text:
Andrea Gomez-Zavaglia
Center for Research and Development in Food, Faculty of Exact Sciences, National University of La Plata, Argentina
E. E. Tymczyszyn
Universidad Nacional de Quilmes (UNQ), Argentina
Jose Antonio Curiel
Research and Development Center of Functional Food (CIDAF), Spain
Front. Microbiol., 30 September 2016 | https://doi.org/10.3389/fmicb.2016.01554
Transcriptomic and Proteomic Analysis of Oenococcus oeni Adaptation to Wine Stress Conditions
Mar Margalef-Català, Isabel Araque, Albert Bordons, Cristina Reguant and Joaquín Bautista-Gallego*
Departament de Bioquímica i Biotecnologia, Facultat d'Enologia, Universitat Rovira i Virgili, Tarragona, Spain
Oenococcus oeni, the main lactic acid bacteria responsible for malolactic fermentation in wine, has to adapt to stressful conditions, such as low pH and high ethanol content. In this study, the changes in the transcriptome and the proteome of O. oeni PSU-1 during the adaptation period before MLF start have been studied. DNA microarrays were used for the transcriptomic analysis and two complementary proteomic techniques, 2-D DIGE and iTRAQ labeling were used to analyze the proteomic response. One of the most influenced functions in PSU-1 due to inoculation into wine-like medium (WLM) was translation, showing the over-expression of certain ribosomal genes and the corresponding proteins. Amino acid metabolism and transport was also altered and several peptidases were up regulated both at gene and protein level. Certain proteins involved in glutamine and glutamate metabolism showed an increased abundance revealing the key role of nitrogen uptake under stressful conditions. A strong transcriptional inhibition of carbohydrate metabolism related genes was observed. On the other hand, the transcriptional up-regulation of malate transport and citrate consumption was indicative of the use of L-malate and citrate associated to stress response and as an alternative energy source to sugar metabolism. Regarding the stress mechanisms, our results support the relevance of the thioredoxin and glutathione systems in the adaptation of O. oeni to wine related stress. Genes and proteins related to cell wall showed also significant changes indicating the relevance of the cell envelop as protective barrier to environmental stress. The differences found between transcriptomic and proteomic data suggested the relevance of post-transcriptional mechanisms and the complexity of the stress response in O. oeni adaptation. Further research should deepen into the metabolisms mostly altered due to wine conditions to elucidate the role of each mechanism in the O. oeni ability to develop MLF.
Malolactic fermentation (MLF) occurs in wine spontaneously and, alternatively, can be induced inoculating selected strains of lactic acid bacterium (LAB), mainly Oenococcus oeni, usually after alcoholic fermentation (Betteridge et al., 2015). The wide range of physiological characteristics and the ability to cope with several environmental stresses make O. oeni the main responsible for MLF. This process consists in the conversion of L-malate to L-(+)-lactate and CO2 and is required in wine, mainly from red grape varieties, because it confers positive sensory traits and improves wine's microbiological stability (Lonvaud-Funel, 1999; Mills et al., 2005).
Wine is a harsh environment for O. oeni due to its physicochemical characteristics, such as low pH, ethanol and SO2 content, which can negatively affect bacterial survival and consequently MLF development. In contrast to the diversity of stress response mechanisms described in Bacillus subtilis (Hecker et al., 1996; Hecker and Völker, 1998), the model organism for Gram-positive bacteria, no gene encoding an alternative sigma factor or any other known regulator of stress response, such as HrcA, could be identified in O. oeni. Grandvalet et al. (2005) described in O. oeni the CtsR as the regulator for most of molecular chaperone genes. Different studies have characterized some of the stress response genes in O. oeni, such as clp, grpE, groES, hsp18, hdc, ftsH, omrA, cfa, atpB, and trxA, among others (Jobin et al., 1997; Guzzo et al., 2000; Bourdineaud et al., 2003, 2004; Beltramo et al., 2004, 2006; Bourdineaud, 2006; Spano and Massa, 2006; Olguín et al., 2009, 2010). These studies revealed that O. oeni has developed cellular mechanisms that make it more resistant to adverse conditions than other LAB species (Beltramo et al., 2006). The knowledge of the stress response machinery of this bacterium is key to understand the ability of adaptation to wine environment of each strain and select the best starter culture.
Thanks to the publication over the recent years of the genomes of different O. oeni strains in the National Center for Biotechnology Information (NCBI), nowadays it is possible the global study of stress response by "omics" technologies. There are two published studies of O. oeni combining transcriptomic and proteomic analysis, applying DNA microarrays and 2DE or 2D-DIGE: Olguín et al. (2015) studied the effect of ethanol addition during growth (after 1 h), and Costantini et al. (2015) studied wine like media adaptation during 24 h. The first proteomic study was from Silveira et al. (2004) and showed that both ethanol stress and adaptation significantly changed the protein profiles of O. oeni cells. Later, Cecconi et al. (2009) performed a proteomic study using 2DE examining O. oeni adaptation to wine conditions. Other authors have studied oenological starters to determine their proteomic profile (Cafaro et al., 2014; Napoli et al., 2014).
In this work, we combined a transcriptomic and proteomic approach to elucidate the changes involved in the adaptation of O. oeni PSU-1 to wine-like conditions, evaluating the period between the inoculation and the beginning of MLF. The transcriptomic analysis was developed using DNA microarrays designed for PSU-1 strain and the results obtained were validated by real-time qPCR. For the proteomic study two complementary techniques were employed: 2D-DIGE and iTRAQ labeling. 2D-DIGE technique (Unlü et al., 1997) relies on a pre-electrophoretic labeling, allowing sample multiplexing in the same gel. This gel-dependent technique has been used in several studies with other LAB species (Mehmeti et al., 2011; Koponen et al., 2012; Genovese et al., 2013). However, the variable reproducibility of this technique along with the difficult automation and detection of low abundance and membrane hydrophobic proteins have led to a wide variety of off-gel methodologies for protein quantification. In order to complement DIGE analysis, in this work it was used a tandem mass spectrometry (MS/MS) coupled with isobaric tags for relative and absolute quantification (iTRAQ) (Ross et al., 2004) labeling to enable the identification and quantification of differentially expressed proteins in specific times. The combination of liquid chromatography (LC) and electrospray ionization MS/MS analysis is an emerging powerful methodology enabling quantification and comparison of protein levels directly from samples with greater efficiency and accuracy. This is the first proteomic analysis using this gel-free technique with O. oeni.
Growth Conditions and MLF Monitoring
The strain used in this study was O. oeni PSU-1, the only in its species with the genome fully annotated (Mills et al., 2005). Stock cultures (kept frozen at −80°C) were grown in MRS broth medium (De Man et al., 1960) supplemented with 4 g/L L-malic acid and 5 g/L fructose at pH 5.0 at 28°C in a 10% CO2 atmosphere. Cells were collected at the end of the exponential phase (OD600nm = 1.4 − 1.6) and inoculated into the medium. Then, cells were harvested at the end of the exponential phase and inoculated (2% v/v) into 5 L screw-cap bottles of wine-like medium (WLM). WLM was prepared following (Bordas et al., 2015) containing 12% ethanol (v/v) at pH 3.4. The bottles were incubated at 20°C. The assays were run in triplicate. Measurements of L-malic acid consumption were performed with the multianalyser Miura One (I.S.E. S.r.l, Guidonia, Italy) and the enzymatic kit ready to use (TDI SL, Barcelona, Spain) in order to determine the beginning and evolution of MLF.
RNA and Protein Extraction
After the inoculation into WLM, samples at different times (0, 0.5, 1, 2, 4, 6, and 8 h) were taken for RNA and protein extraction. For RNA extraction, 20 mL were collected from WLM, or 0.3 mL from MRS culture used for inoculation (0 h). Samples were centrifuged at 10,000 × g for 5 min at 4°C, supernatant was removed and pellet was washed with 10 mM Tris-HCl prepared with diethylpyrocarbonate-treated water (DEPC), and then frozen in liquid nitrogen and kept at −80°C until RNA extraction. High Pure RNA Isolation Kit (Roche, Mannheim, Germany) was used for RNA extraction following the instructions of the manufacturer with some modifications, such as lysis with lysozyme dissolved in 10 mM Tris-HCl buffer DEPC, at 50 mg/mL during 30 min at 37°C. RNA was treated with Turbo DNA-free (Life Technologies, USA). Total acid nucleic concentrations were calculated using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Bremen, Germany).
For protein extract preparation, 800 mL of WLM, or 35 mL from MRS culture used for inoculation (0 h), were centrifuged at 5000 × g rpm for 15 min. Supernatant was removed and pellet was washed twice with 10 mM Tris-HCl buffer at pH 8, frozen in liquid nitrogen and kept at −80°C until protein extraction. Cell pellets were then resuspended to a final OD600 = 30 in a solution of 0.1 M Tris-HCl at pH 7.5, mixed with protease inhibitor cocktail from Roche. Cells were disrupted using One-shot disruptor (Constant Systems Ltd.) at 5°C, applying twice a 2.7 kbar pressure. Protein suspension was centrifuged at 4500 × g for 15 min at 4°C to remove cell debris and the supernatant was frozen in liquid nitrogen until protein analysis.
Transcriptomic Analysis
DNA Microarray Description, Labeling, and Hybridization
Microarrays (090324_Oenococcus oeni expression 4-plex array), based on PSU-1 genome, were developed by Roche NimbleGen (Madison, WI, USA) and samples were analyzed at the Functional Genomics Core of the Institute for Research in Biomedicine (IRB, Barcelona, Spain) as described by Olguín et al. (2015). The results were submitted to GEO (Gene Expression Omnibus Database, NCBI) under accession number GSE85137.
Microarray Results Validation by Real-Time qPCR
Nucleotide sequences of O. oeni strain PSU-1 (NC_008528) were obtained from the NCBI. Several genes were selected for real-time qPCR validation of the microarray data. The primers used for these analyses are shown in Table 1. Some genes were selected due to their involvement in stress response according to previous studies (Jobin et al., 1999b; Beltramo et al., 2006; Olguín et al., 2009; Bordas et al., 2013; Margalef-Català et al., 2017) and others were randomly selected with the sole objective of validating the methodology. Reverse transcription, real-time qPCR and primer design were performed according to Olguín et al. (2009). The Primer Express® Software was used to select primer sequences and analyze secondary structures and dimer formation. The absence of chromosomal DNA contamination was confirmed by qPCR. For the normalization of qPCR data (Vandesompele et al., 2002; Sumby et al., 2012; Cafaro et al., 2014) four genes (ldhD, dpoIII, gyrA, and gyrB) were evaluated as internal controls, using the primers described in Table 1. Of these, ldhD and gyrA genes showed the lowest variation under the experimental conditions used (data not shown) and were chosen as internal controls. The amplification efficiency was calculated as in Olguín et al. (2015).
Table 1. Gene descriptions and the corresponding primer sequences used for validation of microarray results by real-time qPCR.
Proteomic Analysis
2D-DIGE
Protein extracts were analyzed in the Center for Omic Sciences from Servei de Recursos Científics i Tècnics of University Rovira i Virgili (Reus, Spain). Proteins were precipitated using TCA/Acetone, and the pellet was resuspended in 200 μL of rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris-base) at final pH 8.5. The samples were quantified using Bradford and stored at −20°C. Fifty microgram of each protein sample was minimally labeled with 400 pmol of either Cy3 or Cy5 (N-hydroxy succinimidyl ester-derivatives of the cyanine dyes). To facilitate image matching and cross-gel normalization, an internal standard was made pooling all samples and labeling with Cy2 at the same ratio (50 μg:400 pmols). Hence, two samples and the internal standard could be run in the same gel and quantified on multiple 2-DE. Labeling reactions were performed on ice and darkness during 30 min and quenched using an excess of free L-lysine.
Isoelectrofocusing (IEF) was carried out using 24 cm Immobiline Dry-strips (pH interval 4–7, nonlinear, GE Healthcare), and sample was loaded by two rehydration steps (passively for 5 h at 20°C, and actively at 50 V during 12 h in an Ettan IPGphor 3 system from GE Healthcare). IEF migration program started focusing 500 V for 7 h, ramping until 1 KV during 4 h and ramping again until 10 KV during 3 h, and finally a step maintained at 10 KV to reach 70 KVh. Strips were then equilibrated for 15 min in a 50 mM Tris–HCl (pH 8.8) 6 M urea, 30% glycerol and 2% SDS buffer, adding first 1% DTT, and in a second time supplemented with 4% iodoacetamine (Görg et al., 2004).
Imaging and data processing
Gels were scanned using a PharosFX™ Plus Molecular Imager and analyzed using Progenesis Same Spots Analysis Software v4.5 (Totallab). Spots displaying a ≥1 average-fold increase or decrease in abundance with a p < 0.05 were selected for protein identification. Features detected from non-protein sources (e.g., dust particles and dirty backgrounds) were filtered out. Picking analysis was carried loading 720 μg of the internal standard mix without labeling in a 2DE gel as described above. Gels were stained with Coomassie blue G250 and imaged using Pharos FXTM Plus Molecular Imager from BioRad using Quantity One version 4.6.9 software.
In gel-trypsin digestion and MS-Based protein identification
Spots of interest were automatically excised from 2-DE gels using the ExquestTM Spot Cutter with the PDQuestTM Advanced 2D Analysis Software V8.0.1 both from Bio-Rad. Excised spots were de-hydrated by extensive washings with 25 mM ammonium bicarbonate and acetonitrile. All gel pieces were incubated with 15 ng/μL sequencing-grade trypsin in 50 mM ammonium bicarbonate at pH 7.9 overnight at 37°C. Following digestion, the peptides were desalted using C18 zip-tip (Millipore), and eluted with 75% ACN + 0.1% Trifluoroacetic acid.
Peptides were spotted onto an HTP BigAnchor 384 (Bruker) target using α-cyano-4-hydroxy-cinnamic acid as matrix and were analyzed on a MALDI TOF/TOF (Ultraflextreme, Bruker Daltonics, Bremen, Germany) instrument operated in the positive ion mode. All mass spectra were calibrated externally with the Peptide Calibration Standard I from Bruker. The analyzed mass range was 600–3500 Da. MS and MS/MS analyses were performed automatically. For MS analysis, 3100 satisfactory shots were accumulated by recording 100-shot steps at 20 random positions using fuzzy control laser attenuation between 40 and 100% at initial and maximal power respectively. For MS/MS 3100 satisfactory shots were accumulated by recording 100-shot steps and 4000 for the fragment ion spectra.
MS and tandem MS/MS spectra were searched by Protein Scape v: 3.0.0 446 using MASCOT (Matrix Science Inc., MA, 2.4.0) against NCBInr database (46742655 entries) restricted to Bacteria (Eubacteria). The search parameters were set to: MS accuracy 20 ppm, MS/MS accuracy 0.5 Da, two missed cleavage by trypsin allowed, carbamidomethylation of cysteine as fixed modification and oxidation of methionine and N-terminal amino acid conversion of glu and gln to pyroglutamic acid as variable modification. Significant protein hits (for peptide mass fingerprinting p ≤ 0.05 and for MS/MS at least two peptides with p ≤ 0.05, and protein scores greater than 30) were considered significant.
iTRAQ Labeling
Protein digestion and iTRAQ labeling
On a SDS-PAGE gel (12% resolving gel and 4% stacking gel) at 20 mA/gel 40 μg total protein per sample were run. The electrophoresis was stopped when the front dye had barely passed from the stacking gel into the resolving gel, and a unique concentrated band was obtained for every sample, which was stained using Coomassie Brilliant Blue G-250, excised, cut into small pieces and stored at 4°C in ultrapure water.
Protein digestion was performed according to Shevchenko et al. (1996) with minor variations as described before. Proteins were reduced using 5 mM tris(2-carboxyethyl)phosphine (TCEP) in 50 mM triethylammonium bicarbonate pH 7.9 during 1 h at 60°C and alkylated with 10 mM methyl methanethiosulfate (MMTS) in the same buffer during 30 min at room temperature. To digest the samples, they were incubated with 15.4 ng/μL sequencing-grade trypsin in 50 mM triethylammonium bicarbonate at pH 7.9 overnight at 37°C. After digestion, the peptides were extracted from gel by elution in a mixture of 50% acetonitrile 5% formic acid. Tryptic peptides were dried by SpeedVac and re-suspended in 30 μL TEAB 0.5 M at pH 8.5.
iTRAQ-8plex labeling reagents (AB SCIEX) were added to each peptide samples according manufacturer's instructions and incubated at room temperature for 120 min. Mixtures of labeled samples were washed from unreacted reagents using SCX column (Strata® SCX 55 μm, 70 Å, Phenomenex) in 10 mM phosphoric acid, 25% acetonitrile, pH 3 as binding buffer and 5% ammonium hydroxide, 25% acetonitrile for the elution. After the elution, samples were vacuum dried and re-suspended in water for the next step.
Sample fractionation and mass spectrometry analysis (LC-MS/MS)
Pooled peptides were separated in an Agilent 3100 OFFGEL Fractionator (Agilent Technologies, Santa Clara, CA) through 24-well IPG strips (linear gradient from pH 3–10) according to the supplier's protocol. After separation, fractions were desalted and concentrated through C18 Sep-Pak column (Waters, Bedford, MA) previously to LC-MS/MS detection.
A nano LC II coupled to an LTQ-Orbitrap Velos Pro mass analyzer, both from Thermo Scientific (Bremen, Germany), was used for peptide analysis. The chromatographic separation was achieved using a nanoLC C18 trap column (100 μm I.D.; 2 cm length; 5 μm particle diameter, Thermo Fisher Scientific) coupled to a nanoLC C18 analytical column (75 μm I.D.; 15 cm length; 3 μm particle diameter, Nikkyo Technos Co. LTD, Japan) under gradient elution conditions. Ultrapure water with 0.1% HCOOH (solvent A) and acetonitrile with 0.1% HCOOH (solvent B) was the mobile phase and the gradient consisted of 0–5% B during 5 min, 5–35% B 30 min, 35–80% B 15 min and 80–100% B 12 min, and finally is maintained at 100% B during 10 min. A flow rate of 300 nL/min was used to elute peptides for real time ionization on a nanoFlex electrospray ion source from Thermo Fisher Scientific.
MS measurements detected intact peptides in a full scan (m/z 350–2000), with the Orbitrap at FT-resolution spectrum (R = 30,000 FHMW), followed by data dependent MS/MS scan from most intense 10 parent ions with a charge state rejection of one. The signal threshold for triggering an MS/MS event was set to 10,000 counts. The low mass cutoff was set to 100 m/z. Dynamic exclusion of 30 s and activation time of 0.1 s was used. For efficient fragmentation and detection of iTRAQ reporter ions, HCD normalized collision energy of 45 was used. All fragment ions were detected in the Orbitrap (R = 7500 FHMW). Internal calibration was performed using the ion signal of (Si(CH3)2O)6H+ at m/z 445.120025 as a lock mass. Maximal ion accumulation time allowed on the LTQ Orbitrap was 1 s for all scan modes; automatic gain control was used to prevent over-filling of the ion trap.
Database searches and quantitative proteome analysis
Tandem mass spectra were extracted and charge state deconvoluted by Proteome Discoverer v1.4.0.288 (Thermo Fisher Scientific). All MS/MS samples were analyzed using Mascot (v 2.4.1.0) as search engine node. Mascot was set up to search in NCBInr database (46,742,655 entries), following the application of the restriction for Firmicutes taxonomy. Two missed cleavages were allowed for trypsin digestion, and an error of 0.80 Da for fragment ion mass and 10.0 ppm for a parent ion were tolerated. Oxidation of methionine, acetylation of N-termini and ITRAQ 8-plex modifications were specified as variable modifications, whereas methylation of cysteines was set as static modification. The false discovery rate (FDR) and protein probabilities were calculated by Target Decoy PSM Validator working between 0.01 and 0.05 for strict and relaxed, respectively. For proteins identified with only one single peptide meeting these criteria, we required the Mascot score to be at least 30, and visual verification of fragmentation spectra was done. Identified proteins were grouped by the software to minimize redundancy. For quantitative analysis centroided iTRAQ reporter ion signals were computed and only unique peptides were used for relative protein quantification. iTRAQ reporter ion intensities were normalized to sample 113, that were replicated in the two ITRAQ mixtures.
The statistical analysis was performed on Mass Profile Professional software v. 12.6 from Agilent Technologies. To find differential proteins, a paired 1-way ANOVA test was used selecting a p < 0.05 and fold change >1.5 as cut-off values. Principal component analysis (PCA) was used to evaluate variations in the mean quantity of spots.
On-line databases like NCBI information of each gene, Computational Biology at Oak Ridge National Laboratory (ORNL; http://compbio.ornl.gov/public/section/), DAVID database (https://david.ncifcrf.gov/), KOBAS 2.0 (KEGG Orthology Based Annotation System; http://kobas.cbi.pku.edu.cn/) (Xie et al., 2011) were used to assess all the Clusters of Orthologous Groups (COGs) described for O. oeni genes and proteins and metabolic pathways. We analyzed the expression data of arrays with MEV (Multi Experiment View) cluster software using Quality Threshold Clustering (QTC) tool (Heyer et al., 1999). Pearson correlation and a minimum cluster population of the number of genes representative of 10% were used and a maximum cluster diameter of 0.9. For construction of Venn diagrams, Venny (an interactive tool for comparing lists with Venn's diagrams, http://bioinfogp.cnb.csic.es/tools/venny/index.html) was used.
Functional analysis using comparative transcriptomics and proteomics can provide deeper insight into the molecular mechanisms of adaptation of O. oeni to wine stress conditions. The aim of this work was to evaluate which genes and proteins were affected during the adaptation period occurring before the start of MLF. The study was performed with the reference strain PSU-1 using wine-like medium (WLM) at pH 3.4 and with 12% of ethanol (v/v). Under these conditions, PSU-1 showed an adaptation period of 8 h from inoculation until the beginning of MLF. Once L-malate consumption started, MLF was successfully finished in 72 h. The viability of PSU-1 did not decrease during the adaptation period, indicating that there was no detectable cell death of the inoculated population (6.87·107 ± 1.70·107 CFU/mL).
Global Analysis of Functions Affected during Acclimation to WLM
In the transcriptomic analysis 1611 expressed sequence tag (EST) were detected. Among the EST that were differently expressed during adaptation, 27 were classified as discontinued or pseudo genes in NCBI, and they were not included in the analysis. Among the analyzed genes showing significant changes along the assay, 314 were over-expressed, whereas 308 genes were down-regulated. They can be consulted in Table S1 in the Supplementary Data. It is worth noting that 52 over-expressed genes and 51 down-regulated genes were annotated as hypothetical proteins (Table S2 in the Supplementary Data).
These 622 genes differently expressed were classified in Clusters Orthologous Groups (COGs) in order to identify the main biological processes influenced by adaptation to wine conditions (Figures 1A,B). Besides, the QTC analysis grouped the differentially expressed genes into six transcriptional profiles (Figure 2). The main functions transcriptionally activated due to inoculation in WLM in O. oeni PSU-1 were translation, ribosomal structure and biogenesis (J) and amino acid transport and metabolism (E) (Figure 1B). The QTC analysis (Figure 2) showed two expression profiles, I and III (65.9 and 13.6% of over-expressed genes, respectively), in which translation (J) was the most represented function. These two profiles are indicative of an adaptive response since gene transcription increases progressively along adaptation process. The profile II (17.5% of over-expressed genes) included mostly genes of amino acid metabolism showing an increase in their transcription level between 0.5 and 1 h which was later decreased. This behavior is indicative of an early-response to WLM stress conditions. Regarding the genes showing an inhibited transcription, carbohydrate metabolism (G) was the main negatively regulated function in PSU-1 during adaptation to WLM (Figure 1B). The genes related to this metabolism mostly showed a constant down-regulation and were present in profile IV (Figure 2), representing 62.2% of total down-regulated genes. Fewer genes were clustered into profile V (15.3%), showing transcriptional repression only at the beginning of the assay (0.5–1 h). Finally, the profile VI grouped 19.8% of down-regulated genes which had a progressive repression during the adaptation period.
Figure 1. (A) Clusters of Orthologous Groups (COGs) definitions. (B) Percentage of genes of each representative COG significantly over or under-expressed according to transcriptomic analysis. (C) Percentage of proteins of each representative COG showing significant abundance changes, detected by 2D-DIGE or iTRAQ.
Figure 2. Representative gene expression profiles according to Quality Threshold Clustering (QTC) based on transcriptomic data. An example of each profile is shown. Profile I: OEOE_RS07930 (UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase); Profile II: OEOE_RS03595 (amino acid ABC transporter substrate-binding protein); Profile III: OEOE_RS05245 (oligoendopeptidase F); Profile IV: OEOE_RS07040 (glycerol-3-phosphate ABC transporter permease); Profile V: OEOE_RS03155 (F0F1 ATP synthase subunit A); Profile VI: OEOE_RS01045 (PTS sugar transporter subunit IIA).
Proteomic analysis of O. oeni PSU-1 adaptation to WLM conditions was conducted using two techniques: 2D-DIGE and iTRAQ. The PCA analyses to evaluate the variability among samples clearly indicated the presence of three different protein populations 0, 1, and 6 h (data not shown). The 2D-DIGE results showed between 27 and 62 protein spots along the assay exhibiting differential abundance with statistical significance (p ≤ 0.05). The maximum differences were observed comparing the samples from 1 and 6 h after inoculation vs. 0 h. For this reason, the protein identification was carried out only for these samples and also these samples were analyzed using iTRAQ labeling in order to complement 2D-DIGE results. Using 2D-DIGE, 33 different proteins could be determined which were not found with iTRAQ. On the other hand, the off-gel technique detected 71 proteins exclusively. At 1 h there were detected more proteins up regulated than at 6 h, revealing a fast stress proteomic response of the cell against the new environment. All proteomic identification and COG classification from 2D-DIGE and iTRAQ analysis can be consulted in Table S3 (Supplementary Data).
A high percentage of the significantly down-regulated proteins (Figure 1C) belonged to the COG associated to translation, ribosomal structure and biogenesis (J). This is in accordance to previous studies that described the lower abundance of proteins involved in protein synthesis during acid stress in Lactobacillus species (Koponen et al., 2012; Heunis et al., 2014). On the other hand, some ribosomal proteins of 50S and 30S subunits showed an increased abundance in O. oeni PSU-1 after inoculation into WLM. This increase in protein concentration was coincident with an up-regulated gene transcription (Tables 2, 3). Huang et al. (2011) and Koponen et al. (2012) also described the increased abundance of 50S and/or 30S ribosomal proteins in Lactobacillus species as a mechanism of response to acidic stress. Therefore, certain ribosomal proteins involved in the regulation of translation may play a role in stress response as suggested by Dressaire et al. (2010).
Table 2. Selection of genes related with relevant metabolisms or functions, differently regulated after inoculation into WLM from the microarray analysis.
Table 3. Selection of relevant proteins detected by 2D-DIGE and iTRAQ analysis differently regulated after WLM inoculation at 1 and 6 h.
A relevant number of proteins related to amino acid and carbohydrate metabolism (E, G) showed significant variations in abundance (Figure 1C) both increasing or decreasing, being some of them in accordance to the transcriptional response (Tables 2, 3). On the other hand, detected proteins related to defense mechanism (V) and secondary metabolites (Q) showed mostly an increased abundance (Figure 1C).
Main Metabolisms Modified by Wine-Like Conditions
Malate and Citrate Metabolism
Three out of the five malate related genes annotated in O. oeni PSU-1 genome were over-expressed: one of the permeases (mleP), the transporter OEOE_RS06985 -which had a 4-fold expression at 1 h after the inoculation-, and the malate dehydrogenase (mae) (Table 2). The observed transcriptional activation of malate transporters under wine-related conditions were in accordance with previous studies (Labarre et al., 1996) and were indicative of the induction of MLF as a part of stress response. In Augagneur et al. (2007) a significant increase was observed in the abundance of mRNA encoding MleP derived from cells incubated in presence of L-malate at pH 4.5 and 3.2. Similarly, mleP was over-expressed in the microarray performed by Costantini et al. (2015) due to adaptation (1 day) to ethanol 8 and 12%.
In this work it was detected the over-expression of the citrate lyase operon, observing the highest expression 1 h after the inoculation into WLM (Table 2). The transcriptional activation of O. oeni citrate lyase in response to ethanol stress has been previously reported by Olguín et al. (2009). The transcriptional up-regulation of malate transport and citrate consumption could be indicative of the use of L-malate and citrate associated to stress response and as an alternative energy source to sugar metabolism. Significant changes were also observed for genes involved in diacetyl utilization. Diacetyl is the main aromatic compound associated to MLF and is derived from citrate consumption. Diacetyl reductase showed transcriptional inhibition, while its protein abundance increased 6 h after inoculation into WLM. On the other hand, acetoin reductase was inhibited both at gene and protein level. Diacetyl and acetoin reductases are involved in two reactions of transformation of diacetyl, first into acetoin and then into 2,3-butanediol as the final product. These two reactions involve the oxidation of NAD(P)H and would participate in the maintenance of the cofactor redox balance. Diacetyl metabolism has been described as strain-dependent (Bartowsky and Henschke, 2004). In the studied conditions with PSU-1 strain, diacetyl and acetoin reductases would be initially inhibited, however proteomic data showed the increase in abundance of diacetyl reductase toward the beginning of MLF, which could be correlated to the activation of citrate consumption and the consequent production of diacetyl.
ATPase Activity
ATPase activity has been associated to MLF (Salema et al., 1996). Cox and Henick-Kling (1989) proposed a chemiosmotic mechanism where energy is produced by the efflux of L-lactate from L-malate degradation. Fortier et al. (2003) described the increase of F0F1-ATPase β subunit mRNA in response to low pH. However, in this work several genes codifying for other ATPase subunits (α, δ, γ, and ε) were down-regulated before the beginning of MLF (Table 2). Although, in the proteomic study the subunit α was initially down-regulated (1 h), its abundance increased at 6 h. This could indicate that when cells are longer acclimated to WLM (6 h after inoculation), and closer to the start of L-malate consumption, ATPase activity is increased.
Amino Acid Transport and Metabolism
It is worth to note the activation of several genes related with peptidase activity and amino acid transport (Table 2). Also, five peptidases were identified in the proteomic analysis, but their abundance varies depending on the protein and the analyzed time. Liu et al. (2010) reported that many of the peptidases seem to be essential for bacterial growth or survival as they are encoded in all LAB genomes, such as PepC, PepN, and PepM, and proline peptidases PepX and PepQ. Currently in PSU-1 genome there are annotated 29 peptidases. Also, related to nitrogen uptake, three permeases involved in nitrogen compounds transport are strongly over-expressed (Table 2) after the inoculation into WLM, reaching the maximum activation at 2 h. Since peptides account for the largest proportion of total nitrogen in wine (Feuillat et al., 1998), these results suggest the relevance of wine nitrogen composition and the ability of O. oeni to cope with its environment as reported by Manca de Nadra et al. (1999) and Ritt et al. (2008).
Some genes and proteins related to glutamine and glutamate synthesis, involved in the assimilation and re-distribution of nitrogen within the cell, were up-regulated revealing the key role of nitrogen uptake for O. oeni in a poor nutrient media, such as WLM. Glutamine synthetase was up-regulated both at gene and protein level. The 4-aminobutyrate aminotransferase gene (OEOE_RS01860), which transforms GABA into succinate semialdehyde and L-glutamate, was threefold over-expressed during the 8 h of O. oeni PSU-1 adaptation to WLM. GABA can be assimilated as a nitrogen and/or carbon source in bacteria such as Escherichia coli (Bartsch et al., 1990) and Corynebacterium glutamicum (Zhao et al., 2012), but no information is available about LAB in this respect.
Six genes involved in the transport of spermidine/putrescine were over-expressed (Table 2). The uptake of these two polyamines has been associated with an energy-producing state/membrane potential of the cell in E. coli (Kashiwagi et al., 1997). Both putrescine and spermidine protect against oxidative stress (Tkachenko et al., 2001). Olguín et al. (2015) reported that this protective mechanism may also be a target of ethanol damage in an ethanol shock, which would inhibit the uptake of these polyamines. In this case, the adaptation to conditions of WLM resulted in an over-expression of six out of the eight transporters of these polyamines annotated in PSU-1 genome.
Carbohydrate Transport and Metabolism
Microarray data revealed that sugar transport was repressed (Table 2) in response to WLM conditions. In particular, glycerol-3-phosphate ATP-binding cassette ABC transporters and mannose phosphotransferase transporters (PTS) were down-regulated. This inhibition is probably due to the lower availability of sugars in WLM with respect to rich growth medium in which inocula were prepared. A strong transcriptional inhibition of sugar metabolism and transport in response to ethanol was also observed by Olguín et al. (2015).
Enolase, among others, were strongly up-regulated at low pH compared with the optimal growth at pH 6.8 (Lee et al., 2008). In this assay, the enolase protein increased in abundance 1 h after inoculation. On the contrary, the transcriptomic data reported in this work and by Costantini et al. (2015) show the inhibition of enolase gene under wine-like conditions. This suggests that enolase, might be up-regulated at translational level in response to stress. This protein, besides being involved in sugar fermentation, has been related to host tissue adhesion in probiotic bacteria including Lactobacillus plantarum (Castaldo et al., 2009).
Lipid Transport and Metabolism
The regulation at gene and protein level of lipid metabolism was scarce and, in most of the cases, was down-regulated. For instance, the gene cfa (OEOE_RS05660) which is involved in the conversion of monounsaturated fatty acids to cyclopropane fatty acids (CFAs) was inhibited at 1 h. The increase in cfa transcription was observed in acid- and ethanol-grown cells (Grandvalet et al., 2008; Olguín et al., 2010) after a longer period of stress exposure than in this work, once MLF had started.
Cell Wall/Membrane/Envelope Biogenesis
Several genes and proteins related to cell envelope biogenesis were over-expressed. One of the genes annotated as D-alanyl-D-alanine carboxypeptidase (OEOE_RS03435) was 6-fold over-expressed at 2 h and its sharp over-expression started 0.5 h after the inoculation into WLM. This gene and OEOE_RS06975 (for peptidoglycan interpeptide bridge formation protein) were also over-expressed in transcriptomic analysis by Costantini et al. (2015), both after adaptation with 12% of ethanol. However, in the present study, another D-alanyl-D-alanine carboxypeptidase (OEOE_RS07530) showed an opposite behavior, being down-regulated both at gene and protein level. Peptidoglycan (PG) is an essential component of the bacterial cell envelope, required for cell shape and stability (Vollmer et al., 2008). A supply of D-amino acids is essential for peptidoglycan synthesis; moreover, D-Ala is the main constituent of wall teichoic acids and lipoteichoic acids, which are polyanionic polymers exclusively found in Gram-positive bacteria (Wecke et al., 2009). Another activated function, both at gene and protein level, was glucosamine:fructose-6-phosphate aminotransferase, which is also involved in cell wall biosynthesis. Cecconi et al. (2009) found a major concentration of glucosamine 6-phosphate aminotransferase in O. oeni cells acclimated to ethanol than in not acclimated cells. These results point out the specificity and relevance of some enzymes involved in cell envelope protection against stress challenge.
Three proteins related to cell wall biogenesis were down-regulated. Among them, the rod shape-determining protein (MreB), with an actin-like role, was less abundant at 1 and 6 h. These results correlate with the reported information for proteins MreB1 and B2 determining cell shape from L. plantarum 423 which were less abundant in acid-stressed cells (Heunis et al., 2014) and the transcriptional inhibition due to ethanol of other rod shape-determining proteins, such as MreB, in O. oeni (Olguín et al., 2015). Also, microarray analysis revealed the inhibition of three genes (OEOE_RS07265, OEOE_RS03340, and OEOE_RS07010) involved in cell-wall biogenesis, encoding one of them a capsular polysaccharide biosynthesis protein, gene which was described as well by Dimopoulou et al. (2012) as wzd. All these down-regulated functions would be targets of the damage caused by factors such as ethanol and low pH.
Translation, Ribosomal Structure, and Biogenesis
One of the categories showing more significant changes in O. oeni PSU-1 transcriptome and proteome due to WLM conditions was translation related functions. It is worth noting that several 30S and 50S ribosomal genes were over-expressed like their correspondent proteins. Several ribosomal proteins were up-regulated in Lactobacillus rhamnosus under acidic stress (Koponen et al., 2012). Moreover, in O. oeni in agreement with these observations, Cecconi et al. (2009) reported that adaptation in half strength wine-like medium correlates with the up regulation of some transcription/translation proteins as elongation factor Ts and ribosomal protein 30S. A gene codifying for a ribosomal protein was differentially over expressed between 0 and 1 h after ethanol addition in O. oeni PSU-1 (Olguín et al., 2015). As well in samples of O. oeni adapted to 12% ethanol, a ribosomal protein was up-regulated (Costantini et al., 2015). Studies with Lactococcus lactis suggest that the regulation of translation has a major role in stress response (Dressaire et al., 2010). According to our results this would also happen in O. oeni.
Stress Response
As expected, it was observed the activation of chaperon function in response to WLM stress conditions. Some genes, such as grpE, dnaJ, and dnaK, and proteins, like GroEL and GroES (Hsp10), showed up-regulation in PSU-1 after inoculation into WLM (Tables 2, 3). The latter chaperone, Hsp10, is conserved along LAB (Sugimoto et al., 2008). However, Hsp20, the most characterized stress protein in O. oeni (Guzzo et al., 1997, 2000), showed transcriptional inhibition in our assay and no changes in protein concentration. This is in accordance with Costantini et al. (2015), that described the transcriptional activation of hsp20 only in mild ethanol stress (8%) but the under-expression of this gene with 12% ethanol, as found in our work. A cold shock protein (OEOE_RS06620) showed increased abundance 1 h after O. oeni PSU-1 inoculation, but its gene expression was inhibited along the assay. This protein could play a role in the early response to wine-related stress but not in the long term adaptation process.
Our data revealed that wine-like conditions caused an increase of proteins involved in oxidative stress protection, related to thioredoxin and glutathione systems. Two out of the three thioredoxins (trxA) annotated for PSU-1, OEOE_RS07835, and OEOE_RS08215, were up-regulated 6 h after inoculation. Also thioredoxin reductase (trxB), OEOE_RS02695, and a ferredoxin reductase gene (fdr: OEOE_RS00770), annotated in NCBI as trxB until February 2015, were activated under wine stress conditions. Glutathione reductase (GshR) was significantly more abundant in O. oeni PSU-1 after inoculation into WLM. However, transcriptomic data revealed that some of these genes were inhibited; indicating that translational regulation of these functions would be prevalent under the studied conditions. Our results support the relevance of the thioredoxin and glutathione systems in the adaptation of O. oeni to wine related stress. There are few studies regarding thioredoxin in O. oeni (Jobin et al., 1999a; Guzzo et al., 2000; Margalef-Català et al., 2017), thus the role of this mechanism and glutathione system against wine stress is quite unknown.
Among the genes over-expressed related to defense mechanism there were eight multidrug transport genes. ABC transporters are a major part of the efflux systems involved in the transport of harmful-compounds and cell detoxification (Leverrier et al., 2004).
Evaluation of Omic Data Correlation
Real-time qPCR Validation
In order to validate the results obtained from the microarray analysis, real-time qPCR was performed with the same RNA from the original microarray experiment. Twenty-two genes, some related to stress response, were selected, taking the RNA sample of time where maximum over- or under- expression had been observed in microarray (Table 1). There was a general accordance between microarray and real-time qPCR data for all the genes tested. Of the 22 genes, 17 were clearly correlated using both techniques. For hsp18 gene higher values by qPCR were obtained than for microarray data. Finally the four remaining genes (diacylglycerol kinase, PTS sugar, cfa and trxA2) displayed lower numerical values by qPCR, indicating no significant changes using this technique, while with microarray they were slightly inhibited. Overall, the correlation between real-time qPCR and microarray was good, suggesting that the microarray gene expression measurements were valid. Moreover, the validation of two thioredoxins (trxA2 and trxA3) was useful for the proteomic identification.
Integration of Transcriptomic and Proteomic Analysis
It has been largely reported that the correspondence of transcriptomic and proteomic data is low due to the numerous and complex regulatory mechanisms involved in gene transcription and protein synthesis (Dressaire et al., 2010; Haider and Pal, 2013). In this work, 19 genes presented a correlation with proteomic results (Tables 2, 3). The most relevant, in terms of understanding O. oeni stress response, have already been discussed in the text. Venn diagram shown in Figure 3 shows the number of coincident modifications of genes and proteins at different analyzed times vs. time zero. It is worth to note the highest number of coincidences was observed for genes up/down regulated (166 and 158, respectively) 1 and 6 h after inoculation in WLM. This indicates that most of the transcriptional changes were sustained along the 8 h assay, before MLF start. However, some genes were only modified at one of the analyzed times, indicating its specific role in early (1 h) or adaptive (6 h) response, respectively. Regarding protein changes, the observed pattern was different and many proteins showed modifications only at one of the two analyzed times. However, some proteins maintained the up or down regulation along the assay. Altogether, the data reported illustrates the complexity of O. oeni cell regulation and the difficulty of finding specific marker genes and/or proteins associated to stress response.
Figure 3. Venn diagram of the number of proteins and genes showing significant changes in abundance and expression, respectively, according to transcriptomic, and proteomic analysis 1 and 6 h after the inoculation of O. oeni PSU-1 into WLM. (A) over-expressed genes and up-regulated proteins, (B) under-expressed genes and down-regulated protein. The color of diagram petals match with the colored legends in the figure.
The combined transcriptomic and proteomic study was useful to identify the metabolisms mostly altered due to wine-like conditions. The use of two complementary proteomic techniques allowed the detection of a major number of proteins influenced by stress factors. Our results revealed the relevance of translation regulation and nitrogen uptake as key metabolisms involved in the adaptation of O. oeni PSU-1 to wine related stress. Cell wall biosynthesis and redox maintenance mechanisms seem to play also a relevant role in the protection of O. oeni against cell damage. Finally, sugar metabolism is inhibited in contrast to the transcriptional activation of L-malate transport and citrate consumption before the beginning of MLF.
Most of the molecular modifications occurring during O. oeni adaptation to wine will depend on the strain and/or fermentation conditions. However, the omic analysis allows the identification of the most relevant functions affected by wine-related stress, on which should be focused future research.
MM, IA: performed the experiments, participated in the acquisition, analysis and interpretation of the data, approved the final version of the paper. AB, CR, JB: supervised the laboratory work, participated in the analysis and interpretation of the data, drafted the manuscript, and approved the final version of the paper.
The reviewer EET and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.
This work was supported by grants AGL2009-07369ALI and AGL2012-34866ALI from the Spanish Ministry of Economy and Competitiveness. MM is grateful to the pre-doctoral fellowship from the Universitat Rovira i Virgili. JB wish to thank to the Spanish Government for his "Ayuda para la Formación Posdoctoral" research contract.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/article/10.3389/fmicb.2016.01554
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Sugimoto, S., and Abdullah-Al-Mahin, Sonomoto, K. (2008). Molecular chaperones in lactic acid bacteria: physiological consequences and biochemical properties. J. Biosci. Bioeng. 106, 324–336. doi: 10.1263/jbb.106.324
Sumby, K. M., Grbin, P. R., and Jiranek, V. (2012). Validation of the use of multiple internal control genes, and the application of real-time quantitative PCR, to study esterase gene expression in Oenococcus oeni. Appl. Microbiol. Biotechnol. 96, 1039–1047. doi: 10.1007/s00253-012-4409-1
Tkachenko, A. G., Pshenichnov, M. R., and Nesterova, L. Y. (2001). Putrescine as a factor protecting Escherichia coli against oxidative stress. Microbiology 70, 422–428. doi: 10.1023/A:1010430126763
Unlü, M., Morgan, M. E., and Minden, J. S. (1997). Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18, 2071–2077. doi: 10.1002/elps.1150181133
Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., et al. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3:RESEARCH0034. doi: 10.1186/gb-2002-3-7-research0034
Vollmer, W., Joris, B., Charlier, P., and Foster, S. (2008). Bacterial peptidoglycan (murein) hydrolases. FEMS Microbiol. Rev. 32, 259–286. doi: 10.1111/j.1574-6976.2007.00099.x
Wecke, J., Perego, M., and Fischer, W. (2009). d-Alanine deprivation of Bacillus subtilis Teichoic Acids is without effect on cell growth and morphology but affects the autolytic activity. Microb. Drug Resist. 2, 123–129. doi: 10.1089/mdr.1996.2.123
Xie, C., Mao, X., Huang, J., Ding, Y., Wu, J., Dong, S., et al. (2011). KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res. 39(Suppl. 2), W316–W322. doi: 10.1093/nar/gkr483
Zhao, Z., Ding, J. Y., Ma, W. H., Zhou, N. Y., and Liu, S. J. (2012). Identification and characterization of γ-Aminobutyric acid uptake system GabPCg (NCgl0464) in Corynebacterium glutamicum. Appl. Environ. Microbiol. 78, 2596–2601. doi: 10.1128/AEM.07406-11
Keywords: Oenococcus oeni, malolactic fermentation, transcriptomic, proteomic, stress, wine
Citation: Margalef-Català M, Araque I, Bordons A, Reguant C and Bautista-Gallego J (2016) Transcriptomic and Proteomic Analysis of Oenococcus oeni Adaptation to Wine Stress Conditions. Front. Microbiol. 7:1554. doi: 10.3389/fmicb.2016.01554
Received: 08 August 2016; Accepted: 16 September 2016;
Published: 30 September 2016.
Andrea Gomez-Zavaglia, National Scientific and Technical Research Council, Argentina
E. Emma Tymczyszyn, National Scientific and Technical Research Council, Argentina
Jose Antonio Curiel, Instituto de Ciencias de la Vid y del Vino, Spain
Copyright © 2016 Margalef-Català, Araque, Bordons, Reguant and Bautista-Gallego. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Joaquín Bautista-Gallego, joaquin.bautista@urv.cat


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GRETNA – The state of Louisiana is seeking more than $33,000 from a Jefferson Parish resident.
The state of Louisiana Division of Administration Office of Community Development Disaster Recovery Unit filed a suit June 26 against Kelly Schulin Mire in the 24th Judicial District Court alleging sums due.
According to the claim, the plaintiff is seeking repayment of $33,510.90, which was given to the defendant as a grant to complete improvements following Hurricane Katrina. The suit states that the defendant misused the funds.
The plaintiff is seeking all reasonable sums due, attorney fees and court costs. The plaintiff is represented by John C. Walsk of Shows, Cali & Walsh LLP in Baton Rouge.


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redpajama_set_name: RedPajamaGithub


text:
The Škoda Roomster (Typ 5J) is a five-door five-seat multi-purpose vehicle-styled leisure activity vehicle, produced by the Czechautomaker Škoda Auto. It was revealed as a production model in March 2006 at the Geneva Auto Show, with sales starting in June 2006. It is built at the Škoda factory in Kvasiny, Rychnov nad Kněžnou District.
A two-door two-seat panel van version named the Škoda Praktik has been on sale since March 2007.
The Roomster is Škoda's first car since the Volkswagen Group's take over of the brand that is not based entirely on an existing Volkswagen Group platform - although it is fundamentally based on the A4 (PQ34) platform, but with some unique additions. It shares components with the previous Škoda Octavia and the current Škoda Fabia. The name is reportedly a conscious combination of the words 'Room' and 'Roadster', and the split personality theme is played out in the design with a carefully honed 'driving room' ahead of the B-pillars, and a generous 'living room' to the rear. The Roomster therefore competes both in the mini MPV and leisure activity vehicle markets, which it will be sharing with the Fiat Idea, Nissan Note, Opel Meriva, Citroën C3 Picasso and Volkswagen Golf Plus.
The Roomster has also appeared on an episode of the BBC 2 series Top Gear as one of the few inexpensive cars that Jeremy Clarkson liked.
Kvasiny, Czech Republic (until 2011)
Vrchlabí, Czech Republic (since 2011)
Solomonovo, Ukraine (Eurocar)
Kvasiny, Czech Republic (since 2013)
Leisure activity vehicle (Roomster)
Light commercial vehicle (Praktik)
5-door multi-purpose vehicle
2-door panel van
Front-engine, front-wheel-drive
Volkswagen Group A4 (PQ34)
Petrol engines:
1.2 L HTP I3
1.2 L TSI I4
1.4 L 16V I4
Diesel engines:
1.2 L TDI I4
6-speed tiptronic
Thomas Ingenlath
Peter Wouda
The Roomster is based on the concept car of the same name, originally revealed in September 2003 at the Frankfurt Auto Show. The concept was slightly shorter - 4,055 millimetres (159.6 in) against 4,205 mm (165.6 in) - than the production model but has a longer wheelbase - 2,710 mm (106.7 in) against 2,617 mm (103.0 in), and featured a single rear sliding door on the passenger side, which was replaced by two conventionally hinged swinging rear doors. Like the production car it was designed by Thomas Ingenlath and Peter Wouda.
Škoda conceived the Roomster's cabin in a way it can have different configurations at the choice of the user. The rear 'VarioFlex' seatarrangement, which are taller than the front seats, can be folded in a 40-20-40 split configuration, instead of the more usual 40-60, and can also be moved longitudinally and transversally (by removing the middle seat). The rear seats also have variable inclination up to 7° to the back, or completely to the front.
The luggage compartment can have a completely flat surface with the rear seats completely removed, and the surface's height can also be modified with an amplitude of 250 millimetres (9.8 in). With the rear seats removed, the Roomster's luggage compartment has enough room to carry a bicycle.
The Škoda Praktik (Typ 5J8) is a five-door two-seat panel van version of the Roomster. Its cargo space is 1,605 millimetres (63.2 in) long, 1,016 millimetres (40.0 in) wide (minimum), and 900 millimetres (35.4 in) in height - giving a cargo load space of 1,900 litres (67.1 cu ft), and a payload from 550 to 640 kilograms (1,213 to 1,411 lb). It also includes a moveable internal load bulkead, 'hidden' underfloor storage, anti-slip load bay floor covering with six lashing points, and full-length roof rails.
2010 Škoda Praktik front view
2010 Škoda Praktik rear view
The Škoda Roomster takes its internal combustion engines from its Volkswagen Group relatives, all engines are inline four-stroke designs. Petrol engines include the multi-valve inline three cylinder 1.2 litre, with power initially DIN-rated at 47 kilowatts (64 PS; 63 bhp), but now rated at 51 kilowatts (69 PS; 68 bhp), followed by the 1.4 litre and 1.6 litre inline four cylinder EA111 engines, with power of 63 kilowatts (86 PS; 84 bhp) and 77 kilowatts (105 PS; 103 bhp) respectively. The 1.6 litre is also available with a six-speed tiptronic automatic transmission, and is available on the most luxurious models.
Diesel engine propulsion comes from the inline three cylinder 1.4 Turbocharged Direct Injection (TDI), with two power levels, base at 51 kilowatts (69 PS; 68 bhp) and "sport" at 59 kilowatts (80 PS; 79 bhp). The most luxurious models get a four-cylinder 1.9 TDI, with 77 kilowatts (105 PS; 103 bhp).
Disp., config., valvetrain,
fuel system / aspiration
ID code
(family)
at rpm
1.2 HTP
1,198 cc I3, 12v DOHC
47 kW (64 PS; 63 bhp)
at 5,400
112 Nm (83 lbft)
BZG
1,197 cc I4, 8v DOHC
CBZA
160 Nm (118 lbft)
at 1,500 - 3,500
CBZB
77 kW (105 PS; 103 bhp)
at 1550-4100
BXW, CGGB
(EA111)
variable geometry turbocharger
CFWA
1,422 cc I3, 6v SOHC
at 1,600-2,800
1.4 TDI DPF
1.6 TDI CR DPF
CAYB
CAYC
74 kW (101 PS; 99 bhp)
Euro NCAP test results
Škoda Roomster (2006)
Adult occupant:
Child occupant:
Pedestrian:
Back to Škoda
Last updated on 7 June 2013 at 06:25.
Labels: 2000 2010 Current Models Czechoslovakia Car MPV Škoda Auto Škoda Praktik Škoda Roomster Van

redpajama_set_name: RedPajamaCommonCrawl


text:
Combating Bugs in Space
ByAmanda Onion
Oct. 9, 2000 -- When astronaut Norm Thagard spent four months on the Mir space station, his hygiene routine was fairly basic.
He and his crewmates used two dry towels and one wet towel a day to freshen up. And even though Thagard, who was the first American to live on the now 14-year-old station, exercised at least once a day on board, he reports, "I never felt terribly unclean."
One thing Thagard did notice, however, was the way the callused skin on the bottom of his feet began to flake off, since there was no pressure on his soles in microgravity.
"I came back with feet bottoms as smooth as a baby," he says.
Cramped, Damp Places
While sweating and flaking skin may be harmless facts of life here on Earth, in the cramped quarters of a spacecraft or station, such bodily products can invite trouble. Given a moist opportunity, microorganisms inhabiting those shed body parts can flourish, multiply and, particularly if radiation levels are high, mutate.
"Microorganisms grow on everything on Earth or in space," says Ralph Mitchell, a professor of applied biology at Harvard University. "But because a space station is such a tight space, there can be lots of nooks and crannies where organisms can grow."
That reality, plus reports of funky smells and mildew-coated surfaces on the Mir space station, has prompted NASA to investigate how to deal with the problem. Its research, which accelerated with the establishment of an immunology and infection commission three years ago, is likely to take on more significance when two cosmonauts and one astronaut are scheduled to take up long-term residence of the International Space Station for the first time next month.
Learning how to combat infection in space will also become important should NASA launch a human journey to Mars in the future. Scientists now estimate a voyage to the Red Planet would require living in space for about two years.
"I'm sure we can get to Mars from an engineering perspective," says William Shearer, a researcher for the National Space Biomedical Research program commissioned by NASA. "The question is will we be able to return live, healthy astronauts back home."
Mixed Reports From Mir
Duane Pierson, the head of microbiology at NASA's Johnson Space Center, points out that Mir has been a good litmus test for how well spacecraft can keep rampant bacterial and fungal colonies at bay. He says that when he and his team conducted tests on samples taken from the space station five years ago, they found it to be a "healthy environment."
Still, Thagard recalls that at least one module of the Mir station was perpetually damp.
"It would have been a good place to view Earth, but all the windows were always too foggy to see through," he says. "Even when you wiped the window clean, it would fog right up again."
Thagard says he never noticed mildew buildup on the station, but the astronauts who followed him did. Michael Foale, who spent 134 days on the craft, told reporters in 1997 that "Mir has a lot of mold on the walls." And Jerry Linenger, who spent nearly five months on the station, described in his book Off the Planet how the station had taken on a "mushroomy" smell.
Reports from a Russian microbiologist, Natalia Novikova, suggest that micro colonies might have since proliferated. Novikova told a Space.com reporter last June that a green mat of fungus and bacteria had begun eating away at the station's windows and electronic equipment.
Less Immunity
Shearer says such accounts are particularly worrisome because astronauts' immune systems are known to weaken in space.
"The tremendous stress, isolation, lack of sleep and close containment in space can definitely stress the immune system," Shearer says. "So they might be susceptible to microbes that otherwise wouldn't bother them."
He adds that a trip to Mars could pose even more problems since unexpected events like solar flares and gamma ray bursts could expose a crew to high levels of radiation. And radiation, he says, is known to weaken the immune system the same way HIV does, by depleting cells known as T-lymphocytes.
Radiation is also known to cause mutations in bacteria. And some have suggested any mutant bacteria that evolved in space could be difficult to battle, since the human body would have no experience with fighting the bug.
Shearer, whose team works independently of NASA, says he doesn't know if astronauts and cosmonauts have been affected by infection in space since their medical records are kept confidential. Pierson maintains infection has not been a problem.
"I don't known of anyone who reported environmental-related illnesses among crewmembers — and that's the bottom line," Pierson says.
Still, unconfirmed stories have trickled in from Russia describing cosmonauts who have contracted skin boils and infections while living on the Mir. And Shearer and Pierson both admit it would be irresponsible not to look into the matter.
"I know that in individuals we treat on Earth who have compromised immune systems, there are consequences," Shearer says. "And I believe space can pose the equivalent situation."
Clean-Up Time
To combat infection on the International Space Station, Pierson says NASA is installing the most sophisticated air-filtering system available, a so-called hepafilter, on the station. The filter is so sensitive, he says, it can pluck microorganisms from the air. Pierson and other NASA scientists have also encouraged a much more basic approach to fighting scum buildup: required housecleaning.
As Thagard says, "That's one job you can't even avoid in space."


text:
OSA Publishing > Optics Express > Volume 22 > Issue 8 > Page 9334
James Leger, Editor-in-Chief
Efficient design of polarization insensitive polymer optical waveguide devices considering stress-induced effects
Md. Faruque Hossain, Hau Ping Chan, and Abbas Z. Kouzani
Md. Faruque Hossain,1,* Hau Ping Chan,2 and Abbas Z. Kouzani3
1Department of Electronics and Communication Engineering, Khulna University of Engineering and Technology, Fulbari Gate, Khulna 9203, Bangladesh
2Department of Electronic Engineering, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, China
3School of Engineering, Deakin University, Warun Ponds, Victoria 3216, Australia
*Corresponding author: fhossain@ece.kuet.ac.bd
M Hossain
H Chan
A Kouzani
Issue 8,
•https://doi.org/10.1364/OE.22.009334
Md. Faruque Hossain, Hau Ping Chan, and Abbas Z. Kouzani, "Efficient design of polarization insensitive polymer optical waveguide devices considering stress-induced effects," Opt. Express 22, 9334-9343 (2014)
Integrated Optics
Channeled waveguides
Phase matching
Polymer waveguides
Refractive index
Thin films
Integrated optics devices (130.3120)
Polarization-selective devices (130.5440)
Polymer waveguides (130.5460)
Polarization, other optical properties (310.5448)
Original Manuscript: January 8, 2014
Revised Manuscript: March 9, 2014
We present an approach for the efficient design of polarization insensitive polymeric optical waveguide devices considering stress-induced effects. In this approach, the stresses induced in the waveguide during the fabrication process are estimated first using a more realistic model in the finite element analysis. Then we determine the perturbations in the material refractive indices caused by the stress-optic effect. It is observed that the stresses cause non-uniform optical anisotropy in the waveguide materials, which is then incorporated in the modal analysis considering a multilayer structure of waveguide. The approach is exploited in the design of a Bragg grating on strip waveguide. Excellent agreement between calculated and published experimental results confirms the feasibility of our approach in the accurate design of polarization insensitive polymer waveguide devices.
Study of optical anisotropies in benzocyclobutene thin films for the efficient design of optical waveguide devices
Md. Faruque Hossain, Hau Ping Chan, and Mohammad Afsar Uddin
Opt. Express 18(9) 8896-8905 (2010)
Generalized characteristics of photo-elastic birefringence in polymer strip waveguides
Md. Faruque Hossain, Hau Ping Chan, Abbas Z. Kouzani, and Md. Osman Goni
Opt. Mater. Express 5(5) 1030-1044 (2015)
Simultaneous measurement of thermo-optic and stress-optic coefficients of polymer thin films using prism coupler technique
Stress induced birefringence tuning in femtosecond laser fabricated waveguides in fused silica
Luís A. Fernandes, Jason R. Grenier, Peter R. Herman, J. Stewart Aitchison, and Paulo V. S. Marques
Stress-induced birefringence control in optical planar waveguides
Xiuli Zhao, Chunfei Li, and Y. Z. Xu
Opt. Lett. 28(7) 564-566 (2003)
Article Order
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[Crossref] [PubMed]
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M. M. Milosevic, P. S. Matavulj, B. D. Timotijevic, G. T. Reed, and G. Z. Mashanovich, "Design rules for single-mode and polarization-independent silicon-on-insulator rib waveguides using stress engineering," J. Lightwave Technol. 26(13), 1840–1846 (2008).
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M. A. Uddin, H. P. Chan, C. K. Chow, and Y. C. Chan, "Effect of spin coating on the curing rate of epoxy adhesive for the fabrication of a polymer optical waveguide," J. Electron. Mater. 33(3), 224–228 (2004).
K. C. Chan, M. Teo, and Z. W. Zhong, "Characterization of low-k benzocyclobutene dielectric thin film," Microelectron. Int. 20(3), 11–22 (2003).
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M. F. Hossain, H. P. Chan, and M. A. Uddin, "Simultaneous measurement of thermo-optic and stress-optic coefficients of polymer thin films using prism coupler technique," Appl. Opt. 49(3), 403–408 (2010).
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Aitchison, J. S.
Allen, S. A. B.
Antonucci, V.
Azaña, J.
Belhadj, N.
Blech, I. A.
Cada, M.
Chan, H. P.
Chan, K. C.
Chan, Y. C.
Cheben, P.
Cheng, S. Y.
Chiang, K. S.
Chow, C. K.
Cusano, A.
Dalton, L. R.
Dossou, K.
Dumais, P.
Eldada, L.
Fernandes, L. A.
Fetterman, H. R.
Fischer, K.
Gang, S.-J.
Geary, K.
Giordano, M.
Grenier, J. R.
Gu, C.
Herman, P. R.
Ho, P. S.
Hodge, T. C.
Hoffmann, R.
Hossain, M. F.
Huang, M.
Janz, S.
Jen, A. K.-Y.
Jin, G.
Ju, J. J.
Kim, K. H.
Kim, S.-K.
Kohl, P. A.
Koshiba, M.
Lamontagne, B.
Larochelle, S.
Lee, D.-G.
Lee, J.-M.
Lee, M. H.
Li, R. K. Y.
Li, S.
Lin, Y.
Liu, W.
Lohmeyer, M.
Ma, H.
Marques, P. V. S.
Mashanovich, G. Z.
Matavulj, P. S.
McKerrow, A. J.
Milosevic, M. M.
Müller, J.
Nasser, J.
Nicolais, L.
Oh, I.
Park, G.-C.
Park, S.
Park, Y.
Picard, M.-J.
Reed, G. T.
Ryan, T.
Saitoh, K.
Salle, D.
Schriemer, H. P.
Shacklette, L. W.
Shen, Y.-L.
Shi, F. G.
Shih, W.-Y.
Steier, W. H.
Suresh, S.
Tarr, N. G.
Teo, M.
Timotijevic, B. D.
Tsuji, Y.
Uddin, M. A.
Wang, C.
Wasse, F.
Wong, W. P.
Xu, D.-X.
Yan, H.
Yang, C.
Ye, W. N.
Yin, J.
Yuan, W.
Zhang, C.
Zhang, Y.
Zhao, J.-H.
Zhong, Z. W.
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IEEE Trans. Adv. Packag. (1)
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Fig. 1 Schematic of a Bragg grating on strip channel waveguide. The strip width and height are denoted by w and h, respectively, and Λ is the pitch of the grating.
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Fig. 2 Measured and simulated warpage of BCB thin film sample of size 3.5 × 3.5 cm2.
Fig. 3 Contour plot of stress distribution in the strip channel of width, w = 3 µm and height, h = 2.9 µm (a) in-plane stress, σ x and (b) out-of-plane stress, σ y .
Fig. 4 Material in-plane index (nx) as a function of the position (y) along the height of a strip channel (h = 2.9 µm) for different widths (w = 2.25 µm, 3.0 µm and 4.5 µm).
Fig. 5 Variation of material birefringence (nx - ny) along the height of a strip channel (h = 2.9 µm) for different widths (w) of 2.25 µm, 3.0 µm and 4.5 µm.
Fig. 6 Material birefringence along the width at three different heights of 0.5 µm, 1.25 µm, and 2.0 µm of a strip waveguide with dimension of w = 3 µm and h = 2.9 µm.
Fig. 7 Multiple-layered approximation of a strip waveguide.
Fig. 8 Bragg wavelength difference ( Δ λ B ) between TE and TM polarizations as a function of core width ( w ). The core height ( h ) is 2.9 µm.
Table 1 Material properties used in the FE analysis
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(1) n x = n 0 − C 1 σ x − C 2 ( σ y + σ z ), n y = n 0 − C 1 σ y − C 2 ( σ x + σ z ),
(2) σ f = E s (1− ν s ) t s 2 6 t f ( 1 R 2 − 1 R 1 )
Material properties used in the FE analysis
Elastic Modulus (GPa)
CTE (ppm/°C)
Poisson's ratio
Silicon - 130 2.6 0.27
Epoxy Tg = 145 °C Below Tg 2.5 65 0.34
Above Tg 0.25 200 0.34
BCBTg = 350 °C Below Tg 2.9 57.6 0.34


text:
Madhya Pradesh Shocker: Five Cops Suspended After 19-Year-Old Tribal Youth Dies in Police Custody in Indore
September 3, 2022 admin India 0
Bhopal, September 3: Five police personnel, including a sub-inspector (SI), were suspended after a 19-year-old tribal youth, who was arrested as a suspect in a robbery case, died in police custody in Indore on Saturday, the police said. The deceased has been identified as Arjun Singare, a resident of Gittiphoda in Indore's Manpur area.
Singare was arrested late on Friday night in connection with a case of dacoity and illegal arms possession from Manpur. The police claimed that besides dacoity, Singare was also an accused in two other cases. Tamil Nadu Custodial Death: Supreme Court Refuses Urgent Listing Of Bail Plea by Police, Says 'Cop Should Be in Jail for Some Time'.
"He was detained from his home in Manpur for interrogation. However, his health deteriorated after which he was rushed to a nearby hospital where he died during treatment," the police said. However, the kin of the deceased refuted police's claims and accused them of assaulting the youth at the police sation, leading to his death.
Enraged by Singare's death, his family members along with scores of tribals staged a protest and blocked the Agra-Mumbai highway in front of the police station, demanding compensation and government job for a family member.
Later, SP (Rural) Bhagwat Singh Virde ordered suspention of five cops who were handling the case and also recommended judicial probe into the matter. "The body has been sent for post-mortem and the district sessions court has been requested for a judicial probe into the matter," read an order issued by Virde.
The order said that SI Kamal Uike, ASIs Devesh Verma and Nirbhay Singh and constables Gajraj and Sonbir have been suspended and attached to the Mhow police lines pending the probe.
(The above story first appeared on Fresh Headline on Sep 03, 2022 10:10 PM IST. For more news and updates on politics, world, sports, entertainment and lifestyle, log on to our website freshheadline.com).
Arjun Singare
Custodial death
Manpur
Police Custody
police personnel
Tribal Youth
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Our new environmental watchdog backs getting cross-party support for greenhouse-gas targets.
The case for a UK-style independent commission to guide climate policy over coming decades received a boost this month, when the new Parliamentary Commissioner for the Environment, Simon Upton, strongly backed the concept.
Upton, who spent seven years as environmental director for the OECD in Paris, says this model stands out as the one that's most likely to create a stable long-term path to lower emissions.
By throwing his weight behind the idea of a climate commission to provide expert advice on long-term carbon budgets to the government and elevate climate policy above party politics, he has upped the pressure on National to work alongside the Labour-led Government on its plans for a New Zealand version of the system.
The Government plans to bring in a Zero Carbon Bill by the end of this year, which will commit the country to net zero greenhouse-gas emissions by 2050, and it wants to have an independent Climate Change Commission in place by mid-2019.
In December, Climate Change Minister James Shaw announced plans for wide-ranging consultation before the introduction of a bill in October. He made it clear he wants cross-party support. In the meantime, an interim climate committee will be set up to begin research and analysis on climate issues.
National's climate change spokesman, Todd Muller, has so far offered muted support, on one hand accusing the Government of "rhetoric" on climate issues and on the other saying National is "up for" a conversation about the transition to a lower-emissions economy, but that change must be carried out carefully "so as not to shock established sectors".
Upton's report – his first in the role since he took over from former commissioner Jan Wright late last year – says our climate policy must break out of the "stop-go, on-again off-again approach to tackling such a pressing long-term problem".
Although successive governments agreed on an emissions trading scheme, it was watered down soon after it was introduced in 2008, leading to "muted" price signals and having little effect on emissions. Agriculture was left out, and there was "excessive" reliance on buying offshore carbon credits – many of which were cheap "hot air" units with no environmental integrity – to meet international commitments.
New Zealand climate policy has been "dialled back, waiting for the rest of the world to move", Upton says. Meanwhile, there has been very little attention to domestic greenhouse-gas emissions, which have risen by 64% since 1990, with a 68% increase in the transport sector. Per capita, New Zealand's emissions are the fifth-highest in the OECD.
But he is convinced – as was Wright – that the UK model provides a powerful template to overcome the vagaries of the three-year election cycle, by setting long-term targets that provide investors and industry with the certainty that's needed to make the transition to a low-emission economy.
The UK system is underpinned by the Climate Change Act, introduced by David Cameron's opposition Conservative Party in 2008 and passed with a massive cross-party vote of 463 MPs in favour and only three against. It set up an expert climate committee, which develops five-year carbon budgets set 12 years in advance.
These budgets stipulate the total volume of greenhouse gases that can be emitted by the UK in that period. The Government adopts the budgets and is then required to come up with policies that ensure emissions stay within them. The UK is now up to its fifth carbon budget, which requires a 57% cut in emissions from 1990 levels by 2032. The long-term target is for an 80% reduction in carbon emissions by 2050.
The UK easily met the first three of its carbon budgets, although Government policies have so far not been strong enough to meet the fourth and its strategy for meeting the fifth budget also falls short.
Also, the UK was able to focus on carbon dioxide emissions, whereas New Zealand has to deal with three significant greenhouse gases – carbon dioxide from fossil fuels, and methane and nitrous oxide from farming, which together account for half our emissions. This adds significant complexity – there are no proven solutions to emissions from livestock and biological processes, and the gases have different characteristics. Methane is a powerful greenhouse gas but is short-lived in the atmosphere, whereas nitrous oxide is potent and, like carbon dioxide, accumulates in the atmosphere.
Upton points out that all three gases are increasing. "This cannot continue. All sectors will need to play their part to reverse these upward trends." Because of the different characteristics of the three gases, he thinks we should look at having different long-term targets for each.
The first couple of budgets – he suggests they cover six-year periods – need to be ambitious enough to "arrest" the upward trend of emissions and begin a structural adjustment of the economy. But it's the budgets that are set for the 2030s that "really count".