RESUMEN
Understanding the nature of mucus-microbe interactions will provide important information that can help to elucidate the mechanisms underlying probiotic adhesion. This study focused on the adhesive properties of the Lactococcus lactis subsp. cremoris IBB477 strain, previously shown to persist in the gastrointestinal tract of germ-free rats. The shear flow-induced detachment of L. lactis cells was investigated under laminar flow conditions. Such a dynamic approach demonstrated increased adhesion to bare and mucin-coated polystyrene for IBB477, compared to that observed for the MG1820 control strain. To identify potential genetic determinants giving adhesive properties to IBB477, the improved high-quality draft genome sequence comprising chromosome and five plasmids was obtained and analysed. The number of putative adhesion proteins was determined on the basis of surface/extracellular localisation and/or the presence of adhesion domains. To identify proteins essential for the IBB477 specific adhesion property, nine deletion mutants in chromosomal genes have been constructed and analysed using adhesion tests on bare polystyrene as well as mucin-, fibronectin- or collagen IV-coated polystyrene plates in comparison to the wild-type strain. These experiments demonstrated that gene AJ89_07570 encoding a protein containing DUF285, MucBP and four Big_3 domains is involved in adhesion to bare and mucin-coated polystyrene. To summarise, in the present work, we characterised the adhesion of IBB477 under laminar flow conditions; identified the putative adherence factors present in IBB477, which is the first L. lactis strain exhibiting adhesive and mucoadhesive properties to be sequenced and demonstrated that one of the proteins containing adhesion domains contributes to adhesion.
Asunto(s)
Adhesión Bacteriana , Lactococcus lactis/fisiología , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Eliminación de GenRESUMEN
The present work focuses on the role of pili present at the cell surface of Lactococcus lactis in bacterial adhesion to abiotic (hydrophobic polystyrene) and biotic (mucin-coated polystyrene) surfaces. Native pili-displaying strains and isogenic derivatives in which pilins or sortase C structural genes had been modified were used. Surface physico-chemistry, morphology and shear-flow-induced detachment of lactococcal cells were evaluated. The involvement of pili in L. lactis adhesion was clearly demonstrated, irrespective of the surface characteristics (hydrophobic/hydrophilic, presence or not of specific binding sites). The accessory pilin, PilC, and the backbone pilin, PilB, were revealed to play a major role in adhesion, provided that the PilB was present in its polymerized form. Within the population fraction that remained attached to the surface under increasing shear flow, different association behaviors were observed, showing that pili could serve as anchoring sites thus hampering the effect of shear flow on cell orientation and detachment.
Asunto(s)
Aminoaciltransferasas/metabolismo , Adhesión Bacteriana/fisiología , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Lactococcus lactis/fisiología , Poliestirenos/química , Aminoaciltransferasas/genética , Proteínas Bacterianas/genética , Cisteína Endopeptidasas/genética , Proteínas Fimbrias/genética , Humanos , Hidrodinámica , Interacciones Hidrofóbicas e Hidrofílicas , Lactococcus lactis/metabolismo , Mucinas/química , Multimerización de Proteína , Estrés Mecánico , Propiedades de SuperficieRESUMEN
Despite well-known intestinal epithelial barrier impairment and visceral hypersensitivity in irritable bowel syndrome (IBS) patients and IBS-like models, structural and physical changes in the mucus layer remain poorly understood. Using a water avoidance stress (WAS) model, we aimed at evaluating whether 1) WAS modified gut permeability, visceral sensitivity, mucin expression, biochemical structure of O-glycans, and related mucus physical properties, and 2) whether Lactobacillus farciminis treatment prevented these alterations. Wistar rats received orally L. farciminis or vehicle for 14 days; at day 10, they were submitted to either sham or 4-day WAS. Intestinal paracellular permeability and visceral sensitivity were measured in vivo. The number of goblet cells and Muc2 expression were evaluated by histology and immunohistochemistry, respectively. Mucosal adhesion of L. farciminis was determined ex situ. The mucin O-glycosylation profile was obtained by mass spectrometry. Surface imaging of intestinal mucus was performed at nanoscale by atomic force microscopy. WAS induced gut hyperpermeability and visceral hypersensitivity but did not modify either the number of intestinal goblet cells or Muc2 expression. In contrast, O-glycosylation of mucins was strongly affected, with the appearance of elongated polylactosaminic chain containing O-glycan structures, associated with flattening and loss of the mucus layer cohesive properties. L. farciminis bound to intestinal Muc2 and prevented WAS-induced functional alterations and changes in mucin O-glycosylation and mucus physical properties. WAS-induced functional changes were associated with mucus alterations resulting from a shift in O-glycosylation rather than from changes in mucin expression. L. farciminis treatment prevented these alterations, conferring epithelial and mucus barrier strengthening.
Asunto(s)
Mucosa Intestinal/metabolismo , Mucina 2/biosíntesis , Probióticos/uso terapéutico , Estrés Psicológico/fisiopatología , Animales , Colon/metabolismo , Corticosterona/sangre , Glicosilación , Células Caliciformes/fisiología , Mucosa Intestinal/microbiología , Lactobacillus/metabolismo , Masculino , Moco/metabolismo , Permeabilidad , Ratas , Ratas WistarRESUMEN
Staphylococcus aureus is a major cause of food poisoning outbreaks associated with dairy products, because of the ingestion of preformed enterotoxins. The biocontrol of S. aureus using lactic acid bacteria (LAB) offers a promising opportunity to fight this pathogen while respecting the product ecosystem. We had previously established the ability of Lactococcus lactis, a lactic acid bacterium widely used in the dairy industry, to downregulate a major staphylococcal virulence regulator, the accessory gene regulator (agr) system, and, as a consequence, agr-controlled enterotoxins. In the present paper, we have shown that the oxygen-independent reducing properties of L. lactis contribute to agr downregulation. Neutralizing lactococcal reduction by adding potassium ferricyanide or maintaining the oxygen pressure constant at 50% released agr downregulation in the presence of L. lactis. This downregulation still occurred in an S. aureus srrA mutant, indicating that the staphylococcal respiratory response regulator SrrAB was not the only component in the signaling pathway. Therefore, this study clearly demonstrates the ability of L. lactis reducing properties to interfere with the expression of S. aureus virulence, thus highlighting this general property of LAB as a lever to control the virulence expression of this major pathogen in a food context and beyond.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Lactococcus lactis/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/metabolismo , Transactivadores/metabolismo , Proteínas Bacterianas/genética , Regulación hacia Abajo , Humanos , Oxidación-Reducción , Oxígeno/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Transactivadores/genética , VirulenciaRESUMEN
BACKGROUND: The stress response in bacteria involves the multistage control of gene expression but is not entirely understood. To identify the translational response of bacteria in stress conditions and assess its contribution to the regulation of gene expression, the translational states of all mRNAs were compared under optimal growth condition and during nutrient (isoleucine) starvation. RESULTS: A genome-scale study of the translational response to nutritional limitation was performed in the model bacterium Lactococcus lactis. Two measures were used to assess the translational status of each individual mRNA: the fraction engaged in translation (ribosome occupancy) and ribosome density (number of ribosomes per 100 nucleotides). Under isoleucine starvation, half of the mRNAs considered were translationally down-regulated mainly due to decreased ribosome density. This pattern concerned genes involved in growth-related functions such as translation, transcription, and the metabolism of fatty acids, phospholipids and bases, contributing to the slowdown of growth. Only 4% of the mRNAs were translationally up-regulated, mostly related to prophagic expression in response to stress. The remaining genes exhibited antagonistic regulations of the two markers of translation. Ribosome occupancy increased significantly for all the genes involved in the biosynthesis of isoleucine, although their ribosome density had decreased. The results revealed complex translational regulation of this pathway, essential to cope with isoleucine starvation.To elucidate the regulation of global gene expression more generally, translational regulation was compared to transcriptional regulation under isoleucine starvation and to other post-transcriptional regulations related to mRNA degradation and mRNA dilution by growth. Translational regulation appeared to accentuate the effects of transcriptional changes for down-regulated growth-related functions under isoleucine starvation although mRNA stabilization and lower dilution by growth counterbalanced this effect. CONCLUSIONS: We show that the contribution of translational regulation to the control of gene expression is significant in the stress response. Post-transcriptional regulation is complex and not systematically co-directional with transcription regulation. Post-transcriptional regulation is important to the understanding of gene expression control.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Lactococcus lactis/genética , Biosíntesis de Proteínas , Estrés Fisiológico/genética , Adaptación Fisiológica/genética , Proteínas Bacterianas/metabolismo , Análisis por Conglomerados , Isoleucina/deficiencia , Lactococcus lactis/metabolismo , Polirribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , TranscriptomaRESUMEN
The diversity of nine dairy strains of Lactococcus lactis subsp. lactis in fermented milk was investigated by both genotypic and phenotypic analyses. Pulsed-field gel electrophoresis and multilocus sequence typing were used to establish an integrated genotypic classification. This classification was coherent with discrimination of the L. lactis subsp. lactis bv. diacetylactis lineage and reflected clonal complex phylogeny and the uniqueness of the genomes of these strains. To assess phenotypic diversity, 82 variables were selected as important dairy features; they included physiological descriptors and the production of metabolites and volatile organic compounds (VOCs). Principal-component analysis (PCA) demonstrated the phenotypic uniqueness of each of these genetically closely related strains, allowing strain discrimination. A method of variable selection was developed to reduce the time-consuming experimentation. We therefore identified 20 variables, all associated with VOCs, as phenotypic markers allowing discrimination between strain groups. These markers are representative of the three metabolic pathways involved in flavor: lipolysis, proteolysis, and glycolysis. Despite great phenotypic diversity, the strains could be divided into four robust phenotypic clusters based on their metabolic orientations. Inclusion of genotypic diversity in addition to phenotypic characters in the classification led to five clusters rather than four being defined. However, genotypic characters make a smaller contribution than phenotypic variables (no genetic distances selected among the most contributory variables). This work proposes an original method for the phenotypic differentiation of closely related strains in milk and may be the first step toward a predictive classification for the manufacture of starters.
Asunto(s)
Genotipo , Lactococcus lactis/clasificación , Lactococcus lactis/genética , Leche/microbiología , Fenotipo , Compuestos Orgánicos Volátiles/metabolismo , Animales , Biomarcadores/metabolismo , Electroforesis en Gel de Campo Pulsado , Fermentación , Variación Genética , Lactococcus lactis/fisiología , Tipificación de Secuencias Multilocus , Filogenia , Reacción en Cadena de la PolimerasaRESUMEN
Lactococcus lactis subsp. lactis strain A12 was isolated from sourdough. Combined genomic, transcriptomic, and phenotypic analyses were performed to understand its survival capacity in the complex sourdough ecosystem and its role in the microbial community. The genome sequence comparison of strain A12 with strain IL1403 (a derivative of an industrial dairy strain) revealed 78 strain-specific regions representing 23% of the total genome size. Most of the strain-specific genes were involved in carbohydrate metabolism and are potentially required for its persistence in sourdough. Phenotype microarray, growth tests, and analysis of glycoside hydrolase content showed that strain A12 fermented plant-derived carbohydrates, such as arabinose and α-galactosides. Strain A12 exhibited specific growth rates on raffinose that were as high as they were on glucose and was able to release sucrose and galactose outside the cell, providing soluble carbohydrates for sourdough microflora. Transcriptomic analysis identified genes specifically induced during growth on raffinose and arabinose and reveals an alternative pathway for raffinose assimilation to that used by other lactococci.
Asunto(s)
Metabolismo de los Hidratos de Carbono/genética , Microbiología de Alimentos , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Técnicas de Tipificación Bacteriana , ADN Bacteriano/química , ADN Bacteriano/genética , Fermentación , Genoma Bacteriano , Glicósido Hidrolasas/análisis , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , TranscriptomaRESUMEN
BACKGROUND: In bacteria, the weak correlations at the genome scale between mRNA and protein levels suggest that not all mRNAs are translated with the same efficiency. To experimentally explore mRNA translational level regulation at the systemic level, the detailed translational status (translatome) of all mRNAs was measured in the model bacterium Lactococcus lactis in exponential phase growth. RESULTS: Results demonstrated that only part of the entire population of each mRNA species was engaged in translation. For transcripts involved in translation, the polysome size reached a maximum of 18 ribosomes. The fraction of mRNA engaged in translation (ribosome occupancy) and ribosome density were not constant for all genes. This high degree of variability was analyzed by bioinformatics and statistical modeling in order to identify general rules of translational regulation. For most of the genes, the ribosome density was lower than the maximum value revealing major control of translation by initiation. Gene function was a major translational regulatory determinant. Both ribosome occupancy and ribosome density were particularly high for transcriptional regulators, demonstrating the positive role of translational regulation in the coordination of transcriptional networks. mRNA stability was a negative regulatory factor of ribosome occupancy and ribosome density, suggesting antagonistic regulation of translation and mRNA stability. Furthermore, ribosome occupancy was identified as a key component of intracellular protein levels underlining the importance of translational regulation. CONCLUSIONS: We have determined, for the first time in a bacterium, the detailed translational status for all mRNAs present in the cell. We have demonstrated experimentally the high diversity of translational states allowing individual gene differentiation and the importance of translation-level regulation in the complex process linking gene expression to protein synthesis.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/genética , Lactococcus lactis/genética , Biosíntesis de Proteínas/genética , ARN Mensajero/metabolismo , Biología Computacional , Perfilación de la Expresión Génica , Modelos Genéticos , Polirribosomas/metabolismo , ARN Mensajero/genéticaRESUMEN
This work was devoted to probe, at the entire population level, interactions between mucins and Lactococcus lactis, using QCM-D. Real-time monitoring of adsorption on polystyrene of PGM (Pig Gastric Mucin) and subsequent adhesion of L. lactis was performed for IBB477 and MG1820 strains. Measuring simultaneously shifts in resonance frequency and dissipation on the polystyrene-coated crystal demonstrated a two-phase process for PGM adsorption. XPS analysis confirmed the presence of adsorbed mucin. The Voigt-based model was used to describe the QCM-D outputs. The predicted thickness of the PGM layer was consistent with the AFM experimental value. Adhesion of L. lactis to bare or PGM-coated polystyrene was then monitored, in combination with DAPI cell counting. Positive frequency shifts were caused by adhering bacteria. The presence of adsorbed PGM strongly reduced bacterial adhesion. However, adhesion of IBB477 to the PGM coating was greatly increased in comparison with that of MG1820. Muco-adhesion may be a highly variable and valuable phenotypic trait among L. lactis strains.
Asunto(s)
Adhesión Bacteriana , Lactococcus lactis/fisiología , Mucinas/química , Tecnicas de Microbalanza del Cristal de Cuarzo , Adsorción , Animales , Lactococcus lactis/metabolismo , Espectroscopía de Fotoelectrones , Poliestirenos/química , Porcinos , Sustancias ViscoelásticasRESUMEN
In this work we focused on quantifying adhesion between Lactococcus lactis, the model for lactic acid bacteria (LAB) and mucins. Interactions between two strains of L. lactis (IBB477 and MG1820 as control) and pig gastric mucin-based coating were measured and compared with the use of atomic force microscopy. Analysis of retraction force-distance curves shed light on the differential contributions of nonspecific and specific forces. An increased proportion of specific adhesive events was obtained for IBB477 (20% vs. 5% for the control). Blocking assays with free pig gastric mucin and its O-glycan moiety showed that oligosaccharides play a major (but not exclusive) role in L. lactis-mucins interactions. Specific interactions were analyzed in terms of kinetic constants. An increase in the loading rate of atomic force microscope tip led to a higher force between interacting biological entities, which was directly linked to the kinetic dissociation constant (K(off)). Enhancing the contact time between the tip and the sample allowed an increase in the interaction probability, which can be related to the kinetic association constant (K(on)). Variations in the loading rate and contact time enabled us to determine K(on) (3.3 × 10(2) M(-1)·s(-1)) and K(off) (0.46 s(-1)), and the latter was consistent with values given in the literature for sugar-protein interactions.
Asunto(s)
Lactococcus lactis/metabolismo , Sondas Moleculares/metabolismo , Animales , Adhesión Bacteriana , Supervivencia Celular , Humanos , Cinética , Lactococcus lactis/citología , Lactococcus lactis/ultraestructura , Microscopía de Fuerza Atómica , Microscopía Fluorescente , Mucinas , Oligosacáridos/metabolismo , Sus scrofa , Factores de TiempoRESUMEN
The intrasubspecies diversity of six strains of Lactococcus lactis subsp. lactis was investigated at the genomic level and in terms of phenotypic and transcriptomic profiles in an ultrafiltration cheese model. The six strains were isolated from various sources, but all exhibited a dairy phenotype (growth in ultrafiltration cheese model and high acidification rate). The six strains exhibited similar behaviors in terms of growth during cheese ripening, while different acidification capabilities were detected. Even if all strains displayed large genomic similarities, sharing a large core genome of almost 2,000 genes, the expression of this core genome directly in the cheese matrix revealed major strain-specific differences that potentially could account for the observed different acidification capabilities. This work demonstrated that significant transcriptomic polymorphisms exist even among Lactococcus lactis subsp. lactis strains with the same dairy origin.
Asunto(s)
Técnicas de Tipificación Bacteriana , Queso/microbiología , Variación Genética , Lactococcus lactis/clasificación , Lactococcus lactis/genética , Hibridación Genómica Comparativa , Perfilación de la Expresión Génica , Genoma Bacteriano , Genómica , Genotipo , Concentración de Iones de Hidrógeno , Lactococcus lactis/aislamiento & purificación , Lactococcus lactis/metabolismo , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Reacción en Cadena de la Polimerasa , Especificidad de la EspecieRESUMEN
Lactococcus lactis is used extensively for the production of various cheeses. At every stage of cheese fabrication, L. lactis has to face several stress-generating conditions that result from its own modification of the environment as well as externally imposed conditions. We present here the first in situ global gene expression profile of L. lactis in cheeses made from milk concentrated by ultrafiltration (UF-cheeses), a key economical cheese model. The transcriptomic response of L. lactis was analyzed directly in a cheese matrix, starting from as early as 2 h and continuing for 7 days. The growth of L. lactis stopped after 24 h, but metabolic activity was maintained for 7 days. Conservation of its viability relied on an efficient proteolytic activity measured by an increasing, quantified number of free amino acids in the absence of cell lysis. Extensive downregulation of genes under CodY repression was found at day 7. L. lactis developed multiple strategies of adaptation to stressful modifications of the cheese matrix. In particular, expression of genes involved in acidic- and oxidative-stress responses was induced. L. lactis underwent unexpected carbon limitation characterized by an upregulation of genes involved in carbon starvation, principally due to the release of the CcpA control. We report for the first time that in spite of only moderately stressful conditions, lactococci phage is repressed under UF-cheese conditions.
Asunto(s)
Queso/microbiología , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Lactococcus lactis/fisiología , Estrés Fisiológico , Aminoácidos/metabolismo , Animales , Hidrólisis , Lactococcus lactis/genética , Lactococcus lactis/crecimiento & desarrollo , Lactococcus lactis/metabolismo , Viabilidad Microbiana , Leche , Proteínas/metabolismo , Factores de Tiempo , UltrafiltraciónRESUMEN
Batch cultures of Lactococcus lactis NCDO 2118 and IL 1403 were performed in a Couette bioreactor operated in the modulated wavy vortex flow and the turbulent regimes. This study provides an overall analysis taking into account both mechanical stress and mixing in a Couette bioreactor. A unique phenotypic aspect has been proved to occur only in the modulated wavy vortex flow regime for the two studied strains, namely that the cells become entrapped in a filamentous form. No change in the metabolic behavior of the cells has been observed. The polymeric matrix has been microscopically observed through FISH and fluorescent lectin binding, showing cells entrapped in a glycoconjugate matrix. All hypotheses regarding insufficient mixing as a cause of this phenotype have been discarded, leading to the conclusion that this particular phenotypic feature is essentially due a combined effect of mechanical stress and flow structure. Particle size measurement during the fermentation course indicates that formation of filamentous form results from a continuous aggregation started in the early stages of the cultivation. According to our results a minimum shear is required to induce the ability for cells to aggregate. Then, it appears that both flow structure and mechanical stress (shear) are responsible for the appearance of such a filamentous form. As far as the authors know, this is the first experimental evidence of a bio polymerization induced by the flow structure.
Asunto(s)
Biopolímeros/metabolismo , Reactores Biológicos/microbiología , Lactococcus lactis/citología , Lactococcus lactis/crecimiento & desarrollo , Adhesión Bacteriana , Lactococcus lactis/metabolismo , Microscopía , Material ParticuladoRESUMEN
BACKGROUND: Amino acid assimilation is crucial for bacteria and this is particularly true for Lactic Acid Bacteria (LAB) that are generally auxotroph for amino acids. The global response of the LAB model Lactococcus lactis ssp. lactis was characterized during progressive isoleucine starvation in batch culture using a chemically defined medium in which isoleucine concentration was fixed so as to become the sole limiting nutriment. Dynamic analyses were performed using transcriptomic and proteomic approaches and the results were analysed conjointly with fermentation kinetic data. RESULTS: The response was first deduced from transcriptomic analysis and corroborated by proteomic results. It occurred progressively and could be divided into three major mechanisms: (i) a global down-regulation of processes linked to bacterial growth and catabolism (transcription, translation, carbon metabolism and transport, pyrimidine and fatty acid metabolism), (ii) a specific positive response related to the limiting nutrient (activation of pathways of carbon or nitrogen metabolism and leading to isoleucine supply) and (iii) an unexpected oxidative stress response (positive regulation of aerobic metabolism, electron transport, thioredoxin metabolism and pyruvate dehydrogenase). The involvement of various regulatory mechanisms during this adaptation was analysed on the basis of transcriptomic data comparisons. The global regulator CodY seemed specifically dedicated to the regulation of isoleucine supply. Other regulations were massively related to growth rate and stringent response. CONCLUSION: This integrative biology approach provided an overview of the metabolic pathways involved during isoleucine starvation and their regulations. It has extended significantly the physiological understanding of the metabolism of L. lactis ssp. lactis. The approach can be generalised to other conditions and will contribute significantly to the identification of the biological processes involved in complex regulatory networks of micro-organisms.
Asunto(s)
Aminoácidos/metabolismo , Isoleucina/metabolismo , Lactococcus lactis/fisiología , Aminoácidos/genética , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Isoleucina/genética , Lactococcus lactis/genética , Lactococcus lactis/crecimiento & desarrollo , Lactococcus lactis/metabolismo , Proteómica , TranscriptomaRESUMEN
GABA (γ-aminobutyric acid) production has been widely described as an adaptive response to abiotic stress, allowing bacteria to survive in harsh environments. This work aimed to clarify and understand the relationship between GABA production and bacterial growth conditions, with particular reference to osmolarity. For this purpose, Lactococcus lactis NCDO 2118, a GABA-producing strain, was grown in glucose-supplemented chemically defined medium containing 34 mM L-glutamic acid, and different concentrations of salts (chloride, sulfate or phosphate ions) or polyols (sorbitol, glycerol). Unexpectedly, our data demonstrated that GABA production was not directly related to osmolarity. Chloride ions were the most significant factor influencing GABA yield in response to acidic stress while sulfate ions did not enhance GABA production. We demonstrated that the addition of chloride ions increased the glutamic acid decarboxylase (GAD) synthesis and the expression of the gadBC genes. Finally, under fed-batch conditions in a complex medium supplemented with 0.3 M NaCl and after a pH shift to 4.6, L. lactis NCDO 2118 was able to produce up to 413 mM GABA from 441 mM L-glutamic acid after only 56 h of culture, revealing the potential of L. lactis strains for intensive production of this bioactive molecule.
RESUMEN
GABA is a molecule of increasing nutraceutical interest due to its modulatory activity on the central nervous system and smooth muscle relaxation. Potentially probiotic bacteria can produce it by glutamate decarboxylation, but nothing is known about the physiological modifications occurring at the microbial level during GABA production. In the present investigation, a GABA-producing Lactococcus lactis strain grown in a medium supplemented with or without glutamate was studied using a combined transcriptome/proteome analysis. A tenfold increase in GABA production in the glutamate medium was observed only during the stationary phase and at low pH. About 30 genes and/or proteins were shown to be differentially expressed in glutamate-stimulated conditions as compared to control conditions, and the modulation exerted by glutamate on entire metabolic pathways was highlighted by the complementary nature of transcriptomics and proteomics. Most glutamate-induced responses consisted in under-expression of metabolic pathways, with the exception of glycolysis where either over- or under-expression of specific genes was observed. The energy-producing arginine deiminase pathway, the ATPase, and also some stress proteins were down-regulated, suggesting that glutamate is not only an alternative means to get energy, but also a protective agent against stress for the strain studied.
Asunto(s)
Perfilación de la Expresión Génica , Ácido Glutámico/metabolismo , Lactococcus lactis/metabolismo , Proteómica , Ácido gamma-Aminobutírico/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Lactococcus lactis/genética , Lactococcus lactis/crecimiento & desarrolloRESUMEN
This work was devoted to the first AFM investigation of the adhesion force to pig gastric mucin (PGM) using Lactococcus lactis as the model for lactic acid bacteria. The PGM coating on polystyrene was characterized using a complementary set of multiscale analytical methods, including AFM (HarmoniX mode), XPS, and the sessile drop method. The PGM layer, which was mainly composed of C-O, C-N, COOH, CONH, and sulfur-related species (protein core and oligosaccharide side chains), was quite homogeneous and hydrophilic, with an estimated thickness of 3.4 nm. L. lactis cells were immobilized on the AFM tip (lacto probe) and used as a force probe to measure the interaction forces between bacteria and PGM-coated polystyrene on the nanoscale. After mucin adsorption, adhesion force levels were lower because of the interplay of electrostatic, hydrophilic, and steric repulsions. For example, the adhesion forces of the lacto probe to bare and PGM-coated polymer were 0.74 +/- 0.10 and 0.12 +/- 0.06 nN, respectively. The shape analysis of retraction force-distance curves highlighted the contribution of both nonspecific and specific forces (ligand/receptor bonding). The lacto probe concept and the associated AFM measurements may now provide a powerful framework for understanding interaction mechanisms between mucins and lactic acid bacteria.
Asunto(s)
Adhesión Bacteriana/fisiología , Lactococcus lactis/fisiología , Microscopía de Fuerza Atómica , Mucinas/química , Adsorción , Lactococcus lactis/química , Poliestirenos/químicaRESUMEN
This genome-scale study analysed the various parameters influencing protein levels in cells. To achieve this goal, the model bacterium Lactococcus lactis was grown at steady state in continuous cultures at different growth rates, and proteomic and transcriptomic data were thoroughly compared. Ratios of mRNA to protein were highly variable among proteins but also, for a given gene, between the different growth conditions. The modeling of cellular processes combined with a data fitting modeling approach allowed both translation efficiencies and degradation rates to be estimated for each protein in each growth condition. Estimated translational efficiencies and degradation rates strongly differed between proteins and were tested for their biological significance through statistical correlations with relevant parameters such as codon or amino acid bias. These efficiencies and degradation rates were not constant in all growth conditions and were inversely proportional to the growth rate, indicating a more efficient translation at low growth rate but an antagonistic higher rate of protein degradation. Estimated protein median half-lives ranged from 23 to 224 min, underlying the importance of protein degradation notably at low growth rates. The regulation of intracellular protein level was analysed through regulatory coefficient calculations, revealing a complex control depending on protein and growth conditions. The modeling approach enabled translational efficiencies and protein degradation rates to be estimated, two biological parameters extremely difficult to determine experimentally and generally lacking in bacteria. This method is generic and can now be extended to other environments and/or other micro-organisms.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Lactococcus lactis/fisiología , Modelos Biológicos , Proteómica/métodos , Biología de Sistemas/métodos , Aminoácidos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Estabilidad Proteica , Proteoma/genética , Proteoma/metabolismoRESUMEN
The mechanisms of interaction between Lactococcus lactis and the food pathogen Staphylococcus aureus are of crucial importance, as one major role of lactic acid bacteria (LAB) in fermented foods is to inhibit undesirable and pathogenic flora. It was never questioned if the presence of a pathogen can actively modify the gene expression patterns of LAB in a shared environment. In this study, transcriptome and biochemical analyses were combined to assess the dynamic response of L. lactis in a mixed culture with S. aureus. The presence of S. aureus hardly affected the growth of L. lactis but dramatically modified its gene expression profile. The main effect was related to earlier carbon limitation and a concomitantly lower growth rate in the mixed culture due to the consumption of glucose by both species. More specific responses involved diverse cellular functions. Genes associated with amino acid metabolism, ion transport, oxygen response, menaquinone metabolism, and cell surface and phage expression were differentially expressed in the mixed culture. This study led to new insights into possible mechanisms of interaction between L. lactis and S. aureus. Moreover, new and unexpected effects of L. lactis on the virulence of S. aureus were discovered, as described elsewhere (S. Even, C. Charlier, S. Nouaille, N. L. Ben Zakour, M. Cretenet, F. J. Cousin, M. Gautier, M. Cocaign-Bousquet, P. Loubière, and Y. Le Loir, Appl. Environ. Microbiol. 75:4459-4472, 2009).
Asunto(s)
Antibiosis , Perfilación de la Expresión Génica , Lactococcus lactis/fisiología , Staphylococcus aureus/fisiología , Proteínas Bacterianas/biosíntesis , Técnicas de Cocultivo , Regulación Bacteriana de la Expresión Génica , Redes y Vías Metabólicas/genéticaRESUMEN
Staphylococcus aureus is responsible for numerous food poisonings due to the production of enterotoxins by strains contaminating foodstuffs, especially dairy products. Several parameters, including interaction with antagonistic flora such as Lactococcus lactis, a lactic acid bacterium widely used in the dairy industry, can modulate S. aureus proliferation and virulence expression. We developed a dedicated S. aureus microarray to investigate the effect of L. lactis on staphylococcal gene expression in mixed cultures. This microarray was used to establish the transcriptomic profile of S. aureus in mixed cultures with L. lactis in a chemically defined medium held at a constant pH (6.6). Under these conditions, L. lactis hardly affected S. aureus growth. The expression of most genes involved in the cellular machinery, carbohydrate and nitrogen metabolism, and stress responses was only slightly modulated: a short time lag in mixed compared to pure cultures was observed. Interestingly, the induction of several virulence factors and regulators, including the agr locus, sarA, and some enterotoxins, was strongly affected. This work clearly underlines the complexity of L. lactis antagonistic potential for S. aureus and yields promising leads for investigations into nonantibiotic biocontrol of this major pathogen.