John E. Houghton

Copper-induced oxidative stress in Saccharomyces cerevisiae targets enzymes of the glycolytic pathway

Increased cellular levels of reactive oxygen species are known to arise during exposure of organisms to elevated metal concentrations, but the consequences for cells in the context of metal toxicity are poorly characterized. Using two‐dimensional gel electrophoresis, combined with immunodetection of protein carbonyls, we report here that exposure of the yeast Saccharomyces cerevisiae to copper causes a marked increase in cellular protein carbonyl levels, indicative of oxidative protein damage. The response was time dependent, with total‐protein oxidation peaking approximately 15 min after the onset of copper treatment. Moreover, this oxidative damage was not evenly distributed among the expressed proteins of the cell. Rather, in a similar manner to peroxide‐induced oxidative stress, copper‐dependent protein carbonylation appeared to target glycolytic pathway and related enzymes, as well as heat shock proteins. Oxidative targeting of these and other enzymes was isoform‐specific and, in most cases, was also associated with a decline in the proteins’ relative abundance. Our results are consistent with a model in which copper‐induced oxidative stress disables the flow of carbon through the preferred glycolytic pathway, and promotes the production of glucose‐equivalents within the pentose phosphate pathway. Such re‐routing of the metabolic flux may serve as a rapid‐response mechanism to help cells counter the damaging effects of copper‐induced oxidative stress.

Cloning and expression of pca genes from Pseudomonas putida in Escherichia coli.

Beta-Ketoadipate elicits expression of five structural pca genes encoding enzymes that catalyse consecutive reactions in the utilization of protocatechuate by Pseudomonas putida. Three derivatives of P. putida PRS2000 were obtained, each carrying a single copy of Tn5 DNA inserted into a separate region of the genome and preventing expression of different sets of pca genes. Selection of Tn5 in or near the pca genes in these derivatives was used to clone four structural pca genes and to enable their expression as inserts in pUC19 carried in Escherichia coli. Three of the genes were clustered as components of an apparent operon in the order pcaBDC. This observation indicates that rearrangement of the closely linked genes accompanied divergence of their evolutionary homologues, which are known to appear in the order pcaDBC in the Acinetobacter calcoaceticus pcaEFDBCA gene cluster. Additional evidence for genetic reorganization during evolutionary divergence emerged from the demonstration that the P. putida pcaE gene lies more than 15 kilobase pairs (kbp) away from the pcaBDC operon. An additional P. putida gene, pcaR, was shown to be required for expression of the pca structural genes in response to beta-ketoadipate. The regulatory pcaR gene is located about 15 kbp upstream from the pcaBDC operon.

PcaR-mediated activation and repression of pca genes from Pseudomonas putida are propagated by its binding to both the -35 and the -10 promoter elements.

Degradation of protocatechuate in Pseudomonas putida is accomplished by the products of the pca genes (pcaH,G, pcaBDC, pcaI, J and pcaF ). In P. putida, all these genes (with the exception of pcaH,G ) are activated by the regulatory protein PcaR, in association with the pathway intermediate β‐ketoadipate. Having previously cloned and characterized the pcaR locus, we have overexpressed and purified the PcaR protein to homogeneity. The purified PcaR protein was shown to form a homodimer in solution and to bind specifically to its own promoter, as well as to the promoter regions of pcaI, J and pcaF. Subsequent footprint analyses demonstrated that the binding of PcaR to its own promoter occurs within a footprint that extends from the −20 to the +4 position. In contrast, PcaR appeared to interact with the inducible pcaI, J promoter as a dimer of dimers; binding in tandem within a dual footprint encompassing both the ‘−35′ and the ‘−10’ regions of the promoter sequence. The similarities and differences between the two binding patterns correlate well with the very different effects that PcaR has upon transcription at each of the promoter sequences. The interactions at the pcaI, J promoter are reminiscent of those exhibited by the MerR family of regulatory proteins. However, as PcaR bears very little primary sequence homology to any of the regulatory proteins within this family, the results presented here reveal the possible existence of a new series of functionally related transcriptional inducers.

Cadmium induces a heterogeneous and caspase-dependent apoptotic response in Saccharomyces cerevisiae

The toxic metal cadmium is linked to a series of degenerative disorders in humans, in which Cd-induced programmed cell death (apoptosis) may play a role. The yeast, Saccharomyces cerevisiae, provides a valuable model for elucidating apoptosis mechanisms, and this study extends that capability to Cd-induced apoptosis. We demonstrate that S. cerevisiae undergoes a glucose-dependent, programmed cell death in response to low cadmium concentrations, which is initiated within the first hour of Cd exposure. The response was associated with induction of the yeast caspase, Yca1p, and was abolished in a yca1Δ mutant. Cadmium-dependent apoptosis was also suppressed in a gsh1Δ mutant, indicating a requirement for glutathione. Other apoptotic markers, including sub-G1 DNA fragmentation and hyper-polarization of mitochondrial membranes, were also evident among Cd-exposed cells. These responses were not distributed uniformly throughout the cell population, but were restricted to a subset of cells. This apoptotic subpopulation also exhibited markedly elevated levels of intracellular reactive oxygen species (ROS). The heightened ROS levels alone were not sufficient to induce apoptosis. These findings highlight several new perspectives to the mechanism of Cd-dependent apoptosis and its phenotypic heterogeneity, while opening up future analyses to the power of the yeast model system.

Cell cycle- and age-dependent activation of Sod1p drives the formation of stress resistant cell subpopulations within clonal yeast cultures.

Phenotypic heterogeneity describes non‐genetic variation that exists between individual cells within isogenic populations. The basis for such heterogeneity is not well understood, but it is evident in a wide range of cellular functions and phenotypes and may be fundamental to the fitness of microorganisms. Here we use a suite of novel assays applied to yeast, to provide an explanation for the classic example of heterogeneous resistance to stress (copper). Cell cycle stage and replicative cell age, but not mitochondrial content, were found to be principal parameters underpinning differential Cu resistance: cell cycle‐synchronized cells had relatively uniform Cu resistances, and replicative cell‐age profiles differed markedly in sorted Cu‐resistant and Cu‐sensitive subpopulations. From a range of potential Cu‐sensitive mutants, cup1Δ cells lacking Cu‐metallothionein, and particularly sod1Δ cells lacking Cu, Zn‐superoxide dismutase, exhibited diminished heterogeneity. Furthermore, age‐dependent Cu resistance was largely abolished in cup1Δ and sod1Δ cells, whereas cell cycle‐dependent Cu resistance was suppressed in sod1Δ cells. Sod1p activity oscillated ∼fivefold during the cell cycle, with peak activity coinciding with peak Cu‐resistance. Thus, phenotypic heterogeneity in copper resistance is not stochastic but is driven by the progression of individual cells through the cell cycle and ageing, and is primarily dependent on only Sod1p, out of several gene products that can influence the averaged phenotype. We propose that such heterogeneity provides an important insurance mechanism for organisms; creating subpopulations that are pre‐equipped for varied activities as needs may arise (e.g. when faced with stress), but without the permanent metabolic costs of constitutive expression.

Phospholipids Induce Conformational Changes of SecA to Form Membrane-Specific Domains: AFM Structures and Implication on Protein-Conducting Channels

SecA, an essential component of the Sec machinery, exists in a soluble and a membrane form in Escherichia coli. Previous studies have shown that the soluble SecA transforms into pore structures when it interacts with liposomes, and integrates into membranes containing SecYEG in two forms: SecAS and SecAM; the latter exemplified by two tryptic membrane-specific domains, an N-terminal domain (N39) and a middle M48 domain (M48). The formation of these lipid-specific domains was further investigated. The N39 and M48 domains are induced only when SecA interacts with anionic liposomes. Additionally, the N-terminus, not the C-terminus of SecA is required for inducing such conformational changes. Proteolytic treatment and sequence analyses showed that liposome-embedded SecA yields the same M48 and N39 domains as does the membrane-embedded SecA. Studies with chemical extraction and resistance to trypsin have also shown that these proteoliposome-embedded SecA fragments exhibit the same stability and characteristics as their membrane-embedded SecA equivalents. Furthermore, the cloned lipid-specific domains N39 and M48, but not N68 or C34, are able to form partial, but imperfect ring-like structures when they interact with phospholipids. These ring-like structures are characteristic of a SecA pore-structure, suggesting that these domains contribute part of the SecA-dependent protein-conducting channel. We, therefore, propose a model in which SecA alone is capable of forming a lipid-specific, asymmetric dimer that is able to function as a viable protein-conducting channel in the membrane, without any requirement for SecYEG.

SecA inhibitors as potential antimicrobial agents: differential actions on SecA-only and SecA-SecYEG protein-conducting channels

Sec-dependent protein translocation is an essential process in bacteria. SecA is a key component of the translocation machinery and has multiple domains that interact with various ligands. SecA acts as an ATPase motor to drive the precursor protein/peptide through the SecYEG protein translocation channels. As SecA is unique to bacteria and there is no mammalian counterpart, it is an ideal target for the development of new antimicrobials. Several reviews detail the assays for ATPase and protein translocation, as well as the search for SecA inhibitors. Recent studies have shown that, in addition to the SecA-SecYEG translocation channels, there are SecA-only channels in the lipid bilayers, which function independently from the SecYEG machinery. This mini-review focuses on recent advances on the newly developed SecA inhibitors that allow the evaluation of their potential as antimicrobial agents, as well as a fundamental understanding of mechanisms of SecA function(s). These SecA inhibitors abrogate the effects of efflux pumps in both Gram-positive and Gram-negative bacteria. We also discuss recent findings that SecA binds to ribosomes and nascent peptides, which suggest other roles of SecA. A model for the multiple roles of SecA is presented.

Dissecting structures and functions of SecA-only protein-conducting channels: ATPase, pore structure, ion channel activity, protein translocation, and interaction with SecYEG/SecDF•YajC

SecA is an essential protein in the major bacterial Sec-dependent translocation pathways. E. coli SecA has 901 aminoacyl residues which form multi-functional domains that interact with various ligands to impart function. In this study, we constructed and purified tethered C-terminal deletion fragments of SecA to determine the requirements for N-terminal domains interacting with lipids to provide ATPase activity, pore structure, ion channel activity, protein translocation and interactions with SecYEG-SecDF•YajC. We found that the N-terminal fragment SecAN493 (SecA1-493) has low, intrinsic ATPase activity. Larger fragments have greater activity, becoming highest around N619-N632. Lipids greatly stimulated the ATPase activities of the fragments N608-N798, reaching maximal activities around N619. Three helices in amino-acyl residues SecA619-831, which includes the “Helical Scaffold” Domain (SecA619-668) are critical for pore formation, ion channel activity, and for function with SecYEG-SecDF•YajC. In the presence of liposomes, N-terminal domain fragments of SecA form pore-ring structures at fragment-size N640, ion channel activity around N798, and protein translocation capability around N831. SecA domain fragments ranging in size between N643-N669 are critical for functional interactions with SecYEG-SecDF•YajC. In the presence of liposomes, inactive C-terminal fragments complement smaller non-functional N-terminal fragments to form SecA-only pore structures with ion channel activity and protein translocation ability. Thus, SecA domain fragment interactions with liposomes defined critical structures and functional aspects of SecA-only channels. These data provide the mechanistic basis for SecA to form primitive, low-efficiency, SecA-only protein-conducting channels, as well as the minimal parameters for SecA to interact functionally with SecYEG-SecDF•YajC to form high-efficiency channels.

Potential pathogenic factors produced by a clinical nontoxigenicVibrio cholerae O1

A clinical isolate of nontoxigenicVibrio cholerae O1 that caused intestinal fluid accumulation (FA) in adult mice produced proteolytic, hemolytic, and cytotoxic activities in in vitro assays. The linkage of these secreted factors to the FA activity was studied by transposon (TnphoA) mutagenesis. Ten of the 12 TnphoA insertion mutants that were defective for proteolytic activity produced FA, hemolytic and cytotoxic activities; the remaining two mutants lost these latter three activities. These results indicate that FA activity is independent of proteolytic activity but closely associated with cytotoxic and hemolytic activities. Our results with the adult mouse model and a nontoxigenicV. cholerae O1 are in general agreement with previous studies that demonstrated linkage of cytotoxin and hemolysin of toxigenicV. cholerae O1 and non-O1 with FA activity in rabbit ileal loops.