Growth Inhibitory Compounds Research Articles

Decoupling a novel Trichormus variabilis-Synechocystis sp. interaction to boost phycoremediation

To conserve freshwater resources, domestic and industrial wastewater is recycled. Algal systems have emerged as an efficient, low-cost option for treatment (phycoremediation) of nutrient-rich wastewater and environmental protection. However, industrial wastewater may contain growth inhibitory compounds precluding algal use in phycoremediation. Therefore, extremophyte strains, which thrive in hostile environments, are sought-after. Here, we isolated such an alga - a strain of Synechocystis sp. we found to be capable of switching from commensal exploitation of the nitrogen-fixing Trichormus variabilis, for survival in nitrogen-deficient environments, to free-living growth in nitrate abundance. In nitrogen depletion, the cells are tethered to polysaccharide capsules of T. variabilis using nanotubular structures, presumably for nitrate acquisition. The composite culture failed to establish in industrial/domestic waste effluent. However, gradual exposure to increasing wastewater strength over time untethered Synechocystis cells and killed off T. variabilis. This switched the culture to a stress-acclimated monoculture of Synechocystis sp., which rapidly grew and flourished in wastewater, with ammonium and phosphate removal efficiencies of 99.4% and 97.5%, respectively. Therefore, this strain of Synechocystis sp. shows great promise for use in phycoremediation, with potential to rapidly generate biomass that can find use as a green feedstock for valuable bio-products in industrial applications.

Cell-to-cell contact mechanism modulates Starmerella bacillaris death in mixed culture fermentations with Saccharomyces cerevisiae

The use of mixed culture fermentations with selected Starmerella bacillaris and Saccharomyces cerevisiae strains is gaining winemaking attention, mainly due to their ability to enhance particular characteristics in the resulting wines. In this context, yeast interspecies interactions during fermentation have a fundamental role to determine the desired product characteristics, since they may modulate yeast growth and as a consequence metabolite production. In order to get an insight into these interactions, the growth and death kinetics of the abovementioned species were investigated in pure and mixed culture fermentations, using cv. Nebbiolo grape must. Trials were conducted in flasks but also in a double-compartment fermentation system in which cells of the two species were kept separate by a filter membrane. Although the two species had similar growth pattern during the first days of fermentation, Starm. bacillaris died earlier when tested in the flask than in the double-compartment fermentor. The early death of Starm. bacillaris seemed to be not caused by nutrient limitation nor by accumulation of growth inhibitory compounds (which were not measured in the present study). Rather, cell-to-cell contact mechanism, dependent on the presence of viable S. cerevisiae cells, appears to be responsible for the observations made. These results contribute to better understand the factors that influence Starm. bacillaris death during wine fermentations.

Evaluation on Allelopathic Potential of Velvet Bean (Mucuna cochinchinensis) on Germination of Goosegrass (Eleusineindica L.)

The experiment was conducted at the Toxicology laboratory, Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Malaysia in 2013. Allelopathic potential of aqueous methanol and water extracts of M. cochinchinensis leaves, seed and root was investigated on seed germination and seedling growth of goosegrass and biotest crop species: lettuce (Lactuca sativa). The treatments consisted of five concentrations (100, 75, 50, 25, 0 %); plant parts (leaves, seed, root) and extraction solvents (methanol, water) were replicated three times and arranged as a completely randomized block (CRD) design. Germination, radicle and hypocotyl growth of all test plant species were inhibited at concentrations (100, 75, 50 and 25%). The total germination percentage was lowest with the methanolic extracts of leaf, seed and root of M. cochinchinensis at 100 % concentration in the order of 0.00, 30.66 and 4.66%, respectively. Concomitantly, the radicle length inhibition percentages of methanolic extracts at higher concentration of 100% were 100, 88.5 and 94.4% of the leaf, seed and root extracts, respectively. The water extracts recorded the highest germination percentage and lower inhibitory activity of the radicle and hypocotyl length. The study confirmed plant growth-inhibitory compounds of M. cochinchinensis is dependent on the extraction solvents and extract concentrations as expressed that methanolic solvent at higher extract concentration had the stronger inhibitory activity.

Ethanol stress in Oenococcus oeni: transcriptional response and complex physiological mechanisms.

Oenococcus oeni is the dominant species able to cope with a hostile environment of wines, comprising cumulative effects of low pH, high ethanol and SO2 content, nonoptimal growth temperatures and growth inhibitory compounds. Ethanol tolerance is a crucial feature for the activity of O. oeni cells in wine because ethanol acts as a disordering agent of its cell membrane and negatively affects metabolic activity; it damages the membrane integrity, decreases cell viability and, as other stress conditions, delays the start of malolactic fermentation with a consequent alteration of wine quality. The cell wall, cytoplasmic membrane and metabolic pathways are the main sites involved in physiological changes aimed to ensure an adequate adaptive response to ethanol stress and to face the oxidative damage caused by increasing production of reactive oxygen species. Improving our understanding of the cellular impact of ethanol toxicity and how the cell responds to ethanol stress can facilitate the development of strategies to enhance microbial ethanol tolerance; this allows to perform a multidisciplinary endeavour requiring not only an ecological study of the spontaneous process but also the characterization of useful technological and physiological features of the predominant strains in order to select those with the highest potential for industrial applications.

Volatile compounds from beneficial or pathogenic bacteria differentially regulate root exudation, transcription of iron transporters, and defense signaling pathways in Sorghum bicolor.

Our results show that Sorghum bicolor is able to recognize bacteria through its volatile compounds and differentially respond to beneficial or pathogens via eliciting nutritional or defense adaptive traits. Plants establish beneficial, harmful, or neutral relationships with bacteria. Plant growth promoting rhizobacteria (PGPR) emit volatile compounds (VCs), which may act as molecular cues influencing plant development, nutrition, and/or defense. In this study, we compared the effects of VCs produced by bacteria with different lifestyles, including Arthrobacter agilis UMCV2, Bacillus methylotrophicus M4-96, Sinorhizobium meliloti 1021, the plant pathogen Pseudomonas aeruginosa PAO1, and the commensal rhizobacterium Bacillus sp. L2-64, on S. bicolor. We show that VCs from all tested bacteria, except Bacillus sp. L2-64, increased biomass and chlorophyll content, and improved root architecture, but notheworthy A. agilis induced the release of attractant molecules, whereas P. aeruginosa activated the exudation of growth inhibitory compounds by roots. An analysis of the expression of iron-transporters SbIRT1, SbIRT2, SbYS1, and SbYS2 and genes related to plant defense pathways COI1 and PR-1 indicated that beneficial, pathogenic, and commensal bacteria could up-regulate iron transporters, whereas only beneficial and pathogenic species could induce a defense response. These results show how S. bicolor could recognize bacteria through their volatiles profiles and highlight that PGPR or pathogens can elicit nutritional or defensive traits in plants.

Nutrient Stress Small-Molecule Screening Platform for Escherichia coli.

Clinically approved antibiotics target a narrow spectrum of cellular processes, namely cell wall synthesis, DNA replication, and protein synthesis. Numerous screens have been designed to identify inhibitors that target one of these cellular processes. Indeed, this narrow range of drug mechanisms and a reliance on chemical classes discovered many decades ago are thought to be principally responsible for the current crisis of antibiotic drug resistance. Seeking to expand the target base of antibacterial drug discovery, we developed a nutrient stress screening platform that identifies inhibitors of the growth of in Escherichia coli under nutrient limitation. Under nutrient stress, bacteria require an expanded biosynthetic capacity that includes the synthesis of amino acids, vitamins, and nucleobases. Growing evidence suggests that these processes may be indispensable to certain pathogens and at particular sites of infection. Indeed, more than 100 biosynthetic enzymes become indispensable to E. coli grown under nutrient stress in vitro. The screening platform described here puts a focus on these novel targets for new antibiotics and prioritizes growth inhibitory compounds that can be suppressed by individual nutrients and pools thereof.

The carbon consumption pattern of the spoilage yeast Brettanomyces bruxellensis in synthetic wine-like medium

The wine matrix contains limited carbon compounds to sustain microbial life. Brettanomyces bruxellensis is one of very few yeast species that has adapted to this environment. Indeed, the presence of growth-inhibiting compounds and conditions do not prevent its proliferation. Literature regarding the nutritional requirements of this yeast is surprisingly poor, given the observation that B. bruxellensis produces biomass with apparently less nutrients than other yeasts. In this study, various carbon sources were screened in a synthetic wine medium, under anaerobic and semi-aerobic growth conditions, in order to determine which compounds B. bruxellensis assimilates. Slight differences were observed between strains but overall, B. bruxellensis produced biomass from limited nutrients consumed in a specific order regardless of the oxygen conditions. Upon initial consumption of the simple sugars, B. bruxellensis was able to remain viable, by concurrently utilising ethanol (only in the presence of oxygen) and malic acid. Although initially beneficial, oxygen was found detrimental in the long term. Formation of volatile phenols occurred during the consumption of the sugars but not as a mechanism to help correct the redox imbalance. The study confirms that B. bruxellensis is able to survive using limited amount of nutrients, making this yeast a challenge for winemakers.

Cultivation in Space Flight Produces Minimal Alterations in the Susceptibility of Bacillus subtilis Cells to 72 Different Antibiotics and Growth-Inhibiting Compounds.

Past results have suggested that bacterial antibiotic susceptibility is altered during space flight. To test this notion, Bacillus subtilis cells were cultivated in matched hardware, medium, and environmental conditions either in space flight microgravity on the International Space Station, termed flight (FL) samples, or at Earth-normal gravity, termed ground control (GC) samples. The susceptibility of FL and GC samples was compared to 72 antibiotics and growth-inhibitory compounds using the Omnilog phenotype microarray (PM) system. Only 9 compounds were identified by PM screening as exhibiting significant differences (P < 0.05, Student's t test) in FL versus GC samples: 6-mercaptopurine, cesium chloride, enoxacin, lomefloxacin, manganese(II) chloride, nalidixic acid, penimepicycline, rolitetracycline, and trifluoperazine. Testing of the same compounds by standard broth dilution assay did not reveal statistically significant differences in the 50% inhibitory concentrations (IC50s) between FL and GC samples. The results indicate that the susceptibility of B. subtilis cells to a wide range of antibiotics and growth inhibitors is not dramatically altered by space flight.IMPORTANCE This study addresses a major concern of mission planners for human space flight, that bacteria accompanying astronauts on long-duration missions might develop a higher level of resistance to antibiotics due to exposure to the space flight environment. The results of this study do not support that notion.

Coinfection outcome in an opportunistic pathogen depends on the inter-strain interactions

BackgroundIn nature, organisms are commonly coinfected by two or more parasite strains, which has been shown to influence disease virulence. Yet, the effects of coinfections of environmental opportunistic pathogens on disease outcome are still poorly known, although as host-generalists they are highly likely to participate in coinfections. We asked whether coinfection with conspecific opportunistic strains leads to changes in virulence, and if these changes are associated with bacterial growth or interference competition. We infected zebra fish (Danio rerio) with three geographically and/or temporally distant environmental opportunist Flavobacterium columnare strains in single and in coinfection. Growth of the strains was studied in single and in co-cultures in liquid medium, and interference competition (growth-inhibiting ability) on agar.ResultsThe individual strains differed in their virulence, growth and ability for interference competition. Number of coinfecting strains significantly influenced the virulence of infection, with three-strain coinfection differing from the two-strain and single infections. Differences in virulence seemed to associate with the identity of the coinfecting bacterial strains, and their pairwise interactions. This indicates that benefits of competitive ability (production of growth-inhibiting compounds) for virulence are highest when multiple strains co-occur, whereas the high virulence in coinfection may be independent from in vitro bacterial growth.ConclusionsIntraspecific competition can lead to plastic increase in virulence, likely caused by faster utilization of host resources stimulated by the competitive interactions between the strains. However, disease outcome depends both on the characteristics of individual strains and their interactions. Our results highlight the importance of strain interactions in disease dynamics in environments where various pathogen genotypes co-occur.

Microbial Production of Xylitol from Oil Palm Empty Fruit Bunch Hydrolysate: Effects of Inoculum and pH

Considering its high content of hemicellulose, oil palm empty fruit bunch (EFB) lignocellulosic biomass waste from palm oil processing has the potential to be utilized as the raw material for the production of xylitol, a low calorie, low GI, and anti cariogenic alternative sugar with similar sweetness to sucrose. This research explored the possibility of converting EFB to xylitol via green microbial fermentation, in particular the effects of inoculum and initial pH on the fermentation performance. It was observed that the cell concentration in the inoculum and the initial pH affect cell growth and xylitol production. pH 5 was observed to give the best fermentation performance. Further, the fermentation tended to yield more xylitol at higher initial cell concentration. It was also observed that no growth or fermentation inhibitory compounds were found in the EFB hydrolysate obtained from enzymatic hydrolysis of EFB. Thus it can be used directly as substrate for xylitol fermentation.

Identification of Chemical-Genetic Interactions via Parallel Analysis of Barcoded Yeast Strains.

The Yeast Knockout Collection is a complete set of gene deletion strains for the budding yeast, Saccharomyces cerevisiae In each strain, one of approximately 6000 open-reading frames is replaced with a dominant selectable marker flanked by two DNA barcodes. These barcodes, which are unique to each gene, allow the growth of thousands of strains to be individually measured from a single pooled culture. The collection, and other resources that followed, has ushered in a new era in chemical biology, enabling unbiased and systematic identification of chemical-genetic interactions (CGIs) with remarkable ease. CGIs link bioactive compounds to biological processes, and hence can reveal the mechanism of action of growth-inhibitory compounds in vivo, including those of antifungal, antibiotic, and anticancer drugs. The chemogenomic profiling method described here measures the sensitivity induced in yeast heterozygous and homozygous deletion strains in the presence of a chemical inhibitor of growth (termed haploinsufficiency profiling and homozygous profiling, respectively, or HIPHOP). The protocol is both scalable and amenable to automation. After competitive growth of yeast knockout collection cultures, with and without chemical inhibitors, CGIs can be identified and quantified using either array- or sequencing-based approaches as described here.

Components in aqueous Hibiscus rosa-sinensis flower extract inhibit in vitro melanoma cell growth

Skin cancer is extremely common, and melanoma causes about 80% of skin cancer deaths. In fact, melanoma kills over 50 thousand people around the world each year, and these numbers are rising. Clearly, standard treatments are not effectively treating melanoma, and alternative therapies are needed to address this problem. Hibiscus tea has been noted to have medicinal properties, including anticancer effects. Extracts from Hibiscus have been shown to inhibit the growth of a variety of cancer cells. In particular, recent studies found that polyphenols extracted from Hibiscus sabdariffa by organic solvents can inhibit melanoma cell growth. However, effects of aqueous extracts from Hibiscus rosa-sinesis flowers, which are commonly used to make traditional medicinal beverages, have not been examined on melanoma cells. Here, we report that aqueous H. rosa-sinesis flower extract contains compounds that inhibit melanoma cell growth in a dose dependent manner at concentrations that did not affect the growth of nontransformed cells. In addition, these extracts contain low molecular weight growth inhibitory compounds below 3 kD in size that combine with larger compounds to more effectively inhibit melanoma cell growth. Future work should identify these compounds, and evaluate their potential to prevent and treat melanoma and other cancers.

Fusarium Oxysporum Volatiles Enhance Plant Growth Via Affecting Auxin Transport and Signaling.

Volatile organic compounds (VOCs) have well-documented roles in plant-plant communication and directing animal behavior. In this study, we examine the less understood roles of VOCs in plant-fungal relationships. Phylogenetically and ecologically diverse strains of Fusarium oxysporum, a fungal species complex that often resides in the rhizosphere of assorted plants, produce volatile compounds that augment shoot and root growth of Arabidopsis thaliana and tobacco. Growth responses of A. thaliana hormone signaling mutants and expression patterns of a GUS reporter gene under the auxin-responsive DR5 promoter supported the involvement of auxin signaling in F. oxysporum volatile-mediated growth enhancement. In addition, 1-naphthylthalamic acid, an inhibitor of auxin efflux, negated F. oxysporum volatile-mediated growth enhancement in both plants. Comparison of the profiles of volatile compounds produced by F. oxysporum strains that differentially affected plant growth suggests that the relative compositions of both growth inhibitory and stimulatory compounds may determine the degree of plant growth enhancement. Volatile-mediated signaling between fungi and plants may represent a potentially conserved, yet mostly overlooked, mechanism underpinning plant-fungus interactions and fungal niche adaption.

Abstract 3343: Podophyllotoxin acetate (PA) enhancing γ-ionizing radiation-induced apoptosis via the ROS/p38/caspase pathway

Abstract To develop a new radiosensitizer candidate capable of increasing the efficiency of radiotherapy and to investigate the underlying molecular mechanisms in vitro and in vivo. To develop a new radiosensitizer against non-small cell lung cancer cells, we screened a natural product library for growth-inhibitory compounds. Podophyllotoxin acetate (PA) was found to be cytotoxic toward NCI-H460 cells, and its IC50 value was determined. The radiosensitizer effects of PA were tested at its IC50 value in clonogenic and cell-counting assays. The intracellular mechanism underlying this effect was determined by immunoblotting and by measuring propidium iodide uptake and ROS generation. The radiosensitizer activity of PA in vivo was tested in nude mice by treating with PA and IR, and measuring tumor volume and assessing apoptosis. PA, tested at its experimentally determined IC50 value (12 nM), enhanced γ-ionizing radiation (IR)-induced death of NCI-H460 cells by increasing apoptosis, yielding a mean calculated dose-enhancement ratio of 3.8. Combination with PA and IR also increased the production of reactive oxygen species (ROS), which subsequently induced phosphorylation of p38, suppressed phosphorylation of ERK, and activated caspase-3, -8, and -9. Notably, inhibition of ROS production prevented p38 phosphorylation, and inhibition of ROS production or p38 activation blocked caspase activation and apoptosis. In a xenograft assay, combination with PA and IR delayed tumor growth by 11.4 days compared with controls, yielding an enhancement factor of 1.48. Collectively, these results indicate that PA functions as a radiosensitizer by enhancing apoptosis through activation of a ROS/p38/caspase pathway and suppression of ERK. Citation Format: Jae Yeon Choi, Sang-Gu Hwang, Hong-Duck Um, Jong Kuk Park. Podophyllotoxin acetate (PA) enhancing γ-ionizing radiation-induced apoptosis via the ROS/p38/caspase pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3343. doi:10.1158/1538-7445.AM2015-3343

Identification of self-growth-inhibiting compounds lauric acid and 7-(Z)-tetradecenoic acid from Helicobacter pylori.

Helicobacter pylori growth medium is usually supplemented with horse serum (HS) or FCS. However, cyclodextrin derivatives or activated charcoal can replace serum. In this study, we purified self-growth-inhibiting (SGI) compounds from H. pylori growth medium. The compounds were recovered from porous resin, Diaion HP-20, which was added to the H. pylori growth medium instead of known supplements. These SGI compounds were also identified from 2,6-di-O-methyl-β-cyclodextrin, which was supplemented in a pleuropneumonia-like organisms broth. The growth-inhibiting compounds were identified as lauric acid (LA) and 7-(Z)-tetradecenoic acid [7-(Z)-TDA]. Although several fatty acids had been identified in H. pylori, these specific compounds were not previously found in this species. However, we confirmed that these fatty acids were universally present in the cultivation medium of the H. pylori strains examined in this study. A live/dead assay carried out without HS indicated that these compounds were bacteriostatic; however, no significant growth-inhibiting effect was observed against other tested bacterial species that constituted the indigenous bacterial flora. These findings suggested that LA and 7-(Z)-TDA might play important roles in the survival of H. pylori in human stomach epithelial cells.

Podophyllotoxin acetate enhances γ-ionizing radiation-induced apoptotic cell death by stimulating the ROS/p38/caspase pathway

To develop a new radiosensitizer against non-small cell lung cancer cells, we screened a natural product library for growth-inhibitory compounds. PA was found to be cytotoxic toward NCI-H460 cells, and its IC50 value was determined. The radiosensitizer effects of PA were tested at its IC50 value in clonogenic and cell-counting assays. The intracellular mechanism underlying this effect was determined by immunoblotting and by measuring propidium iodide uptake and ROS generation. The radiosensitizer activity of PA in vivo was tested in nude mice by treating with PA and IR, and measuring tumor volume and assessing apoptosis. PA, tested at its experimentally determined IC50 value (12nM), enhanced IR-induced death of NCI-H460 cells by increasing apoptosis, yielding a mean calculated dose-enhancement ratio of 1.67. Combination with PA and IR also increased the production of ROS, which subsequently induced phosphorylation of p38, suppressed phosphorylation of ERK, and activated caspase-3, -8, and -9. Notably, inhibition of ROS production prevented p38 phosphorylation, and inhibition of ROS production or p38 activation blocked caspase activation and apoptosis. In a xenograft assay, combination with PA and IR delayed tumor growth by 11.4days compared with controls, yielding an enhancement factor of 1.48. Collectively, these results indicate that PA functions as a radiosensitizer by enhancing apoptosis through activation of a ROS/p38/caspase pathway and suppression of ERK.

Determination of compound kill kinetics against Mycobacterium tuberculosis.

In this chapter, we describe how to determine the kill kinetics and minimum bactericidal concentration (MBC) of a compound against Mycobacterium tuberculosis. Techniques are described for three conditions: actively growing aerobic bacteria, and non-replicating bacteria induced by nutrient starvation and/or low pH. Each technique involves determining the number of viable bacteria in the presence of several concentrations of compound over 3 weeks. Guidelines for how to interpret the results, to determine if growth-inhibitory compounds are bactericidal or bacteriostatic and also whether compounds exhibit time-dependent or concentration-dependent kill are provided.

Synthesis of 6-O-decanoyl-d-altrose and 6-O-decanoyl-d-gulose and evaluation of their biological activity on plant growth

Two new growth inhibitory compounds, 6-O-decanoyl-d-altrose and 6-O-decanoyl-d-gulose, were synthesized from d-altrose (C-2 epimer of d-allose) and d-gulose (C-4 epimer of d-allose), respectively, via lipase-catalyzed transesterification using vinyl decanoate. Furthermore, the inhibitory activity of 6-O-decanoyl-d-altrose and 6-O-decanoyl-d-gulose on different plant growths were examined. These two new rare sugar derivatives exhibited the inhibitory activity on the growth of cress, Italian rye grass, and rice seedlings similar to 6-O-decanoyl-d-allose, but in the case of lettuce, they showed the weaker inhibitory activity than 6-O-decanoyl-d-allose. In addition, we found that the co-addition of gibberellins (GAs) with a test solution of 6-O-decanoyl-d-altrose or 6-O-decanoyl-d-gulose on rice seedlings brought the recovery of inhibition. These results suggest that the d-altrose and d-gulose esters as well as the d-allose ester inhibited the biosynthesis of GAs to exhibit the plant growth inhibitory activity.

Novel phenotypes related to the breeding of purple-fruited tomatoes and effect of peel extracts on human cancer cell proliferation

The production of anthocyanins in the tomato (Solanum lycopersicum L.) fruit is normally absent or poor, but a number of mutants or introgression lines are known to increase anthocyanin levels in vegetative and reproductive tissues. Through conventional breeding, a genetic combination was obtained with the remarkable phenotype of a deep purple fruit pigmentation, due to an accumulation of anthocyanins on the peel. Such a genotype was named Sun Black (SB) as a consequence of its sensitivity to light induction. When characterized for morpho-agronomic traits, SB plants showed increased fertility. Purple fruits displayed an arrangement of the epicarp cells different from normal tomatoes, a feature that could account for different mechanical properties and shelf-life potential. The SB genotype and, to a lesser extent, its single mutant parents showed the capacity to accumulate anthocyanins in the seedling root when grown under light. This phenotype, which was greatly improved by the addition of sucrose to the germination medium, proved to be useful as selection index and gave new insights for in vitro production of anthocyanin extracts. To assess the nutraceutical potential of purple tomatoes, we tested the activity of SB skin extracts on the proliferation of two human cancer cells lines. Cell proliferation was significantly inhibited by SB extract in a dose-dependent manner. When the bioactivity of SB extracts was compared with that of other anthocyanin-containing fruits or vegetables, a significant "Extract*Line" interaction was evidenced, suggesting a crucial role for the extract composition in terms of anthocyanidins and other eventual cell growth-inhibiting compounds.

Competitive advantage and tolerance of selected shochu yeast in barley shochu mash

A shochu yeast strain, Saccharomyces cerevisiae BAW-6, was previously isolated from Kagoshima yeast strain Ko, and has since been utilized in shochu production. The BAW-6 strain carries pho3/pho3 homozygous genes in contrast to the heterozygous PHO3/pho3 genes in the parental Ko strain. However, absence of the PHO3 gene per se cannot explain the fermentation superiority of BAW-6. Here, we demonstrate the growth advantage of the BAW-6 strain over the Ko strain by competitive cultivation in barley shochu preparation, where alcohol yield and nihonshudo of the former strain were higher than those of the latter strain. In addition, the maximum growth rate of BAW-6 was less affected than that of Ko by high Brix values of barley koji medium, suggesting that BAW-6 is less sensitive to growth inhibitory compounds derived from barley or barley koji. The tolerance of BAW-6 to growth inhibitory compounds, cerulenin and diethylstilbestrol (an H⁺-ATPase inhibitor), was also higher than that of other yeast strains. Consistent with BAW-6's tolerance to diethylstilbestrol in the presence of 8% ethanol (pH 4.5), H⁺-ATPase activity, but not transcription of its gene, was higher in BAW-6 than in Ko. We conclude that the BAW-6 strain is associated with certain gene alterations other than PHO3, such that it can maintain cellular ion homeostasis under conditions of ethanol stress during the latter phase of fermentation.