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.

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.

Hsp90/Hsp70 chaperone machine regulation of the Saccharomyces MAL-activator as determined in vivo using noninducible and constitutive mutant alleles.

The Hsp90/Hsp70 chaperone machine is an essential regulator of cell growth and division. It is required for activation of select client proteins, chiefly protein kinases and transcription activators and thus plays a major role in regulating intracellular signaling and gene expression. This report demonstrates, in vivo, the association of the Saccharomyces cerevisiae maltose-responsive transcription activator Mal63 (MAL-activator) with the yeast Hsp70 (Ssa1), Hsp90 (Hsp82), and Hop (Sti1) homologs, using a collection of inducible, constitutive, and noninducible alleles. Each class of mutant activator forms a distinctly different stable multichaperone complex in the absence of maltose. Inducible Mal63p associates with Ssa1, Hsp82, and Sti1 and is released in the presence of maltose. Noninducible mal63 mutant proteins bind to Ssa1 alone and do not stably associate with Hsp82 or Sti1. Constitutive MAL-activators bind well to Hsp82 and poorly to Ssa1 and Sti1, but deletion of STI1 restores Ssa1 binding. Taken together, Mal63p regulation requires the formation of Hsp90/Hsp70 subcomplexes comparable to, yet distinct from those observed with previously characterized Hsp90 clients including glucocorticoid receptor and yeast Hap1p. Thus, comparative studies of different client proteins highlight functional diversity in the operation of the Hsp90/Hsp70 chaperone machine.

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.

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.

Mutations in SIN4 and RGR1 cause constitutive expression of MAL structural genes in Saccharomyces cerevisiae.

Transcription of the Saccharomyces MAL structural genes is induced 40-fold by maltose and requires the MAL-activator and maltose permease. To identify additional players involved in regulating MAL gene expression, we carried out a genetic selection for MAL constitutive mutants. Strain CMY4000 containing MAL1 and integrated copies of MAL61promoter-HIS3 and MAL61promoter-lacZ reporter genes was used to select constitutive mutants. The 29 recessive mutants fall into at least three complementation groups. Group 1 and group 2 mutants exhibit pleiotropic phenotypes and represent alleles of Mediator component genes RGR1 and SIN4, respectively. The rgr1 and sin4 constitutive phenotype does not require either the MAL-activator or maltose permease, indicating that Mediator represses MAL basal expression. Further genetic analysis demonstrates that RGR1 and SIN4 work in a common pathway and each component of the Mediator Sin4 module plays a distinct role in regulating MAL gene expression. Additionally, the Swi/Snf chromatin-remodeling complex is required for full induction, suggesting a role for chromatin remodeling in the regulation of MAL gene expression. A sin4Delta mutation is unable to suppress the defects in MAL gene expression resulting from loss of the Swi/Snf complex component Snf2p. The role of the Mediator in MAL gene regulation is discussed.

The Hsp90 molecular chaperone complex regulates maltose induction and stability of the Saccharomyces MAL gene transcription activator Mal63p.

Induction of the Saccharomyces MAL structural genes encoding maltose permease and maltase requires the MAL activator, a DNA-binding transcription activator. Genetic analysis of MAL activator mutations suggested that protein folding and stability play an important role in MAL activator regulation and led us to explore the role of the Hsp90 molecular chaperone complex in the regulation of the MAL activator. Strains carrying mutations in genes encoding components of the Hsp90 chaperone complex, hsc82 Delta hsp82-T101I and hsc82 Delta cpr7 Delta, are defective for maltase induction and exhibit significantly reduced growth rates on media containing a limiting concentration of maltose (0.05%). This growth defect is suppressed by providing maltose in excess. Using epitope-tagged alleles of the MAL63 MAL activator, we showed that Mal63p levels are drastically reduced following depletion of cellular Hsp90. Overexpression ( approximately 3-fold) of Mal63p in the hsc82 Delta hsp82-T101I and hsc82 Delta cpr7 Delta strains suppresses their Mal- growth phenotype, suggesting that Mal63p levels are limiting for maltose utilization in strains with abrogated Hsp90 activity. Consistent with this, the half-life of Mal63p is significantly shorter in the hsc82 Delta cpr7 Delta strain (reduced about 6-fold) and modestly affected in the Hsp90-ts strain (reduced about 2-fold). Most importantly, triple hemagglutinin-tagged Mal63p protein is found in association with Hsp90 as demonstrated by co-immunoprecipitation. Taken together, these results identify the inducible MAL activator as a client protein of the Hsp90 molecular chaperone complex and point to a critical role for chaperone function in alternate carbon source utilization in Saccharomyces cerevisiae.

Clustered-charge to alanine scanning mutagenesis of the Mal63 MAL-activator C-terminal regulatory domain.

The MAL-activator genes of Saccharomyces cerevisiae encode regulatory proteins required for the expression of the structural genes encoding maltose permease and maltase. Residues within the C-terminal region of the Mal63 protein required for negative regulation were previously identified. Evidence suggested that the C-terminal domain is also involved in positive regulatory functions, such as inducer responsiveness and transactivation in the context of a full-length protein. Charged-cluster to alanine scanning mutagenesis of the regulatory domain of MAL63 and the constitutive MAL43-C were undertaken to identify distinct regions within Mal63p involved in positive functions and to define their roles in induction. Mutations that affect the ability to activate transcription in the inducible MAL63 but have no effect in the constitutive MAL43-C define regions that function in induction. Those that affect both the inducible and constitutive alleles define regions involved in activation more generally. Mutations in MAL63 fell into three classes, those that have little or no impact on activity, those that decrease activity, and those that enhance function. Mutations from these classes mapped to distinct regions of the protein, identifying a region of approximately 90 residues (residues 331-423) involved in maltose sensing and an approximately 50-residue region at the extreme C-terminus (residues 420-470) required for activation, such as the formation and/or maintenance of an active state. These studies support a model for MAL-activator function which involves complex protein-protein interactions and overlapping negative and positive regulatory regions.

Intracellular maltose is sufficient to induce MAL gene expression in Saccharomyces cerevisiae.

The presence of maltose induces M4L gene expression in Saccharomyces cells, but little is known abouthow maltose is sensed. Strains with all maltose permease genes deleted are unable to induce MAL geneexpression. In this study, we examined the role of maltose permease in maltose sensing by substituting a heterologous transporter for the native maltose permease. PmSUC2 encodes a sucrose transporter from the dicot plant Plantago major that exhibits no significant sequence homology to maltose permease. When expressed in Saccharomyces cerevisiae, PmSUC2 is capable of transporting maltose, albeit at a reduced rate. We showed that introduction of PmSUC2 restores maltose-inducible MAL gene expression to a maltose permease-null mutant and that this induction requires the MAL activator. These data indicate that intracellular maltose is sufficient to induce MAL gene expression independently of the mechanism of maltose transport. By usingstrains expressing defective mal61 mutant alleles, we demonstrated a correlation between the rate of maltose transport and the level of the induction, which is particularly evident in medium containing very limiting concentrations of maltose. Moreover, our results indicate that a rather low concentration of intracellular maltose is needed to trigger MAL gene expression. We also showed that constitutive overexpression of either MAL61 maltose permease or PmSUC2 suppresses the noninducible phenotype of a defective mal13 MAL-activator allele, suggesting that this suppression is solely a function of maltose transport activity and is not specific to the sequence of the permease. Our studies indicate that maltose permease does not function as the maltose sensor in S. cerevisiae.