FNR is a well-studied global regulator of anaerobiosis, which is widely conserved across bacteria. Despite the importance of FNR and anaerobiosis in microbial lifestyles, the factors that influence its function on a genome-wide scale are poorly understood. Here, we report a functional genomic analysis of FNR action. We find that FNR occupancy at many target sites is strongly influenced by nucleoid-associated proteins (NAPs) that restrict access to many FNR binding sites. At a genome-wide level, only a subset of predicted FNR binding sites were bound under anaerobic fermentative conditions and many appeared to be masked by the NAPs H-NS, IHF and Fis. Similar assays in cells lacking H-NS and its paralog StpA showed increased FNR occupancy at sites bound by H-NS in WT strains, indicating that large regions of the genome are not readily accessible for FNR binding. Genome accessibility may also explain our finding that genome-wide FNR occupancy did not correlate with the match to consensus at binding sites, suggesting that significant variation in ChIP signal was attributable to cross-linking or immunoprecipitation efficiency rather than differences in binding affinities for FNR sites. Correlation of FNR ChIP-seq peaks with transcriptomic data showed that less than half of the FNR-regulated operons could be attributed to direct FNR binding. Conversely, FNR bound some promoters without regulating expression presumably requiring changes in activity of condition-specific transcription factors. Such combinatorial regulation may allow Escherichia coli to respond rapidly to environmental changes and confer an ecological advantage in the anaerobic but nutrient-fluctuating environment of the mammalian gut. Regulation of gene expression by transcription factors (TFs) is key to adaptation to environmental changes. Our comprehensive, genome-scale analysis of a prototypical global TF, the anaerobic regulator FNR from Escherichia coli, leads to several novel and unanticipated insights into the influences on FNR binding genome-wide and the complex structure of bacterial regulons. We found that binding of NAPs restricts FNR binding at a subset of sites, suggesting that the bacterial genome is not freely accessible for FNR binding. Our finding that less than half of the predicted FNR binding sites were occupied in vivo further challenges the utility of using bioinformatic searches alone to predict regulon structure, reinforcing the need for experimental determination of TF binding. By correlating the occupancy data with transcriptomic data, we confirm that FNR serves as a global signal of anaerobiosis but expression of some operons in the FNR regulon require other regulators sensitive to alternative environmental stimuli. Thus, FNR binding and regulation appear to depend on both the nucleoprotein structure of the chromosome and on combinatorial binding of FNR with other regulators. Both of these phenomena are typical of TF binding in eukaryotes; our results establish that they are also features of bacterial TF binding.

The inability of native Saccharomyces cerevisiae to convert xylose from plant biomass into biofuels remains a major challenge for the production of renewable bioenergy. Despite extensive knowledge of the regulatory networks controlling carbon metabolism in yeast, little is known about how to reprogram S. cerevisiae to ferment xylose at rates comparable to glucose. Here we combined genome sequencing, proteomic profiling, and metabolomic analyses to identify and characterize the responsible mutations in a series of evolved strains capable of metabolizing xylose aerobically or anaerobically. We report that rapid xylose conversion by engineered and evolved S. cerevisiae strains depends upon epistatic interactions among genes encoding a xylose reductase (GRE3), a component of MAP Kinase (MAPK) signaling (HOG1), a regulator of Protein Kinase A (PKA) signaling (IRA2), and a scaffolding protein for mitochondrial iron-sulfur (Fe-S) cluster biogenesis (ISU1). Interestingly, the mutation in IRA2 only impacted anaerobic xylose consumption and required the loss of ISU1 function, indicating a previously unknown connection between PKA signaling, Fe-S cluster biogenesis, and anaerobiosis. Proteomic and metabolomic comparisons revealed that the xylose-metabolizing mutant strains exhibit altered metabolic pathways relative to the parental strain when grown in xylose. Further analyses revealed that interacting mutations in HOG1 and ISU1 unexpectedly elevated mitochondrial respiratory proteins and enabled rapid aerobic respiration of xylose and other non-fermentable carbon substrates. Our findings suggest a surprising connection between Fe-S cluster biogenesis and signaling that facilitates aerobic respiration and anaerobic fermentation of xylose, underscoring how much remains unknown about the eukaryotic signaling systems that regulate carbon metabolism. The yeast Saccharomyces cerevisiae is being genetically engineered to produce renewable biofuels from sustainable plant material. Efficient biofuel production from plant material requires conversion of the complex suite of sugars found in plant material, including the five-carbon sugar xylose. Because it does not efficiently metabolize xylose, S. cerevisiae has been engineered with a minimal set of genes that should overcome this problem; however, additional genetic changes are required for optimal fermentative conversion of xylose into biofuel. Despite extensive knowledge of the regulatory networks controlling glucose metabolism, less is known about the regulation of xylose metabolism and how to rewire these networks for effective biofuel production. Here we report genetic mutations that enabled the conversion of xylose into bioethanol by a previously ineffective yeast strain. By comparing altered protein and metabolite abundance within yeast cells containing these mutations, we determined that the mutations synergistically alter metabolic pathways to improve the rate of xylose conversion. One change in a gene with well-characterized aerobic mitochondrial functions was found to play an unexpected role in anaerobic conversion of xylose into ethanol. The results of this work will allow others to rapidly generate yeast strains for the conversion of xylose into biofuels and other products.

Iron, a major protein cofactor, is essential for most organisms. Despite the well-known effects of O₂on the oxidation state and solubility of iron, the impact of O₂on cellular iron homeostasis is not well understood. Here we report that inEscherichia coliK-12, the lack of O₂dramatically changes expression of genes controlled by the global regulators of iron homeostasis, the transcription factor Fur and the small RNA RyhB. Using chromatin immunoprecipitation sequencing (ChIP-seq), we found anaerobic conditions promote Fur binding to more locations across the genome. However, by expression profiling, we discovered that the major effect of anaerobiosis was to increase the magnitude of Fur regulation, leading to increased expression of iron storage proteins and decreased expression of most iron uptake pathways and several Mn-binding proteins. This change in the pattern of gene expression also correlated with an unanticipated decrease in Mn in anaerobic cells. Changes in the genes posttranscriptionally regulated by RyhB under aerobic and anaerobic conditions could be attributed to O₂-dependent changes in transcription of the target genes: aerobic RyhB targets were enriched in iron-containing proteins associated with aerobic energy metabolism, whereas anaerobic RyhB targets were enriched in iron-containing anaerobic respiratory functions. Overall, these studies showed that anaerobiosis has a larger impact on iron homeostasis than previously anticipated, both by expanding the number of direct Fur target genes and the magnitude of their regulation and by altering the expression of genes predicted to be posttranscriptionally regulated by the small RNA RyhB under iron-limiting conditions.IMPORTANCEMicrobes and host cells engage in an “arms race” for iron, an essential nutrient that is often scarce in the environment. Studies of iron homeostasis have been key to understanding the control of iron acquisition and the downstream pathways that enable microbes to compete for this valuable resource. Here we report that O₂availability affects the gene expression programs of twoEscherichia colimaster regulators that function in iron homeostasis: the transcription factor Fur and the small RNA regulator RyhB. Fur appeared to be more active under anaerobic conditions, suggesting a change in the set point for iron homeostasis. RyhB preferentially targeted iron-containing proteins of respiration-linked pathways, which are differentially expressed under aerobic and anaerobic conditions. Such findings may be relevant to the success of bacteria within their hosts since zones of reduced O₂may actually reduce bacterial iron demands, making it easier to win the arms race for iron.

The genome sequences of more than 100 strains of the yeast Saccharomyces cerevisiae have been published. Unfortunately, most of these genome assemblies contain dozens to hundreds of gaps at repetitive sequences, including transposable elements, tRNAs, and subtelomeric regions, which is where novel genes generally reside. Relatively few strains have been chosen for genome sequencing based on their biofuel production potential, leaving an additional knowledge gap. Here, we describe the nearly complete genome sequence of GLBRCY22-3 (Y22-3), a strain of S. cerevisiae derived from the stress-tolerant wild strain NRRL YB-210 and subsequently engineered for xylose metabolism. After benchmarking several genome assembly approaches, we developed a pipeline to integrate Pacific Biosciences (PacBio) and Illumina sequencing data and achieved one of the highest quality genome assemblies for any S. cerevisiae strain. Specifically, the contig N50 is 693 kbp, and the sequences of most chromosomes, the mitochondrial genome, and the 2-micron plasmid are complete. Our annotation predicts 92 genes that are not present in the reference genome of the laboratory strain S288c, over 70% of which were expressed. We predicted functions for 43 of these genes, 28 of which were previously uncharacterized and unnamed. Remarkably, many of these genes are predicted to be involved in stress tolerance and carbon metabolism and are shared with a Brazilian bioethanol production strain, even though the strains differ dramatically at most genetic loci. The Y22-3 genome sequence provides an exceptionally high-quality resource for basic and applied research in bioenergy and genetics.