Maria Luz Mohedano

Role of Tyramine Synthesis by Food-Borne Enterococcus durans in Adaptation to the Gastrointestinal Tract Environment

ABSTRACT Biogenic amines in food constitute a human health risk. Here we report that tyramine-producing Enterococcus durans strain IPLA655 (from cheese) was able to produce tyramine under conditions simulating transit through the gastrointestinal tract. Activation of the tyramine biosynthetic pathway contributed to binding and immunomodulation of enterocytes.

Comparative Proteomic Analysis of Lactobacillus plantarum WCFS1 and ΔctsR Mutant Strains Under Physiological and Heat Stress Conditions

Among Gram-positive bacteria, CtsR (Class Three Stress gene Repressor) mainly regulates the expression of genes encoding the Clp ATPases and the ClpP protease. To gain a better understanding of the biological significance of the CtsR regulon in response to heat-shock conditions, we performed a global proteomic analysis of Lactobacillus plantarum WCFS1 and ΔctsR mutant strains under optimal or heat stress temperatures. Total protein extracts from bacterial cells were analyzed by two-dimensional gel fractionation. By comparing maps from different culture conditions and different L. plantarum strains, image analysis revealed 23 spots with altered levels of expression. The proteomic analysis of L. plantarum WCFS1 and ctsR mutant strains confirms at the translational level the CtsR-mediated regulation of some members of the Clp family, as well as the heat induction of typical stress response genes. Heat activation of the putative CtsR regulon genes at transcriptional and translational levels, in the ΔctsR mutant, suggests additional regulative mechanisms, as is the case of hsp1. Furthermore, isoforms of ClpE with different molecular mass were found, which might contribute to CtsR quality control. Our results could add new outlooks in order to determine the complex biological role of CtsR-mediated stress response in lactic acid bacteria.

Dextran production by Lactobacillus sakei MN1 coincides with reduced autoagglutination, biofilm formation and epithelial cell adhesion

In this work we have investigated two dextran-producing lactic acid bacteria, Lactobacillus sakei MN1 and Leuconostoc mesenteroides RTF10, isolated from fermented meat products. These bacteria synthesise dextran when sucrose, but not glucose, is present in the growth medium. The influence of dextran on bacterial aggregation, adhesion and biofilm formation was investigated in cultures challenged with sucrose or glucose. For Lb. sakei MN1, the synthesis of the dextran drastically impaired the three processes; in contrast it had no effect on Lc. mesenteroides RTF10. Therefore, the influence of dextran on probiotic properties of Lb. sakei MN1 was tested in vivo using gnotobiotic zebrafish models. The bacterium efficiently colonised the fish gut and inhibited the killing activity of Vibrio anguillarum NB10[pOT11]. Furthermore, under conditions of dextran synthesis, the adhesion of Lb. sakei MN1 to the epithelial cells decreased, without greatly affecting its anti V. anguillarum activity.

A partial proteome reference map of the wine lactic acid bacterium Oenococcus oeni ATCC BAA-1163

Oenococcus oeni is the main lactic acid bacterium that carries out the malolactic fermentation in virtually all red wines and in some white and sparkling wines. Oenococcus oeni possesses an array of metabolic activities that can modify the taste and aromatic properties of wine. There is, therefore, industrial interest in the proteins involved in these metabolic pathways and related transport systems of this bacterium. In this work, we report the characterization of the O. oeni ATCC BAA-1163 proteome. Total and membrane protein preparations from O. oeni were standardized and analysed by two-dimensional gel electrophoresis. Using tandem mass spectrometry, we identified 224 different spots corresponding to 152 unique proteins, which have been classified by their putative function and subjected to bioinformatics analysis.

Construction and validation of a mCherry protein vector for promoter analysis in Lactobacillus acidophilus

Lactobacilli are widespread in natural environments and are increasingly being investigated as potential health modulators. In this study, we have adapted the broad-host-range vector pNZ8048 to express the mCherry protein (pRCR) to expand the usage of the mCherry protein for analysis of gene expression in Lactobacillus. This vector is also able to replicate in Streptococcus pneumoniae and Escherichia coli. The usage of pRCR as a promoter probe was validated in Lactobacillus acidophilus by characterizing the regulation of lactacin B expression. The results show that the regulation is exerted at the transcriptional level, with lbaB gene expression being specifically induced by co-culture of the L. acidophilus bacteriocin producer and the S. thermophilus STY-31 inducer bacterium.

A specific immunological method to detect and quantify bacterial 2-substituted (1,3)- β-D-glucan

Exopolysaccharides synthesized by lactic acid bacteria have prebiotic properties and contribute to the rheology and texture of fermented foods. Here, we have standardized an immunological method for the specific detection of 2-substituted (1,3)-β-D-glucans. The method allows direct detection and quantification of this exopolysaccharide in culture supernatants containing other mono- and poly-saccharides. Moreover, it allows specific detection of the biomolecules synthesized in vitro in enzymatic reactions. Thus, this method allows the fast identification of producing bacteria, as well as biochemical characterization of the glycosyltransferases responsible for their synthesis.

Controlling the formation of biogenic amines in fermented foods

Abstract In this chapter, biogenic amine (BA) production in dairy products, beverages and sausages is described, and their potential harm to human health is addressed. To control the production of toxic compounds it is necessary to know how they are produced, and under which environmental conditions their synthesis is induced. Therefore, the molecular bases for BA production are discussed here. Techniques for detection of the BAs as well as their producing organisms are also described in this chapter, as are current and future strategies to control production. Finally, legislation relating to BA content in food is presented.

The response regulator YycF inhibits expression of the fatty acid biosynthesis repressor FabT in Streptococcus pneumoniae

The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation.

In Situ β-Glucan Fortification of Cereal-Based Matrices by Pediococcus parvulus 2.6: Technological Aspects and Prebiotic Potential

Bacterial exopolysaccharides produced by lactic acid bacteria are of increasing interest in the food industry, since they might enhance the technological and functional properties of some edible matrices. In this work, Pediococcus parvulus 2.6, which produces an O2-substituted (1,3)-β-d-glucan exopolysaccharide only synthesised by bacteria, was proposed as a starter culture for the production of three cereal-based fermented foods. The obtained fermented matrices were naturally bio-fortified in microbial β-glucans, and used to investigate the prebiotic potential of the bacterial exopolysaccharide by analysing the impact on the survival of a probiotic Lactobacillus plantarum strain under starvation and gastrointestinal simulated conditions. All of the assays were performed by using as control of the P. parvulus 2.6’s performance, the isogenic β-glucan non-producing 2.6NR strain. Our results showed a differential capability of P. parvulus to ferment the cereal flours. During the fermentation step, the β-glucans produced were specifically quantified and their concentration correlated with an increased viscosity of the products. The survival of the model probiotic L. plantarum WCFS1 was improved by the presence of the bacterial β-glucans in oat and rice fermented foods under starvation conditions. The probiotic bacteria showed a significantly higher viability when submitted to a simulated intestinal stress in the oat matrix fermented by the 2.6 strain. Therefore, the cereal flours were a suitable substrate for in situ bio-fortification with the bacterial β-glucan, and these matrices could be used as carriers to enhance the beneficial properties of probiotic bacteria.

Dextransucrase Expression Is Concomitant with that of Replication and Maintenance Functions of the pMN1 Plasmid in Lactobacillus sakei MN1

The exopolysaccharide synthesized by Lactobacillus sakei MN1 is a dextran with antiviral and immunomodulatory properties of potential utility in aquaculture. In this work we have investigated the genetic basis of dextran production by this bacterium. Southern blot hybridization experiments demonstrated the plasmidic location of the dsrLS gene, which encodes the dextransucrase involved in dextran synthesis. DNA sequencing of the 11,126 kbp plasmid (pMN1) revealed that it belongs to a family which replicates by the theta mechanism, whose prototype is pUCL287. The plasmid comprises the origin of replication, repA, repB, and dsrLS genes, as well as seven open reading frames of uncharacterized function. Lb. sakei MN1 produces dextran when sucrose, but not glucose, is present in the growth medium. Therefore, plasmid copy number and stability, as well as dsrLS expression, were investigated in cultures grown in the presence of either sucrose or glucose. The results revealed that pMN1 is a stable low-copy-number plasmid in both conditions. Gene expression studies showed that dsrLS is constitutively expressed, irrespective of the carbon source present in the medium. Moreover, dsrLS is expressed from a monocistronic transcript as well as from a polycistronic repA-repB-orf1-dsrLS mRNA. To our knowledge, this is the first report of a plasmid-borne dextransucrase-encoding gene, as well as the first time that co-transcription of genes involved in plasmid maintenance and replication with a gene encoding an enzyme has been established.