Heterologous expression of Dehalobacter spp. respiratory reductive dehalogenases in Escherichia coli

Reductive dehalogenases (RDases) are a family of redox enzymes that are required for anaerobic organohalide respiration, a microbial process that is useful in bioremediation. Structural and mechanistic studies of these enzymes have been greatly impeded due to challenges in RDase heterologous expression, potentially because of their cobamide-dependence. There have been a few successful attempts at RDase production in unconventional heterologous hosts, but a robust method has yet to be developed. Here we outline a novel respiratory RDase expression system using Escherichia coli . The overexpression of E. coli ’s cobamide transport system, btu , and anaerobic expression conditions were found to be essential for production of active RDases from Dehalobacter - an obligate organohalide respiring bacterium. The expression system was validated on six enzymes with amino acid sequence identities as low as 28%. Dehalogenation activity was verified for each RDase by assaying cell-free extracts of small-scale expression cultures on various chlorinated substrates including chloroalkanes, chloroethenes, and hexachlorocyclohexanes. Two RDases, TmrA from Dehalobacter sp. UNSWDHB and HchA from Dehalobacter sp. HCH1, were purified by nickel affinity chromatography. Incorporation of the cobamide and iron-sulfur cluster cofactors was verified; though, the precise cobalamin incorporation could not be determined due to variance between methodologies, and the specific activity of TmrA was consistent with that of the native enzyme. The heterologous expression of respiratory RDases, particularly from obligate organohalide respiring bacteria, has been extremely challenging and unreliable. Here we present a relatively straightforward E. coli expression system that has performed well for a variety of Dehalobacter spp. RDases. IMPORTANCE Understanding microbial reductive dehalogenation is important to refine the global halogen cycle and to improve bioremediation of halogenated contaminants; however, studies of the family of enzymes responsible are limited. Characterization of reductive dehalogenase enzymes has largely eluded researchers due to the lack of a reliable and high-yielding production method. We are presenting an approach to express reductive dehalogenase enzymes from Dehalobacter , a key group of organisms used in bioremediation, in E. coli . This expression system will propel the study of reductive dehalogenases by facilitating their production and isolation, allowing researchers to pursue more in-depth questions about the activity and structure of these enzymes. This platform will also provide a starting point to improve the expression of reductive dehalogenases from many other organisms.

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

Journal of Bacteriology ◽

10.1128/jb.188.6.2163-2172.2006 ◽

2006 ◽

Vol 188 (6) ◽

pp. 2163-2172 ◽

Cited By ~ 212

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.

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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 ◽

10.1099/mic.0.27476-0 ◽

2005 ◽

Vol 151 (5) ◽

pp. 1421-1431 ◽

Cited By ~ 34

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.

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

Genes ◽

10.3390/genes10010034 ◽

2019 ◽

Vol 10 (1) ◽

pp. 34 ◽

Cited By ~ 4

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00867-17 ◽

2017 ◽

Vol 83 (16) ◽

Cited By ~ 21

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.

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

Journal of Bacteriology ◽

10.1128/jb.00496-19 ◽

2019 ◽

Vol 202 (3) ◽

Cited By ~ 2

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.

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Reactivity of nitric oxide with the [4Fe–4S] cluster of dihydroxyacid dehydratase from Escherichia coli

Biochemical Journal ◽

10.1042/bj20081423 ◽

2009 ◽

Vol 417 (3) ◽

pp. 783-789 ◽

Cited By ~ 49

Author(s):

Xuewu Duan ◽

Juanjuan Yang ◽

Binbin Ren ◽

Guoqiang Tan ◽

Huangen Ding

Keyword(s):

Nitric Oxide ◽

Escherichia Coli ◽

Enzyme Activity ◽

Anaerobic Conditions ◽

Iron Sulfur Clusters ◽

E Coli ◽

Endonuclease Iii ◽

Iron Sulfur ◽

Aerobic And Anaerobic ◽

Aerobic And Anaerobic Conditions

Although the NO (nitric oxide)-mediated modification of iron–sulfur proteins has been well-documented in bacteria and mammalian cells, specific reactivity of NO with iron–sulfur proteins still remains elusive. In the present study, we report the first kinetic characterization of the reaction between NO and iron–sulfur clusters in protein using the Escherichia coli IlvD (dihydroxyacid dehydratase) [4Fe–4S] cluster as an example. Combining a sensitive NO electrode with EPR (electron paramagnetic resonance) spectroscopy and an enzyme activity assay, we demonstrate that NO is rapidly consumed by the IlvD [4Fe–4S] cluster with the concomitant formation of the IlvD-bound DNIC (dinitrosyl–iron complex) and inactivation of the enzyme activity under anaerobic conditions. The rate constant for the initial reaction between NO and the IlvD [4Fe–4S] cluster is estimated to be (7.0±2.0)×106 M−2·s−1 at 25 °C, which is approx. 2–3 times faster than that of the NO autoxidation by O2 in aqueous solution. Addition of GSH failed to prevent the NO-mediated modification of the IlvD [4Fe–4S] cluster regardless of the presence of O2 in the medium, further suggesting that NO is more reactive with the IlvD [4Fe–4S] cluster than with GSH or O2. Purified aconitase B [4Fe–4S] cluster from E. coli has an almost identical NO reactivity as the IlvD [4Fe–4S] cluster. However, the reaction between NO and the endonuclease III [4Fe–4S] cluster is relatively slow, apparently because the [4Fe–4S] cluster in endonuclease III is less accessible to solvent than those in IlvD and aconitase B. When E. coli cells containing recombinant IlvD, aconitase B or endonuclease III are exposed to NO using the Silastic tubing NO delivery system under aerobic and anaerobic conditions, the [4Fe–4S] clusters in IlvD and aconitase B, but not in endonuclease III, are efficiently modified forming the protein-bound DNICs, confirming that NO has a higher reactivity with the [4Fe–4S] clusters in IlvD and aconitase B than with O2 or GSH. The results suggest that the iron–sulfur clusters in proteins such as IlvD and aconitase B may constitute the primary targets of the NO cytotoxicity under both aerobic and anaerobic conditions.

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Contribution of Cysteine Desulfurase (NifS Protein) to the Biotin Synthase Reaction of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.182.10.2879-2885.2000 ◽

2000 ◽

Vol 182 (10) ◽

pp. 2879-2885 ◽

Cited By ~ 25

Author(s):

Tatsuya Kiyasu ◽

Akira Asakura ◽

Yoshie Nagahashi ◽

Tatsuo Hoshino

Keyword(s):

Escherichia Coli ◽

Reaction System ◽

Cysteine Desulfurase ◽

Biotin Synthase ◽

Iron Sulfur Cluster ◽

Free System ◽

E Coli ◽

Iron Sulfur

ABSTRACT The contribution of cysteine desulfurase, the NifS protein ofKlebsiella pneumoniae and the IscS protein ofEscherichia coli, to the biotin synthase reaction was investigated in in vitro and in vivo reaction systems with E. coli. When the nifS and nifU genes ofK. pneumoniae were coexpressed in E. coli, NifS and NifU proteins in complex (NifU/S complex) and NifU monomer forms were observed. Both the NifU/S complex and the NifU monomer stimulated the biotin synthase reaction in the presence of l-cysteine in an in vitro reaction system. The NifU/S complex enhanced the production of biotin from dethiobiotin by the cells growing in an in vivo reaction system. Moreover, the IscS protein of E. colistimulated the biotin synthase reaction in the presence ofl-cysteine in the cell-free system. These results strongly suggest that cysteine desulfurase participates in the biotin synthase reaction, probably by supplying sulfur to the iron-sulfur cluster of biotin synthase.

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Cadmium Toxicity in Glutathione Mutants of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00272-08 ◽

2008 ◽

Vol 190 (15) ◽

pp. 5439-5454 ◽

Cited By ~ 79

Author(s):

Kerstin Helbig ◽

Cornelia Grosse ◽

Dietrich H. Nies

Keyword(s):

Escherichia Coli ◽

Cadmium Toxicity ◽

Complex Compounds ◽

Wild Type ◽

Cellular Functions ◽

Iron Sulfur Cluster ◽

E Coli ◽

Reverse Transcription Pcr ◽

Iron Sulfur ◽

Damage Repair

ABSTRACTThe higher affinity of Cd2+for sulfur compounds than for nitrogen and oxygen led to the theoretical consideration that cadmium toxicity should result mainly from the binding of Cd2+to sulfide, thiol groups, and sulfur-rich complex compounds rather than from Cd2+replacement of transition-metal cations from nitrogen- or oxygen-rich biological compounds. This hypothesis was tested by usingEscherichia colifor a global transcriptome analysis of cells synthesizing glutathione (GSH; wild type), γ-glutamylcysteine (ΔgshBmutant), or neither of the two cellular thiols (ΔgshAmutant). The resulting data, some of which were validated by quantitative reverse transcription-PCR, were sorted using the KEGG (Kyoto Encyclopedia of Genes and Genomes) orthology system, which groups genes hierarchically with respect to the cellular functions of their respective products. The main difference among the three strains concerned tryptophan biosynthesis, which was up-regulated in wild-type cells upon cadmium shock and strongly up-regulated in ΔgshAcells but repressed in ΔgshBcells containing γ-glutamylcysteine instead of GSH. Overall, however, all threeE. colistrains responded to cadmium shock similarly, with the up-regulation of genes involved in protein, disulfide bond, and oxidative damage repair; cysteine and iron-sulfur cluster biosynthesis; the production of proteins containing sensitive iron-sulfur clusters; the storage of iron; and the detoxification of Cd2+by efflux. General energy conservation pathways and iron uptake were down-regulated. These findings indicated that the toxic action of Cd2+indeed results from the binding of the metal cation to sulfur, lending support to the hypothesis tested.

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Soluble Expression and Characterization of Biologically Active Bacillus anthracis Protective Antigen in Escherichia coli

Molecular Biology International ◽

10.1155/2016/4732791 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11 ◽

Cited By ~ 1

Author(s):

Nagendra Suryanarayana ◽

Vanlalhmuaka ◽

Bharti Mankere ◽

Monika Verma ◽

Kulanthaivel Thavachelvam ◽

...

Keyword(s):

Escherichia Coli ◽

Bacillus Anthracis ◽

Protective Antigen ◽

Expression System ◽

Lethal Factor ◽

Biologically Active ◽

Expression Vectors ◽

Soluble Form ◽

Small Scale ◽

E Coli

Bacillus anthracis secretory protein protective antigen (PA) is primary candidate for subunit vaccine against anthrax. Attempts to obtain large quantity of PA from Escherichia coli expression system often result in the formation of insoluble inclusion bodies. Therefore, it is always better to produce recombinant proteins in a soluble form. In the present study, we have obtained biologically active recombinant PA in small scale E. coli shake culture system using three different expression constructs. The PA gene was cloned in expression vectors bearing trc, T5, and T7 promoters and transformed into their respective E. coli hosts. The growth conditions were optimized to obtain maximum expression of PA in soluble form. The expression construct PA-pET32c in DE3-pLysS E. coli host resulted in a maximum production of soluble PA (15 mg L−1) compared to other combinations. Purified PA was subjected to trypsin digestion and binding assay with lethal factor to confirm the protein’s functionality. Biological activity was confirmed by cytotoxicity assay on J774.1 cells. Balb/c mice were immunized with PA and the immunogenicity was tested by ELISA and toxin neutralization assay. This study highlights the expression of soluble and biologically active recombinant PA in larger quantity using simpler E. coli production platform.

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