Impact of Anaerobiosis on Expression of the Iron-Responsive Fur and RyhB Regulons

Abstract

Iron, a major protein cofactor, is essential for most organisms. Despite the well-known effects of O2 on the oxidation state and solubility of iron, the impact of O2 on cellular iron homeostasis is not well understood. Here we report that in Escherichia coli K-12, the lack of O2 dramatically 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.

Importance: Microbes 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 O2 availability affects the gene expression programs of two Escherichia coli master 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 O2 may actually reduce bacterial iron demands, making it easier to win the arms race for iron.

FIG 1

Genome-wide Fur DNA binding. Fur binding across the genome was compared under either aerobic (track A, red) or anaerobic (track B, blue) iron-sufficient (10 µM FeSO₄) growth conditions in the wild-type strain, MG1655, or under anaerobic iron-limiting (1.0 µM FeSO₄) growth conditions (track C, black) in an iron-uptake-deficient strain (ΔtonB Δfeo ΔzupT) by ChIP-seq. The x axis indicates the genomic position of the ChIP-seq peaks using the MG1655 genome coordinates (version U00096.2), and the y axis indicates the read count after each data set was normalized to 20 million reads; enrichment of Fur DNA binding is indicated by the height of the lines in each track. An asterisk following the gene name indicates the read count extends beyond 250,000. A subset of peaks has been labelled with the corresponding downstream gene for comparison.

FIG 2

Fur binds less-conserved sequences under anaerobic growth conditions. DNA sequences from iron-dependent Fur ChIP-seq peaks were analyzed by the motif-finding algorithm MEME-ChIP. The height of the letters (in bits on the y axis) represents the degree of conservation at a given position within the aligned sequence set, with perfect conservation being 2 bits. The motif in the top panel was constructed from 90 sequences bound by Fur under both aerobic and anaerobic growth conditions. The motif in the bottom panel was constructed from 157 sequences bound by Fur only under anaerobic growth conditions. Arrows indicate the presence of an inverted repeat.

FIG 3

Only a few newly identified Fur binding regions lead to transcription regulation. Representative ChIP-seq plots of Fur binding to preT, dps, or appY promoter regions are shown for aerobic (red) or anaerobic (blue) growth conditions (top panel). Plots of preT, dps, or appY RNA levels from transcriptomic data are shown for wild-type (red, blue) and Δfur (yellow, green) strains grown under aerobic (red or yellow) or anaerobic (blue or green) growth conditions (middle panel). Strains bearing promoter-lacZ fusions to the preT, dps, or appY promoters were assayed for β-galactosidase activity in the presence or absence of Fur under aerobic or anaerobic growth conditions and normalized by cell density as a measure of promoter activity (bottom panel). The error bars represent the standard errors from at least three biological replicates.

FIG 4

Anaerobiosis impacts the expression of the Fur and RyhB regulons. The expression pattern of operons regulated directly by Fur (top panel [data from Table S3 in the supplemental material]) and operons that are candidates for RyhB regulation (bottom panel [data from Table S4 in the supplemental material]) were compared and grouped by cellular iron functions. In the top panel, green squares indicate operons repressed by Fur, whereas purple squares indicate operons induced by Fur. Diamonds indicate operons exhibiting more Fur regulation under anaerobic conditions, and underlined operons are bound by Fur only under anaerobic conditions. In the bottom panel, green squares indicate operons whose transcript levels are decreased by RyhB, whereas purple squares indicate operons that are increased by RyhB. Operons exhibiting ≥2-fold-increased wild-type expression under aerobic or anaerobic conditions are marked with a circle or triangle, respectively, and operons where upstream Fur DNA binding was mapped are underlined. Newly identified Fur- or RyhB-regulated genes are in red text. Proteins known to contain an Fe cofactor are indicated.

FIG 5

Effect of O₂ on expression of select Fur regulon members. Strains bearing promoter-reporter gene (lacZ) fusions to several Fur-regulated promoters (fhuA, bfd, nrdH, fepA, fhuE, feoA, fiu, and mntH) were assayed for β-galactosidase activity in the presence or absence of Fur under aerobic or anaerobic growth conditions. Promoter activity was normalized to cell density. ND indicates that the promoter activity was below our detection limit. The error bars represent the standard errors from at least three biological replicates.

FIG 6

Growth of strains lacking Fur and RyhB. Cell density was measured over time for the wild-type (black diamonds), Δfur (red circles), ΔryhB (open squares), and Δfur ΔryhB (orange triangles) strains under aerobic or anaerobic growth conditions in MOPS glucose minimal medium with 10 µM FeSO₄. Trend lines were added for ease of visualization. This representative growth curve was replicated several times.

FIG 7

Iron homeostasis pathways are reprogrammed in response to O₂ availability: a model. Under both aerobic and anaerobic conditions, we propose that iron is distributed to iron storage complexes, Fur, iron-binding proteins, or Fe-S biogenesis and heme synthesis pathways. However, under anaerobic growth conditions, iron uptake is shifted to ferrous transport systems, expression of iron storage proteins is increased, and Fe-S biogenesis occurs primarily by the housekeeping Isc pathway. When expressed under anaerobic iron-limiting conditions, we propose that RyhB targets anaerobically induced Fe-S-binding and iron-binding proteins of anaerobic respiratory pathways in addition to other constitutively expressed RyhB targets. Whereas under aerobic growth conditions, iron uptake is shifted to ferric transport systems, iron storage gene expression is decreased, and Fe-S biogenesis is still mainly by the housekeeping Isc pathway, but expression of the stress-induced Suf pathway is increased. When expressed under aerobic iron-limiting conditions, RyhB switches from targeting Fe-S-binding and iron-binding proteins of anaerobic respiratory pathways to those of aerobic respiratory pathways and the TCA cycle. The abundance of Mn, Mn proteins, and Mn efflux systems also increases under aerobic growth conditions.