Histamine production by wine lactic acid bacteria: isolation of a histamine-producingstrain of Leuconostoc oenos

Populations of Leuconostoc œnos were harvested from wines containing a relatively high concentration of biogenic amines. Cultivation of the biomass in synthetic media and wine showed that it consisted of histamine-producing strains. Histamine levels after culture depended on the quantity of precursor available and on the presence of yeast lees, which certainly enriched the medium in histidine. Ethanol and pH, which control bacterial growth rate and total population, were also significant factors: pH and low ethanol concentration enhanced histamine production.
Strain Leuc. œnos 9204 was isolated and studied since it retained its ability to produce histamine after several transfers. In synthetic medium this strain produced large amounts of histamine especially in the poorest nutritional conditions (no glucose, no L-malic acid). These results clearly demonstrate that Leuc. œnos involvedin wine-making might play a role in biogenic amine production. The vinification method might also influence the final amine concentration in wine.     

Malolactic fermentation of wine: study of the influence of some physico-chemical factors by experimental design assays

The ability of selected lactic acid bacteria to carry out malolactic fermentation depends on the level of numerous wine characteristics. A Hadamard's experimental matrix was used to determine the main effects of 11 physico-chemical factors on malolactic activity of three Leuconostoc œnos strains and one Lactobacillus plantarum strain. Ethanol had the greatest inhibitory effect on the achievement of malolactic fermentation for all Leuc. œnos strains. An inhibitory effect of the L-malic acid was also found in the operating conditions. These strains show different degrees of sensitivity to pH. One of these strains was inhibited by SO₂. Malolactic activity of the Lact. plantarum strain is mainly affected by a low pH, and this strain is often less efficient than Leuc. œnos strains. This methodology could be used for the selection of strains for malolactic starters. Further work is in progress using factorial design in order to determine the interactions between influential factors.     

Development of a detection system for histidine decarboxylating lactic acid bacteria based on DNA probes, PCR and activity test

On the basis of the comparison of the nucleotide sequences of the histidine decarboxylase genes ( hdc A) of Lactobacillus 30A and Clostridium perfringens and the amino acid sequences of these histidine decarboxylases and those of Lactobacillus buchneri and Micrococcus , oligonucleotides unique to the hdc A genes were synthesized and used in PCR. All histidine-decarboxylating lactic acid bacteria gave a signal with primer set JV16HC/JV17HC in PCR. In addition to this primer set, CL1/CL2 and CL1/JV17HC were also useful for the detection of histamine-forming Leuconostoc œnos strains in PCR. The 150 base pair amplification product of the decarboxylating Leuc. œnos strain generated with primer set CL1/CL2 was sequenced. Alignment studies showed a high degree of relatedness among the hdc A gene products of Gram-positive bacteria.
The amplification products of the hdc A genes from Lact. buchneri and Leuc. œnos were used to serve as a DNA probe in hybridization studies. All histidine-decarboxylating lactic acid bacteria gave a hybridization signal with the DNA probes. In hybridization only one false-positive signal with a Lactobacillus lindneri strain was observed, which was anticipated to contain a truncated hdc A gene.
In addition to these DNA probe tests, a simple and reliable activity test is presented, which can be used during starter selection to test strains for histidine decarboxylase activity.     

Histidine carboxylase of Leuconostoc œnos 9204: purification, kinetic properties, cloning and nucleotide sequence of the hdc gene

Histidine decarboxylase (HDC) was purified to homogeneity from Leuconostoc ' nos 9204, a wine lactic acid bacterium. Histidine decarboxylase comprised two subunits, respectively α and β. The hdc gene was cloned and sequenced. The gene encodes a single polypeptide of 315 amino acids, demonstrating that Leuc. ' nos 9204 HDC was synthesized as a precursor proHDC π₆ (Mr 205 000). A cleavage between Ser-81 and Ser-82 generated the α (Mr 25 380) and β (Mr 8840) chains, which suggested that the holoenzyme exists as a hexameric structure (αβ)₆. At the optimal pH of 4·8, the HDC activity exhibited a simple Michaelis-Menten kinetic ( K _m = 0·33 mmol l⁻¹, V _max = 17·8 μmol CO₂ min⁻¹ mg⁻¹), while at pH 7·6 it was sigmoidal (cooperativity index of 2). Histamine acted as a competitive inhibitor ( K _i = 32 mmol l⁻¹). The similarities of these results with those described for other bacterial HDC support the assumption that the pyruvoyl enzymes evolved from a common ancestral protein and have similar catalytic mechanisms. These results also confirmed that the main lactic acid bacterial species responsible for malolactic fermentation in red wine is able to produce histamine. Bacteria carrying the HDC activity must be avoided during selection of strains for the production of malolactic starters.     

Identification of Outer Membrane Proteins Altered in Response to UVC-Radiation in Vibrio parahaemolyticus and Vibrio alginolyticus

Vibrio parahaemolyticus and V. alginolyticus, marine foodborne pathogens, were treated with UVC-radiation (240 J/m²) to evaluate alterations in their outer membrane protein profiles. Outer membrane protein patterns of UVC-irradiated bacteria were found altered when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Altered proteins were identified by mass spectrometry (MS and MS/MS) and analysis revealed that OmpW, OmpA, Long-chain fatty acid transport protein, Outer membrane receptor protein, Putative uncharacterized protein VP0167, Maltoporin (lamB), Polar flagellin B/D, Agglutination protein Peptidoglycan-associated lipoprotein and MltA-interacting protein MipA were appeared, thereby they can be considered as UVC-stress proteins in some vibrios. In addition, expression of OmpK decreased to non-detectable level. Furthermore, we observed a decrease or an increase in the expression level of other outer membrane proteins.     

Viability of dried vegetative cells and the formation and germination of reproductive structures inPithophora œdogonia, Cladophora glomerata andRhizoclonium hieroglyphicum under water stress

All dried vegetative cells ofPithophora œdogonia died within 1 h, while those ofCladophora glomerata andRhizoclonium hieroglyphicum retain viability to some extent for 1 and 8 d, respectively, under similar storage conditions. The viability of dried vegetative cells of eitherC. glomerata orR. hieroglyphicum decreased more or less equally when stored either at 20 °C. in light or dark or at 12 °C in dark, but was lost rapidly and drastically when stored at 0 °C in dark. Both dried and wet akinetes ofP. œdogonia were equally more viable when stored at 20 °C in dark than in light, but they lost germination ability when stored either at 12 or 0 °C in dark; this might be either due to loss of viability or dormancy induction at low temperatures. The water stress imposed by growing vegetative filaments either on highly agarized media, in NaCl-supplemented liquid media or in media undergoing progressive air-drying to complete dryness did not induce, but reduced akinete formation inP. œdogonia, decreased zoosporangium formation inC. glomerata andR. hieroglyphicum, decreased or totally suppressed akinete germination inP. œdogonia and zoospore germination inC. glomerata andR. hieroglyphicum. Akinetes ofP. œdogonia formed under water stress were equally viable, while zoosporangia ofC. glomerata andR. hieroglyphicum formed under water stress were comparatively less viable than those formed without any water stress. Akinete germination inP. œdogonia and zoospore germination inC. glomerata andR. hieroglyphicum were comparatively more sensitive to water stress than the formation of akinetes and zoosporangia. The akinete germination inP. œdogonia was more sensitive to water stress than zoospore germination inC. glomerata andR. hieroglyphicum and it might be either due to their large size, thick wall or dense content.     

Proteomics approach to identify wound-response related proteins from rice leaf sheath

In order to avoid the complex conditions of the intact plant for simple analysis of proteins in wound-response stress, we used the detached rice leaf sheath which is a very active part of the rice seedling. Proteins were extracted from rice leaf sheath at 0, 12, 24, 48 h after cutting and separated by two-dimensional (2-D) polyacrylamide gel electrophoresis. Changes in differentially displayed proteins were found in leaf sheaths after cutting in the 0-48 h time course. Ten proteins were up-regulated, while 19 proteins were down-regulated compared with those on the four 2-D gels. Among them, 14 proteins were analyzed by N-terminal, or internal amino acid sequence. The clear functions of nine proteins could be identified. Six proteins did not yield amino acid sequence information due to their blocked N-termini. Furthermore, 11 proteins were determined by matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and identified protein database matching. It was shown that the down-regulated proteins were calreticulin (nos. 5, 6), histone H1 (no. 15) and hemoglobin (no. 17), putative peroxidase (no. 19); the up-regulated proteins were Bowman-Birk trypsin inhibitor (no. 23), putative receptor-like protein kinase (nos. 24, 25), calmodulin-related protein (no. 26), small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (no. 27), mannose-binding rice lectin (nos. 28, 29). Among all the above proteins, four (nos. 23, 24, 25, 26) have been confirmed to be wound-response proteins. The others cannot be excluded as also being related to wound-responses, such as the signal transduction-related proteins (nos. 5, 6), photosynthesis-related protein (no. 27), and stress-response proteins (nos. 19, 28, 29). This is the first time protein changes in response to wounding in rice leaf sheath have been shown.     

Molecular and biochemical properties of the S-layer protein from the wine bacterium <Emphasis Type="Italic">Lactobacillus hilgardii</Emphasis> B706

Different strains of the genus Lactobacillus can be regularly isolated from must and wine samples. By various physiological activities, they can improve or reduce the wine quality. Lactobacillus hilgardii that is known to survive under harsh wine conditions is classified as a spoilage bacterium, e.g. due to the production of histamine. Many lactobacilli form an S-layer as the outermost cell wall component which has been found to facilitate the colonization of special ecological niches. A detailed understanding of the properties related to their S-layer proteins is necessary to improve the knowledge of the interactions between different bacterial cells and with the surrounding environments. The S-layer protein from the wine-related L. hilgardii strain B706 has been isolated and its gene sequence determined. The deduced amino acid sequence corresponds to a 41 kDa protein with an isoelectric point of 9.6 without additional posttranslational modifications after splitting off the leader peptide. The complete protein is organized in a 32 amino acids signal sequence for membrane translocation, a positively charged N-terminal domain that binds to the cell wall and a negatively charged C-terminal domain. When the S-layer was removed, the corresponding L. hilgardii B706 cells became more sensitive to bacteriolytic enzymes and some wine-related stress conditions. From a practical point of view, the S-layer may be considered as a target for the inhibition of food-spoiling lactobacilli.     

Proteomic analysis of malignant lymphocyte membrane microparticles using double ionization coverage optimization

Shed membrane microparticles (MPs) are microvesicles generated from the plasma membrane when cells are submitted to stress conditions. Although MPs reflect the cell state (at least in vitro), little is known on their protein composition. We describe the first set of experiments aiming to characterize the MP proteome. Two ways of triggering MP formation from a T-lymphocytic cell line were analyzed using a 1-D gel approach coupled with LC-MS/MS and the results were compared with those obtained from a classic membrane preparation. In total, 390 proteins were identified in MPs, among which 34% were localized to the plasma membrane. The MPs revealed a broad representation of plasma membrane proteins including 17 hematopoietic clusters of differentiation. This approach was successfully applied to one human chronic B-cell lymphoid malignancy. In all, 413 proteins were identified, including 117 membrane proteins, many of them being pathology associated. The sequence coverage in identified proteins was improved combining both nano-LC-MS/MS and MALDI-MS data. The suppression effect, observed on very complex peptide mixtures, was remediated by chromatographic fractionation. MPs may represent a new tool for studying plasma membrane proteins, displaying the advantages of reproducibility, minimal organelle contamination, and being potentially applicable to most cell types.     

The deletion of Ffh in Escherichia coli induces adverse physiological and morphological changes

The Escherichia coli Ffh protein is homologous to the SRP54 subunit of the eukaryotic signal recognition particle (SRP) that is involved in targeting and translocation of membrane proteins. The functions of Ffh in E. coli were investigated using the mutant with the Ffh deficiency. The mutant showed lower growth rate at 30°C and rapidly lost viability at the non-permissive temperature of 42°C. In addition, the amount of the total membrane proteins decreased sharply in the mutant. The mutant cells cultured at either 30 or 42°C appeared to have an elongated shape as compared to the wild type cells. Transmission electron microscopy revealed that the membrane layer of the mutant cells was thinner than that of the wild type cells. The article is published in the original.     

ROS accumulation and oxidative damage to cell structures in Saccharomyces cerevisiae wine strains during fermentation of high-sugar-containing medium

To further elucidate the impact of fermentative stress on Saccharomyces cerevisiae wine strains, we have here evaluated markers of oxidative stress, oxidative damage and antioxidant response in four oenological strains of S. cerevisiae, relating these to membrane integrity, ethanol production and cell viability during fermentation in high-sugar-containing medium. The cells were sampled at different fermentation stages and analysed by flow cytometry to evaluate membrane integrity and accumulation of reactive oxygen species (ROS). At the same time, catalase and superoxide dismutase activities, trehalose accumulation, and protein carbonylation and degradation were measured. The results indicate that the stress conditions occurring during hypoxic fermentation in high-sugar-containing medium result in the production of ROS and trigger an antioxidant response. This involves superoxide dismutase and trehalose for the protection of cell structures from oxidative damage, and protein catabolism for the removal of damaged proteins. Cell viability, membrane integrity and ethanol production depend on the extent of oxidative damage to cellular components. This is, in turn, related to the ‘fitness’ of each strain, which depends on the contribution of individual cells to ROS accumulation and scavenging. These findings highlight that the differences in individual cell resistances to ROS contribute to the persistence of wine strains during growth under unfavourable culture conditions, and they provide further insights into our understanding of yeast behaviour during industrial fermentation.     

Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism

Grapes (Vitis vinifera) are a valuable fruit crop and wine production is a major industry. Global warming and expanded range of cultivation will expose grapes to more temperature stresses in future. Our study investigated protein level responses to abiotic stresses, with particular reference to proteomic changes induced by the impact of four different temperature stress regimes, including both hot and cold temperatures, on cultured grape cells. Cabernet Sauvignon cell suspension cultures grown at 26°C were subjected to 14 h of exposure to 34 and 42°C for heat stress, and 18 and 10°C for cold stress. Cells from the five temperatures were harvested in biological triplicates and label‐free quantitative shotgun proteomic analysis was performed. A total of 2042 non‐redundant proteins were identified from the five temperature points. Fifty‐five proteins were only detected in extreme heat stress conditions (42°C) and 53 proteins were only detected at extreme cold stress conditions (10°C). Gene Ontology (GO) annotations of differentially expressed proteins provided insights into the metabolic pathways that are involved in temperature stress in grape cells. Sugar metabolism displayed switching between alternative and classical pathways during temperature stresses. Additionally, nine proteins involved in the phenylpropanoid pathway were greatly increased in abundance at extreme cold stress, and were thus found to be cold‐responsive proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD000977 ( http://proteomecentral.proteomexchange.org/dataset/PXD000977 ).     

Factors affecting the induction of malolactic fermentation in red wines with Leuconostoc oenos

Malolactic fermentation was induced in red wines by inoculation with several strains of Leuconostoc oenos . The progress of Malolactic Fermentation was monitored by following the kinetics of bacterial growth and degradation of malic acid. These kinetics varied significantly depending on the strain of Leuc. oenos inoculated, the strain of Saccharomyces cerevisiae used to conduct the alcoholic fermentation, and the wine properties of pH and concentrations of ethanol and sulphur dioxide. Rapid, predictable malolactic fermentation was achieved by inoculating a high density (> 10⁶ cfu/ml) of Leuc. oenos , whereby malic acid degradation was not connected to the growth of the bacterial cells. Wines after malolactic fermentation were not bacteriologically stable and supported the growth of Leuc. oenos inoculated into the wines.     

ROS accumulation and oxidative damage to cell structures in Saccharomyces cerevisiae wine strains during fermentation of high-sugar-containing medium

To further elucidate the impact of fermentative stress on Saccharomyces cerevisiae wine strains, we have here evaluated markers of oxidative stress, oxidative damage and antioxidant response in four oenological strains of S. cerevisiae, relating these to membrane integrity, ethanol production and cell viability during fermentation in high-sugar-containing medium. The cells were sampled at different fermentation stages and analysed by flow cytometry to evaluate membrane integrity and accumulation of reactive oxygen species (ROS). At the same time, catalase and superoxide dismutase activities, trehalose accumulation, and protein carbonylation and degradation were measured. The results indicate that the stress conditions occurring during hypoxic fermentation in high-sugar-containing medium result in the production of ROS and trigger an antioxidant response. This involves superoxide dismutase and trehalose for the protection of cell structures from oxidative damage, and protein catabolism for the removal of damaged proteins. Cell viability, membrane integrity and ethanol production depend on the extent of oxidative damage to cellular components. This is, in turn, related to the 'fitness' of each strain, which depends on the contribution of individual cells to ROS accumulation and scavenging. These findings highlight that the differences in individual cell resistances to ROS contribute to the persistence of wine strains during growth under unfavourable culture conditions, and they provide further insights into our understanding of yeast behaviour during industrial fermentation.     

Changes of plasma membrane proteins during prespore differentiation in Dictyostelium discoideum

By the use of a shake culture system, we have previously shown (Oyama, M., Okamoto, K., & Takeuchi, I. (1982) J. Cell Sci. 56, 223-232) that both cAMP and cAMP-dependent cell contact are required for prespore differentiation in Dictyostelium discoideum. The present study was undertaken to examine changes of the plasma membrane proteins during prespore differentiation in the shake culture system. Rabbit antibodies prepared against the plasma membrane fraction of the differentiated cells inhibited the reaggregation of the differentiated cells but not that of aggregation-competent cells. This result indicates that new contact sites are formed in the differentiated cells. By the combined use of the antibody-conjugated immuno-adsorbent with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, changes of membrane proteins were analyzed with the cells incubated under various conditions. Three proteins were found to be present specifically in the differentiated cells only in the presence of cAMP, one of which (105K protein) appeared when cells became adhesive, but before prespore specific proteins were detected. Two others (80K and 58K proteins) appeared during prespore differentiation after cells formed agglomerates.     

Interaction of isolated components from mammalian sperm and egg

In order to identify those proteins from the plasma membrane of hamster spermatozoa that exhibit an affinity for components of the zona pellucida we have used the Western blot technique. Zonae pellucidae from postovulatory hamster oocytes were solubilized by exposure to an acidic pH and then radiolabeled using the Bolton-Hunter reagent. These ¹²⁵I-zona pellucida proteins retain their immunoreactivity and migrate in three heterogeneous bands when submitted to SDS-PAGE electrophoresis. Membrane proteins from epididymal spermatozoa of mature hamsters were extracted by treatment with Nonidet P-40 and then submitted to electrophoresis (SDS-PAGE). The proteins in the gel were electrophoretically transferred to a nitrocellulose membrane and then probed with the radiolabelled zone pellucida proteins. ¹²⁵I-zona pellucida proteins bind preferentially to a sperm protein with a molecular weight of 26,400 ± 1,400 daltons (n = 9). Using a similar procedure it was shown that this protein also binds ¹²⁵I-Concanavalin A. The interaction between the sperm protein and the ¹²⁵I-zona pellucida proteins shows species specificity as demonstrated by the fact that the hamster ¹²⁵I-zona pellucida proteins do not bind to proteins extracted from ram, bull, and stallion spermatozoa. Whether this sperm protein could be implicated be implicated in the process of sperm-egg interaction is under investigation.     

Impact of oxidative stress on the function,abundance, and turnover of the Arabidopsis 80S cytosolic ribosome

Abiotic stress in plants causes accumulation of reactive oxygen species (ROS) leading to the need for new protein synthesis to defend against ROS and to replace existing proteins that are damaged by oxidation. Functional plant ribosomes are critical for these activities, however we know little about the impact of oxidative stress on plant ribosome abundance, turnover, and function. Using Arabidopsis cell culture as a model system, we induced oxidative stress using 1 µm of H₂O₂ or 5 µm menadione to more than halve cell growth rate and limit total protein content. We show that ribosome content on a total cell protein basis decreased in oxidatively stressed cells. However, overall protein synthesis rates on a ribosome abundance basis showed the resident ribosomes retained their function in oxidatively stressed cells. ¹⁵N progressive labelling was used to calculate the rate of ribosome synthesis and degradation to track the fate of 62 r‐proteins. The degradation rates and the synthesis rates of most r‐proteins slowed following oxidative stress leading to an ageing population of ribosomes in stressed cells. However, there were exceptions to this trend; r‐protein RPS14C doubled its degradation rate in both oxidative treatments. Overall, we show that ribosome abundance decreases and their age increases with oxidative stress in line with loss of cell growth rate and total cellular protein amount, but ribosome function of the ageing ribosomes appeared to be maintained concomittently with differences in the turnover rate and abundance of specific ribosomal proteins. Data are available via ProteomeXchange with identifier PXD012840.     

Analysis of Proteins Expressed by an Abiotic Stress Tolerant Pseudomonas putida (NBAII-RPF9) Isolate Under Saline and High Temperature Conditions

Pseudomonas putida (NBAII-RPF9) was identified as an abiotic stress tolerant bacterium capable of growing at 45 °C as well as in 1 M NaCl. The proteins expressed by this bacterium when subjected to these two stresses were analyzed by 2D gel and MALDI-TOF/MS. Two parameters viz., heat/saline shock (20 min at 45 °C/1 M solid NaCl added at mid log phase and incubated for 1 h) and heat/saline tolerance (24 h growth at 45 °C/in 1 M NaCl) were studied. Under heat shock 13 upregulated proteins and 1 downregulated protein were identified and under tolerance 6 upregulated proteins were identified. GroES and GroEL proteins were expressed under both tolerance and shock. Under saline shock 11 upregulated proteins were identified whereas under saline tolerance 6 upregulated proteins were identified and all these proteins had pI between 3 and 10 with molecular weights ranging from 14.3 to 97 kDa. Aspartate carbamoyltransferase was common under both the saline conditions studied. The analysis revealed involvement of heat stress responsive molecular chaperones and membrane proteins during heat stress. During salt stress, proteins involved in metabolic processes were found to be upregulated to favor growth and adaptation of the bacterium. Heat shock chaperones viz., DnaK and DnaJ were expressed under both saline and heat stress. This is the first report of protein profile obtained from a single bacterium under saline and heat stress and the studies reveal the complex mechanisms adapted by the organism to survive under high temperature or saline conditions.