Membrane lipid peroxidation in copper alloy-mediated contact killing of Escherichia coli.

Copper alloy surfaces are passive antimicrobial sanitizing agents that kill bacteria, fungi, and some viruses. Studies of the mechanism of contact killing in Escherichia coli implicate the membrane as the target, yet the specific component and underlying biochemistry remain unknown. This study explores the hypothesis that nonenzymatic peroxidation of membrane phospholipids is responsible for copper alloy-mediated surface killing. Lipid peroxidation was monitored with the thiobarbituric acid-reactive substances (TBARS) assay. Survival, TBARS levels, and DNA degradation were followed in cells exposed to copper alloy surfaces containing 60 to 99.90% copper or in medium containing CuSO(4). In all cases, TBARS levels increased with copper exposure levels. Cells exposed to the highest copper content alloys, C11000 and C24000, exhibited novel characteristics. TBARS increased immediately at a very rapid rate but peaked at about 30 min. This peak was associated with the period of most rapid killing, loss in membrane integrity, and DNA degradation. DNA degradation is not the primary cause of copper-mediated surface killing. Cells exposed to the 60% copper alloy for 60 min had fully intact genomic DNA but no viable cells. In a fabR mutant strain with increased levels of unsaturated fatty acids, sensitivity to copper alloy surface-mediated killing increased, TBARS levels peaked earlier, and genomic DNA degradation occurred sooner than in the isogenic parental strain. Taken together, these results suggest that copper alloy surface-mediated killing of E. coli is triggered by nonenzymatic oxidative damage of membrane phospholipids that ultimately results in the loss of membrane integrity and cell death.

Hsp90 cochaperone Aha1 is a negative regulator of the Saccharomyces MAL activator and acts early in the chaperone activation pathway.

Aha1 is a ubiquitous cochaperone of the Hsp90/Hsp70 chaperone machine. It binds the middle domain of Hsp90 and stimulates ATPase activity, suggesting a function late in the chaperone pathway. Saccharomyces Mal63 MAL activator is a DNA-binding transcription factor and Hsp90 client protein. This study utilizes several MAL activator mutants to investigate Aha1 function in vivo. Deletion of AHA1 enhances induced Mal63-dependent maltase activity levels 2-fold, whereas overproduction of Aha1 represses expression. Maltase expression in strains carrying constitutive and super-inducible mutant activators with alterations near the C terminus (particularly residues 433-463) is unaffected by either aha1Delta or Aha1 overproduction. However, another constitutive activator with alterations outside of this C-terminal region is sensitive to Aha1 regulation. Previously, we showed that in the absence of inducer, Mal63 forms a stable intermediate complex with Hsp70, Hsp90, and Sti1, whereas noninducible mutant activators bind only with Hsp70 in an apparent early complex. Here, we find that triple Myc-tagged Aha1/Myc3 copurifies with all noninducible Mal63 mutant activators tested. Aha1/Myc3 association with inducible Mal63 is observed only in a sti1Delta strain, in which Hsp90 binding and intermediate complex formation are defective. Constitutive and super-inducible mutant activators with C-terminal alterations do not bind Aha1 even in a sti1Delta strain. Mal63 binding to Hsp90 and Hsp70 is enhanced 3-fold by loss of Aha1. These results suggest an interaction between Aha1 and residues near the C terminus of Mal63 thereby regulating Hsp90 association. A novel mechanism for the negative regulation of the MAL activator by Aha1 cochaperone is proposed.

Glc7-Reg1 phosphatase signals to Yck1,2 casein kinase 1 to regulate transport activity and glucose-induced inactivation of Saccharomyces maltose permease.

The Saccharomyces casein kinase 1 isoforms encoded by the essential gene pair YCK1 and YCK2 control cell growth and morphogenesis and are linked to the endocytosis of several membrane proteins. Here we define roles for the Yck1,2 kinases in Mal61p maltose permease activation and trafficking, using a yck1delta yck2-2(ts) (yck(ts)) strain with conditional Yck activity. Moreover, we provide evidence that Glc7-Reg1 phosphatase acts as an upstream activator of Yck1,2 kinases in a novel signaling pathway that modulates kinase activity in response to carbon source availability. The yck(ts) strain exhibits significantly reduced maltose transport activity despite apparently normal levels and cell surface localization of maltose permease protein. Glucose-induced internalization and rapid loss of maltose transport activity of Mal61/HAp-GFP are not observed in the yck(ts) strain and maltose permease proteolysis is blocked. We show that a reg1delta mutant exhibits a phenotype remarkably similar to that conferred by yck(ts). The reg1delta phenotype is not enhanced in the yck(ts) reg1delta double mutant and is suppressed by increased Yck1,2p dosage. Further, although Yck2p localization and abundance do not change in the reg1delta mutant, Yck1,2 kinase activity, as assayed by glucose-induced HXT1 expression and Mth1 repressor stability, is substantially reduced in the reg1delta strain.

Antimicrobial copper alloy surfaces are effective against vegetative but not sporulated cells of gram-positive Bacillus subtilis.

This study explores the role of membrane phospholipid peroxidation in the copper alloy mediated contact killing of Bacillus subtilis, a spore-forming gram-positive bacterial species. We found that B. subtilis endospores exhibited significant resistance to copper alloy surface killing but vegetative cells were highly sensitive to copper surface exposure. Cell death and lipid peroxidation occurred in B. subtilis upon copper alloy surface exposure. In a sporulation-defective strain carrying a deletion of almost the entire SpoIIA operon, lipid peroxidation directly correlated with cell death. Moreover, killing and lipid peroxidation initiated immediately and at a constant rate upon exposure to the copper surface without the delay observed previously in E. coli. These findings support the hypothesis that membrane lipid peroxidation is the initiating event causing copper surface induced cell death of B. subtilis vegetative cells. The findings suggest that the observed differences in the kinetics of copper-induced killing compared to E. coli result from differences in cell envelop structure. As demonstrated in E. coli, DNA degradation was shown to be a secondary effect of copper exposure in a B. subtilis sporulation-defective strain.

Sequences in the N-terminal cytoplasmic domain of Saccharomyces cerevisiae maltose permease are required for vacuolar degradation but not glucose-induced internalization.

In Saccharomyces cerevisiae, glucose addition to maltose fermenting cells causes a rapid loss of maltose transport activity and ubiquitin-mediated vacuolar proteolysis of maltose permease. GFP-tagged Mal61 maltose permease was used to explore the role of the N-terminal cytoplasmic domain in glucose-induced inactivation. In maltose-grown cells, Mal61/HA-GFP localizes to the cell surface and, surprisingly, to the vacuole. Studies of end3Delta and doa4Delta mutants indicate that a slow constitutive internalization of Mal61/HA-GFP is required for its vacuolar localization. Site-specific mutagenesis of multiple serine/threonine residues in a putative PEST sequence of the N-terminal cytoplasmic domain of maltose permease blocks glucose-induced Mal61p degradation but does not affect the rapid loss of maltose transport activity associated with glucose-induced internalization. The internalized multiple Ser/Thr mutant protein co-localizes with Snf7p in a putative late endosome or E-compartment. Further, alteration of a putative dileucine [D/EExxxLL/I] motif at residues 64-70 causes a significant defect in maltose transport activity and mislocalization to an E-compartment but appears to have little impact on glucose-induced internalization. We conclude that the N-terminal cytoplasmic domain of maltose permease is not the target of the signaling pathways leading to glucose-induced internalization of Mal61 permease but is required for its subsequent delivery to the vacuole for degradation.