During fermentation, increased ethanol concentration is a major stress for yeast cells. Vacuolar H؉ -ATPase (V-ATPase), which plays an important role in the maintenance of intracellular pH homeostasis through vacuolar acidification, has been shown to be required for tolerance to straight-chain alcohols, including ethanol. Since ethanol is known to increase membrane permeability to protons, which then promotes intracellular acidification, it is possible that the V-ATPase is required for recovery from alcohol-induced intracellular acidification. In this study, we show that the effects of straight-chain alcohols on membrane permeabilization and acidification of the cytosol and vacuole are strongly dependent on their lipophilicity. These findings suggest that the membrane-permeabilizing effect of straight-chain alcohols induces cytosolic and vacuolar acidification in a lipophilicity-dependent manner. Surprisingly, after ethanol challenge, the cytosolic pH in ⌬vma2 and ⌬vma3 mutants lacking V-ATPase activity was similar to that of the wild-type strain. It is therefore unlikely that the ethanol-sensitive phenotype of vma mutants resulted from severe cytosolic acidification. Interestingly, the vma mutants exposed to ethanol exhibited a delay in cell wall remodeling and a significant increase in intracellular reactive oxygen species (ROS). These findings suggest a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress in response to ethanol. IMPORTANCEThe yeast Saccharomyces cerevisiae has been widely used in the alcoholic fermentation industry. Among the environmental stresses that yeast cells encounter during the process of alcoholic fermentation, ethanol is a major stress factor that inhibits yeast growth and viability, eventually leading to fermentation arrest. This study provides evidence for the molecular mechanisms of ethanol tolerance, which is a desirable characteristic for yeast strains used in alcoholic fermentation. The results revealed that straight-chain alcohols induced cytosolic and vacuolar acidification through their membrane-permeabilizing effects. Contrary to expectations, a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress, but not in the maintenance of intracellular pH, seems to be important for protecting yeast cells against ethanol stress. These findings will expand our understanding of the mechanisms of ethanol tolerance and provide promising clues for the development of ethanol-tolerant yeast strains. The budding yeast Saccharomyces cerevisiae has been widely used in industrial fermentation, such as in the production of alcoholic beverages and ethanol fuel. During fermentation, yeast cells encounter several environmental stresses, such as increased alcohol concentration, high osmolarity, and temperature fluctuations. Among these, ethanol is a major stress factor that affects the vitality and viability of yeast cells, thereby leading to an arrest of the fe...

The Agrobacterium tumefaciens C58 genome harbors an operon containing the dmeR (Atu0890) and dmeF (Atu0891) genes, which encode a transcriptional regulatory protein belonging to the RcnR/CsoR family and a metal efflux protein belonging to the cation diffusion facilitator (CDF) family, respectively. The dmeRF operon is specifically induced by cobalt and nickel, with cobalt being the more potent inducer. Promoter-lacZ transcriptional fusion, an electrophoretic mobility shift assay, and DNase I footprinting assays revealed that DmeR represses dmeRF transcription through direct binding to the promoter region upstream of dmeR. A strain lacking dmeF showed increased accumulation of intracellular cobalt and nickel and exhibited hypersensitivity to these metals; however, this strain displayed full virulence, comparable to that of the wild-type strain, when infecting a Nicotiana benthamiana plant model under the tested conditions. Cobalt, but not nickel, increased the expression of many iron-responsive genes and reduced the induction of the SoxR-regulated gene sodBII. Furthermore, control of iron homeostasis via RirA is important for the ability of A. tumefaciens to cope with cobalt and nickel toxicity. IMPORTANCEThe molecular mechanism of the regulation of dmeRF transcription by DmeR was demonstrated. This work provides evidence of a direct interaction of apo-DmeR with the corresponding DNA operator site in vitro. The recognition site for apo-DmeR consists of 10-bp AT-rich inverted repeats separated by six C bases (5=-ATATAGTATACCCCCCTATAGTATAT-3=). Cobalt and nickel cause DmeR to dissociate from the dmeRF promoter, which leads to expression of the metal efflux gene dmeF. This work also revealed a connection between iron homeostasis and cobalt/nickel resistance in A. tumefaciens. Cobalt is required by coenzyme B 12 -dependent enzymes and several proteins (1, 2). However, cobalt overload can cause cellular toxicity by catalyzing the generation of reactive oxygen species (3, 4), which leads to iron and glutathione depletion, and thus disturbing iron homeostasis (4-6). Cobalt competes with iron in heme proteins (7) and inhibits the activity of iron-sulfur (Fe-S) proteins as shown in Escherichia coli (5) and Salmonella enterica (6). To avoid cobalt toxicity, levels of intracellular cobalt must be properly controlled. Cobalt trafficking systems in the cell, including uptake systems, efflux systems, and metallochaperones, help maintain cobalt at levels suitable for growth (8).To prevent intracellular cobalt overload-mediated toxicity, excessive amounts of cobalt are eliminated by efflux systems involving components such as the major facilitator superfamily (MFS), P 1B-4 -ATPase, resistance nodulation cell division (RND), cation/ proton antiporter, and cation diffusion facilitator (CDF). The E. coli RcnA (resistance to cobalt and nickel) efflux pump belongs to a unique family that is responsible for the detoxification of cobalt and nickel (9). The expression of rcnA is negatively regulated by RcnR (10). CoaT is a P 1B-...

Agrobacterium tumefaciens has a cluster of genes (Atu3178, Atu3179, and Atu3180) encoding an ABC-type transporter, here named troA, troB, and troC, respectively, which is shown here to be a zinc-specific uptake system. Reverse transcription (RT)-PCR analysis confirmed that troA, troB, and troC are cotranscribed, with troC as the first gene of the operon. The yciC (Atu3181) gene is transcribed in the opposite orientation to that of the troCBA operon and belongs to a metal-binding GTPase family. Expression of troCBA and yciC was inducible under zinc-limiting conditions and was controlled by the zinc uptake regulator, Zur. Compared to the wild type, the mutant strain lacking troC was hypersensitive to a metal chelator, EDTA, and the phenotype could be rescued by the addition of zinc, while the strain with a single yciC mutation showed no phenotype. However, yciC was important for survival under zinc limitation when either troC or zinT was inactivated. The periplasmic zinc-binding protein, ZinT, could not function when TroC was inactivated, suggesting that ZinT may interact with TroCBA in zinc uptake. Unlike many other bacteria, the ABC-type transporter ZnuABC was not the major zinc uptake system in A. tumefaciens. However, the important role of A. tumefaciens ZnuABC was revealed when TroCBA was impaired. The strain containing double mutations in the znuA and troC genes exhibited a growth defect in minimal medium. A. tumefaciens requires cooperation of zinc uptake systems and zinc chaperones, including TroCBA, ZnuABC, ZinT, and YciC, for survival under a wide range of zinc-limiting conditions. IMPORTANCEBoth host and pathogen battle over access to essential metals, including zinc. In low-zinc environments, physiological responses that make it possible to acquire enough zinc are important for bacterial survival and could determine the outcome of hostpathogen interactions. A. tumefaciens was found to operate a novel pathway for zinc uptake in which ZinT functions in concert with the high-affinity zinc importer TroCBA. Zinc is an essential metal for bacteria because it is required for the functions of many enzymes and proteins (1, 2). However, zinc overload is toxic to cells (3-7). Bacteria have mechanisms to maintain zinc homeostasis via the coordinated response of genes involved in zinc uptake, efflux, and storage (8-13). The zinc uptake regulator Zur is a transcriptional regulator belonging to the Fur family and functions as a repressor of zinc uptake genes, including znuABC and zinT (10). To prevent excessive amounts of zinc in cells under high-zinc conditions, Zur uses Zn 2ϩ as its cofactor to bind to a conserved AT-rich sequence, called the Zur box (14), found in the promoter region of the zinc uptake genes, leading to inhibition of gene expression (15)(16)(17). ZnuA is a periplasmic protein that binds zinc and transfers it to the membrane permease ZnuB and the ATPase ZnuC (15). The ZinT protein is a periplasmic zinc-binding protein (18-21) that has been shown to directly interact with and assist ZnuABC i...

Agrobacterium tumefaciens AcrR is the transcriptional repressor of the acrABR operon. The AcrAB efflux pump confers resistance to various toxic compounds, including antibiotics [ciprofloxacin (CIP), nalidixic acid (NAL), novobiocin (NOV) and tetracycline (TET)], a detergent [sodium dodecyl sulfate (SDS)] and a biocide [triclosan (TRI)]. The sequence to which AcrR specifically binds in the acrA promoter region was determined by EMSA and DNase I footprinting. The AcrR-DNA interaction was abolished by adding NAL, SDS and TRI. Quantitative real time-PCR analysis showed that induction of the acrA transcript occurred when wild-type cells were exposed to NAL, SDS and TRI. Indole is a signaling molecule that increases the antibiotic resistance of bacteria, at least in part, through activation of efflux pumps. Expression of the A. tumefaciens acrA transcript was also inducible by indole in a dose-dependent manner. Indole induced protection against CIP, NAL and SDS but enhanced susceptibility to NOV and TRI. Additionally, the TET resistance of A. tumefaciens was not apparently modulated by indole. A. tumefaciens AcrAB played a dominant role and was required for tolerance to high levels of the toxic compounds. Understanding the regulation of multidrug efflux pumps and bacterial adaptive responses to intracellular and extracellular signaling molecules for antibiotic resistance is essential. This information will be useful for the rational design of effective treatments for bacterial infection to overcome possible multidrug-resistant pathogens.