scholarly journals Minor Alterations in Core Promoter Element Positioning Reveal Functional Plasticity of a Bacterial Transcription Factor

mBio ◽  
2021 ◽  
Author(s):  
Wamiah P. Chowdhury ◽  
Kenneth A. Satyshur ◽  
James L. Keck ◽  
Patricia J. Kiley
Keyword(s):  
Transcription Factor ◽  
Core Promoter ◽  
Promoter Element ◽  
Functional Plasticity ◽  
Iron Sulfur Cluster ◽  
Content Type ◽  
E Coli ◽  
Bacterial Transcription ◽  
Iron Sulfur ◽  
Living Organisms

Transcription regulation is a key process in all living organisms, involving a myriad of transcription factors. In E. coli , the regulator of the iron-sulfur cluster biogenesis pathway, IscR, acts as a global transcription factor, activating the transcription of some pathways and repressing others.

scholarly journals Zinc Toxicity and Iron-Sulfur Cluster Biogenesis inEscherichia coli

2019 ◽  
Vol 85 (9) ◽  
Author(s):  
Jianghui Li ◽  
Xiaojun Ren ◽  
Bingqian Fan ◽  
Zhaoyang Huang ◽  
Wu Wang ◽  
...  
Keyword(s):  
Binding Activity ◽  
Zinc Toxicity ◽  
Cluster Assembly ◽  
Intracellular Zinc ◽  
Iron Sulfur Cluster ◽  
Content Type ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Assembly Proteins

ABSTRACTWhile zinc is an essential trace metal in biology, excess zinc is toxic to organisms. Previous studies have shown that zinc toxicity is associated with disruption of the [4Fe-4S] clusters in various dehydratases inEscherichia coli. Here, we report that the intracellular zinc overload inE. colicells inhibits iron-sulfur cluster biogenesis without affecting the preassembled iron-sulfur clusters in proteins. Among the housekeeping iron-sulfur cluster assembly proteins encoded by the gene clusteriscSUA-hscBA-fdx-iscXinE. colicells, the scaffold IscU, the iron chaperone IscA, and ferredoxin have strong zinc binding activity in cells, suggesting that intracellular zinc overload inhibits iron-sulfur cluster biogenesis by binding to the iron-sulfur cluster assembly proteins. Mutations of the conserved cysteine residues to serine in IscA, IscU, or ferredoxin completely abolish the zinc binding activity of the proteins, indicating that zinc can compete with iron or iron-sulfur cluster binding in IscA, IscU, and ferredoxin and block iron-sulfur cluster biogenesis. Furthermore, intracellular zinc overload appears to emulate the slow-growth phenotype of theE. colimutant cells with deletion of the iron-sulfur cluster assembly proteins IscU, IscA, and ferredoxin. Our results suggest that intracellular zinc overload inhibits iron-sulfur cluster biogenesis by targeting the iron-sulfur cluster assembly proteins IscU, IscA, and ferredoxin inE. colicells.IMPORTANCEZinc toxicity has been implicated in causing various human diseases. High concentrations of zinc can also inhibit bacterial cell growth. However, the underlying mechanism has not been fully understood. Here, we report that zinc overload inEscherichia colicells inhibits iron-sulfur cluster biogenesis by targeting specific iron-sulfur cluster assembly proteins. Because iron-sulfur proteins are involved in diverse physiological processes, the zinc-mediated inhibition of iron-sulfur cluster biogenesis could be largely responsible for the zinc-mediated cytotoxicity. Our finding provides new insights on how intracellular zinc overload may inhibit cellular functions in bacteria.

scholarly journals Anaerobic Copper Toxicity and Iron-Sulfur Cluster Biogenesis in Escherichia coli

2017 ◽  
Vol 83 (16) ◽  
Author(s):  
Guoqiang Tan ◽  
Jing Yang ◽  
Tang Li ◽  
Jin Zhao ◽  
Shujuan Sun ◽  
...  
Keyword(s):  
Copper Toxicity ◽  
Anaerobic Conditions ◽  
Iron Sulfur Cluster ◽  
Aerobic Conditions ◽  
Content Type ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Iron Sulfur Proteins ◽  
Intracellular Copper

ABSTRACT While copper is an essential trace element in biology, pollution of groundwater from copper has become a threat to all living organisms. Cellular mechanisms underlying copper toxicity, however, are still not fully understood. Previous studies have shown that iron-sulfur proteins are among the primary targets of copper toxicity in Escherichia coli under aerobic conditions. Here, we report that, under anaerobic conditions, iron-sulfur proteins in E. coli cells are even more susceptible to copper in medium. Whereas addition of 0.2 mM copper(II) chloride to LB (Luria-Bertani) medium has very little or no effect on iron-sulfur proteins in wild-type E. coli cells under aerobic conditions, the same copper treatment largely inactivates iron-sulfur proteins by blocking iron-sulfur cluster biogenesis in the cells under anaerobic conditions. Importantly, proteins that do not have iron-sulfur clusters (e.g., fumarase C and cysteine desulfurase) in E. coli cells are not significantly affected by copper treatment under aerobic or anaerobic conditions, indicating that copper may specifically target iron-sulfur proteins in cells. Additional studies revealed that E. coli cells accumulate more intracellular copper under anaerobic conditions than under aerobic conditions and that the elevated copper content binds to the iron-sulfur cluster assembly proteins IscU and IscA, which effectively inhibits iron-sulfur cluster biogenesis. The results suggest that the copper-mediated inhibition of iron-sulfur proteins does not require oxygen and that iron-sulfur cluster biogenesis is the primary target of anaerobic copper toxicity in cells. IMPORTANCE Copper contamination in groundwater has become a threat to all living organisms. However, cellular mechanisms underlying copper toxicity have not been fully understood up to now. The work described here reveals that iron-sulfur proteins in Escherichia coli cells are much more susceptible to copper in medium under anaerobic conditions than they are under aerobic conditions. Under anaerobic conditions, E. coli cells accumulate excess intracellular copper, which specifically targets iron-sulfur proteins by blocking iron-sulfur cluster biogenesis. Since iron-sulfur proteins are involved in diverse and vital physiological processes, inhibition of iron-sulfur cluster biogenesis by copper disrupts multiple cellular functions and ultimately inhibits cell growth. The results from this study illustrate a new interplay between intracellular copper toxicity and iron-sulfur cluster biogenesis in bacterial cells under anaerobic conditions.

scholarly journals Elevated Expression of a Functional Suf Pathway in Escherichia coli BL21(DE3) Enhances Recombinant Production of an Iron-Sulfur Cluster-Containing Protein

2019 ◽  
Vol 202 (3) ◽  
Author(s):  
Elliot I. Corless ◽  
Erin L. Mettert ◽  
Patricia J. Kiley ◽  
Edwin Antony
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Biochemical Characterization ◽  
Protein A ◽  
Fold Increase ◽  
Iron Sulfur Cluster ◽  
Content Type ◽  
E Coli ◽  
Protein Levels ◽  
Iron Sulfur

ABSTRACT Structural and spectroscopic analysis of iron-sulfur [Fe-S] cluster-containing proteins is often limited by the occupancy and yield of recombinantly produced proteins. Here we report that Escherichia coli BL21(DE3), a strain routinely used to overproduce [Fe-S] cluster-containing proteins, has a nonfunctional Suf pathway, one of two E. coli [Fe-S] cluster biogenesis pathways. We confirmed that BL21(DE3) and commercially available derivatives carry a deletion that results in an in-frame fusion of sufA and sufB genes within the sufABCDSE operon. We show that this fusion protein accumulates in cells but is inactive in [Fe-S] cluster biogenesis. Restoration of an intact Suf pathway combined with enhanced suf operon expression led to a remarkable (∼3-fold) increase in the production of the [4Fe-4S] cluster-containing BchL protein, a key component of the dark-operative protochlorophyllide oxidoreductase complex. These results show that this engineered “SufFeScient” derivative of BL21(DE3) is suitable for enhanced large-scale synthesis of an [Fe-S] cluster-containing protein. IMPORTANCE Large quantities of recombinantly overproduced [Fe-S] cluster-containing proteins are necessary for their in-depth biochemical characterization. Commercially available E. coli strain BL21(DE3) and its derivatives have a mutation that inactivates the function of one of the two native pathways (Suf pathway) responsible for cluster biogenesis. Correction of the mutation, combined with sequence changes that elevate Suf protein levels, can increase yield and cluster occupancy of [Fe-S] cluster-containing enzymes, facilitating the biochemical analysis of this fascinating group of proteins.

scholarly journals Identification of an Intermediate Form of Ferredoxin That Binds Only Iron Suggests That Conversion to Holo-Ferredoxin Is Independent of the ISC System in Escherichia coli

2021 ◽  
Vol 87 (10) ◽  
Author(s):  
Xiaojun Ren ◽  
Feng Liang ◽  
Zhengfen He ◽  
Bingqian Fan ◽  
Zhirong Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cold Stress ◽  
Stress Conditions ◽  
Iron Binding ◽  
Cluster Assembly ◽  
Iron Sulfur Cluster ◽  
Content Type ◽  
Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur

ABSTRACT Escherichia coli [2Fe-2S]-ferredoxin and other ISC proteins encoded by the iscRSUA-hscBA-fdx-iscX (isc) operon are responsible for the assembly of iron-sulfur clusters. It is proposed that ferredoxin (Fdx) donates electrons from its reduced [2Fe-2S] center to iron-sulfur cluster biogenesis reactions. However, the underlying mechanisms of the [2Fe-2S] cluster assembly in Fdx remain elusive. Here, we report that Fdx preferentially binds iron, but not the [2Fe-2S] cluster, under cold stress conditions (≤16°C). The iron binding in Fdx is characterized by a unique absorption peak at 320 nm based on UV-visible spectroscopy. In addition, the iron-binding form of Fdx could be converted to the [2Fe-2S] cluster-bound form after transferring cold-stressed cells to normal cultivation temperatures above 25°C. In vitro experiments also revealed that Fdx could utilize bound iron to assemble the [2Fe-2S] cluster by itself. Furthermore, inactivation of the genes encoding IscS, IscU, and IscA did not limit [2Fe-2S] cluster assembly in Fdx, which was also observed by inactivating the isc or suf operon, indicating that iron-sulfur cluster biogenesis in Fdx arose from a unique pathway in E. coli. Our results suggest that the intracellular assembly of [2Fe-2S] clusters in Fdx is susceptible to environmental temperatures. The iron binding form of Fdx (Fe-Fdx) is a precursor during its maturation to a cluster binding form ([2Fe-2S]-Fdx), and reassembly of the [2Fe-2S] clusters during temperature increases is not strictly reliant on other specific iron donors and scaffold proteins within the Isc or Suf system. IMPORTANCE Fdx is an electron carrier that is required for the maturation of many other iron-sulfur proteins. Its function strictly depends on its [2Fe-2S] center that bonds with the cysteinyl S atoms of four cysteine residues within Fdx. However, the assembly mechanism of the [2Fe-2S] clusters in Fdx remains controversial. This study reports that Fdx fails to form its [2Fe-2S] cluster under cold stress conditions but instead binds a single Fe atom at the cluster binding site. Moreover, when temperatures increase, Fdx can assemble clusters by itself from its iron-only binding form in E. coli cells. The possibility remains that Fdx can effectively accept clusters from multiple sources. Nevertheless, our results suggest that Fdx has a strong iron binding activity that contributes to the assembly of its own [2Fe-2S] cluster and that Fdx acts as a temperature sensor to regulate Isc system-mediated iron-sulfur cluster biogenesis.

scholarly journals Functional Studies of [FeFe] Hydrogenase Maturation in an Escherichia coli Biosynthetic System

2006 ◽  
Vol 188 (6) ◽  
pp. 2163-2172 ◽  
Author(s):  
Paul W. King ◽  
Matthew C. Posewitz ◽  
Maria L. Ghirardi ◽  
Michael Seibert
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Expression System ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
E Coli ◽  
Functional Studies ◽  
Hydrogenase Maturation ◽  
Iron Sulfur ◽  
And Function

ABSTRACT Maturation of [FeFe] hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study [FeFe] hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 [FeFe] hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable [FeFe] hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial [FeFe] hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex [FeFe] hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active [FeFe] hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic [FeFe] hydrogenases.

The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1421-1431 ◽  
Author(s):  
Patrice Bruscella ◽  
Laure Cassagnaud ◽  
Jeanine Ratouchniak ◽  
Gaël Brasseur ◽  
Elisabeth Lojou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Acidithiobacillus Ferrooxidans ◽  
Biochemical Properties ◽  
Gene Encoding ◽  
Iron Sulfur Cluster ◽  
E Coli ◽  
Iron Sulfur ◽  
Sulfur Protein ◽  
Tat System ◽  
Micro Organisms

The gene encoding a putative high-potential iron–sulfur protein (HiPIP) from the strictly acidophilic and chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 33020 has been cloned and sequenced. This potential HiPIP was overproduced in the periplasm of the neutrophile and heterotroph Escherichia coli. As shown by optical and EPR spectra and by electrochemical studies, the recombinant protein has all the biochemical properties of a HiPIP, indicating that the iron–sulfur cluster was correctly inserted. Translocation of this protein in the periplasm of E. coli was not detected in a ΔtatC mutant, indicating that it is dependent on the Tat system. The genetic organization of the iro locus in strains ATCC 23270 and ATCC 33020 is different from that found in strains Fe-1 and BRGM. Indeed, in A. ferrooxidans ATCC 33020 and ATCC 23270 (the type strain), iro was not located downstream from purA but was instead downstream from petC2, encoding cytochrome c 1 from the second A. ferrooxidans cytochrome bc 1 complex. These findings underline the genotypic heterogeneity within the A. ferrooxidans species. The results suggest that Iro transfers electrons from a cytochrome bc 1 complex to a terminal oxidase, as proposed for the HiPIP in photosynthetic bacteria.

scholarly journals The mitochondrial iron exporter genes MMT1 and MMT2 in yeast are transcriptionally regulated by Aft1 and Yap1

2020 ◽  
Vol 295 (6) ◽  
pp. 1716-1726 ◽  
Author(s):  
Liangtao Li ◽  
Sophie Bertram ◽  
Jerry Kaplan ◽  
Xuan Jia ◽  
Diane M. Ward
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Promoter Region ◽  
Iron Transport ◽  
Mitochondrial Proteins ◽  
Upstream Region ◽  
Iron Sulfur Cluster ◽  
High Affinity ◽  
Iron Sulfur ◽  
Mitochondrial Iron

Budding yeast (Saccharomyces cerevisiae) responds to low cytosolic iron by up-regulating the expression of iron import genes; iron import can reflect iron transport into the cytosol or mitochondria. Mmt1 and Mmt2 are nuclearly encoded mitochondrial proteins that export iron from the mitochondria into the cytosol. Here we report that MMT1 and MMT2 expression is transcriptionally regulated by two pathways: the low-iron-sensing transcription factor Aft1 and the oxidant-sensing transcription factor Yap1. We determined that MMT1 and MMT2 expression is increased under low-iron conditions and decreased when mitochondrial iron import is increased through overexpression of the high-affinity mitochondrial iron importer Mrs3. Moreover, loss of iron-sulfur cluster synthesis induced expression of MMT1 and MMT2. We show that exposure to the oxidant H2O2 induced MMT1 expression but not MMT2 expression and identified the transcription factor Yap1 as being involved in oxidant-mediated MMT1 expression. We defined Aft1- and Yap1-dependent transcriptional sites in the MMT1 promoter that are necessary for low-iron- or oxidant-mediated MMT1 expression. We also found that the MMT2 promoter contains domains that are important for regulating its expression under low-iron conditions, including an upstream region that appears to partially repress expression under low-iron conditions. Our findings reveal that MMT1 and MMT2 are induced under low-iron conditions and that the low-iron regulator Aft1 is required for this induction. We further uncover an Aft1-binding site in the MMT1 promoter sufficient for inducing MMT1 transcription and identify an MMT2 promoter region required for low iron induction.

scholarly journals AVibrio choleraeBolA-Like Protein Is Required for Proper Cell Shape and Cell Envelope Integrity

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Aurore Fleurie ◽  
Abdelrahim Zoued ◽  
Laura Alvarez ◽  
Kelly M. Hines ◽  
Felipe Cava ◽  
...  
Keyword(s):  
Vibrio Cholerae ◽  
Cell Morphology ◽  
Cell Shape ◽  
Cell Envelope ◽  
Intestinal Colonization ◽  
Iron Sulfur Cluster ◽  
Content Type ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Morphological Defects

ABSTRACTBolA family proteins are conserved in Gram-negative bacteria and many eukaryotes. While diverse cellular phenotypes have been linked to this protein family, the molecular pathways through which these proteins mediate their effects are not well described. Here, we investigated the roles of BolA family proteins inVibrio cholerae, the cholera pathogen. LikeEscherichia coli,V. choleraeencodes two BolA proteins, BolA and IbaG. However, in marked contrast toE. coli, wherebolAis linked to cell shape andibaGis not, inV. cholerae,bolAmutants lack morphological defects, whereasibaGproved critical for the generation and/or maintenance of the pathogen’s morphology. Notably, the bizarre-shaped, multipolar, elongated, and wide cells that predominated in exponential-phase ΔibaGV. choleraecultures were not observed in stationary-phase cultures. TheV. choleraeΔibaGmutant exhibited increased sensitivity to cell envelope stressors, including cell wall-acting antibiotics and bile, and was defective in intestinal colonization. ΔibaGV. choleraehad reduced peptidoglycan and lipid II and altered outer membrane lipids, likely contributing to the mutant’s morphological defects and sensitivity to envelope stressors. Transposon insertion sequencing analysis ofibaG’s genetic interactions suggested thatibaGis involved in several processes involved in the generation and homeostasis of the cell envelope. Furthermore, copurification studies revealed that IbaG interacts with proteins containing iron-sulfur clusters or involved in their assembly. Collectively, our findings suggest thatV. choleraeIbaG controls cell morphology and cell envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins.IMPORTANCEBolA-like proteins are conserved across prokaryotes and eukaryotes. These proteins have been linked to a variety of phenotypes, but the pathways and mechanisms through which they act have not been extensively characterized. Here, we unraveled the role of the BolA-like protein IbaG in the cholera pathogenVibrio cholerae. The absence of IbaG was associated with dramatic changes in cell morphology, sensitivity to envelope stressors, and intestinal colonization defects. IbaG was found to be required for biogenesis of several components of theV. choleraecell envelope and to interact with numerous iron-sulfur cluster-containing proteins and factors involved in their assembly. Thus, our findings suggest that IbaG governsV. choleraecell shape and cell envelope homeostasis through its effects on iron-sulfur proteins and associated pathways. The diversity of processes involving iron-sulfur-containing proteins is likely a factor underlying the range of phenotypes associated with BolA family proteins.

scholarly journals Using a Chemical Genetic Screen to Enhance Our Understanding of the Antimicrobial Properties of Gallium against Escherichia coli

Genes ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. 34 ◽  
Author(s):  
Natalie Gugala ◽  
Kate Chatfield-Reed ◽  
Raymond J. Turner ◽  
Gordon Chua
Keyword(s):  
Escherichia Coli ◽  
Cell Envelope ◽  
Antimicrobial Properties ◽  
Iron Sulfur Cluster ◽  
Nucleotide Biosynthesis ◽  
E Coli ◽  
Chemical Genetic ◽  
Iron Sulfur ◽  
Insight Into ◽  
Mutant Collection

The diagnostic and therapeutic agent gallium offers multiple clinical and commercial uses including the treatment of cancer and the localization of tumors, among others. Further, this metal has been proven to be an effective antimicrobial agent against a number of microbes. Despite the latter, the fundamental mechanisms of gallium action have yet to be fully identified and understood. To further the development of this antimicrobial, it is imperative that we understand the mechanisms by which gallium interacts with cells. As a result, we screened the Escherichia coli Keio mutant collection as a means of identifying the genes that are implicated in prolonged gallium toxicity or resistance and mapped their biological processes to their respective cellular system. We discovered that the deletion of genes functioning in response to oxidative stress, DNA or iron–sulfur cluster repair, and nucleotide biosynthesis were sensitive to gallium, while Ga resistance comprised of genes involved in iron/siderophore import, amino acid biosynthesis and cell envelope maintenance. Altogether, our explanations of these findings offer further insight into the mechanisms of gallium toxicity and resistance in E. coli.

Sign in / Sign up

Export Citation Format

Share Document