Lethality of MalE-LacZ hybrid protein shares mechanistic attributes with oxidative component of antibiotic lethality

Downstream metabolic events can contribute to the lethality of drugs or agents that interact with a primary cellular target. In bacteria, the production of reactive oxygen species (ROS) has been associated with the lethal effects of a variety of stresses including bactericidal antibiotics, but the relative contribution of this oxidative component to cell death depends on a variety of factors. Experimental evidence has suggested that unresolvable DNA problems caused by incorporation of oxidized nucleotides into nascent DNA followed by incomplete base excision repair contribute to the ROS-dependent component of antibiotic lethality. Expression of the chimeric periplasmic-cytoplasmic MalE-LacZ[subscript 72 – 47] protein is an historically important lethal stress originally identified during seminal genetic experiments that defined the SecY-dependent protein translocation system. Multiple, independent lines of evidence presented here indicate that the predominant mechanism for MalE-LacZ lethality shares attributes with the ROS-dependent component of antibiotic lethality. MalE-LacZ lethality requires molecular oxygen, and its expression induces ROS production. The increased susceptibility of mutants sensitive to oxidative stress to MalE-LacZ lethality indicates that ROS contribute causally to cell death rather than simply being produced by dying cells. Observations that support the proposed mechanism of cell death include MalE-LacZ expression being bacteriostatic rather than bactericidal in cells that over-express MutT, a nucleotide sanitizer that hydrolyzes 8-oxo-dGTP to the monophosphate, or that lack MutM and MutY, DNA glycosylases that process base pairs involving 8-oxo-dGTP. Our studies suggest stress-induced physiological changes that favor this mode of ROS-dependent death.National Institutes of Health (U.S.) (Grant R01CA021615)Defense Threat Reduction Agency (DTRA) (Grant HDTRA1-15-1-0051)National Science Foundation (U.S.) (Grant 1336493)National Institutes of Health (U.S.) (Grant K99GM118907

Mechanisms of iron- and O2-sensing by the [4Fe-4S] cluster of the global iron regulator RirA

RirA is a global regulator of iron homeostasis in Rhizobium and related α-proteobacteria. In its [4Fe-4S] cluster-bound form it represses iron uptake by binding to IRO Box sequences upstream of RirA-regulated genes. Under low iron and/or aerobic conditions, [4Fe-4S] RirA undergoes cluster conversion/degradation to apo-RirA, which can no longer bind IRO Box sequences. Here, we apply time-resolved mass spectrometry and electron paramagnetic resonance spectroscopy to determine how the RirA cluster senses iron and O2. The data indicate that the key iron-sensing step is the O2-independent, reversible dissociation of Fe2+ from [4Fe-4S]2+ to form [3Fe-4S]0. The dissociation constant for this process was determined as Kd = ~3 µM, which is consistent with the sensing of ‘free’ iron in the cytoplasm. O2-sensing occurs through enhanced cluster degradation under aerobic conditions, via O2-mediated oxidation of the [3Fe-4S]0 intermediate to form [3Fe-4S]1+. This work provides a detailed mechanistic/functional view of an iron-responsive regulator

Sensing iron availability via the fragile [4Fe-4S] cluster of the bacterial transcriptional repressor RirA

Rhizobial iron regulator A (RirA) is a global regulator of iron homeostasis in many nitrogen-fixing Rhizobia and related species of α-proteobacteria. It belongs to the widespread Rrf2 super-family of transcriptional regulators and features three conserved Cys residues that characterise the binding of an iron–sulfur cluster in other Rrf2 family regulators. Here we report biophysical studies demonstrating that RirA contains a [4Fe–4S] cluster, and that this form of the protein binds RirA-regulated DNA, consistent with its function as a repressor of expression of many genes involved in iron uptake. Under low iron conditions, [4Fe–4S] RirA undergoes a cluster conversion reaction resulting in a [2Fe–2S] form, which exhibits much lower affinity for DNA. Under prolonged low iron conditions, the [2Fe–2S] cluster degrades to apo-RirA, which does not bind DNA and can no longer function as a repressor of the cell's iron-uptake machinery. [4Fe–4S] RirA was also found to be sensitive to O2, suggesting that both iron and O2 are important signals for iron metabolism. Consistent with this, in vivo data showed that expression of RirA-regulated genes is also affected by O2. These data lead us to propose a novel regulatory model for iron homeostasis, in which RirA senses iron via the incorporation of a fragile iron–sulfur cluster that is sensitive to iron and O2 concentrations

Structure and function of the vacuolar Ccc1/VIT1 family of iron transporters and its regulation in fungi

Iron is an essential micronutrient for most living beings since it participates as a redox active cofactor in many biological processes including cellular respiration, lipid biosynthesis, DNA replication and repair, and ribosome biogenesis and recycling. However, when present in excess, iron can participate in Fenton reactions and generate reactive oxygen species that damage cells at the level of proteins, lipids and nucleic acids. Organisms have developed different molecular strategies to protect themselves against the harmful effects of high concentrations of iron. In the case of fungi and plants, detoxification mainly occurs by importing cytosolic iron into the vacuole through the Ccc1/VIT1 iron transporter. New sequenced genomes and bioinformatic tools are facilitating the functional characterization, evolution and ecological relevance of metabolic pathways and homeostatic networks across the Tree of Life. Sequence analysis shows that Ccc1/VIT1 homologs are widely distributed among organisms with the exception of animals. The recent elucidation of the crystal structure of a Ccc1/VIT1 plant ortholog has enabled the identification of both conserved and species-specific motifs required for its metal transport mechanism. Moreover, recent studies in the yeast Saccharomyces cerevisiae have also revealed that multiple transcription factors including Yap5 and Msn2/Msn4 contribute to the expression of CCC1 in high-iron conditions. Interestingly, Malaysian S. cerevisiae strains express a partially functional Ccc1 protein that renders them sensitive to iron. Different regulatory mechanisms have been described for non-Saccharomycetaceae Ccc1 homologs. The characterization of Ccc1/VIT1 proteins is of high interest in the development of biofortified crops and the protection against microbial-derived diseases

The Role of Vacuole and Vacuolar H+-ATPase in Tolerance to Cadmium in Saccharomyces Cerevisiae

รายงานการวิจัย--มหาวิทยาลัยเทคโนโลยีราชมงคลพระนคร, 2553Heavy metal is one of the major environmental pollutants. However, the protective cellular mechanisms in response to heavy metal stress are not well-understood yet. Previously, it has been found that the yeast deletion mutants lacking vacuolar H+-ATPase (V-ATPase) activity, which functions in vacuolar acidification, exhibited growth defects under cadmium stress conditions. In addition, several genes involved in H+-ATPase have been recently found to be required for resistance to aluminum. To investigate the roles of vacuolar H+-ATPase, plasma membrane H+-ATPase, and mitochondrial H+-ATPase in tolerance to cadmium, we examined the growth of yeast deletion mutants lacking different types of H+-ATPase in YPD media containing 80 μM of cadmium. Our results showed that a number of mutants lacking V-ATPase activity were sensitive to cadmium, suggesting the important role of V-ATPase in cadmium detoxification. We next examined the growth of mutants lacking genes responsible for different vacuolar functions in order to investigate the functional activities of vacuoles involved in cadmium detoxification mechanism. We found that the mutants lacking vacuolar protein-sorting genes exhibited high sensitivity to cadmium as well, suggesting the importance of protein transportation during cadmium stress.Rajamangala University of Technology Phra Nakho