentations can be seriously compromised by bacteriophages infecting lactic acid bacteria (LAB), and particularly Lactococcus lactis, which is a commonly used starter culture for cheese making (Garneau & Moineau, 2011). Lactococcal phages are currently classified in 10 groups, eight of them belonging to the Siphoviridae family of the order Caudovirales. Those of the c2, 936 and P335 groups are the most commonly found in dairy plants (Deveau et al., 2006).A considerable effort has been made to improve and select phage-resistant starter strains based on the knowledge of resistance mechanisms developed by LAB and a better understanding of phage-host interactions (Sturino & Klaenhammer, 2006). Described mechanisms of phage resistance expand from blocking adsorption, restriction/ modification enzymes, to abortive infection systems that interfere with phage DNA replication, morphogenesis and release. Clustered regularly interspaced short palindromic repeats (CRISPRs) located in the genomes of host bacteria together with a group of associated proteins can also confer resistance to phages (Labrie et al., 2010). Recently, the response to phage infection in L. lactis has been approached by genome-wide transcriptomics (Fallico et al., 2011;Ainsworth et al., 2013). L. lactis appears to sense bacteriophage infection as a perturbation of its cell envelope and mounts a response targeted to the cell wall, activating regulators of the cell envelope stress response such as the two-component system CesSR.One of the members of the CesSR regulon in L. lactis is the ftsH gene (Martínez et al., 2007). ftsH encodes a conserved AAA (ATPase associated with various cellular activities)-type membrane protease involved in stress response and protein quality control (Ito & Akiyama, 2005;Narberhaus et al., 2009). In this work, we analysed the impact of the ftsH null mutation on the life cycle of the P335 temperate phage TP712, which is able to infect and lysogenize L. lactis MG1363 (Gasson, 1983) and derivatives thereof. The complete TP712 nt sequence has been determined (GenBank accession number AY766464) essentially as described by Wegmann et al. (2012).Preliminary experiments revealed that in standard plaque assays (Lillehaug, 1997), the efficiency of plaquing (EOP) of this phage on an available non-polar and in-frame L. lactis mutant lacking ftsH (Pinto et al., 2011) was reduced to 1.5610 25 when compared to its parent L. lactis NZ9000. Upon complementation with the plasmid pUK200 : : ftsH, ...

BackgroundLactococcus lactis is widely used as a dairy starter and has been extensively studied. Based on the acquired knowledge on its physiology and metabolism, new applications have been envisaged and there is an increasing interest of using L. lactis as a cell factory. Plasmids constitute the main toolbox for L. lactis genetic engineering and most rely on antibiotic resistant markers for plasmid selection and maintenance. In this work, we have assessed the ability of the bacteriocin Lactococcin 972 (Lcn972) gene cluster to behave as a food-grade post-segregational killing system to stabilize recombinant plasmids in L. lactis in the absence of antibiotics. Lcn972 is a non-lantibiotic bacteriocin encoded by the 11-kbp plasmid pBL1 with a potent antimicrobial activity against Lactococcus.ResultsAttempts to clone the full lcn972 operon with its own promoter (P972), the structural gene lcn972 and the immunity genes orf2-orf3 in the unstable plasmid pIL252 failed and only plasmids with a mutated promoter were recovered. Alternatively, cloning under other constitutive promoters was approached and achieved, but bacteriocin production levels were lower than those provided by pBL1. Segregational stability studies revealed that the recombinant plasmids that yielded high bacteriocin titers were maintained for at least 200 generations without antibiotic selection. In the case of expression vectors such as pTRL1, the Lcn972 gene cluster also contributed to plasmid maintenance without compromising the production of the fluorescent mCherry protein. Furthermore, unstable Lcn972 recombinant plasmids became integrated into the chromosome through the activity of insertion sequences, supporting the notion that Lcn972 does apply a strong selective pressure against susceptible cells. Despite of it, the Lcn972 gene cluster was not enough to avoid the use of antibiotics to select plasmid-bearing cells right after transformation.ConclusionsInserting the Lcn972 cluster into segregational unstable plasmids prevents their lost by segregation and probable could be applied as an alternative to the use of antibiotics to support safer and more sustainable biotechnological applications of genetically engineered L. lactis.

bLactococcin 972 (Lcn972) is a cell wall-active bacteriocin that inhibits cell wall biosynthesis in Lactococcus lactis. In this work, the transcriptomes of the Lcn972-resistant (Lcn r ) mutant L. lactis D1 and its parent strain were compared to identify factors involved in Lcn972 resistance. Upregulated genes included members of the cell envelope stress (CesSR) regulon, the penicillinbinding protein pbpX gene and gene llmg2447, which may encode a putative extracytoplasmic function (ECF) anti-sigma factor. The gene llmg2447 is located downstream of the nonfunctional ECF gene sigX pseudo . Nisin-controlled expression of llmg2447 led to high Lcn972 resistance in L. lactis, with no cross-resistance to other cell wall-active antimicrobials. Upregulation of llmg2447 in L. lactis D1 (Lcn r ) was linked to the integration of insertion element IS981 into the llmg2447 promoter region, replacing the native ؊35 box and activating the otherwise silent promoter P 2447 . This is the first example of an orphan ECF anti-sigma factor involved in bacteriocin resistance. This new role in neutralizing cell wall-active compounds (e.g., Lcn972) could have evolved from a putative primary function of Llmg2447 in sensing cell envelope stress.

Lactococcus lactis is one of the main components of the starter cultures used in cheese manufacture. As starter, L. lactis must tolerate harsh conditions encountered either during their production in bulk quantities or during dairy products processing. To face these hostile conditions, bacteria monitor the environment and respond by modifying gene expression appropriately. Previous transcriptomic studies showed that the two component system CesSR is the main pathway that triggers the cell envelope stress response in L. lactis treated with lactococcin 972 (Lcn972), a cell wall synthesis inhibiting bacteriocin. Among the CesR-regulated genes, llmg0169 and the operon llmg2164-2163, encoding proteins of unknown function, are among the highest up-regulated genes after activation of CesSR. In this study, we have assessed the contribution of these genes to the survival of L. lactis to different technologically-relevant stresses. Overexpressing and knock-out mutants of the genes were generated and their viability to low pH, heat, freeze-drying, presence of NaCl, cell wall antimicrobials and lytic phages attack was compared to the wild type strain. The genes llmg0169 and llmg2164-2163 contributed differently to L. lactis fitness. L. lactis Deltallmg0169 was very sensitive to heat treatment while L. lactis Deltallmg2164 was more sensitive to NaCl. Absence of both genes also compromised viability at low pH. On the contrary, higher expression levels of llmg0169 and llmg2164-2163, up to 26- and 14-fold increase determined by qRT-PCR, respectively, did not enhance L. lactis survival in any of the above stressful conditions (heat, pH and NaCl) or after freeze-drying. All the mutants displayed a similar phage susceptibility profile. Overexpression of llmg2164-2163 seemed to specifically protect L. lactis against the bacteriocin Lcn972 but not against other cell wall active antimicrobials. Based on our phenotypic analysis, the investigated genes are required to mount a proper response to guarantee survival of L. lactis under technologically-relevant stresses and their functionality could be a useful marker to select robust dairy starters.