ABSTRACTMany strains have been used and selected by the food industry for their capacities to ferment, produce flavors, or produce heterologous molecules. Very little is known about the diversity of foodborne yeasts and their potential effect on gut microbiota and gut health. We initiated a complete characterization of five strains belonging to five species with a long history of safe use in food: Cyberlindnera jadinii, Debaryomyces hansenii, Kazachstania unispora, Kluyveromyces lactis, and Pichia membranifaciens, with a focus on their capacity to protect against gut inflammation using an in vivo dextran sodium sulfate-induced colitis model in mice. C. jadinii and K. lactis living cells showed a clear reduction in mouse sensitivity to colitis in vivo. Interestingly, we observed that C. jadinii had the capacity to survive transit in the gut, while K. lactis did not. We demonstrated that C. jadinii was unable to efficiently adhere to epithelial cells and did not survive more than 24 to 48 h in the gut. Transcriptomic analysis using NanoString technology suggested a potential role of IL-8 through Mif and Fkbp5 in the effect of C. jadinii on the immune system. Bacterial and fungal microbiota characterization showed a modification of both microbiota after C. jadinii treatment, with a significant increase in positive microorganisms and a decrease in pathobionts. Altogether, these data suggest that both C. jadinii and K. lactis strains have potential as probiotic yeast strains to fight against inflammation in the gut, but further studies are needed to understand the mechanisms by which these strains act on gut health.IMPORTANCEThe food industry has always used many strains of microorganisms including fungi in their production processes. These strains have been widely characterized for their biotechnological value, but we still know very little about their interaction capacities with the host at a time when the intestinal microbiota is at the center of many pathologies. In this study, we characterized five yeast strains from food production which allowed us to identify two new strains with high probiotic potential and beneficial effects in a model of intestinal inflammation.

Food processes use different microorganisms, from bacteria to fungi. Yeast strains have been extensively studied, especially Saccharomyces cerevisiae. However, to date, very little is known about the potential beneficial effects of molds on gut health as part of gut microbiota. We undertook a comprehensive characterization of five mold strains, Penicillium camemberti, P. nalgiovense, P. roqueforti, Fusarium domesticum, and Geotrichum candidum used in food processes, on their ability to trigger or protect intestinal inflammation using in vitro human cell models and in vivo susceptibility to sodium dextran sulfate-induced colitis. Comparison of spore adhesion to epithelial cells showed a very wide disparity in results, with F. domesticum and P. roqueforti being the two extremes, with almost no adhesion and 20% adhesion, respectively. Interaction with human immune cells showed mild pro-inflammatory properties of all Penicillium strains and no effect of the others. However, the potential anti-inflammatory abilities detected for G. candidum in vitro were not confirmed in vivo after oral gavage to mice before and during induced colitis. According to the different series of experiments carried out in this study, the impact of the spores of these molds used in food production is limited, with no specific beneficial or harmful effect on the gut.

ABSTRACT Lactococcus lactis is one of the most commonly used lactic acid bacteria in the dairy industry. Activation of competence for natural DNA transformation in this species would greatly improve the selection of novel strains with desired genetic traits. Here, we investigated the activation of natural transformation in L. lactis subsp. cremoris KW2, a strain of plant origin whose genome encodes the master competence regulator ComX and the complete set of proteins usually required for natural transformation. In the absence of knowledge about competence regulation in this species, we constitutively overproduced ComX in a reporter strain of late competence phase activation and showed, by transcriptomic analyses, a ComX-dependent induction of all key competence genes. We further demonstrated that natural DNA transformation is functional in this strain and requires the competence DNA uptake machinery. Since constitutive ComX overproduction is unstable, we alternatively expressed comX under the control of an endogenous xylose-inducible promoter. This regulated system was used to successfully inactivate the adaptor protein MecA and subunits of the Clp proteolytic complex, which were previously shown to be involved in ComX degradation in streptococci. In the presence of a small amount of ComX, the deletion of mecA, clpC, or clpP genes markedly increased the activation of the late competence phase and transformability. Altogether, our results report the functionality of natural DNA transformation in L. lactis and pave the way for the identification of signaling mechanisms that trigger the competence state in this species. IMPORTANCE Lactococcus lactis is a lactic acid bacterium of major importance, which is used as a starter species for milk fermentation, a host for heterologous protein production, and a delivery platform for therapeutic molecules. Here, we report the functionality of natural transformation in L. lactis subsp. cremoris KW2 by the overproduction of the master competence regulator ComX. The developed procedure enables a flexible approach to modify the chromosome with single point mutation, sequence insertion, or sequence replacement. These results represent an important step for the genetic engineering of L. lactis that will facilitate the design of strains optimized for industrial applications. This will also help to discover natural regulatory mechanisms controlling competence in the genus Lactococcus.

Streptococcus thermophilus is a lactic acid bacterium of major importance to the dairy industry as it is found in numerous cheeses and is one of the two bacterial species involved in the fermentation of yogurt. Bacterial two-component signal transduction systems (TCSs) play important roles in the process of bacterial environmental adaptation. S. thermophilus LMD-9 possesses eight such TCS systems; however, their functions have thus far been only poorly investigated. Here, we focused on two of the TCSs in LMD-9, TCS06 and TCS07, whose encoding genes are located close to each other on the chromosome, and are associated with those of ABC transporters. TCS06 homologs are frequently found in Lactobacillales, but their function has not yet been determined, while TCS07 and its upstream potential ABC transporter are homologous to the BceRS/AB system, which is involved in bacitracin resistance in Bacillus and Streptococcus species. To investigate the function(s) of TCS06 and TCS07, we constructed and characterized deletion mutants and performed transcriptional analysis in the presence and absence of bacitracin. We show here that both TCS06 and TCS07 regulate the genes in their close vicinity, in particular those encoding ABC transporters. We propose that the response of S. thermophilus to bacitracin includes i) a bacitracin export system, regulated by TCS07 and constituting a BceRS/AB-like detoxification module, and ii) the modification of cell-envelope properties via modulation of rhamnose–glucose polysaccharide synthesis, at least partially regulated by TCS06.

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CRISPR (clustered regularly interspaced short palindromic repeats) together with cas (CRISPR-associated) genes form the CRISPR–Cas immune system, which provides sequence-specific adaptive immunity against foreign genetic elements in bacteria and archaea. Immunity is acquired by the integration of short stretches of invasive DNA as novel ‘spacers’ into CRISPR loci. Subsequently, these immune markers are transcribed and generate small non-coding interfering RNAs that specifically guide nucleases for sequence-specific cleavage of complementary sequences. Among the four CRISPR–Cas systems present in Streptococcus thermophilus, CRISPR1 and CRISPR3 have the ability to readily acquire new spacers following bacteriophage or plasmid exposure. In order to investigate the impact of building CRISPR-encoded immunity on the host chromosome, we determined the genome sequence of a BIM (bacteriophage-insensitive mutant) derived from the DGCC7710 model organism, after four consecutive rounds of bacteriophage challenge. As expected, active CRISPR loci evolved via polarized addition of several novel spacers following exposure to bacteriophages. Although analysis of the draft genome sequence revealed a variety of SNPs (single nucleotide polymorphisms) and INDELs (insertions/deletions), most of the in silico differences were not validated by Sanger re-sequencing. In addition, two SNPs and two small INDELs were identified and tracked in the intermediate variants. Overall, building CRISPR-encoded immunity does not significantly affect the genome, which allows the maintenance of important functional properties in isogenic CRISPR mutants. This is critical for the development and formulation of sustainable and robust next-generation starter cultures with increased industrial lifespans.

We evaluated menaquinone contents in a large set of 62 fermented dairy products samples by using a new liquid chromatography method for accurate quantification of lipo-soluble vitamin K(2), including distribution of individual menaquinones. The method used a simple and rapid purification step to remove matrix components in various fermented dairy products 3 times faster than a reference preparation step. Moreover, the chromatography elution time was significantly shortened and resolution and efficiency were optimized. We observed wide diversity of vitamin K(2) contents in the set of fermented dairy products, from undetectable to 1,100 ng/g of product, and a remarkable diversity of menaquinone forms among products. These observations relate to the main microorganism species currently in the different fermented product technologies. The major form in this large set of fermented dairy products was menaquinone (MK)-9, and contents of MK-9 and MK-8 forms were correlated, that of MK-9 being around 4 times that of MK-8, suggesting that microorganisms able to produce MK-9 also produce MK-8. This was not the case for the other menaquinones, which were produced independently of each other. Finally, no obvious link was established between MK-9 content and fat content or pH of the fermented dairy products.

Vitamin K exists in the food supply as phylloquinone, a plant-based form and as menaquinones (MKs), a collection of isoprenologues mostly originating from bacterial synthesis. Although multiple bacterial species used as starter cultures for food fermentations synthesize MK, relatively little is known about the presence and distribution of MK in the food supply and the relative contribution of MK to total dietary vitamin K intake. Dairy products may be a predominant source of dietary MK in many regions of the world, and there is recent interest in enhancing the MK content of dairy products through identification and selection of MK-producing bacteria in dairy fermentations. This interest is increased by emerging evidence that current dietary recommendations based on the classic role of vitamin K as an enzyme cofactor for coagulation proteins may not be optimal for supporting vitamin K requirements in extrahepatic tissues and that MK may have unique bioactivity beyond that as an enzyme cofactor. Observational studies have reported favorable associations between MK intake and bone and cardiovascular health. Although randomized trials have provided some evidence to support the beneficial effects of MK on bone, the evidence to date is not definitive, and randomized trials have not yet examined MK intake in relation to cardiovascular outcomes. Food production practices provide a means to enhance dietary MK availability and intake. However, parallel research is needed to optimize these production practices, develop comprehensive food MK content databases, and test hypotheses of unique beneficial physiological roles of MK beyond that achieved by phylloquinone.

Lactococcus lactis is a biotechnological workhorse for food fermentations and potentially therapeutic products and is therefore widely consumed by humans. It is predominantly used as a starter microbe for fermented dairy products, and specialized strains have adapted from a plant environment through reductive evolution and horizontal gene transfer as evidenced by the association of adventitious traits with mobile elements. Specifically, L. lactis has armed itself with a myriad of plasmid-encoded bacteriophage defensive systems to protect against viral predation. This known arsenal had not included CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins), which forms a remarkable microbial immunity system against invading DNA. Although CRISPR/Cas systems are common in the genomes of closely related lactic acid bacteria (LAB), none was identified within the eight published lactococcal genomes. Furthermore, a PCR-based search of the common LAB CRISPR/Cas systems (Types I and II) in 383 industrial L. lactis strains proved unsuccessful. Here we describe a novel, Type III, self-transmissible, plasmid-encoded, phage-interfering CRISPR/Cas discovered in L. lactis. The native CRISPR spacers confer resistance based on sequence identity to corresponding lactococcal phage. The interference is directed at phages problematic to the dairy industry, indicative of a responsive system. Moreover, targeting could be modified by engineering the spacer content. The 62.8-kb plasmid was shown to be conjugally transferrable to various strains. Its mobility should facilitate dissemination within microbial communities and provide a readily applicable system to naturally introduce CRISPR/Cas to industrially relevant strains for enhanced phage resistance and prevention against acquisition of undesirable genes.

Background In industrial fermentation processes, the rate of milk acidification by Streptococcus thermophilus is of major technological importance. The cell-envelope proteinase PrtS was previously shown to be a key determinant of the milk acidification activity in this species. The PrtS enzyme is tightly anchored to the cell wall via a mechanism involving the typical sortase A (SrtA) and initiates the breakdown of milk casein into small oligopeptides. The presence or absence of PrtS divides the S. thermophilus strains into two phenotypic groups i.e. the slow and the fast acidifying strains. The aim of this study was to improve the milk acidification rate of slow S. thermophilus strains, and hence optimise the fermentation process of dairy products. Results In the present work, we developed for the first time a strategy based on natural transformation to confer the rapid acidification phenotype to slow acidifying starter strains of S. thermophilus . First, we established by gene disruption that (i) prtS , encoding the cell-envelope proteinase, is a key factor responsible for rapid milk acidification in fast acidifying strains, and that (ii) srtA , encoding sortase A, is not absolutely required to express the PrtS activity. Second, a 15-kb PCR product encompassing the prtS genomic island was transfered by natural transformation using the competence-inducing peptide in three distinct prtS -defective genetic backgrounds having or not a truncated sortase A gene. We showed that in all cases the milk acidification rate of transformants was significantly increased, reaching a level similar to that of wild-type fast acidifying strains. Furthermore, it appeared that the prtS -encoded activity does not depend on the prtS copy number or on its chromosomal integration locus. Conclusion We have successfully used natural competence to transfer the prtS locus encoding the cell-envelope proteinase in three slow acidifying strains of S. thermophilus , allowing their conversion into fast acidifying derivatives. The efficient protocol developed in this article will provide the dairy industry with novel and optimised S. thermophilus starter strains.

The lactic acid bacterium Streptococcus thermophilus (S. thermophilus) is widely used in the dairy industry. As a food bacterium, it has to cope with changing environments such as milk, yogurt, as well as the digestive tract, after the product has been ingested. In bacteria, two-component systems (TCS) are one of the most prevalent mechanisms to sense and respond appropriately to a wide range of signals. They are typically composed of a sensor kinase (HK) that detects a stimulus and a response regulator (RR) which acts as a transcriptional regulator. Our objective was to make an inventory of the TCS present in S. thermophilus LMD-9 and investigate the contribution of each TCS to LMD-9 growth in milk. For that purpose, we performed in silico, transcriptomic as well as functional analysis. The LMD-9 genome presented 6 complete TCS with both HK and RR (TCS 2, 4, 5, 6, 7, and 9) and 2 orphan RRs (RR01 and 08) with truncated HK. Our in silico analysis revealed that for 5 TCS out of the 8, orthologs with known functions were found in other bacterial species whereas for TCS02, 4 and 6 the function of the orthologs are unidentified. Transcriptomic studies (using quantitative PCR) revealed that all S. thermophilus LMD-9 response regulator genes were expressed in milk; they were expressed at different levels and with different profiles during growth. In mixed culture with Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus), the S. thermophilus partner in yogurt, the expression of four S. thermophilus LMD-9 response regulator increased; two of them, rr02 and rr09, increased by a factor of 6. These results indicate that the presence of L. bulgaricus induces regulatory changes in S. thermophilus. We also demonstrated that a response regulator (rr02) can exert its regulatory function on its target genes even when expressed at very low levels. We showed that RR05—an ortholog of Bacillus subtilis YycF or Staphylococcus aureus WalR—was essential for the growth of S. thermophilus. For the 7 other RRs, the absence of a single response regulator gene was insufficient to notably impact the growth of LMD-9 in milk, with or without supplementation with purines, formate, or stress agents (lactate, H2O2). We demonstrated here that the 8 response regulators of LMD-9 are expressed—and thus potentially active—during growth in milk and suggested that the response regulators have possibly overlapping regulons and/or functions not essential under the conditions tested.

ABSTRACT A versatile natural transformation protocol was established for and successfully applied to 18 of the 19 Streptococcus thermophilus strains tested. The efficiency of the protocol enables the use of in vitro-amplified mutagenesis fragments to perform deletion or insertion of large genetic fragments. Depending on the phenotype linked to the mutation, markerless mutants can be selected either in two steps, i.e., resistance marker insertion and excision using an adapted Cre-loxP system, or in one step using a powerful positive screening procedure as illustrated here for histidine prototrophy.

The casein fractions solubility in 45% ethanol at T = 293 K as a function of pH and the electrostatic surface energy (V-E) can be represented by an equation describing the ionisation process. The half solubility of alpha(S1)-, beta- and kappa-caseins is observed respectively at pH: 6.54, 6.85 and 6.85 at V-E: 38, 19 and 0.3 k(B).T. The solubility increases from 10% to 90% for the change in pH by 0.78, 0.84 and 1.08, and in V-E by 22, 11 and 0.6 k(B).T, respectively. For the solubility presented as a function of the reduced V-E, the behaviour of kappa-casein is different from alpha(S1)-, beta-caseins. The fraction of an industrial sodium caseinate, soluble in 45% ethanol at pH 6.4 and 293K is enriched to about 60% in alpha(S1)-casein. This fraction could be used as an emulsifying agent or for cream liqueur production.

The casein fractions solubility in 45% ethanol at T = 293 K as a function of pH and the electrostatic surface energy (V-E) can be represented by an equation describing the ionisation process. The half solubility of alpha(S1)-, beta- and kappa-caseins is observed respectively at pH: 6.54, 6.85 and 6.85 at V-E: 38, 19 and 0.3 k(B).T. The solubility increases from 10% to 90% for the change in pH by 0.78, 0.84 and 1.08, and in V-E by 22, 11 and 0.6 k(B).T, respectively. For the solubility presented as a function of the reduced V-E, the behaviour of kappa-casein is different from alpha(S1)-, beta-caseins. The fraction of an industrial sodium caseinate, soluble in 45% ethanol at pH 6.4 and 293K is enriched to about 60% in alpha(S1)-casein. This fraction could be used as an emulsifying agent or for cream liqueur production

Département de biochimie, de microbiologie et de bio-informatique, Faculté des sciences et de génie, Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Félix d’Hérelle Reference Center for Bacterial Viruses, Université Laval, Quebec City, Quebec, Canada, G1V 0A6. 2 Danisco USA Inc., 3329 Agriculture Drive, Madison, WI 53716, USA. 3 Danisco France SAS, Boîte Postale 10, F-86220 Dangé-Saint-Romain, France.