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, copperdependent protein carbonylation appeared to target glycolytic pathway and related enzymes, as well as heat shock proteins. Oxidative targeting of these and other enzymes was isoformspeci¢c 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 £ow 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 £ux may serve as a rapid-response mechanism to help cells counter the damaging e¡ects of copper-induced oxidative stress. ß

Oxidative damage in microbial cells occurs during exposure to the toxic metal chromium, but it is not certain whether such oxidation accounts for the toxicity of Cr. Here, a Saccharomyces cerevisiae sod1D mutant (defective for the Cu,Zn-superoxide dismutase) was found to be hypersensitive to Cr(VI) toxicity under aerobic conditions, but this phenotype was suppressed under anaerobic conditions. Studies with cells expressing a Sod1p variant (Sod1 H46C ) showed that the superoxide dismutase activity rather than the metal-binding function of Sod1p was required for Cr resistance. To help identify the macromolecular target(s) of Cr-dependent oxidative damage, cells deficient for the reduction of phospholipid hydroperoxides (gpx3D and gpx1D/gpx2D/gpx3D) and for the repair of DNA oxidation (ogg1D and rad30D/ogg1D) were tested, but were found not to be Cr-sensitive. In contrast, S. cerevisiae msraD (mxr1D) and msrbD (ycl033cD) mutants defective for peptide methionine sulfoxide reductase (MSR) activity exhibited a Cr sensitivity phenotype, and cells overexpressing these enzymes were Cr-resistant. Overexpression of MSRs also suppressed the Cr sensitivity of sod1D cells. The inference that protein oxidation is a primary mechanism of Cr toxicity was corroborated by an observed~20-fold increase in the cellular levels of protein carbonyls within 30 min of Cr exposure. Carbonylation was not distributed evenly among the expressed proteins of the cells; certain glycolytic enzymes and heat-shock proteins were specifically targeted by Cr-dependent oxidative damage. This study establishes an oxidative mode of Cr toxicity in S. cerevisiae, which primarily involves oxidative damage to cellular proteins.

Soil bacteria comprise a largely untapped resource with only 1-10% of bacterial species predicted to live in soil being culturable in the laboratory. Establishing culture-dependent protocols that identify unique operational taxonomic units (OTUs) is an important research topic in soil bacterial ecology. The culturability of soil bacteria may be improved by employing different culture media due to inherent preferences of growth substrate utilization. Soil-extract agar, R-2A agar, and 1% nutrient agar were used in this study to isolate bacteria obtained from soil samples collected in winter months to increase the understanding of bacterial diversity in Abernathy Field Station, a Marcellus shale temperate forest in Washington, Pennsylvania. Changes in bacterial diversity can be used to assess the early impact of anthropological factors, such as hydraulic fracturing in the Marcellus shale region, which may lead to severe environmental problems. For the purpose of long term ecological monitoring, data obtained from this year’s sample collection were analyzed in conjunction with previous years’ assessments. Bacterial isolates were analyzed taxonomically and phylogenetically. Unique OTUs were identified through comparative analysis of 16S rDNA. The Shannon-Weaver and Simpson’s diversity indices ranked isolates on soil-extract agar highest for species richness, and rarefaction analysis suggests that sampling saturation of OTUs identified on soil-extract agar has not yet been reached. Each culture medium studied supported isolates of four common phyla: Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. Soil-extract agar supported the greatest proportion of pigmented colonies including a Cyanobacterium which exhibited intra-16S rDNA polymorphism. Each culture medium supported the growth of unique OTUs and genera with Bacillus, Flavobacterium, Pseudomonas, Rhizobium, and Streptomyces found on each. This study suggests that utilizing different culture media can increase the culturability of soil bacteria.