Translating the genome in time and space: specialized ribosomes, RNA regulons, and RNA-binding proteins.

A central question in cell and developmental biology is how the information encoded in the genome is differentially interpreted to generate a diverse array of cell types. A growing body of research on posttranscriptional gene regulation is revealing that both global protein synthesis rates and the translation of specific mRNAs are highly specialized in different cell types. How this exquisite translational regulation is achieved is the focus of this review. Two levels of regulation are discussed: the translation machinery and cis-acting elements within mRNAs. Recent evidence shows that the ribosome itself directs how the genome is translated in time and space and reveals surprising functional specificity in individual components of the core translation machinery. We are also just beginning to appreciate the rich regulatory information embedded in the untranslated regions of mRNAs, which direct the selective translation of transcripts. These hidden RNA regulons may interface with a myriad of RNA-binding proteins and specialized translation machinery to provide an additional layer of regulation to how transcripts are spatiotemporally expressed. Understanding this largely unexplored world of translational codes hardwired in the core translation machinery is an exciting new research frontier fundamental to our understanding of gene regulation, organismal development, and evolution.

Sfp1 regulates transcriptional networks driving cell growth and division through multiple promoter-binding modes.

The yeast Sfp1 protein regulates both cell division and growth but how it coordinates these processes is poorly understood. We demonstrate that Sfp1 directly controls genes required for ribosome production and many other growth-promoting processes. Remarkably, the complete set of Sfp1 target genes is revealed only by a combination of ChIP (chromatin immunoprecipitation) and ChEC (chromatin endogenous cleavage) methods, which uncover two promoter binding modes, one requiring a cofactor and the other a DNA-recognition motif. Glucose-regulated Sfp1 binding at cell cycle "START" genes suggests that Sfp1 controls cell size by coordinating expression of genes implicated in mass accumulation and cell division.

Translational control of furina by an RNA regulon is important for left-right patterning, heart morphogenesis and cardiac valve function.

Heart development is a complex process that requires asymmetric positioning of the heart, cardiac growth and valve morphogenesis. The mechanisms controlling heart morphogenesis and valve formation are not fully understood. The pro-convertase FurinA functions in heart development across vertebrates. How FurinA activity is regulated during heart development is unknown. Through computational analysis of the zebrafish transcriptome, we identified an RNA motif in a variant FurinA transcript harbouring a long 3' untranslated region (3'UTR). The alternative 3'UTR furina isoform is expressed prior to organ positioning. Somatic deletions in the furina 3'UTR lead to embryonic left-right patterning defects. Reporter localisation and RNA-binding assays show that the furina 3'UTR forms complexes with the conserved RNA-binding translational repressor, Ybx1. Conditional ybx1 mutant embryos show premature and increased Furin reporter expression, abnormal cardiac morphogenesis and looping defects. Mutant ybx1 hearts have an expanded atrioventricular canal, abnormal sino-atrial valves and retrograde blood flow from the ventricle to the atrium. This is similar to observations in humans with heart valve regurgitation. Thus, the furina 3'UTR element/Ybx1 regulon is important for translational repression of FurinA and regulation of heart development.

Molecular design of hypothalamus development.

A wealth of specialized neuroendocrine command systems intercalated within the hypothalamus control the most fundamental physiological needs in vertebrates1,2. Nevertheless, we lack a developmental blueprint that integrates the molecular determinants of neuronal and glial diversity along temporal and spatial scales of hypothalamus development3. Here we combine single-cell RNA sequencing of 51,199 mouse cells of ectodermal origin, gene regulatory network (GRN) screens in conjunction with genome-wide association study-based disease phenotyping, and genetic lineage reconstruction to show that nine glial and thirty-three neuronal subtypes are generated by mid-gestation under the control of distinct GRNs. Combinatorial molecular codes that arise from neurotransmitters, neuropeptides and transcription factors are minimally required to decode the taxonomical hierarchy of hypothalamic neurons. The differentiation of γ-aminobutyric acid (GABA) and dopamine neurons, but not glutamate neurons, relies on quasi-stable intermediate states, with a pool of GABA progenitors giving rise to dopamine cells4. We found an unexpected abundance of chemotropic proliferation and guidance cues that are commonly implicated in dorsal (cortical) patterning5 in the hypothalamus. In particular, loss of SLIT-ROBO signalling impaired both the production and positioning of periventricular dopamine neurons. Overall, we identify molecular principles that shape the developmental architecture of the hypothalamus and show how neuronal heterogeneity is transformed into a multimodal neural unit to provide virtually infinite adaptive potential throughout life.

The mRNA stability factor Khd4 defines a specific mRNA regulon for membrane trafficking in the pathogen Ustilago maydis.

Fungal pathogens depend on sophisticated gene expression programs for successful infection. A crucial component is RNA regulation mediated by RNA-binding proteins (RBPs). However, little is known about the spatiotemporal RNA control mechanisms during fungal pathogenicity. Here, we discover that the RBP Khd4 defines a distinct mRNA regulon to orchestrate membrane trafficking during pathogenic development of Ustilago maydis. By establishing hyperTRIBE for fungal RBPs, we generated a comprehensive transcriptome-wide map of Khd4 interactions in vivo. We identify a defined set of target mRNAs enriched for regulatory proteins involved, e.g., in GTPase signaling. Khd4 controls the stability of target mRNAs via its cognate regulatory element AUACCC present in their 3' untranslated regions. Studying individual examples reveals a unique link between Khd4 and vacuole maturation. Thus, we uncover a distinct role for an RNA stability factor defining a specific mRNA regulon for membrane trafficking during pathogenicity.

Perfect adaptation achieved by transport limitations governs the inorganic phosphate response in S. cerevisiae.

Cells cope with and adapt to ever-changing environmental conditions. Sophisticated regulatory networks allow cells to adjust to these fluctuating environments. One such archetypal system is the Saccharomyces cerevisiae Pho regulon. When external inorganic phosphate (Pi) concentration is low, the Pho regulon activates, expressing genes that scavenge external and internal Pi. However, the precise mechanism controlling this regulon remains elusive. We conducted a systems analysis of the Pho regulon on the single-cell level under well-controlled environmental conditions. This analysis identified a robust, perfectly adapted Pho regulon state in intermediate Pi conditions, and we identified an intermediate nuclear localization state of the transcriptional master regulator Pho4p. The existence of an intermediate nuclear Pho4p state unifies and resolves outstanding incongruities associated with the Pho regulon, explains the observed programmatic states of the Pho regulon, and improves our general understanding of how nature evolves and controls sophisticated gene regulatory networks. We further propose that robustness and perfect adaptation are not achieved through complex network-centric control but by simple transport biophysics. The ubiquity of multitransporter systems suggests that similar mechanisms could govern the function of other regulatory networks as well.

Inferred regulons are consistent with regulator binding sequences in E. coli.

The transcriptional regulatory network (TRN) of E. coli consists of thousands of interactions between regulators and DNA sequences. Regulons are typically determined either from resource-intensive experimental measurement of functional binding sites, or inferred from analysis of high-throughput gene expression datasets. Recently, independent component analysis (ICA) of RNA-seq compendia has shown to be a powerful method for inferring bacterial regulons. However, it remains unclear to what extent regulons predicted by ICA structure have a biochemical basis in promoter sequences. Here, we address this question by developing machine learning models that predict inferred regulon structures in E. coli based on promoter sequence features. Models were constructed successfully (cross-validation AUROC > = 0.8) for 85% (40/47) of ICA-inferred E. coli regulons. We found that: 1) The presence of a high scoring regulator motif in the promoter region was sufficient to specify regulatory activity in 40% (19/47) of the regulons, 2) Additional features, such as DNA shape and extended motifs that can account for regulator multimeric binding, helped to specify regulon structure for the remaining 60% of regulons (28/47); 3) investigating regulons where initial machine learning models failed revealed new regulator-specific sequence features that improved model accuracy. Finally, we found that strong regulatory binding sequences underlie both the genes shared between ICA-inferred and experimental regulons as well as genes in the E. coli core pan-regulon of Fur. This work demonstrates that the structure of ICA-inferred regulons largely can be understood through the strength of regulator binding sites in promoter regions, reinforcing the utility of top-down inference for regulon discovery.

Molecular architecture of lineage allocation and tissue organization in early mouse embryo.

During post-implantation development of the mouse embryo, descendants of the inner cell mass in the early epiblast transit from the naive to primed pluripotent state1. Concurrently, germ layers are formed and cell lineages are specified, leading to the establishment of the blueprint for embryogenesis. Fate-mapping and lineage-analysis studies have revealed that cells in different regions of the germ layers acquire location-specific cell fates during gastrulation2-5. The regionalization of cell fates preceding the formation of the basic body plan-the mechanisms of which are instrumental for understanding embryonic programming and stem-cell-based translational study-is conserved in vertebrate embryos6-8. However, a genome-wide molecular annotation of lineage segregation and tissue architecture of the post-implantation embryo has yet to be undertaken. Here we report a spatially resolved transcriptome of cell populations at defined positions in the germ layers during development from pre- to late-gastrulation stages. This spatiotemporal transcriptome provides high-resolution digitized in situ gene-expression profiles, reveals the molecular genealogy of tissue lineages and defines the continuum of pluripotency states in time and space. The transcriptome further identifies the networks of molecular determinants that drive lineage specification and tissue patterning, supports a role of Hippo-Yap signalling in germ-layer development and reveals the contribution of visceral endoderm to the endoderm in the early mouse embryo.

The Escherichia coli Fur pan-regulon has few conserved but many unique regulatory targets.

While global transcription factors (TFs) have been studied extensively in Escherichia coli model strains, conservation and diversity in TF regulation between strains is still unknown. Here we use a combination of ChIP-exo-to define ferric uptake regulator (Fur) binding sites-and differential gene expression-to define the Fur regulon in nine E. coli strains. We then define a pan-regulon consisting of 469 target genes that includes all Fur target genes in all nine strains. The pan-regulon is then divided into the core regulon (target genes found in all the strains, n = 36), the accessory regulon (target found in two to eight strains, n = 158) and the unique regulon (target genes found in one strain, n = 275). Thus, there is a small set of Fur regulated genes common to all nine strains, but a large number of regulatory targets unique to a particular strain. Many of the unique regulatory targets are genes unique to that strain. This first-established pan-regulon reveals a common core of conserved regulatory targets and significant diversity in transcriptional regulation amongst E. coli strains, reflecting diverse niche specification and strain history.

Competence shut-off by intracellular pheromone degradation in salivarius streptococci.

Competence for DNA transformation is a major strategy for bacterial adaptation and survival. Yet, this successful tactic is energy-consuming, shifts dramatically the metabolism, and transitory impairs the regular cell-cycle. In streptococci, complex regulatory pathways control competence deactivation to narrow its development to a sharp window of time, a process known as competence shut-off. Although characterized in streptococci whose competence is activated by the ComCDE signaling pathway, it remains unclear for those controlled by the ComRS system. In this work, we investigate competence shut-off in the major human gut commensal Streptococcus salivarius. Using a deterministic mathematical model of the ComRS system, we predicted a negative player under the control of the central regulator ComX as involved in ComS/XIP pheromone degradation through a negative feedback loop. The individual inactivation of peptidase genes belonging to the ComX regulon allowed the identification of PepF as an essential oligoendopeptidase in S. salivarius. By combining conditional mutants, transcriptional analyses, and biochemical characterization of pheromone degradation, we validated the reciprocal role of PepF and XIP in ComRS shut-off. Notably, engineering cleavage site residues generated ultra-resistant peptides producing high and long-lasting competence activation. Altogether, this study reveals a proteolytic shut-off mechanism of competence in the salivarius group and suggests that this mechanism could be shared by other ComRS-containing streptococci.

Integrated analysis of single-cell and bulk RNA sequencing data reveals a myeloid cell-related regulon predicting neoadjuvant immunotherapy response across cancers.

BACKGROUND: Immunotherapy has brought about a paradigm shift in the treatment of cancer. However, the majority of patients exhibit resistance or become refractory to immunotherapy, and the underlying mechanisms remain to be explored. METHODS: Sing-cell RNA sequencing (scRNA­seq) datasets derived from 1 pretreatment and 1 posttreatment achieving pathological complete response (pCR) patient with lung adenocarcinoma (LUAD) who received neoadjuvant immunotherapy were collected, and pySCENIC was used to find the gene regulatory network (GRN) between cell types and immune checkpoint inhibitor (ICI) response. A regulon predicting ICI response was identified and validated using large­scale pan-cancer data, including a colorectal cancer scRNA­seq dataset, a breast cancer scRNA­seq dataset, The Cancer Genome Atlas (TCGA) pan-cancer cohort, and 5 ICI transcriptomic cohorts. Symphony reference mapping was performed to construct the myeloid cell map. RESULTS: Thirteen major cluster cell types were identified by comparing pretreatment and posttreatment patients, and the fraction of myeloid cells was higher in the posttreatment group (19.0% vs. 11.8%). A PPARG regulon (containing 23 target genes) was associated with ICI response, and its function was validated by a colorectal cancer scRNA­seq dataset, a breast cancer scRNA­seq dataset, TCGA pan-cancer cohort, and 5 ICI transcriptomic cohorts. Additionally, a myeloid cell map was developed, and cluster I, II, and III myeloid cells with high expression of PPARG were identified. Moreover, we constructed a website called PPARG ( https://pparg.online/PPARG/ or http://43.134.20.130:3838/PPARG/ ), which provides a powerful discovery tool and resource value for researchers. CONCLUSIONS: The PPARG regulon is a predictor of ICI response. The myeloid cell map enables the identification of PPARG subclusters in public scRNA-seq datasets and provides a powerful discovery tool and resource value.

Regulon active landscape reveals cell development and functional state changes of human primary osteoblasts in vivo.

BACKGROUND: While transcription factor (TF) regulation is known to play an important role in osteoblast development, differentiation, and bone metabolism, the molecular features of TFs in human osteoblasts at the single-cell resolution level have not yet been characterized. Here, we identified modules (regulons) of co-regulated genes by applying single-cell regulatory network inference and clustering to the single-cell RNA sequencing profiles of human osteoblasts. We also performed cell-specific network (CSN) analysis, reconstructed regulon activity-based osteoblast development trajectories, and validated the functions of important regulons both in vivo and in vitro. RESULTS: We identified four cell clusters: preosteoblast-S1, preosteoblast-S2, intermediate osteoblasts, and mature osteoblasts. CSN analysis results and regulon activity-based osteoblast development trajectories revealed cell development and functional state changes of osteoblasts. CREM and FOSL2 regulons were mainly active in preosteoblast-S1, FOXC2 regulons were mainly active in intermediate osteoblast, and RUNX2 and CREB3L1 regulons were most active in mature osteoblasts. CONCLUSIONS: This is the first study to describe the unique features of human osteoblasts in vivo based on cellular regulon active landscapes. Functional state changes of CREM, FOSL2, FOXC2, RUNX2, and CREB3L1 regulons regarding immunity, cell proliferation, and differentiation identified the important cell stages or subtypes that may be predominantly affected by bone metabolism disorders. These findings may lead to a deeper understanding of the mechanisms underlying bone metabolism and associated diseases.

A metabolic regulon reveals early and late acting enzymes in neuroactive Lycopodium alkaloid biosynthesis.

Plants synthesize many diverse small molecules that affect function of the mammalian central nervous system, making them crucial sources of therapeutics for neurological disorders. A notable portion of neuroactive phytochemicals are lysine-derived alkaloids, but the mechanisms by which plants produce these compounds have remained largely unexplored. To better understand how plants synthesize these metabolites, we focused on biosynthesis of the Lycopodium alkaloids that are produced by club mosses, a clade of plants used traditionally as herbal medicines. Hundreds of Lycopodium alkaloids have been described, including huperzine A (HupA), an acetylcholine esterase inhibitor that has generated interest as a treatment for the symptoms of Alzheimer's disease. Through combined metabolomic profiling and transcriptomics, we have identified a developmentally controlled set of biosynthetic genes, or potential regulon, for the Lycopodium alkaloids. The discovery of this putative regulon facilitated the biosynthetic reconstitution and functional characterization of six enzymes that act in the initiation and conclusion of HupA biosynthesis. This includes a type III polyketide synthase that catalyzes a crucial imine-polyketide condensation, as well as three Fe(II)/2-oxoglutarate-dependent dioxygenase (2OGD) enzymes that catalyze transformations (pyridone ring-forming desaturation, piperidine ring cleavage, and redox-neutral isomerization) within downstream HupA biosynthesis. Our results expand the diversity of known chemical transformations catalyzed by 2OGDs and provide mechanistic insight into the function of noncanonical type III PKS enzymes that generate plant alkaloid scaffolds. These data offer insight into the chemical logic of Lys-derived alkaloid biosynthesis and demonstrate the tightly coordinated coexpression of secondary metabolic genes for the biosynthesis of medicinal alkaloids.

A protease-mediated mechanism regulates the cytochrome c6/plastocyanin switch in Synechocystis sp. PCC 6803.

After the Great Oxidation Event (GOE), iron availability was greatly decreased, and photosynthetic organisms evolved several alternative proteins and mechanisms. One of these proteins, plastocyanin, is a type I blue-copper protein that can replace cytochrome c6 as a soluble electron carrier between cytochrome b6f and photosystem I. In most cyanobacteria, expression of these two alternative proteins is regulated by copper availability, but the regulatory system remains unknown. Herein, we provide evidence that the regulatory system is composed of a BlaI/CopY-family transcription factor (PetR) and a BlaR-membrane protease (PetP). PetR represses petE (plastocyanin) expression and activates petJ (cytochrome c6), while PetP controls PetR levels in vivo. Using whole-cell extracts, we demonstrated that PetR degradation requires both PetP and copper. Transcriptomic analysis revealed that the PetRP system regulates only four genes (petE, petJ, slr0601, and slr0602), highlighting its specificity. Furthermore, the presence of petE and petRP in early branching cyanobacteria indicates that acquisition of these genes could represent an early adaptation to decreased iron bioavailability following the GOE.

Mining RNA-seq data reveals the massive regulon of GcvB small RNA and its physiological significance in maintaining amino acid homeostasis in Escherichia coli.

Bacterial small RNAs regulate the expression of multiple genes through imperfect base-pairing with target mRNAs mediated by RNA chaperone proteins such as Hfq. GcvB is the master sRNA regulator of amino acid metabolism and transport in a wide range of Gram-negative bacteria. Recently, independent RNA-seq approaches identified a plethora of transcripts interacting with GcvB in Escherichia coli. In this study, the compilation of RIL-seq, CLASH, and MAPS data sets allowed us to identify GcvB targets with high accuracy. We validated 21 new GcvB targets repressed at the posttranscriptional level, raising the number of direct targets to >50 genes in E. coli. Among its multiple seed sequences, GcvB utilizes either R1 or R3 to regulate most of these targets. Furthermore, we demonstrated that both R1 and R3 seed sequences are required to fully repress the expression of gdhA, cstA, and sucC genes. In contrast, the ilvLXGMEDA polycistronic mRNA is targeted by GcvB through at least four individual binding sites in the mRNA. Finally, we revealed that GcvB is involved in the susceptibility of peptidase-deficient E. coli strain (Δpeps) to Ala-Gln dipeptide by regulating both Dpp dipeptide importer and YdeE dipeptide exporter via R1 and R3 seed sequences, respectively.

Inactivation of RNase P in Escherichia coli significantly changes post-transcriptional RNA metabolism.

Ribonuclease P (RNase P), which is required for the 5'-end maturation of tRNAs in every organism, has been shown to play a limited role in other aspects of RNA metabolism in Escherichia coli. Using RNA-sequencing (RNA-seq), we demonstrate that RNase P inactivation affects the abundances of ~46% of the expressed transcripts in E. coli and provide evidence that its essential function is its ability to generate pre-tRNAs from polycistronic tRNA transcripts. The RNA-seq results agreed with the published data and northern blot analyses of 75/83 transcripts (mRNAs, sRNAs, and tRNAs). Changes in transcript abundances in the RNase P mutant also correlated with changes in their half-lives. Inactivating the stringent response did not alter the rnpA49 phenotype. Most notably, increases in the transcript abundances were observed for all genes in the cysteine regulons, multiple toxin-antitoxin modules, and sigma S-controlled genes. Surprisingly, poly(A) polymerase (PAP I) modulated the abundances of ~10% of the transcripts affected by RNase P. A comparison of the transcriptomes of RNase P, RNase E, and RNase III mutants suggests that they affect distinct substrates. Together, our work strongly indicates that RNase P is a major player in all aspects of post-transcriptional RNA metabolism in E. coli.

The oxidative stress response of pathogenic Leptospira is controlled by two peroxide stress regulators which putatively cooperate in controlling virulence.

Pathogenic Leptospira are the causative agents of leptospirosis, the most widespread zoonotic infectious disease. Leptospirosis is a potentially severe and life-threatening emerging disease with highest burden in sub-tropical areas and impoverished populations. Mechanisms allowing pathogenic Leptospira to survive inside a host and induce acute leptospirosis are not fully understood. The ability to resist deadly oxidants produced by the host during infection is pivotal for Leptospira virulence. We have previously shown that genes encoding defenses against oxidants in L. interrogans are repressed by PerRA (encoded by LIMLP_10155), a peroxide stress regulator of the Fur family. In this study, we describe the identification and characterization of another putative PerR-like regulator (LIMLP_05620) in L. interrogans. Protein sequence and phylogenetic analyses indicated that LIMLP_05620 displayed all the canonical PerR amino acid residues and is restricted to pathogenic Leptospira clades. We therefore named this PerR-like regulator PerRB. In L. interrogans, the PerRB regulon is distinct from that of PerRA. While a perRA mutant had a greater tolerance to peroxide, inactivating perRB led to a higher tolerance to superoxide, suggesting that these two regulators have a distinct function in the adaptation of L. interrogans to oxidative stress. The concomitant inactivation of perRA and perRB resulted in a higher tolerance to both peroxide and superoxide and, unlike the single mutants, a double perRAperRB mutant was avirulent. Interestingly, this correlated with major changes in gene and non-coding RNA expression. Notably, several virulence-associated genes (clpB, ligA/B, and lvrAB) were repressed. By obtaining a double mutant in a pathogenic Leptospira strain, our study has uncovered an interplay of two PerRs in the adaptation of Leptospira to oxidative stress with a putative role in virulence and pathogenicity, most likely through the transcriptional control of a complex regulatory network.

RpoN Regulon in Erwinia amylovora Revealed by Transcriptional Profiling and In Silico Binding Site Analysis.

Erwinia amylovora causes a devastating fire blight disease in apples and pears. One of the main virulence determinants in E. amylovora is the hypersensitive response (HR) and pathogenicity (hrp)-type III secretion system (T3SS), which is activated by the RpoN-HrpL sigma factor cascade. However, the RpoN regulon in E. amylovora has not been investigated. In this study, we determined the RpoN regulon in E. amylovora by combining RNA-seq transcriptomic analysis with in silico binding site analysis. RNA-seq revealed that 262 genes, approximately 7.5% genes in the genome of E. amylovora, were differentially transcribed in the rpoN mutant as compared with the wild type. Specifically, genes associated with virulence, motility, nitrogen assimilation, the PspF system, stress response, and arginine biosynthesis are positively regulated by RpoN, whereas genes associated with biosynthesis of amino acids and sorbitol transport are negatively regulated by RpoN. In silico binding site analysis identified 46 potential target genes with a putative RpoN binding site, and the upstream sequences of six, three, and three genes also contain putative GlnG, PspF, and YfhA binding sites, respectively. Overall, RpoN directly regulates genes associated with virulence, nitrogen assimilation, the PspF system, motility and the YfhA/YfhK two-component regulatory system.

Rewiring the specificity of extracytoplasmic function sigma factors.

Bacterial genomes are being sequenced at an exponentially increasing rate, but our inability to decipher their transcriptional wiring limits our ability to derive new biology from these sequences. De novo determination of regulatory interactions requires accurate prediction of regulators' DNA binding and precise determination of biologically significant binding sites. Here we address these challenges by solving the DNA-specificity code of extracytoplasmic function sigma factors (ECF σs), a major family of bacterial regulators, and determining their putative regulons. We generated an aligned collection of ECF σs and their promoters by leveraging the autoregulatory nature of ECF σs as a means of promoter discovery and analyzed it to identify and characterize the conserved amino acid-nucleotide interactions that determine promoter specificity. This enabled de novo prediction of ECF σ specificity, which we combined with a statistically rigorous phylogenetic footprinting pipeline based on precomputed orthologs to predict the direct targets of ∼67% of ECF σs. This global survey indicated that some ECF σs are conserved global regulators controlling many genes throughout the genome, which are important under many conditions, while others are local regulators, controlling a few closely linked genes in response to specific stimuli in select species. This analysis reveals important organizing principles of bacterial gene regulation and presents a conceptual and computational framework for deciphering gene regulatory networks.

Revisiting Fur Regulon Leads to a Comprehensive Understanding of Iron and Fur Regulation.

Iron is an essential element because it functions as a cofactor of many enzymes, but excess iron causes cell damage. Iron hemostasis in Escherichia coli was transcriptionally maintained by the ferric uptake regulator (Fur). Despite having been studied extensively, the comprehensive physiological roles and mechanisms of Fur-coordinated iron metabolism still remain obscure. In this work, by integrating a high-resolution transcriptomic study of the Fur wild-type and knockout Escherichia coli K-12 strains in the presence or absence of iron with high-throughput ChIP-seq assay and physiological studies, we revisited the regulatory roles of iron and Fur systematically and discovered several intriguing features of Fur regulation. The size of the Fur regulon was expanded greatly, and significant discrepancies were observed to exist between the regulations of Fur on the genes under its direct repression and activation. Fur showed stronger binding strength to the genes under its repression, and genes that were repressed by Fur were more sensitive to Fur and iron regulation as compared to the genes that were activated by Fur. Finally, we found that Fur linked iron metabolism to many essential processes, and the systemic regulations of Fur on carbon metabolism, respiration, and motility were further validated or discussed. These results highlight how Fur and Fur-controlled iron metabolism affect many cellular processes in a systematic way.