Ethanol Stress Research Articles

The Combined Effects of Perinatal Ethanol and Early-Life Stress on Cognition and Risk-Taking Behavior through Oxidative Stress in Rats.

Both prenatal ethanol and early-life stress have been shown to induce reduced risk-taking and explorative behavior as well as cognitive dysfunction in the offspring. In this study, we examined the effect of combined exposure to ethanol and early stress on maternal care, exploratory behavior, memory performances, and oxidative stress in male offspring.Pregnant rats were exposed to ethanol (4g/kg) from gestational day (GD) 6-to postnatal day (PND) 14 and limited nesting material (LNS) from PND0-PND14 individually or in combination. Maternal behavior was evaluated during diurnal cycle. The level of corticosterone hormone and markers of oxidative stress were evaluated in the pups. Risk-taking and explorative behavior were assessed with the elevated-plus maze (EPM) test and cognitive behavior with the Morris water maze (MWM), novel object recognition (NORT), and object location memory (OLM) tests.In the mothers, perinatal alcohol or LNS either alone or in combination decreased maternal behavior. In the offspring, the combination of the two factors significantly increased the pup's plasma corticosterone concentration in comparison with ethanol and LNS alone. Reduced risk-taking behavior was observed in the ethanol, LNS and ethanol + LNS groups compared with the control group, and this was amplified in the co-exposure of ethanol and LNS groups. The MWM, NORT, and OLM tests revealed spatial and recognition memory impairment in the ethanol and LNS groups. This impairment was more profound in the co-exposure of ethanol and LNS. Also, we observed a significant decrease in superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and an increase in malondialdehyde (MDA) level in the hippocampus of ethanol and LNS co-exposed animals as compared with individual exposure of ethanol and LNS.While each factor independently produced similar outcomes, the results indicate that the dual exposure paradigm could significantly strengthen the outcomes.

Wheat Gluten Peptides Enhance Ethanol Stress Tolerance by Regulating the Membrane Lipid Composition in Yeast.

Wheat gluten peptides (WGPs), identified as Leu-Leu (LL), Leu-Leu-Leu (LLL), and Leu-Met-Leu (LML), were tested for their impacts on cell growth, membrane lipid composition, and membrane homeostasis of yeast under ethanol stress. The results showed that WGP supplementation could strengthen cell growth and viability and enhance the ethanol stress tolerance of yeast. WGP supplementation increased the expressions of OLE1 and ERG1 and enhanced the levels of oleic acid (C18:1) and ergosterol in yeast cell membranes. Moreover, LLL and LML exhibited a better protective effect for yeast under ethanol stress compared to LL. LLL and LML supplementation led to 20.3 ± 1.5% and 18.9 ± 1.7% enhancement in cell membrane fluidity, 21.8 ± 1.6% and 30.5 ± 1.1% increase in membrane integrity, and 26.3 ± 4.8% and 27.6 ± 4.6% decrease in membrane permeability in yeast under ethanol stress, respectively. The results from scanning electron microscopy (SEM) elucidated that WGP supplementation is favorable for the maintenance of yeast cell morphology under ethanol stress. All of these results revealed that WGP is an efficient enhancer for improving the ethanol stress tolerance of yeast by regulating the membrane lipid composition.

Severe ethanol stress induces the preferential synthesis of mitochondrial disaggregase Hsp78 and formation of DUMPs in Saccharomyces cerevisiae

Severe ethanol stress (>9% v/v) induces pronounced translation repression in yeast cells. However, some proteins, which are exceptionally synthesized even under translation repression, play important roles in ethanol tolerance. These proteins are expected to provide important clues for elucidating the survival strategies of yeast cells under severe ethanol stress. In this study, we identified Hsp78 as a protein effectively synthesized under severe ethanol stress. As Hsp78 is involved in mitochondrial protein quality control, we investigated the effect of severe ethanol stress on mitochondrial proteins and found that Ilv2, Kgd1, and Aco1 aggregated with Hsp78 under severe ethanol stress, forming mitochondrial deposition sites for denatured proteins, called DUMPs (Deposits of Unfolded Mitochondrial Proteins). Aggregation of mitochondrial proteins and formation of DUMPs were accelerated in hsp78∆ cells compared with those in wild-type cells. During the recovery process after ethanol removal, aggregated Ilv2 and DUMP levels rapidly decreased in wild-type cells but were maintained for a long time (>180 min) in hsp78Δ cells. Furthermore, the frequency of respiration-deficient mutants caused by severe ethanol stress was higher in hsp78∆ cells than in wild-type cells. These results indicate that severe ethanol stress damaged mitochondrial proteins and that Hsp78 was preferentially synthesized to cope with the damage, thereby suppressing the rapid increase in aggregated protein levels under stress and achieving proper clearance of aggregated proteins during the recovery process. This study provides novel insights into the adverse effects of ethanol on mitochondria and yeast response to severe ethanol stress.

Ethanol Productivity of Ethanol-Tolerant Mutant Strain Pichia kudriavzevii R-T3 in Monoculture and Co-culture Fermentation with Saccharomyces cerevisiae

We previously developed ethanol-tolerant P. kudriavzevii R-T3 (P.ku R-T3) mutant from its parental strain P.ku R-WT by evolutionary adaptation method. Hence, we further analyze the ethanol productivity of the particular isolates in a monoculture and co-culture with industrial yeast Saccharomyces cerevisiae BY4741 at various inoculum ratio. Based on the spot assay, R3 mutant yeast showed better cell viability under 10% ethanol stress than the wild type, potentially due to the high expression level of PKINO1 gene involved in the synthesis of inositol. In the monoculture fermentation, S. cerevisiae could use glucose, while P.ku could use mixed glucose and xylose as carbon sources for ethanol fermentation. P.ku R-T3 performed the most potential ethanol kinetics parameters, including the highest ethanol production (10.10 g/L), ethanol productivity (0.21 g/L/h), and fermentation efficiency (84.36%). Upscaling the inoculum of P.ku R-T3 by ten times resulted in 10% higher ethanol production. However, the highest substrate utilization rate did not indicate an increase in ethanol production. Indeed, P.ku R-T3 showed a low mixed substrate use but produced higher ethanol production than S. cerevisiae, as much as 21-31%, depending on the initial inoculum. Interestingly, the co-culture of P.ku R-T3 and BY4741 did not substantially produce higher ethanol production than the monoculture technique. About 30% reduction of ethanol production was found by co-culturing BY4741 with P.ku R-T3 than P.ku R-T3 alone. Taken together, the monoculture fermentation of P.ku R-T3 remains the promising fermentation technique than that of the co-culture with industrial yeast S. cerevisiae.

Etp1 confers arsenite resistance by affecting ACR3 expression.

In a high-throughput yeast two-hybrid screen of predicted coiled-coil motif interactions in the Saccharomyces cerevisiae proteome, the protein Etp1 was found to interact with the yeast AP-1-like transcription factors Yap8, Yap1 and Yap6. Yap8 plays a crucial role during arsenic stress since it regulates expression of the resistance genes ACR2 and ACR3. The function of Etp1 is not well understood but the protein has been implicated in transcription and protein turnover during ethanol stress, and the etp1∆ mutant is sensitive to ethanol. In this current study, we investigated whether Etp1 is implicated in Yap8-dependent functions. We show that Etp1 is required for optimal growth in the presence of trivalent arsenite and for optimal expression of the arsenite export protein encoded by ACR3. Since Yap8 is the only known transcription factor that regulates ACR3 expression, we investigated whether Etp1 regulates Yap8. Yap8 ubiquitination, stability, nuclear localization and ACR3 promoter association were unaffected in etp1∆ cells, indicating that Etp1 affects ACR3 expression independently of Yap8. Thus, Etp1 impacts gene expression under arsenic and other stress conditions but the mechanistic details remain to be elucidated.

Development of high temperature simultaneous saccharification and fermentation by thermosensitive Saccharomyces cerevisiae and Bacillus amyloliquefaciens

Scarcity of energy and pollution are two major challenges that have become a threat to all living things worldwide. Bioethanol is a renewable, ecological-friendly clean energy that may be utilized to address these issues. This study aimed to develop simultaneous saccharification and fermentation (SSF) process through high temperature-substrate adaptation and co-cultivation of S. cerevisiae with other potential amylolytic strains. In this study, we adapted our previously screened thermosensitive Saccharomyces cerevisiae Dj-3 strain up-to 42 °C and also screened three potential thermotolerant amylolytic strains based on their starch utilization capability. We performed SSF fermentation at high temperature by adapted Dj-3 and amylolytic strains using 10.0% starch feedstock. Interestingly, we observed significant ethanol concentration [3.86% (v/v)] from high temperature simultaneous saccharification and fermentation (HSSF) of adapted Bacillus amyloliquefaciens (C-7) and Dj-3. We attribute the significant ethanol concentration from starch of this HSSF process to C-7’s high levels of glucoamylase activity (4.01 U/ml/min) after adaptation in starch (up-to 42 °C) as well as Dj-3's strong glucose fermentation capacity and also their ethanol stress tolerance capability. This study suggests the significant feasibility of our HSSF process.

Formation of Biofilm by Tetragenococcus halophilus Benefited Stress Tolerance and Anti-biofilm Activity Against S. aureus and S. Typhimurium.

Tetragenococcus halophilus, a halophilic lactic acid bacterium (LAB), plays an important role in the production of high-salt fermented foods. Generally, formation of biofilm benefits the fitness of cells when faced with competitive and increasingly hostile fermented environments. In this work, the biofilm-forming capacity of T. halophilus was investigated. The results showed that the optimal conditions for biofilm formation by T. halophilus were at 3–9% salt content, 0–6% ethanol content, pH 7.0, 30°C, and on the surface of stainless steel. Confocal laser scanning microscopy (CLSM) analysis presented a dense and flat biofilm with a thickness of about 24 μm, and higher amounts of live cells were located near the surface of biofilm and more dead cells located at the bottom. Proteins, polysaccharides, extracellular-DNA (eDNA), and humic-like substances were all proved to take part in biofilm formation. Higher basic surface charge, greater hydrophilicity, and lower intracellular lactate dehydrogenase (LDH) activities were detected in T. halophilus grown in biofilms. Atomic force microscopy (AFM) imaging revealed that biofilm cultures of T. halophilus had stronger surface adhesion forces than planktonic cells. Cells in biofilm exhibited higher cell viability under acid stress, ethanol stress, heat stress, and oxidative stress. In addition, T. halophilus biofilms exhibited aggregation activity and anti-biofilm activity against Staphylococcus aureus and Salmonella Typhimurium. Results presented in the study may contribute to enhancing stress tolerance of T. halophilus and utilize their antagonistic activities against foodborne pathogens during the production of fermented foods.

Characterization and Role of Sterols in Saccharomyces cerevisiae during White Wine Alcoholic Fermentation

Responsible for plasma membrane structure maintenance in eukaryotic organisms, sterols are essential for yeast development. The role of two sterol sources in Saccharomyces cerevisiae during wine fermentation is highlighted in this review: ergosterol (yeast sterol produced by yeast cells under aerobic conditions) and phytosterols (plant sterols imported by yeast cells from grape musts in the absence of oxygen). These compounds are responsible for the maintenance of yeast cell viability during white wine fermentation under stress conditions, such as ethanol stress and sterol starvation, to avoid sluggish and stuck fermentations.

Ethanol stress responses in Kluyveromyces marxianus: current knowledge and perspectives

The rising concern with the emission of greenhouse gases has boosted new incentives for biofuels production, which are less polluting than fossil fuels. Special attention has been given to the second-generation ethanol, as it is produced from abundant feedstocks which do not compete with food production, such as lignocellulosic biomass and whey. Kluyveromyces marxianus stands out in second-generation ethanol production due to its capacity of assimilating lactose, the sugar found in whey, and tolerating high temperatures used in simultaneous saccharification processes. Nonetheless, contrary to Saccharomyces cerevisiae, K. marxianus does not tolerate high ethanol concentrations. Ethanol causes a broad range of toxic effects on yeasts, acting on cell membrane and proteins, as well as inducing the generation of reactive oxygen species (ROS). The ethanol stress responses are not fully understood, mainly in non-conventional yeasts such as K. marxianus. Indeed, many molecular responses to ethanol stress are still inferred from S. cerevisiae. As such, a better understanding of the ethanol stress responses in K. marxianus may provide the basis for improving its use in the biofuel industry. Additionally, the selection of ethanol-tolerant strains by metabolic engineering is useful to provide strains with improved capacity to withstand stressful conditions, as well as to obtain new insights about the ethanol stress responses. Key points • It is still not totally clear why K. marxianus is less tolerant to ethanol than S. cerevisiae. • Understanding the ethanol stress response in K. marxianus is pivotal for improving its application in the biofuel industry. • The Metabolic engineering is expected to improve the ethanol tolerance in K. marxianus.

Isolation, Selection and Characterization of Wild Yeasts with Potential for Brewing

Ninety-two wild yeasts were isolated from different fruits, for possible use in brewing, using the technological traits of carbohydrate fermentation and hydrogen sulfide production. Twelve yeasts were selected for identification by sequencing (D1/D2 domain or ITS1-5.8S-ITS2 region) and their volatile compounds production analyzed using gas chromatography. From these analyses, three yeasts were chosen for further characterization and beer production. Strains were characterized by their tolerance to osmotic and ethanol stress, their flocculation profile, growth at different temperatures, apparent attenuation (percentage of sugars converted into alcohol), estimated ABV (alcohol by volume) and reducing sugar profile. Beers were analyzed for alcohol content and sensorial analysis. The isolates CL011 and PB111 (Saccharomyces cerevisiae) and CB341 (Wickerhamomyces anomalus) showed promising properties for brewing, which included high production of interesting volatile compounds (esters, terpenes, lactones, and C13-norisoprenoids) by CL011, a neutral profile by PB111, and the production of non-classic beer volatile compounds by CB341 in a mixed culture. Supplemental data for this article is available online at https://doi.org/10.1080/03610470.2022.2031777 .

Effect of Cell Membrane Integrity of Pichia Anomala by the Excessive ROS Under Ethanol Stress

Effect of Cell Membrane Integrity of Pichia Anomala by the Excessive ROS Under Ethanol Stress

Proteostasis in Yeast Cells Under Ethanol Stress and During Wine Making Process

Proteostasis in Yeast Cells Under Ethanol Stress and During Wine Making Process

Germ cell apoptosis is critical to maintain Caenorhabditis elegans offspring viability in stressful environments.

Maintaining reproduction in highly variable, often stressful, environments is an essential challenge for all organisms. Even transient exposure to mild environmental stress may directly damage germ cells or simply tax the physiology of an individual, making it difficult to produce quality gametes. In Caenorhabditis elegans, a large fraction of germ cells acts as nurse cells, supporting developing oocytes before eventually undergoing so-called physiological germ cell apoptosis. Although C. elegans apoptosis has been extensively studied, little is known about how germline apoptosis is influenced by ecologically relevant environmental stress. Moreover, it remains unclear to what extent germline apoptosis contributes to maintaining oocyte quality, and thus offspring viability, in such conditions. Here we show that exposure to diverse environmental stressors, likely occurring in the natural C. elegans habitat (starvation, ethanol, acid, and mild oxidative stress), increases germline apoptosis, consistent with previous reports on stress-induced apoptosis. Using loss-of-function mutant alleles of ced-3 and ced-4, we demonstrate that eliminating the core apoptotic machinery strongly reduces embryonic survival when mothers are exposed to such environmental stressors during early adult life. In contrast, mutations in ced-9 and egl-1 that primarily block apoptosis in the soma but not in the germline, did not exhibit such reduced embryonic survival under environmental stress. Therefore, C. elegans germ cell apoptosis plays an essential role in maintaining offspring fitness in adverse environments. Finally, we show that ced-3 and ced-4 mutants exhibit concomitant decreases in embryo size and changes in embryo shape when mothers are exposed to environmental stress. These observations may indicate inadequate oocyte provisioning due to the absence of germ cell apoptosis. Taken together, our results show that the central genes of the apoptosis pathway play a key role in maintaining gamete quality, and thus offspring fitness, under ecologically relevant environmental conditions.

Transcriptomic analysis of bacteriocin synthesis and stress response in Lactobacillus paracasei HD1.7 under acetic acid stress

Bacteriocins from lactic acid bacteria are natural preservatives that inhibit foodborne pathogenic microorganisms and have recently attracted much attention. Acetic acid metabolism is part of the core of central metabolism. To elucidate the acetic acid stress response of L. paracasei HD1.7, we performed high-throughput RNA-seq to compare gene expression under different concentrations of acetic acid stress. The results showed that with increasing acetic acid stress, the concentration of bacteriocin was increased, its production rate was accelerated, and the number of differentially expressed genes also increased. The continuously upregulated and downregulated KEGG pathways adjusted to the increase in acetic acid stress. The late-stage regulatory system of bacteriocin synthesis was related to ABC transporters (OppABCDF system). Purine and pyrimidine metabolism may provide ATP for ABC transporters. Acetic acid stress reduced glutamate biosynthesis, but arginine biosynthesis increased to attenuate acid stress. In addition, the KEGG enrichment analysis results of different environmental stresses (acetic acid, HCl, citric acid and ethanol stress) indicated that ABC transporters and fatty acid metabolism were the keys to bacteria adapting to environmental stress. This experiment lays the foundation for further research on bacteriocins.

Protective Effects of Melatonin on Saccharomyces cerevisiae under Ethanol Stress.

During alcoholic fermentation, Saccharomyces cerevisiae is subjected to several stresses, among which ethanol is of capital importance. Melatonin, a bioactive molecule synthesized by yeast during alcoholic fermentation, has an antioxidant role and is proposed to contribute to counteracting fermentation-associated stresses. The aim of this study was to unravel the protective effect of melatonin on yeast cells subjected to ethanol stress. For that purpose, the effect of ethanol concentrations (6 to 12%) on a wine strain and a lab strain of S. cerevisiae was evaluated, monitoring the viability, growth capacity, mortality, and several indicators of oxidative stress over time, such as reactive oxygen species (ROS) accumulation, lipid peroxidation, and the activity of catalase and superoxide dismutase enzymes. In general, ethanol exposure reduced the cell growth of S. cerevisiae and increased mortality, ROS accumulation, lipid peroxidation and antioxidant enzyme activity. Melatonin supplementation softened the effect of ethanol, enhancing cell growth and decreasing oxidative damage by lowering ROS accumulation, lipid peroxidation, and antioxidant enzyme activities. However, the effects of melatonin were dependent on strain, melatonin concentration, and growth phase. The results of this study indicate that melatonin has a protective role against mild ethanol stress, mainly by reducing the oxidative stress triggered by this alcohol.

Possible Role for Allelic Variation in Yeast MED15 in Ecological Adaptation.

The propensity for Saccharomyces cerevisiae yeast to ferment sugars into ethanol and CO2 has long been useful in the production of a wide range of food and drink. In the production of alcoholic beverages, the yeast strain selected for fermentation is crucial because not all strains are equally proficient in tolerating fermentation stresses. One potential mechanism by which domesticated yeast may have adapted to fermentation stresses is through changes in the expression of stress response genes. MED15 is a general transcriptional regulator and RNA Pol II Mediator complex subunit which modulates the expression of many metabolic and stress response genes. In this study, we explore the role of MED15 in alcoholic fermentation. In addition, we ask whether MED15 alleles from wine, sake or palm wine yeast improve fermentation activity and grape juice fermentation stress responses. And last, we investigate to what extent any differences in activity are due to allelic differences in the lengths of three polyglutamine tracts in MED15. We find that strains lacking MED15 are deficient in fermentation and fermentation stress responses and that MED15 alleles from alcoholic beverage yeast strains can improve both the fermentation capacity and the response to ethanol stresses when transplanted into a standard laboratory strain. Finally, we find that polyglutamine tract length in the Med15 protein is one determinant in the efficiency of the alcoholic fermentation process. These data lead to a working model in which polyglutamine tract length and other types of variability within transcriptional hubs like the Mediator subunit, Med15, may contribute to a reservoir of transcriptional profiles that may provide a fitness benefit in the face of environmental fluctuations.

Honey Mead Fermentation from Thai Stingless Bee (Tetragonula leaviceps) Honey using Ethanol Tolerant Yeast

Honey mead is a well-known conventional alcoholic beverage made by microbial fermentation of diluted honey. The selection of prospective yeasts for inoculation of honey-must with regard to honey mead quality determines the quality of mead production. The yeast consortium tolerant to ethanol stress was selected for this study using an enrichment technique. The activity of the invertase enzyme and the level of ethanol tolerance have been investigated. Thai stingless bee honey was used as a substrate, and the selected ethanol tolerant yeast consortium was used for mead fermentation. The results revealed that the PP03 had the highest invertase activity of 75.13±9.16 U/mL and the highest ethanol tolerance level of 12%. This is the first study using an ethanol tolerant yeast consortium to ferment honey mead from Thai stingless bee honey.

Cisplatin-based Electrochemotherapy Significantly Downregulates Key Heat Shock Proteins in MDA-MB-231-Human Triple-Negative Breast Cancer Cells.

Heat shock proteins (HSPs) are available and/or induced for the survival of all organisms, including eukaryotic, prokaryotic, and plants, from higher temperature stresses. They are the chaperone proteins that protect all cells against heat, as the name implies. In addition to thermal stress, they also protect them from chemical, physical, and other stresses, including exposure to oxidative stress, nutritional deficiencies, ultraviolet radiation, ethanol, viral infection, ischemia-reperfusion injury, and cancer-related stresses. They are classified based on their molecular weights in kDa, such as HSP90 and HSP70. In our label-free, high-throughput, quantitative LC-MS/MS-based proteomic studies of MDA-MB-231, human, triple-negative breast cancer cells, treated with electrical pulses (EP) and cisplatin (CsP), we identified a number of HSPs, such as HSP90AA1, and others to be significantly downregulated in EP + CsP, compared to CsP alone. This indicates that cells will undergo apoptotic cell death and hence could cause effective cancer cure/treatment. Considering that over 2 million new cases and over 600,000 deaths in 2020, of which ~ 15% are TNBC, heat shock proteins could be the untapped resources, available for the next biomarkers and/or inhibitors for new/additional therapies.

Analysis of Transcriptomic Response to SO2 by Oenococcus oeni Growing in Continuous Culture.

ABSTRACTTo successfully complete malolactic fermentation (MLF), Oenococcus oeni must overcome wine stress conditions of low pH, high ethanol, and the presence of SO2. Failure to complete MLF may result in detrimental effects to the quality and stability of the resulting wines. Research efforts to date have focused on elucidating the mechanisms and genetic features that confer the ability to withstand low pH and high ethanol concentrations on O. oeni; however, the responses to SO2 stress are less well defined. This study focused on characterizing the transcriptional response of O. oeni to SO2 challenge during cultivation in a continuous system at wine-like pH (3.5). This experimental design allowed the precise discrimination of transcriptional changes linked to SO2 stress from responses associated with growth stage and cultivation parameters. Differential gene expression analysis revealed major transcriptional changes following SO2 exposure and suggested that this compound primarily interacts with intracellular proteins, DNA, and the cell envelope of O. oeni. The molecular chaperone hsp20, which has a demonstrated function in the heat, ethanol, and acid stress response, was highly upregulated, confirming its additional role in the response of this species to SO2 stress. This work also reports the first nanopore-based complete genome assemblies for O. oeni.IMPORTANCE Malolactic fermentation is an indispensable step in the elaboration of most wines and is generally performed by Oenococcus oeni, a Gram-positive heterofermentative lactic acid bacterium species. While O. oeni is tolerant to many of the wine stresses, including low pH and high ethanol concentrations, it has high sensitivity to SO2, an antiseptic and antioxidant compound regularly used in winemaking. Understanding the physiological changes induced in O. oeni by SO2 stress is essential for the development of more robust starter cultures and methods for their use. This study describes the main transcriptional changes induced by SO2 stress in the wine bacterium O. oeni and provides foundational understanding on how this compound interacts with the cellular components and the induced protective mechanisms of this species.

Stress adaptation and virulence in Vibrio alginolyticus is mediated by two (p)ppGpp synthetase genes, relA and spoT

Vibrio alginolyticus belongs to gram-negative opportunistic pathogen realm infecting humans and aquatic animals causing severe economic losses. The (p)ppGpp-mediated stringent response is corroborated to stress adaptation and virulence of pathogenic mechanisms. Limited reports are documented for the intricate assessment of (p)ppGpp synthetase genes in combating various stress adaptation and elucidation of virulence in V. alginolyticus remains unraveled. The present assessment comprises of generation of deletion mutants in the (p)ppGpp-deficient strains, ΔrelA (relA gene single mutant) and ΔrelAΔspoT (relA and spoT genes double mutant), and the complemented strains, ΔrelA+ and ΔrelAΔspoT+, were constructed to investigate the pivotal roles of (p)ppGpp synthetase genes in V. alginolyticus, respectively. Amino acid sequence alignment analysis initially revealed that RelA and SpoT possess relatively conserved domains and synthetase activity. Hydrolase activity was emancipated by SpoT alone showing variant mode of action. Compared with the wild type and complemented strains, the relA-deficient strain was more sensitive to amino acid starvation and mupirocin. Interestingly, the deletion of spoT resulted in a significant growth deficiency supplemented with bile salts, 3 % ethanol and heat shock. Rapid growth was observed in the stationary phase upon exposure to cold stress and lower doses of ethanol. Subsequently, disruption of (p)ppGpp synthetase genes caused the decline in swimming motility, enhanced biofilm formation, cell aggregation of V. alginolyticus, and reduced mortality of Litopenaeus vannamei. The expression levels of some virulence-associated genes were quantified affirming consistency established by pleiotropic phenotypes. The results are evident for putative roles of (p)ppGpp synthetase genes attributing essential roles for environmental adaption and virulence regulation in V. alginolyticus.