High Hydrostatic Pressure Stress Research Articles

Pressure-tolerant survival mechanism of Schizophyllum commune 20R-7-F01 isolated from deep sediments 2 kilometers below the seafloor

In anaerobic high hydrostatic pressure (HHP) sedimentary environments below the seafloor, fungi are found to dominate the eukaryotic communities, playing crucial ecological roles. However, the specific mechanisms by which fungi adapt to anaerobic HHP environments remain unclear. In this study, we investigated Schizophyllum commune 20R-7-F01 isolated from coal-bearing sediments at a depth of 2 km below the seafloor. By assessing the cell viability, biomass, and cell wall thickness changes of strain 20-7-1 under different HHP conditions, we observed that, compared to 0.1 MPa, strain 20-7-1 exhibited slower growth rates and decreased cell viability at 15 MPa and 35 MPa, yet demonstrated significant pressure tolerance. Transcriptomic and metabolomic analyses revealed that this strain activated the carbohydrate metabolic process to simultaneously utilize ethanol and lactic acid fermentation pathway. Additionally, it activates the oxidoreductase activity and hydrolase activity pathways to detoxify intracellular reactive oxygen species (ROS). Activation of the metal ion binding pathway increases the proportion of unsaturated fatty acids in the cell membrane, while instigation of the integral component of membrane pathway maintains cell wall structural stability. Furthermore, activation of the DNA repair pathway repairs DNA damage, demonstrating its comprehensive adaptive mechanisms against the HHP stress. These research findings deepen our understanding of fungal survival strategies and adaptation mechanisms in extreme environments, laying the groundwork for further exploration of their roles in cycling of carbon, nitrogen, sulfur, and other elements in the deep biosphere.

Mechanisms of nucleic acid degradation and high hydrostatic pressure tolerance of a novel deep-sea wall-less bacterium.

Wall-less bacteria are broadly distributed in diverse habitats. They evolved from a common ancestor within the Firmicutes phylum through reductive evolution. Here, we report the cultivation, characterization, and polyphasic taxonomic analysis of the novel free-living wall-less bacterium, Hujiaoplasma nucleasis zrk29. We demonstrated that strain zrk29 had a strong ability to degrade DNA and RNA both under laboratory conditions and in the deep sea. We found that nucleic acids induced strain zrk29 to release chronic bacteriophages which supported strain zrk29 and other marine bacteria to metabolize nucleic acids without lysing host cells. We also showed that strain zrk29 tolerated high hydrostatic pressure via two pathways: (i) by transporting cations into its cells to increase intracellular osmotic pressure and (ii) by adjusting the unsaturated fatty acid chain content in its cell membrane phospholipids to increase cell membrane fluidity. This study extends our understanding of free-living wall-less bacteria and provides a useful model to explore the unique adaptation mechanisms of deep-sea microbes. IMPORTANCE The unique physiology and survival strategies of the Tenericutes bacterium-a typical wall-less bacterium-have fascinated scientists and the public, especially in extreme deep-sea environments where there is high hydrostatic pressure (HHP) and limited availability of nutrients. Here, we have isolated a novel free-living Tenericutes strain from deep-sea sediment and have found that it metabolizes nucleic acids with the support of chronic bacteriophages. This Tenericutes strain tolerates HHP stress by increasing intracellular osmotic pressure and the unsaturated fatty acid chain content of phospholipids in its cell membrane. Our results provide insights into the unique physiology of deep-sea free-living Tenericutes bacteria and highlight the significant role that chronic bacteriophages play in assisting wall-less bacteria to adapt to harsh conditions.

Impact of high hydrostatic pressure on the single nucleotide polymorphism of stress-related dnaK, hrcA, and ctsR in the Lactobacillus strains

Lactic acid bacteria (LAB) are widespread in environments and can either have a positive impact because their ability to survive in harsh conditions and influence the product (probiotic properties, change of structure-EPS [exopolysaccharides], etc.), or a negative impact, (so not needed) because of their spoilage ability (beer, juices). High hydrostatic pressure (HHP), one of the non-thermal preservation methods used in the food industry, can force the LAB to activate the adaptative mechanisms. Under pressurization, the changes in the bacteria cells can occur at the transcriptional or translational level. This study evaluated the HHP on the single nucleotide polymorphism (SNP) changes in three genes, dnaK, ctsR, and hrcA, related to the stress-response mechanism in LAB. The correlation between the DNA polymorphism and the gene expression under HHP stress was assessed. The applied pressure of 300 MPa resulted in a low ratio of nonsynonymous substitutions to the synonymous substitutions (0 to 1.12), and a lower number of mutations was observed for pressurized strains (from 6 in hrcA to 11 in dnaK) than in controlled (from 3 in ctsR to 92 in hrcA). In all pressurized strains, the expression of genes was observed, whereas, in control strains, the gene expression was detected in three out of five strains. Although there was a noticeable change in stress-related gene expression after HHP, there was no correlation with SNPs. At the same time, with a high frequency of synonymous changes in nucleotide and high diversity for hrcA and dnaK, a very low diversity was found in ctsR sequences. The LAB strains stress response mechanisms are much more complex. The study requires information on the general mechanism and changes in the membranes’ composition, proteome changes, and gene expression patterns. The mutations in genes related to stress can have important implications for the strains’ fitness effect and adaptive ability of LAB strains, especially considering their food industry implication where the HHP techniques are used.

Impact of high hydrostatic pressure on the single nucleotide polymorphism of stress-related dnaK, hrcA, and ctsR in the Lactobacillus strains

Lactic acid bacteria (LAB) are widespread in environments and can either have a positive impact because their ability to survive in harsh conditions and influence the product (probiotic properties, change of structure-EPS [exopolysaccharides], etc.), or a negative impact, (so not needed) because of their spoilage ability (beer, juices). High hydrostatic pressure (HHP), one of the non-thermal preservation methods used in the food industry, can force the LAB to activate the adaptative mechanisms. Under pressurization, the changes in the bacteria cells can occur at the transcriptional or translational level. This study evaluated the HHP on the single nucleotide polymorphism (SNP) changes in three genes, dnaK, ctsR, and hrcA, related to the stress-response mechanism in LAB. The correlation between the DNA polymorphism and the gene expression under HHP stress was assessed. The applied pressure of 300 MPa resulted in a low ratio of nonsynonymous substitutions to the synonymous substitutions (0 to 1.12), and a lower number of mutations was observed for pressurized strains (from 6 in hrcA to 11 in dnaK) than in controlled (from 3 in ctsR to 92 in hrcA). In all pressurized strains, the expression of genes was observed, whereas, in control strains, the gene expression was detected in three out of five strains. Although there was a noticeable change in stress-related gene expression after HHP, there was no correlation with SNPs. At the same time, with a high frequency of synonymous changes in nucleotide and high diversity for hrcA and dnaK, a very low diversity was found in ctsR sequences. The LAB strains stress response mechanisms are much more complex. The study requires information on the general mechanism and changes in the membranes’ composition, proteome changes, and gene expression patterns. The mutations in genes related to stress can have important implications for the strains’ fitness effect and adaptive ability of LAB strains, especially considering their food industry implication where the HHP techniques are used.

Response mechanism of Vibrio parahaemolyticus at high pressure revealed by transcriptomic analysis.

Vibrio parahaemolyticus is a common pathogen in aquatic products, such as shellfishes. Laboratory-based simulated studies demonstrated that V. parahaemolyticus can tolerate high hydrostatic pressure (HHP) up to 20MPa. However, the molecular mechanisms of high-pressure adaptation remain unclear. Herein, we analyzed the physiological changes and transcriptomic responses of V. parahaemolyticus ATCC 17,802 under HHP conditions to determine the possible survival mechanisms. Under HHP conditions, the morphology of V. parahaemolyticus was notably changed exhibiting the coccoid microbial cells. The transcriptome analysis revealed that there were 795 differentially expressed genes (DEGs) under the 20MPa condition, including 406 upregulated DEGs and 389 downregulated DEGs. Most of the downregulated DEGs encoded proteins related to energy metabolism, such as citrate synthase (gltA), pyruvate kinase (pyk), and glyceraldehyde-3-phosphate dehydrogenase (gapA). Many of the upregulated DEGs encoded proteins related to adhesion and virulence factors, such as RNA polymerase σ factor (rpoE), L-threonine 3-dehydrogenase, and bacterial nucleotide signal c-di-GMP (WU75_RS02745 and WU75_RS07185). In our proposed mechanism model, V. parahaemolyticus responds to HHP stress through RNA polymerase σ factor RpoE. These findings indicate that V. parahaemolyticus cells may adopt a complex adaptation strategy to cope with HHP stress. KEY POINTS: •The transcriptomic response of Vibrio parahaemolyticus under HHP conditions was studied for the first time. •V. parahaemolyticus may adopt a complex adaptation strategy to cope with HHP stress. •ToxRS and RpoE played an important role in sensing and responding the HHP signal.

High hydrostatic pressure treatment of Arthrospira (Spirulina) platensis extracts and the baroprotective effect of sugars on phycobiliproteins

The effect of High Hydrostatic Pressure (HHP) (600 MPa; 300 s) upon phycobiliproteins from Spirulina platensis extract, in aqueous systems, has been investigated along with the potential baroprotective ability of several sugars (sucrose, trehalose and glucose) at different concentrations (20%, 40% w/w). Phycobiliproteins stability and technological functionality were assessed through spectroscopical and colour analysis, and radical scavenging ability.HHP treatment resulted in a decrease of the phycobiliproteins concentration with allophycocyanin being the most sensitive towards HHP stress (p ≤ 0.05). Addition of saccharides had a positive effect on phycocyanin stability with a higher residual concentration after HHP treatment (p ≤ 0.05) irrespective of the type and concentration of sugars added in the system, whilst baroprotective effects on allophycocyanin and phycoerythrin were observed only at the highest (40%) concentrations tested (p ≤ 0.05). After the HHP treatment, colouring ability of Spirulina platensis extract resulted in being directly correlated to phycocyanin concentration variation (R2 = 0.75). Addition of sugars preserved the antioxidant activity of the extract subject to high pressure regardless of the sugar type. Industrial relevanceSpirulina platensis has become a popular ingredient among manufacturers and consumers, especially for its technological functionality and nutritional properties. Nevertheless, processing and storage may result detrimental for its performance in foods. High hydrostatic pressure is a successful, non-thermal technology that is finding increasing applications in the food industry though processing costs of food products stabilized by HHP result, nowadays, being 7 to 15-fold higher than thermal pasteurization. However, consumers today are willing to pay higher prices for higher quality products with better sensory profiles and nutritional properties. Moreover, compared to thermal pasteurization, HHP is a rather new technology whose cost-effectiveness and environmental impact is expected to improve over time, with a consequent reduction of the gap among performance of these two technologies. Hence, this study focused on the effects of HHP treatments on aqueous Spirulina platensis extracts, alone or added with small saccharides to evaluate their potential baroprotective abilities, providing results of interest for application in food product design and development.

Oxidation of trimethylamine to trimethylamine N-oxide facilitates high hydrostatic pressure tolerance in a generalist bacterial lineage.

High hydrostatic pressure (HHP) is a characteristic environmental factor of the deep ocean. However, it remains unclear how piezotolerant bacteria adapt to HHP. Here, we identify a two-step metabolic pathway to cope with HHP stress in a piezotolerant bacterium. Myroides profundi D25T, obtained from a deep-sea sediment, can take up trimethylamine (TMA) through a previously unidentified TMA transporter, TmaT, and oxidize intracellular TMA into trimethylamine N-oxide (TMAO) by a TMA monooxygenase, MpTmm. The produced TMAO is accumulated in the cell, functioning as a piezolyte, improving both growth and survival at HHP. The function of the TmaT-MpTmm pathway was further confirmed by introducing it into Escherichia coli and Bacillus subtilis Encoded TmaT-like and MpTmm-like sequences extensively exist in marine metagenomes, and other marine Bacteroidetes bacteria containing genes encoding TmaT-like and MpTmm-like proteins also have improved HHP tolerance in the presence of TMA, implying the universality of this HHP tolerance strategy in marine Bacteroidetes.

Hydrostatic pressure boost rate and mode to enhance sterilization mediated by GroEL-interacting proteins

High hydrostatic pressure (HHP) is a new type of non-thermal food processing technology that is very important for ensuring the safety and quality of food. However, the biological molecular mechanism of HHP sterilization remains unclear. Here, our study explored the sterilizing effects of different HHP-boosting processes (boost mode and rate) on Escherichia coli O157:H7 and elucidated the biological mechanism based on the GroEL protein and its interacting proteins. First, the effects of different boost modes and rates on the bactericidal effect of E. coli O157:H7 were analyzed. The results showed that the bactericidal effect of the linear and fast boosting treatment was the most significant, and the expression of the GroEL protein in E. coli O157:H7 also increased under all four different boost modes and rates. The proteins that interacted with GroEL under different boost modes and rates were obtained and analyzed by immunoprecipitation and mass spectrometry (MS). The differences in the metabolic pathways of the E. coli O157:H7 stress response induced by different boosting methods and rates were analyzed. The results of the KEGG pathway analysis indicated that these interacting proteins were involved in 42 metabolic pathways mainly concentrated in ribosome, ABC transporter, oxidative phosphorylation and sulfate assimilation and cysteine biosynthesis. According to the difference in the stress-related metabolic pathway, the key interacting proteins NuoB, NuoF, CysD, ZnuC and RplX were screened, and their interaction with GroEL was verified in vivo by co-immunoprecipitation (CoIP). Finally, the linear and fast boost mode could reduce the defense capacity of E. coli O157:H7 cells against HHP stress, which is not conducive to the survival of E. coli O157:H7 under HHP, resulting in the best bactericidal effect.

An untargeted metabolomic insight into the high-pressure stress effect on the germination of wholegrain Oryza sativa L.

High hydrostatic pressure (HHP) technique is used as a novel abiotic stress factor for efficiently enhancing the biosynthesis of selected bioactive phytochemicals in germinated wholegrain, but the information about HHP stress-induced metabolic changes remains rather limited. Thus, the current work employed an untargeted gas chromatography-mass spectrometry-based metabolomic approach combining with multivariate models to analyze the effect of mild HHP stress (30 MPa/5 min) on the overall metabolome shifts of wholegrain brown rice (WBR) during germination. Simultaneously, major phenolics in germinated WBR (GBR) were detected by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry, to explore the potential relationship between HHP stress-induced rice metabolome alternations and the biotransformation of bioactive components. The results demonstrated that the influence of HHP stress on GBR metabolite profiles was defined by germination durations, as revealed by the differentiation of the stressed grains from the naturally germinated grains at different germination points according to principal component analysis. This was further confirmed by the results of orthogonal projections to latent structures discriminant analysis, in which the discriminating metabolites between naturally germinated and HHP-stressed grains varied across the germination process. The metabolite signatures differentiating natural and HHP-stressed germination included glycerol-3-phosphate, monosaccharides, gamma-aminobutyric acid, 2,3-butanediol, glyceryl-glycoside, amino acids and myo-inositol. Besides, HHP stress led to the increase in ribose, arabinitol, salicylic acid, azelaic acid and gamma-aminobutyric acid, as well as the reduced phenolic acids. These results demonstrated that HHP stress before germination matched with appropriate process parameters could be used as a promising technology to tailor metabolic features of germinated products, thus exerting targeted nutrition and health implications.

Differences in gene modulation in Saccharomyces cerevisiae indicate that maturity plays an important role in the high hydrostatic pressure stress response and resistance

There is a strong relationship between the regulatory pathways to oxidative stress, longevity, and aging. High hydrostatic pressure (HHP) induces oxidative stress and activates cellular defense mechanisms. The understanding of these mechanisms is a strategy to delay damage associated with aging. Addressing resistance to stress and aging in Saccharomyces cerevisiae is a well-accepted approach since pathways involved in energy balance, damage accumulation and stress response are preserved among eukaryotes. The purpose of this study was to correlate the environmental stress response to cell maturity. HHP stress response on S. cerevisiae mother and daughter cells was evaluated through survival, reactive oxygen species (ROS) accumulation and gene expression. Mature cells were yeasts that had budded and originated at least one descendant, and young cells were the ones that did not form a bud. Mature cells were more resistant to HHP, although they showed a decrease in expression of antioxidants enzymes genes, and a higher intracellular levels of ROS. Young cells had less resistance to HHP despite a tendency of positively regulating these same antioxidant encoders. The TOR1 gene, related to aging and apoptosis, was unchanged in mother cells and showed a tendency toward increased expression in daughter cells submitted to HHP. The gene modulation differences of the mother and daughter cells indicates that maturity plays an important role in the HHP stress response and resistance. Thus, even accumulating high levels of ROS, mature cells were more tolerant to HHP stress and survived better, despite aging.

Effect of high hydrostatic pressure on mitochondrial activity, reactive oxygen species level and developmental competence of cultured pig embryos

High hydrostatic pressure (HHP) has been previously used to increase mammalian oocyte and embryo tolerance on subsequent stresses related with different assisted reproductive technologies. Nevertheless, the mechanisms for HHP-induced stress responses in early embryos have not been yet well understood. Previous studies focused mainly on HHP-modified gene expression while possible changes in cellular functions, including modification of energy metabolism and oxidative stress were neglected. Therefore, we aimed to analyze the effect of HHP treatment on the efficiency of subsequent in vitro pig embryos culture in NCSU-23 medium, on mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) level during their pre-implantation development. Porcine embryos were exposed to the hydrostatic pressure of 20 MPa and their quick response to such stress was analyzed 1 h later. In comparison with control embryos, we detected lower ΔΨm by ∼13% only in expanded blastocysts as well as decreased ROS level by ∼30% and ∼42% at the morula and expanded blastocyst stages, respectively. After HHP-treatment at transcriptionally inactive zygote stage and subsequent embryo culture, long-time responses were found: (1) at expanded blastocyst stage manifesting by ΔΨm decrease by ∼16%, (2) at the morula and expanded blastocyst stages in the form of ROS level reduction by ∼38% and ∼33% respectively. Following HHP stress applied at the transcriptionally active morula stage the long-time response in the expanded blastocysts as a decrease of ΔΨm by ∼19% and ROS level by ∼37% was observed. The percentage of obtained expanded blastocysts was higher after culture of HHP-treated zygotes in comparison to the control. Moreover, expanded blastocysts developed in vitro from both HHP-treated zygotes or morulae, exhibited higher total number of cells per blastocyst, higher number of cells in the inner cell mass as well as lower number of TUNEL-positive nuclei per blastocyst and lower TUNEL index, when compared to untreated embryos. Therefore, the HHP stress applied at the zygote stage, enhances developmental potential and quality of in vitro obtained porcine blastocysts due to the both decreased ΔΨm and ROS level. Our findings may contribute to better understanding of the mechanism of HHP-mediated modifications of energy metabolism and oxidative stress during in vitro development of pig embryos.

Mild high hydrostatic pressure pretreatments applied before soaking process to modulate wholegrain brown rice germination: An examination on embryo growth and physicochemical properties

This investigation aimed to examine the effects of a novel processing pattern, combining high hydrostatic pressure (HHP) with germination, on the embryo growth and physicochemical characteristics of wholegrain brown rice (WBR). WBR grains were firstly subjected to mild HHP stress (30–90 MPa/5 min) and then incubated at 37 °C for 52 h for obtaining germinated samples (GBR). The results showed that HHP shock resulted in a delayed embryo growth of WBR grains, maintaining acceptable sprouting rates ranging from 65% to 76% when germination was finished. The contents of gama-aminobutyric acid in GBR were greatly increased responding to HHP stress, showing pressure intensities dependent. Total digestible and resistant starch contents in samples stressed at 60/90 MPa were decreased, mainly associated with high pressure-induced amorphization as revealed by SEM imaging and FTIR, which promoted starch hydrolysis during germination. Besides, the levels of zinc and iron were influenced by HHP pretreatments due to the high pressure-mediated degradation behavior for phytic acids. The storability of HHP-stressed GBR grains was significantly enhanced through reducing free fatty acids formation and maintaining color stability during a storage testing. These results obtained from the current work demonstrated that mild HHP stress pretreatment prior to germination process could be used as a promising strategy to modulate certain physicochemical characteristics of WBR products.

A transcriptome resource for the deep-sea bacterium Shewanella piezotolerans WP3 under cold and high hydrostatic pressure shock stress

Low temperature and high hydrostatic pressure (HHP) are two of the most remarkable environmental factors influencing deep-sea ecosystem. The adaptive mechanisms of microorganisms which live in these extreme environments to low temperature and high pressure warrant investigation. In this study, the global gene expression patterns of the deep-sea bacterium Shewanella piezotolerans WP3 in response to cold (0 °C) and HHP (50 MPa) shock were evaluated through DNA microarray analysis. Results revealed that 22, 66, and 106 genes were differentially expressed after WP3 was respectively exposed to cold shock for 30, 60, and 90 min. Of these genes, 16 genes were identified as common differentially expressed genes (DEGs). After 30 min and 120 min of HHP shock, 5 and 10 genes were respectively identified as DEGs. The hierarchical clustering analysis of the DEG pattern indicated that WP3 may employ different adaptive strategies to cope with cold and HHP shock stress. Taken together, our study provided a transcriptome resource for deep-sea bacterial responses to cold and HHP stress. This study also established a basis for further investigations on environmental adaptive mechanisms utilized by benthic bacteria.

High hydrostatic pressure leads to free radicals accumulation in yeast cells triggering oxidative stress.

Saccharomyces cerevisiae is a unicellular organism that during the fermentative process is exposed to a variable environment; hence, resistance to multiple stress conditions is a desirable trait. The stress caused by high hydrostatic pressure (HHP) in S. cerevisiae resembles the injuries generated by other industrial stresses. In this study, it was confirmed that gene expression pattern in response to HHP displays an oxidative stress response profile which is expanded upon hydrostatic pressure release. Actually, reactive oxygen species (ROS) concentration level increased in yeast cells exposed to HHP treatment and an incubation period at room pressure led to a decrease in intracellular ROS concentration. On the other hand, ethylic, thermic and osmotic stresses did not result in any ROS accumulation in yeast cells. Microarray analysis revealed an upregulation of genes related to methionine metabolism, appearing to be a specific cellular response to HHP, and not related to other stresses, such as heat and osmotic stresses. Next, we investigated whether enhanced oxidative stress tolerance leads to enhanced tolerance to HHP stress. Overexpression of STF2 is known to enhance tolerance to oxidative stress and we show that it also leads to enhanced tolerance to HHP stress.

Effect of high hydrostatic pressure on seeds Carica papaya

The breaking of seed dormancy induced by stress [1] is extremely important with regard to the acceleration of germination process [1,2]. Abiotic stress processes, just like high hydrostatic pressure (HHP), are applied in several biotechnological studies, mainly because HHP induces changes in physiological and biochemical processes [3]. Pretreatment with a stress induction tool can be lethal or induce the synthesis of protective factors. When the stress does not kill the organism, it is called sublethal stress, and it can even increase the resistance to other stresses. High hydrostatic pressure is an important model in stress response study, for it has the ability to change the plasma membrane fluidity so as to keep the organism alive, which allows studies on the metabolic changes. Water is used as the medium of fluid pressure transmission since it has lower compressibility and greater compatibility, resulting in less risk of contamination. In our experiment, Carica Papaya seeds were put into a polyethylene tube containing water, the system is then closed in a capsule of high resistance steel, and suffers pressures between 10 and 400 MPa. The seeds were set to germinate in vitro, while the pressure transfer fluid present in the capsule was used to analyze abscisic acid per HPLC. The HHP generated positive effects due the seed hydration, which reflected in the percentage and speed of germination [4]. The begin of the seeds germination subjected to HHP was anticipated to 70 hours due to greater water retention, characterized by the increasing of weight (about 2.8 times greater when compared to dry weight), followed by germination values between 70-90%, while the seeds subjected to ambient pressure (control group) showed hydration of about 2.4 times and germination around 55-65%. The ambient pressure (0.1 MPa) showed lower water retention, resulting in lower mean percentage and germination speed when compared to HHP treatments. The abscisic acid (ABA) is closely related to tolerance to stress conditions (HHP) and its relation to gibberellin (GA) is crucial in the process of germination. The pressure transfer fluid has proven useful for the detection of abscisic acid, characterized by the presence of chromatograph peak with retention time equals to analyses of samples fortified with industrial ABA. The analyses of the HPLC chromatograms demonstrate changes in the composition of the water used as medium of pressure transfer. The seeds undergoing HHP pressure showed through gas chromatogram, differences between the concentrations of protein, peptides and phenol, when compared to the seeds subjected to hydrostatic control group. The balance between the concentrations of abscisic acid and gibberelin is related to germination. The presence of ABA in fluid of pressure transfer indicates a factor of germination capacity of seeds subjected to HHP stress. HHP stress is an effective biotechnological tool in the search for new mechanisms of stimulation of Carica Papaya seeds in order to provide improved germination and breaking of dormancy. Some data obtained in this experiment were omitted because due to patent submission.

Effect of high hydrostatic pressure on the biosynthesis of sulfur amino acids in Saccharomyces cerevisiae

High hydrostatic pressure (HHP) is successfully applied in several industrial segments, as in vaccine production and food conservation. The response of microorganisms to HHP treatment resemble the responses of other stresses with industrial relevance, like osmotic, temperature and ethanol, which make the HHP a valuable tool in biotechnology research, as in the ethanol production [1]. Amino acids play a key role in central metabolism besides being the building blocks of proteins, and they are important to the HHP stress response. In this study, the Saccharomyces cerevisiae BT0510 was exposed to 50 MPa for 30 min at room temperature, followed by incubation at room pressure with aeration for 15 min. Samples of total RNA were collected every 5 min for transcriptional analysis by DNA microarray technique. Bioinformatics analysis demonstrated the upregulation (≥ 2 fold) by HHP treatment of genes related to the sulfur amino acids synthesis, methionine and cysteine. The HHP treatment induced the genes MET3, MET10, MET14 and MET16, which are correlated with the conversion of intracellular sulfate in sulfide. MET2, related to the conversion of homoserine to O-acetylhomoserine, was also induced by HHP, as well as the gene that codes for Met17p, responsible for the incorporation of sulfide in O-acetylhomoserine to form homocysteine, that will be directed to methionine or cysteine synthesis. These amino acids are directly correlated with sulfur assimilation in yeast cells. Methionine is the S-adenosylmethionine precursor, which participates in the biosynthesis of lipids and polyamines, and is also involved in methylation reactions, being a methyl group donor [2,3]. Cysteine is part of iron-sulfur proteins and is the glutathione biosynthesis precursor. Glutathione maintain the redox state in cytoplasm, therefore, playing an important role in cell response to oxidative stress [2,3]. The key gene related to the biosynthesis of methionine (MET6) was upregulated by HHP, while the gene related to the biosynthesis of cysteine (CYS4) was unaffected. Five minutes after pressure release MET6 was repressed. The genes related to the conversion of methionine to S-adenosylmethionine, SAM1 and SAM2, were downregulated. Methionine residues are important against reactive oxygen species (ROS) [4], and genes associated with the reduction of methionine sulfoxide (MXR1 and MXR2) were induced by HHP treatment, suggesting that methionine plays an important role in the reduction of ROS resulting from stress caused by HHP [5]. Concerning the regulation of sulfur amino acids metabolism, MET28 was strongly induced during the entire HHP and post treatment. Other factors, such as the transcription factor encoded by MET4 were not affected by HHP, and also MET30 that negatively regulates Met4p. Met28p appears to play an important role in the biosynthesis of sulfur amino acids in response to HHP. It seems that this protein participates in the Met4 complexes-DNA stabilization. Methionine biosynthesis upregulation is not related to other stresses, such as heat and osmotic stresses, and appears to be specific to HHP, which reinforces the use of this treatment to study the stress response in microorganisms.

Proteomic Analysis by Two-Dimensional Differential Gel Electrophoresis (2D DIGE) of a High-Pressure Effect in Bacillus cereus

High hydrostatic pressure (HHP) is a new method used to reduce or eliminate microorganisms that are present in food. Proteins are known to be the most important target of high pressure in living organisms. The main goal of this investigation was focused on the changes that occur on the proteins of Bacillus cereus under HHP stress conditions. The two-dimensional differential gel electrophoresis (2D DIGE) technique allows for a simultaneous resolution of thousands of proteins based on fluorescent prelabeling of the samples with spectrally resolvable fluorescent CyDyes. The results of proteomics profiling show an average of 1300 spots being detected. The analysis revealed 75 spot proteins whose abundance is modified after the application of high pressure, of which 66 were decreased after the HHP treatment. Among them, flagellin was the protein that changed the most. The differential expression of some proteins after HHP treatment at 700 MPa may suggest a reduction of virulence and protective response against oxidative stress in flagellated Bacillus .

Comparative proteome approach to characterize the high‐pressure stress response of Lactobacillus sanfranciscensis DSM 20451T

High hydrostatic pressure (HHP) exerts diverse effects on microorganisms, leading to stress response and cell death. While inactivation of microorganisms by lethal HHP is well investigated in the context of food preservation and the hygienic safety of minimal food processes, sublethal HHP stress response and its effect on adaptation and cross-protection is less understood. In this study, the HHP stress response of Lactobacillus sanfranciscensis was characterized and compared with cold, heat, salt, acid and starvation stress at the proteome level by using 2-DE so as to provide insight into general versus specific stress responses. Sixteen proteins were found to be affected by HHP and were identified by using N-terminal amino acid sequencing and MS. Only one slightly increased protein was specific to the HHP response and showed homology to a clp protease. The other proteins were influenced by most of the investigated stresses in a similar way as HHP. The highest similarity in the HHP proteome was found to be with cold- and NaCl-stressed cells, with 11 overlapping proteins. At the proteome level, L. sanfranciscensis appears to use overlapping subsets of stress-inducible proteins rather than stereotype responses. Our data suggest that a specific pressure response does not exist in this bacteria.