DNA arrays were used to investigate the functional role of Rox1 in mediating acclimatization to anaerobic conditions in Saccharomyces cerevisiae. Multiple growth conditions for wild-type and rox1 null strains were used to identify open reading frames with a statistically robust response to this repressor. These results were compared to those obtained for a wild-type strain in response to oxygen availability. Transcripts of nearly one-sixth of the genome were differentially expressed (P < 0.05) with respect to oxygen availability, the majority (>65%) being down-regulated under anoxia. Of the anaerobically induced genes, about one-third (106) contain putative Rox1-binding sites in their promoters and were significantly (P < 0.05) up-regulated in the rox1 null strains under aerobiosis. Additional promoter searches revealed that nearly one-third of the anaerobically induced genes contain an AR1 site(s) for the Upc2 transcription factor, suggesting that Upc2 and Rox1 regulate the majority of anaerobically induced genes in S. cerevisiae. Functional analyses indicate that a large fraction of the anaerobically induced genes are involved in cell stress (ϳ1/3), cell wall maintenance (ϳ1/8), carbohydrate metabolism (ϳ1/10), and lipid metabolism (ϳ1/12), with both Rox1 and Upc2 predominating in the regulation of this latter group and Upc2 predominating in cell wall maintenance. Mapping the changes in expression of functional regulons onto metabolic pathways has provided novel insight into the role of Rox1 and other trans-acting factors in mediating the physiological response of S. cerevisiae to anaerobic conditions.

Variation in gene content has been hypothesized to be the primary mode of adaptive evolution in microorganisms; however, very little is known about the spatial and temporal distribution of variable genes. Through population-scale comparative genomics of 7 Sulfolobus islandicus genomes from 3 locations, we demonstrate the biogeographical structure of the pan-genome of this species, with no evidence of gene flow between geographically isolated populations. The evolutionary independence of each population allowed us to assess genome dynamics over very recent evolutionary time, beginning ≈910,000 years ago. On this time scale, genome variation largely consists of recent strain-specific integration of mobile elements. Localized sectors of parallel gene loss are identified; however, the balance between the gain and loss of genetic material suggests that S. islandicus genomes acquire material slowly over time, primarily from closely related Sulfolobus species. Examination of the genome dynamics through population genomics in S. islandicus exposes the process of allopatric speciation in thermophilic Archaea and brings us closer to a generalized framework for understanding microbial genome evolution in a spatial context.

Oxygen availability affects the transcription of a number of genes in nearly all organisms. Although the molecular mechanisms for sensing oxygen are not precisely known, heme is thought to play a pivotal role. Here, we address the possibility that oxygen sensing in yeast, as in mammals, involves a redox-sensitive hemoprotein. We have found that carbon monoxide (CO) completely blocks the anoxia-induced expression of two hypoxic genes, OLE1 and CYC7, partially blocks the induction of a third gene, COX5b, and has no effect on the expression of other hypoxic or aerobic genes. In addition, transition metals (Co and Ni) induce the expression of OLE1 and CYC7 in a concentration-dependent manner under aerobic conditions. These findings suggest that the redox state of an oxygen-binding hemoprotein is involved in controlling the expression of at least two hypoxic yeast genes. By using mutants deficient in each of the two major yeast CO-binding hemoproteins (cytochrome c oxidase and f lavohemoglobin), respiratory inhibitors, and cob1 and 0 mutants, we have found that the respiratory chain is involved in the anoxic induction of these two genes and that cytochrome c oxidase is likely the hemoprotein ''sensor.'' Our findings also indicate that there are at least two classes of hypoxic genes in yeast (CO sensitive and CO insensitive) and imply that multiple pathways͞mechanisms are involved in modulating the expression of hypoxic yeast genes.

We conducted a comprehensive genomic analysis of the temporal response of yeast to anaerobiosis (six generations) and subsequent aerobic recovery (Ϸ2 generations) to reveal metabolic-state (galactose versus glucose)-dependent differences in gene network activity and function. Analysis of variance showed that far fewer genes responded (raw P value of <10 ؊8 ) to the O 2 shifts in glucose (1,603 genes) than in galactose (2,388 genes). Gene network analysis reveals that this difference is due largely to the failure of "stress"-activated networks controlled by Msn2/4, Fhl1, MCB, SCB, PAC, and RRPE to transiently respond to the shift to anaerobiosis in glucose as they did in galactose. After Ϸ1 generation of anaerobiosis, the response was similar in both media, beginning with the deactivation of Hap1 and Hap2/3/4/5 networks involved in mitochondrial functions and the concomitant derepression of Rox1-regulated networks for carbohydrate catabolism and redox regulation and ending (>2 generations) with the activation of Upc2-and Mot3-regulated networks involved in sterol and cell wall homeostasis. The response to reoxygenation was rapid (<5 min) and similar in both media, dominated by Yap1 networks involved in oxidative stress/redox regulation and the concomitant activation of heme-regulated ones. Our analyses revealed extensive networks of genes subject to combinatorial regulation by both heme-dependent (e.g., Hap1, Hap2/3/4/5, Rox1, Mot3, and Upc2) and heme-independent (e.g., Yap1, Skn7, and Puf3) factors under these conditions. We also uncover novel functions for several cis-regulatory sites and trans-acting factors and define functional regulons involved in the physiological acclimatization to changes in oxygen availability.Although the majority of yeasts cannot grow in the absence of oxygen, many of the Saccharomyces sensu stricto yeasts, as well as other Saccharomyces members, are facultative anaerobes capable of sustained anaerobic growth (3,4,75,86). From genomic comparisons of obligate aerobic and facultative anaerobic yeasts, it would appear that this capacity coincides with a genome duplication event that occurred Ϸ100 to 150 million years ago in the Saccharomyces lineage (75, 83), followed by the subsequent evolution of new protein variants and the rewiring of transcriptional networks (44, 93). Interestingly, even under oxygen-replete conditions, these Crabtree-positive yeasts preferentially dissimilate hexoses to the C 3 and C 2 compounds pyruvate and ethanol. This is due in part to the evolution of a glucose repression circuit, which represses the transcription of respiratory genes in the presence of high concentrations of glucose (reviewed in reference 31). Although thermodynamically less efficient, glucose fermentation provides a much higher power output (ATP · min Ϫ1 · glycosyl unit Ϫ1 ) than glucose oxidation, which confers an obvious selective advantage to these fast-growing, ethanol-producing yeasts in certain environments (84). In addition to maximizing fermentation capacity, facultative anaerobic ye...

In contrast to previous steady-state analyses of the O 2 -responsive transcriptome, here we examined the dynamics of the response to short-term anaerobiosis (2 generations) in both catabolite-repressed (glucose) and derepressed (galactose) cells, assessed the specific role that Msn2 and Msn4 play in mediating the response, and identified gene networks using a novel clustering approach. Upon shifting cells to anaerobic conditions in galactose medium, there was an acute (ϳ10 min) yet transient (<45 min) induction of Msn2-and/or Msn4-regulated genes associated with the remodeling of reserve energy and catabolic pathways during the switch from mixed respiro-fermentative to strictly fermentative growth. Concomitantly, MCB-and SCB-regulated networks associated with the G 1 /S transition of the cell cycle were transiently down-regulated along with rRNA processing genes containing PAC and RRPE motifs. Remarkably, none of these gene networks were differentially expressed when cells were shifted in glucose, suggesting that a metabolically derived signal arising from the abrupt cessation of respiration, rather than O 2 deprivation per se, elicits this "stress response." By ϳ0.2 generation of anaerobiosis in both media, more chronic, heme-dependent effects were observed, including the down-regulation of Hap1-regulated networks, derepression of Rox1-regulated networks, and activation of Upc2-regulated ones. Changes in these networks result in the functional remodeling of the cell wall, sterol and sphingolipid metabolism, and dissimilatory pathways required for long-term anaerobiosis. Overall, this study reveals that the acute withdrawal of oxygen can invoke a metabolic state-dependent "stress response" but that acclimatization to oxygen deprivation is a relatively slow process involving complex changes primarily in heme-regulated gene networks.