Natural variation in yeast reveals multiple paths for acquiring higher stress resistance.

BACKGROUND: Organisms frequently experience environmental stresses that occur in predictable patterns and combinations. For wild Saccharomyces cerevisiae yeast growing in natural environments, cells may experience high osmotic stress when they first enter broken fruit, followed by high ethanol levels during fermentation, and then finally high levels of oxidative stress resulting from respiration of ethanol. Yeast have adapted to these patterns by evolving sophisticated "cross protection" mechanisms, where mild 'primary' doses of one stress can enhance tolerance to severe doses of a different 'secondary' stress. For example, in many yeast strains, mild osmotic or mild ethanol stresses cross protect against severe oxidative stress, which likely reflects an anticipatory response important for high fitness in nature. RESULTS: During the course of genetic mapping studies aimed at understanding the mechanisms underlying natural variation in ethanol-induced cross protection against H2O2, we found that a key H2O2 scavenging enzyme, cytosolic catalase T (Ctt1p), was absolutely essential for cross protection in a wild oak strain. This suggested the absence of other compensatory mechanisms for acquiring H2O2 resistance in that strain background under those conditions. In this study, we found surprising heterogeneity across diverse yeast strains in whether CTT1 function was fully necessary for acquired H2O2 resistance. Some strains exhibited partial dispensability of CTT1 when ethanol and/or salt were used as mild stressors, suggesting that compensatory peroxidases may play a role in acquired stress resistance in certain genetic backgrounds. We leveraged global transcriptional responses to ethanol and salt stresses in strains with different levels of CTT1 dispensability, allowing us to identify possible regulators of these alternative peroxidases and acquired stress resistance in general. CONCLUSIONS: Ultimately, this study highlights how superficially similar traits can have different underlying molecular foundations and provides a framework for understanding the diversity and regulation of stress defense mechanisms.

Independent Mechanisms for Acquired Salt Tolerance versus Growth Resumption Induced by Mild Ethanol Pretreatment in Saccharomyces cerevisiae.

All living organisms must recognize and respond to various environmental stresses throughout their lifetime. In natural environments, cells frequently encounter fluctuating concentrations of different stressors that can occur in combination or sequentially. Thus, the ability to anticipate an impending stress is likely ecologically relevant. One possible mechanism for anticipating future stress is acquired stress resistance, where cells preexposed to a mild sublethal dose of stress gain the ability to survive an otherwise lethal dose of stress. We have been leveraging wild strains of Saccharomyces cerevisiae to investigate natural variation in the yeast ethanol stress response and its role in acquired stress resistance. Here, we report that a wild vineyard isolate possesses ethanol-induced cross protection against severe concentrations of salt. Because this phenotype correlates with ethanol-dependent induction of the ENA genes, which encode sodium efflux pumps already associated with salt resistance, we hypothesized that variation in ENA expression was responsible for differences in acquired salt tolerance across strains. Surprisingly, we found that the ENA genes were completely dispensable for ethanol-induced survival of high salt concentrations in the wild vineyard strain. Instead, the ENA genes were necessary for the ability to resume growth on high concentrations of salt following a mild ethanol pretreatment. Surprisingly, this growth acclimation phenotype was also shared by the lab yeast strain despite lack of ENA induction under this condition. This study underscores that cross protection can affect both viability and growth through distinct mechanisms, both of which likely confer fitness effects that are ecologically relevant.IMPORTANCE Microbes in nature frequently experience "boom or bust" cycles of environmental stress. Thus, microbes that can anticipate the onset of stress would have an advantage. One way that microbes anticipate future stress is through acquired stress resistance, where cells exposed to a mild dose of one stress gain the ability to survive an otherwise lethal dose of a subsequent stress. In the budding yeast Saccharomyces cerevisiae, certain stressors can cross protect against high salt concentrations, though the mechanisms governing this acquired stress resistance are not well understood. In this study, we took advantage of wild yeast strains to understand the mechanism underlying ethanol-induced cross protection against high salt concentrations. We found that mild ethanol stress allows cells to resume growth on high salt, which involves a novel role for a well-studied salt transporter. Overall, this discovery highlights how leveraging natural variation can provide new insights into well-studied stress defense mechanisms.

Linkage mapping of yeast cross protection connects gene expression variation to a higher-order organismal trait.

Gene expression variation is extensive in nature, and is hypothesized to play a major role in shaping phenotypic diversity. However, connecting differences in gene expression across individuals to higher-order organismal traits is not trivial. In many cases, gene expression variation may be evolutionarily neutral, and in other cases expression variation may only affect phenotype under specific conditions. To understand connections between gene expression variation and stress defense phenotypes, we have been leveraging extensive natural variation in the gene expression response to acute ethanol in laboratory and wild Saccharomyces cerevisiae strains. Previous work found that the genetic architecture underlying these expression differences included dozens of "hotspot" loci that affected many transcripts in trans. In the present study, we provide new evidence that one of these expression QTL hotspot loci affects natural variation in one particular stress defense phenotype-ethanol-induced cross protection against severe doses of H2O2. A major causative polymorphism is in the heme-activated transcription factor Hap1p, which we show directly impacts cross protection, but not the basal H2O2 resistance of unstressed cells. This provides further support that distinct cellular mechanisms underlie basal and acquired stress resistance. We also show that Hap1p-dependent cross protection relies on novel regulation of cytosolic catalase T (Ctt1p) during ethanol stress in a wild oak strain. Because ethanol accumulation precedes aerobic respiration and accompanying reactive oxygen species formation, wild strains with the ability to anticipate impending oxidative stress would likely be at an advantage. This study highlights how strategically chosen traits that better correlate with gene expression changes can improve our power to identify novel connections between gene expression variation and higher-order organismal phenotypes.

Do biofilm communities respond to the chemical signatures of fracking? A test involving streams in North-central Arkansas.

BACKGROUND: Unconventional natural gas (UNG) extraction (fracking) is ongoing in 29 North American shale basins (20 states), with ~6000 wells found within the Fayetteville shale (north-central Arkansas). If the chemical signature of fracking is detectable in streams, it can be employed to bookmark potential impacts. We evaluated benthic biofilm community composition as a proxy for stream chemistry so as to segregate anthropogenic signatures in eight Arkansas River catchments. In doing so, we tested the hypothesis that fracking characteristics in study streams are statistically distinguishable from those produced by agriculture or urbanization. RESULTS: Four tributary catchments had UNG-wells significantly more dense and near to our sampling sites and were grouped as 'potentially-impacted catchment zones' (PICZ). Four others were characterized by significantly larger forested area with greater slope and elevation but reduced pasture, and were classified as 'minimally-impacted' (MICZ). Overall, 46 bacterial phyla/141 classes were identified, with 24 phyla (52%) and 54 classes (38%) across all samples. PICZ-sites were ecologically more variable than MICZ-sites, with significantly greater nutrient levels (total nitrogen, total phosphorous), and elevated Cyanobacteria as bioindicators that tracked these conditions. PICZ-sites also exhibited elevated conductance (a correlate of increased ion concentration) and depressed salt-intolerant Spartobacteria, suggesting the presence of brine as a fracking effect. Biofilm communities at PICZ-sites were significantly less variable than those at MICZ-sites. CONCLUSIONS: Study streams differed by Group according to morphology, land use, and water chemistry but not in biofilm community structure. Those at PICZ-sites covaried according to anthropogenic impact, and were qualitatively similar to communities found at sites disturbed by fracking. The hypothesis that fracking signatures in study streams are distinguishable from those produced by other anthropogenic effects was statistically rejected. Instead, alterations in biofilm community composition, as induced by fracking, may be less specific than initially predicted, and thus more easily confounded by agriculture and urbanization effects (among others). Study streams must be carefully categorized with regard to the magnitude and extent of anthropogenic impacts. They must also be segregated with statistical confidence (as herein) before fracking impacts are monitored.

Phylogenetic and amino acid conservation analyses of bacterial L-aspartate-α-decarboxylase and of its zymogen-maturation protein reveal a putative interaction domain.

BACKGROUND: All organisms must synthesize the enzymatic cofactor coenzyme A (CoA) from the precursor pantothenate. Most bacteria can synthesize pantothenate de novo by the condensation of pantoate and ß-alanine. The synthesis of ß-alanine is catalyzed by L-aspartate-α-decarboxylase (PanD), a pyruvoyl enzyme that is initially synthesized as a zymogen (pro-PanD). Active PanD is generated by self-cleavage of pro-PanD at Gly24-Ser25 creating the active-site pyruvoyl moiety. In Salmonella enterica, this cleavage requires PanM, an acetyl-CoA sensor related to the Gcn5-like N-acetyltransferases. PanM does not acetylate pro-PanD, but the recent publication of the three-dimensional crystal structure of the PanM homologue PanZ in complex with the PanD zymogen of Escherichia coli provides validation to our predictions and provides a framework in which to further examine the cleavage mechanism. In contrast, PanD from bacteria lacking PanM efficiently cleaved in the absence of PanM in vivo. RESULTS: Using phylogenetic analyses combined with in vivo phenotypic investigations, we showed that two classes of bacterial L-aspartate-α-decarboxylases exist. This classification is based on their posttranslational activation by self-cleavage of its zymogen. Class I L-aspartate-α-decarboxylase zymogens require the acetyl-CoA sensor PanM to be cleaved into active PanD. This class is found exclusively in the Gammaproteobacteria. Class II L-aspartate-α-decarboxylase zymogens self cleave efficiently in the absence of PanM, and are found in a wide number of bacterial phyla. Several members of the Euryarchaeota and Crenarchaeota also contain Class II L-aspartate-α-decarboxylases. Phylogenetic and amino acid conservation analyses of PanM revealed a conserved region of PanM distinct from conserved regions found in related Gcn5-related acetyltransferase enzymes (Pfam00583). This conserved region represents a putative domain for interactions with L-aspartate-α-decarboxylase zymogens. This work may inform future biochemical and structural studies of pro-PanD-PanM interactions. CONCLUSIONS: Experimental results indicate that S. enterica and C. glutamicum L-aspartate-α-decarboxylases represent two different classes of homologues of these enzymes. Class I homologues require PanM for activation, while Class II self cleave in the absence of PanM. Computer modeling of conserved amino acids using structure coordinates of PanM and L-aspartate-α-decarboxylase available in the protein data bank (RCSB PDB) revealed a putative site of interactions, which may help generate models to help understand the molecular details of the self-cleavage mechanism of L-aspartate-α-decarboxylases.

PanM, an acetyl-coenzyme A sensor required for maturation of L-aspartate decarboxylase (PanD).

UNLABELLED: Coenzyme A (CoA) is essential for cellular chemistry in all forms of life. The pantothenate moiety of CoA is generated from the condensation of pantoate and ß-alanine. ß-Alanine is formed by decarboxylation of l-aspartate catalyzed by PanD, a pyruvoyl enzyme that is synthesized by the cell as an inactive precursor (pro-PanD). Maturation of pro-PanD into PanD occurs via a self-cleavage event at residue Ser25, which forms the catalytic pyruvoyl moiety. We recently reported that Salmonella enterica PanM was necessary for pro-PanD maturation, both in vitro and in vivo. Notably, PanM is annotated as a Gcn5-like N-acetyltransferase (GNAT), which suggested that lysine acetylation might be part of the mechanism of maturation. Here we show that PanM lacks acetyltransferase activity and that acetyl-CoA stimulates its activity. Results of experiments with nonhydrolyzable ethyl-CoA and genetically encoded acetyl-lysine-containing PanD support the conclusion that PanM-dependent pro-PanD maturation does not involve an acetyl transfer event. We also show that CoA binding to PanM is needed for in vivo activity and that disruption of CoA binding prevents PanM from interacting with PanD. We conclude that PanM is a GNAT homologue that lost its acetyltransferase activity and evolved a new function as an acetyl-CoA sensor that can trigger the maturation of pro-PanD. IMPORTANCE: Nε-lysine acetylation is increasingly being recognized as a widespread and important form of posttranslational regulation in bacteria. The acetyltransferases that catalyze these reactions are poorly characterized in bacteria. Based on annotation, most bacterial genomes contain several acetyltransferases, but the physiological roles of only a handful have been determined. Notably, a subset of putative acetyltransferases lack residues that are critical for activity in most biochemically characterized acetyltransferases. We show that one such putative acetyltransferase, PanM (formerly YhhK), lacks acetyltransferase activity but functions instead as an acetyl-coenzyme A (CoA) sensor. This work establishes the possibility that, like PanM, other putative acetyltransferases may have evolved new functions while retaining the ability to sense acetyl-CoA.

The missing link in coenzyme A biosynthesis: PanM (formerly YhhK), a yeast GCN5 acetyltransferase homologue triggers aspartate decarboxylase (PanD) maturation in Salmonella enterica.

Coenzyme A (CoA) is an essential cofactor for all forms of life. The biochemistry underpinning the assembly of CoA in Escherichia coli and other enterobacteria is well understood, except for the events leading to maturation of the L-aspartate-α-decarboxylase (PanD) enzyme that converts pantothenate to ß-alanine. PanD is synthesized as pro-PanD, which undergoes an auto-proteolytic cleavage at residue Ser25 to yield the catalytic pyruvoyl moiety of the enzyme. Since 1990, it has been known that E. coli yhhK strains are pantothenate auxotrophs, but the role of YhhK in pantothenate biosynthesis remained an enigma. Here we show that Salmonella enterica yhhK strains are also pantothenate auxotrophs. In vivo and in vitro evidence shows that YhhK interacts directly with PanD, and that such interactions accelerate pro-PanD maturation. We also show that S. enterica yhhK strains accumulate pro-PanD, and that not all pro-PanD proteins require YhhK for maturation. For example, the Corynebacterium glutamicum panD(+) gene corrected the pantothenate auxotrophy of a S. enterica yhhK strain, supporting in vitro evidence obtained by others that some pro-PanD proteins autocleave at faster rates. We propose the name PanM for YhhK to reflect its role as a trigger of pro-PanD maturation by stabilizing pro-PanD in an autocleavage-prone conformation.