Analytical combinations to evaluate the macromolecular composition of extracellular substances (ECS) from Lactobacillus plantarum cell culture media.

Sugar-enriched media are used to produce extracellular substances (ECS) by Lactobacillus plantarum WCSF1, with a focus on growing stages and carbon source substrates. Combination of size exclusion chromatography and ATR-FTIR spectroscopy provides physicochemical patterns of bulk ECS produced along culture growing time. Secreted biopolymers present polydisperse and high molecular weight distributions, with significant amounts of carbohydrates and proteins. Results, supported by a multivariate statistical analysis, enable to differentiate the macromolecular content of bacterial ECS along the growing stages regardless of the growing media, highlighting a higher production of proteinaceous materials compared to polysaccharides. At the end of the exponential phase, common exoproteins were present in all the tested sugar-enriched media such as transglycosylases between 20 and 35 kDa, a muropeptidase at 36.9 kDa and a cell wall hydrolase. Additionally, L. plantarum WCFS1 secretes ECS with a greater diversity of proteins, when growing in the sucrose-enriched media. Graphical abstract.

Adaptation of the wine bacterium Oenococcus oeni to ethanol stress: role of the small heat shock protein Lo18 in membrane integrity.

Malolactic fermentation in wine is often carried out by Oenococcus oeni. Wine is a stressful environment for bacteria because ethanol is a toxic compound that impairs the integrity of bacterial membranes. The small heat shock protein (sHsp) Lo18 is an essential actor of the stress response in O. oeni. Lo18 prevents the thermal aggregation of proteins and plays a crucial role in membrane quality control. Here, we investigated the interaction between Lo18 and four types of liposomes: one was prepared from O. oeni grown under optimal growth conditions (here, control liposomes), one was prepared from O. oeni grown in the presence of 8% ethanol (here, ethanol liposomes), one was prepared from synthetic phospholipids, and one was prepared from phospholipids from Bacillus subtilis or Lactococcus lactis. We observed the strongest interaction between Lo18 and control liposomes. The lipid binding activity of Lo18 required the dissociation of oligomeric structures into dimers. Protein protection experiments carried out in the presence of the liposomes from O. oeni suggested that Lo18 had a higher affinity for control liposomes than for a model protein. In anisotropy experiments, we mimicked ethanol action by temperature-dependent fluidization of the liposomes. Results suggest that the principal determinant of Lo18-membrane interaction is lipid bilayer phase behavior rather than phospholipid composition. We suggest a model to describe the ethanol adaptation of O. oeni. This model highlights the dual role of Lo18 in the protection of proteins from aggregation and membrane stabilization and suggests how modifications of phospholipid content may be a key factor determining the balance between these two functions.

The oligomer plasticity of the small heat-shock protein Lo18 from Oenococcus oeni influences its role in both membrane stabilization and protein protection.

The ability of the small Hsp (heat-shock protein) Lo18 from Oenococcus oeni to modulate the membrane fluidity of liposomes or to reduce the thermal aggregation of proteins was studied as a function of the pH in the range 5-9. We have determined by size-exclusion chromatography and analytical ultracentrifugation that Lo18 assembles essentially as a 16-mer at acidic pH. Its quaternary structure evolves to a mixture of lower molecular mass oligomers probably in dynamic equilibrium when the pH increases. The best Lo18 activities are observed at pH 7 when the particle distribution contains a major proportion of dodecamers. At basic pH, particles corresponding to a dimer prevail and are thought to be the building blocks leading to oligomerization of Lo18. At acidic pH, the dimers are organized in a double-ring of stacked octamers to form the 16-mer as shown by the low-resolution structure determined by electron microscopy. Experiments performed with a modified protein (A123S) shown to preferentially form dimers confirm these results. The α-crystallin domain of Methanococcus jannaschii Hsp16.5, taken as a model of the Lo18 counterpart, fits with the electron microscopy envelope of Lo18.

Is the lipochaperone activity of sHSP a key to the stress response encoded in its primary sequence?

Several strategies have been put in place by organisms to adapt to their environment. One of these strategies is the production of stress proteins such as sHSPs, which have been widely described over the last 30 years for their role as molecular chaperones. Some sHSPs have, in addition, the particularity to exert a lipochaperone role by interacting with membrane lipids to maintain an optimal membrane fluidity. However, the mechanisms involved in this sHSP-lipid interaction remain poorly understood and described rather sporadically in the literature. This review gathers the information concerning the structure and function of these proteins available in the literature in order to highlight the mechanism involved in this interaction. In addition, analysis of primary sequence data of sHSPs available in database shows that sHSPs can interact with lipids via certain amino acid residues present on some ß sheets of these proteins. These residues could have a key role in the structure and/or oligomerization dynamics of sHPSs, which is certainly essential for interaction with membrane lipids and consequently for maintaining optimal cell membrane fluidity.

Molecular chaperone function of three small heat-shock proteins from a model probiotic species.

Small heat-shock proteins (sHSP) are ubiquitous ATP-independent chaperones that prevent irreversible aggregation of heat-damaged denaturing proteins. Lactiplantibacillus plantarum is a widespread Gram-positive bacterium with probiotic claims and vast potential for agro-food, biotechnological and biomedical applications. L. plantarum possesses a family of three sHSP, which were previously demonstrated to be involved in its stress tolerance mechanisms. Here, the three L. plantarum sHSP were heterologously expressed, purified and shown to have a chaperone activity in vitro, measuring their capacity to suppress protein aggregation, as assayed spectrophotometrically by light scattering. Their anti-aggregative capacity was found to be differently influenced by pH. Differences were also found relative to their holdase function and their capacity to modulate liposome membrane fluidity, suggesting interplays between them and indicating diversified activities. This is the first study assessing the chaperone action of sHSP from a probiotic model. The different roles of the three sHSP can increase L. plantarum's capabilities to survive the various types of stress characterising the diverse habitats of this highly adaptable species. Reported evidence supports the interest in L. plantarum as one of the model species for bacteria that have three different sHSP-encoding genes in their genomes.

Significant influence of four highly conserved amino-acids in lipochaperon-active sHsps on the structure and functions of the Lo18 protein.

To cope with environmental stresses, bacteria have developed different strategies, including the production of small heat shock proteins (sHSP). All sHSPs are described for their role as molecular chaperones. Some of them, like the Lo18 protein synthesized by Oenococcus oeni, also have the particularity of acting as a lipochaperon to maintain membrane fluidity in its optimal state following cellular stresses. Lipochaperon activity is poorly characterized and very little information is available on the domains or amino-acids key to this activity. The aim in this paper is to investigate the importance at the protein structure and function level of four highly conserved residues in sHSP exhibiting lipochaperon activity. Thus, by combining in silico, in vitro and in vivo approaches the importance of three amino-acids present in the core of the protein was shown to maintain both the structure of Lo18 and its functions.

Tyrosine-containing peptides are precursors of tyramine produced by Lactobacillus plantarum strain IR BL0076 isolated from wine.

BACKGROUND: Biogenic amines are molecules with allergenic properties. They are found in fermented products and are synthesized by lactic acid bacteria through the decarboxylation of amino acids present in the food matrix. The concentration of biogenic amines in fermented foodstuffs is influenced by many environmental factors, and in particular, biogenic amine accumulation depends on the quantity of available precursors. Enological practices which lead to an enrichment in nitrogen compounds therefore favor biogenic amine production in wine. Free amino acids are the only known precursors for the synthesis of biogenic amines, and no direct link has previously been demonstrated between the use of peptides by lactic acid bacteria and biogenic amine synthesis. RESULTS: Here we demonstrate for the first time that a Lactobacillus plantarum strain isolated from a red wine can produce the biogenic amine tyramine from peptides containing tyrosine. In our conditions, most of the tyramine was produced during the late exponential growth phase, coinciding with the expression of the tyrDC and tyrP genes. The DNA sequences of tyrDC and tyrP in this strain share 98% identity with those in Lactobacillus brevis consistent with horizontal gene transfer from L. brevis to L. plantarum. CONCLUSION: Peptides amino acids are precursors of biogenic amines for Lactobacillus plantarum strain IR BL0076.

The production of preconditioned freeze-dried Oenococcus oeni primes its metabolism to withstand environmental stresses encountered upon inoculation into wine.

Oenococcus oeni is the most resistant lactic acid bacteria species to the environmental stresses encountered in wine, particularly the acidity, presence of ethanol and phenolic compounds. Indigenous strains develop spontaneously following the yeast-driven alcoholic fermentation and may perform the malolactic fermentation whereby improving taste, aroma, and the microbial stability of wine. However, spontaneous fermentation is sometimes delayed, prolonged or incomplete. In order to better control its timing and quality, O. oeni strains are selected and developed to be used as malolactic starters. They are prepared under proprietary manufacturing processes to survive direct inoculation and are predominantly provided as freeze-dried preparations. In this study, we have investigated the physiological and molecular alterations occurring in O. oeni cells prepared by an industrial process that consists of preconditioning protocols and freeze-drying, and compared them to the same strain grown in a grape juice medium. We found that compared to cultured cells, the industrial production process improved survival under extreme conditions, i. e. at low pH or high tannin concentrations. In contrast, cultured cells resumed active growth more quickly and strongly than freeze-dried preparations in standard pH wines. A proteomic analysis showed that during the industrial production most non-essential metabolic processes are shut down and components of the general and the stringent stress response are upregulated. The presence of major components of the stress response facilitates protein homeostasis and physiological changes that further ensure the integrity of cells.

Prediction of Genetic Groups within Brettanomyces bruxellensis through Cell Morphology Using a Deep Learning Tool.

Brettanomyces bruxellensis is described as a wine spoilage yeast with many mainly strain-dependent genetic characteristics, bestowing tolerance against environmental stresses and persistence during the winemaking process. Thus, it is essential to discriminate B. bruxellensis isolates at the strain level in order to predict their stress resistance capacities. Few predictive tools are available to reveal intraspecific diversity within B. bruxellensis species; also, they require expertise and can be expensive. In this study, a Random Amplified Polymorphic DNA (RAPD) adapted PCR method was used with three different primers to discriminate 74 different B. bruxellensis isolates. High correlation between the results of this method using the primer OPA-09 and those of a previous microsatellite analysis was obtained, allowing us to cluster the isolates among four genetic groups more quickly and cheaply than microsatellite analysis. To make analysis even faster, we further investigated the correlation suggested in a previous study between genetic groups and cell polymorphism using the analysis of optical microscopy images via deep learning. A Convolutional Neural Network (CNN) was trained to predict the genetic group of B. bruxellensis isolates with 96.6% accuracy. These methods make intraspecific discrimination among B. bruxellensis species faster, simpler and less costly. These results open up very promising new perspectives in oenology for the study of microbial ecosystems.

Characterization of the CtsR stress response regulon in Lactobacillus plantarum.

Lactobacillus plantarum ctsR was characterized. ctsR was found to be cotranscribed with clpC and induced in response to various abiotic stresses. ctsR deletion conferred a heat-sensitive phenotype with peculiar cell morphological features. The transcriptional pattern of putative CtsR regulon genes was examined in the Delta ctsR mutant. Direct CtsR-dependent regulation was demonstrated by DNA-binding assays using recombinant CtsR and the promoters of the ctsR-clpC operon and hsp1.

New advances on the Brettanomyces bruxellensis biofilm mode of life.

The wine spoilage yeast Brettanomyces bruxellensis can be found at several steps in the winemaking process due to its resistance to multiple stress conditions. The ability to form biofilm is a potential resistance strategy, although it has been given little attention so far for this yeast. In this work, the capacity to form biofilm and its structure were explored in YPD medium and in wine. Using microsatellite analysis, 65 isolates were discriminated into 5 different genetic groups from which 12 strains were selected. All 12 strains were able to form biofilm in YPD medium on a polystyrene surface. The presence of microcolonies, filamentous cells and extracellular polymeric substances, constituting the structure of the biofilm despite a small thickness, were highlighted using confocal and electronic microscopy. Moreover, different cell morphologies according to genetic groups were highlighted. The capacity to form biofilm in wine was also revealed for two selected strains. The impact of wine on biofilms was demonstrated with firstly considerable biofilm cell release and secondly growth of these released biofilm cells, both in a strain dependent manner. Finally, B. bruxellensis has been newly described as a producer of chlamydospore-like structures in wine, for both planktonic and biofilm lifestyles.

Inactivation of PadR, the repressor of the phenolic acid stress response, by molecular interaction with Usp1, a universal stress protein from Lactobacillus plantarum, in Escherichia coli.

The phenolic acid decarboxylase gene padA is involved in the phenolic acid stress response (PASR) in gram-positive bacteria. In Lactobacillus plantarum, the padR gene encodes the negative transcriptional regulator of padA and is cotranscribed with a downstream gene, usp1, which encodes a putative universal stress protein (USP), Usp1, of unknown function. The usp1 gene is overexpressed during the PASR. However, the role and the mechanism of action of the USPs are unknown in gram-positive bacteria. Therefore, to gain insights into the role of USPs in the PASR; (i) a usp1 deletion mutant was constructed; (ii) the two genes padR and usp1 were coexpressed with padA under its own promoter as a reporter gene in Escherichia coli; and (iii) molecular in vitro interactions between the PadR, Usp1, and the padA promoter were studied. Although the usp1 mutant strain retained phenolic acid-dependent PAD activity, it displayed a greater sensitivity to strong acidic conditions compared to that of the wild-type strain. PadR cannot be inactivated directly by phenolic acid in E. coli recombinant cultures but is inactivated by Usp1 when the two proteins are coexpressed in E. coli. The PadR inactivation observed in recombinant E. coli cells was supported by electrophoretic mobility shift assays. Although Usp1 seems not to be absolutely required for the PASR, its capacity to inactivate PadR indicates that it could serve as an important mediator in acid stress response mechanisms through its capacity to interact with transcriptional regulators.

Chemical Transfers Occurring Through Oenococcus oeni Biofilm in Different Enological Conditions.

Chardonnay wine malolactic fermentations were carried out to evaluate the chemical transfers occurring at the wood/wine interface in the presence of two different bacterial lifestyles. To do this, Oenococcus oeni was inoculated into must and wine in its planktonic and biofilm lifestyles, whether adhering or not to oak chips, leading to three distinct enological conditions: (i) post-alcoholic fermentation inoculation in wine in the absence of oak chips, (ii) post-alcoholic fermentation inoculation in wine in the presence of oak chips, and (iii) co-inoculation of both Saccharomyces cerevisiae and O. oeni directly in Chardonnay musts in the presence of oak chips. Classical microbiological and physico-chemical parameters analyzed during the fermentation processes confirmed that alcoholic fermentation was completed identically regardless of the enological conditions, and that once O. oeni had acquired a biofilm lifestyle in the presence or absence of oak, malolactic fermentation occurred faster and with better reproducibility compared to planktonic lifestyles. Analyses of volatile components (higher alcohols and wood aromas) and non-volatile components (Chardonnay grape polyphenols) carried out in the resulting wines revealed chemical differences, particularly when bacterial biofilms were present at the wood interface. This study revealed the non-specific trapping activity of biofilm networks in the presence of wood and grape compounds regardless of the enological conditions. Changes of concentrations in higher alcohols reflected the fermentation bioactivity of bacterial biofilms on wood surfaces. These chemical transfers were statistically validated by an untargeted approach using Excitation Emission Matrices of Fluorescence combined with multivariate analysis to discriminate innovative enological practices during winemaking and to provide winemakers with an optical tool for validating the biological and chemical differentiations occurring in wine that result from their decisions.

The Phenotypic Analysis of Lactobacillus plantarum shsp Mutants Reveals a Potential Role for hsp1 in Cryotolerance.

Small heat shock proteins (sHSPs) are ubiquitous, low molecular weight (MW) proteins that share a conserved alpha-crystallin domain. sHSPs oligomers exhibit chaperon-like activities by interacting with unfolded substrates, thereby preventing their aggregation and precipitation. Unlike most lactobacilli, which have single shsp genes, three different sHSP-encoding genes, i.e., hsp1, hsp2, and hsp3, were previously identified in the probiotic Lactobacillus plantarum WCFS1. Early studies, including the characterization of the knock out (KO) mutant for hsp2, indicated a different organization and transcriptional regulation of these genes and suggested that the three L. plantarum sHSPs might accomplish different tasks in stress response. To unravel the role of sHSPs, KO mutants of hsp1 and hsp3 were generated using a Cre-lox based system. Mutation of either genes resulted in impaired growth capacity under normal conditions, heat-stress and stresses typically found during host interactions and food technological process. However, survival to heat shock and the level of thermal stabilization of cytoplasmic proteins were similar between mutants and parental strain. Transcriptional analysis revealed that in the mutant genetic backgrounds there is an upregulated basal expression of the un-mutated mate hsps and other stress-related genes, which may compensate for the loss of HSP function, hence possibly accounting for the lack of a remarkable susceptibility to heat challenge. HSP3 seemed relevant for the induction of thermotolerance, while HSP1 was required for improved cryotolerance. Cell surface properties and plasma membrane fluidity were investigated to ascertain the possible membrane association of sHSP. Intriguingly, the loss of hsp1 was associated to a lower level of maximal membrane fluidity upon heat stress. A role for HSP1 in controlling and improving membrane fluidity is suggested which may pertains its cryoprotective function.

Production of the small heat shock protein Lo18 from Oenococcus oeni in Lactococcus lactis improves its stress tolerance.

Lactococcus lactis is a lactic acid bacterium widely used in cheese and fermented milk production. During fermentation, L. lactis is subjected to acid stress that impairs its growth. The small heat shock protein (sHsp) Lo18 from the acidophilic species Oenococcus oeni was expressed in L. lactis. This sHsp is known to play an important role in protein protection and membrane stabilization in O. oeni. The role of this sHsp could be studied in L. lactis, since no gene encoding for sHsp has been detected in this species. L. lactis subsp. cremoris strain MG1363 was transformed with the pDLhsp18 plasmid, which is derived from pDL278 and contains the hsp18 gene (encoding Lo18) and its own promoter sequence. The production of Lo18 during stress conditions was checked by immunoblotting and the cellular distribution of Lo18 in L. lactis cells after heat shock was determined. Our results clearly indicated a role for Lo18 in cytoplasmic protein protection and membrane stabilization during stress. The production of sHsp in L. lactis improved tolerance to heat and acid conditions in this species. Finally, the improvement of the L. lactis survival in milk medium thanks to Lo18 was highlighted, suggesting an interesting role of this sHsp. These findings suggest that the expression of a sHsp by a L. lactis strain results in greater resistance to stress, and, can consequently enhance the performances of industrial strains.

Effect of Biofilm Formation by Oenococcus oeni on Malolactic Fermentation and the Release of Aromatic Compounds in Wine.

The winemaking process involves the alcoholic fermentation of must, often followed by malolactic fermentation (MLF). The latter, mainly carried out by the lactic acid bacterium Oenococcus oeni, is used to improve wine quality when acidity reduction is required. Moreover, it prevents microbial spoilage and improves the wine's organoleptic profile. Prior observations showed that O. oeni is able to resist several months in harsh wine conditions when adhered on oak barrels. Since biofilm is a prevailing microbial lifestyle in natural environments, the capacity of O. oeni to form biofilms was investigated on winemaking material such as stainless steel and oak chips. Scanning Electron Microscopy and Confocal Laser Scanning Microscopy showed that O. oeni was able to adhere to these surfaces and form spatially organized microcolonies embedded in extracellular substances. To assess the competitive advantage of this mode of life in wine, the properties of biofilm and planktonic cells were compared after inoculation in a fermented must (pH 3.5 or 3.2 and 12% ethanol) The results indicated that the biofilm culture of O. oeni conferred (i) increased tolerance to wine stress, and (ii) functional performance with effective malolactic activities. Relative gene expression focusing on stress genes and genes involved in EPS synthesis was investigated in a mature biofilm and emphasized the role of the matrix in increased biofilm resistance. As oak is commonly used in wine aging, we focused on the O. oeni biofilm on this material and its contribution to the development of wine color and the release of aromatic compounds. Analytical chromatography was used to target the main oak aging compounds such as vanillin, gaiacol, eugenol, whisky-lactones, and furfural. The results reveal that O. oeni biofilm developed on oak can modulate the wood-wine transfer of volatile aromatic compounds during MLF and aging by decreasing furfural, gaiacol, and eugenol in particular. This work showed that O. oeni forms biofilms consisting of stress-tolerant cells capable of efficient MLF under winemaking conditions. Therefore surface-associated behaviors should be considered in the development of improved strategies for the control of MLF in wine.

Protective role of glutathione addition against wine-related stress in Oenococcus oeni.

Oenococcus oeni is the main species responsible for the malolactic fermentation (MLF) of wine due to its ability to survive in this environment. Some wine-related stress factors, such as ethanol and low pH, may alter the cell redox balance of O. oeni. For the first time, the ability to uptake glutathione (GSH), an almost universal tripeptide with antioxidant properties, has been associated to the improvement of stress response in O. oeni. Despite the inability of O. oeni to synthesize GSH, this bacterium can capture it from the media. The ability of 30 O. oeni strains to uptake GSH was assessed in this study. Although all of the strains tested were able to import GSH, substantial variability among them was detected. To assess the physiological function of GSH, three strains with different GSH-import capacities were selected. Significant changes in membrane fatty acids composition were observed due to GSH addition. The most relevant was the increase of cyclopropane fatty acids in cell membrane, in both the exponential and the stationary phases. Cells grown with GSH showed an improved survival against ethanol shock (14% v/v). GSH addition also increased biomass production during the adaptation to wine stress conditions (pH4, pH3.4 and 6% ethanol). The results suggest that GSH enrichment could improve the resistance to stress to O. oeni, which could be useful for the adaptation of MLF starter cultures.

Fungal elicitation of signal transduction-related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula.

Suppressive subtractive hybridization and expressed sequence tag sequencing identified 29 plant genes which are upregulated during the appressorium stage of mycorrhiza establishment between Medicago truncatula J5 (Myc+) and Glomus mosseae. Eleven genes coding plant proteins with predicted functions in signal transduction, transcription, and translation were investigated in more detail for their relation to early events of symbiotic interactions. Expression profiling showed that the genes are activated not only from the appressorium stage up to the fully established symbiosis in the Myc+ genotype of M. truncatula, but also when the symbionts are not in direct cell contact, suggesting that diffusible fungal molecules (Myc factors) play a, role in the induction of a signal-transduction pathway. Transcript accumulation in roots of a mycorrhiza-defective Myc- dmi3 mutant of M. truncatula is not modified by appressorium formation or diffusible fungal molecules, indicating that the signal transduction pathway is required for a successful G. mosseae-M. truncatula interaction leading to symbiosis development. The symbiotic nodulating bacterium Sinorhizobium meliloti does not activate the 11 genes, which supposes early discrimination by plant roots between the microbial symbionts.

Quantification of HSP27 and HSP70 molecular chaperone activities.

Stress-inducible heat-shock proteins (HSPs, like HSP70 and HSP27) are molecular chaperones that -protect cells from stress damage by keeping cellular proteins in a folding competent state and preventing them from irreversible aggregation. HSP27 and HSP70 chaperone activities are useful indicators to test chemical products and physical stress impact on protein denaturation, to select HSP inhibitors, or to -determine the implication of the chaperone function in other HSP activities, such as apoptosis. We have developed two simple and fast chaperone activity tests for HSP27 and HSP70 that we initially set up to test the effect of potential HSP inhibitors obtained after screening of chemical and small molecule libraries. These chaperone quantification tests are based on the capacity of HSP to counteract chemical or thermal protein aggregation.

Inactivation of a small heat shock protein affects cell morphology and membrane fluidity in Lactobacillus plantarum WCFS1.

A small heat shock gene of Lactobacillus plantarum strain WCFS1 was deleted using a Cre-lox based system. Compared to the wild type, the ∆hsp 18.55 mutant strain displayed a similar growth rate when cultivated either under optimal temperature or under different stress conditions such as heat, low pH and salt stress. However, a longer lag phase was observed when the ∆hsp 18.55 mutant strain was cultivated under short intense heat stress (50 °C). This suggests that the hsp 18.55 gene of L. plantarum may be involved in recovery of L. plantarum stressed cells in the early stage of high temperature stress. In addition, morphology of the mutant cells, investigated by scanning electron microscopy, revealed that cells clumped together and had rough surfaces, and that some of the cells had a shrunken empty appearance, which clearly contrasted with the characteristic rod-shaped, smooth-surface morphology of control L. plantarum cells. Furthermore, inactivation of the hsp 18.55 gene affected membrane fluidity and physicochemical surface properties of L. plantarum WCFS1.