scholarly journals Recapitulating the frataxin activation mechanism in an engineered bacterial cysteine desulfurase supports the architectural switch model

2020 ◽  
Author(s):  
Shachin Patra ◽  
Cheng-Wei Lin ◽  
Manas K. Ghosh ◽  
Steven M. Havens ◽  
Seth A. Cory ◽  
...  
Keyword(s):  
Life Forms ◽  
Activation Mechanism ◽  
Cysteine Desulfurase ◽  
Biochemical Processes ◽  
E Coli ◽  
A Subunit ◽  
Dimeric Form ◽  
Quaternary Structures ◽  
Low Activity

ABSTRACTIron-sulfur (Fe-S) clusters have a key role in many biochemical processes and are essential for most life forms. Despite recent mechanistic advances in understanding the Fe-S cluster biosynthetic pathway, critical questions remain unresolved. Although human NFS1 and E. coli IscS share ∼60% sequence identity, NFS1 exhibits low activity and requires activation by the Friedreich’s ataxia protein frataxin (FXN) for in vivo function. Surprisingly, structures of the human complex reveal three distinct quaternary structures with one form exhibiting the same subunit interactions as IscS. An architectural switch model has been proposed in which evolutionarily lost interactions between NFS1 subunits results in the formation of low-activity architectures; FXN binding compensates for these lost interactions and facilitates a subunit rearrangement to activate the complex. Here, we used a structure and evolution-guided approach to identify three conserved residues proposed to weaken interactions between NFS1 subunits and transplanted these amino acids into IscS. Compared to native IscS, the engineered variant had a 4000-fold weaker dimer interface and diminished activity that correlated with the absence of the second catalytic subunit. Remarkably, the addition of the FXN homolog to the engineered variant stimulated the decay of the Cys-quinonoid pyridoxal 5’-phosphate intermediate, shifted IscS from the monomeric to dimeric form, and increased the cysteine desulfurase activity, reproducing results from the human system and supporting the architectural switch model. Overall, these studies indicate a weakening of the homodimeric interface was a key development during the evolution of the eukaryotic system and provide new insights into the role of FXN.

scholarly journals Cardiolipin is an Optimal Phospholipid for the Assembly, Stability, and Proper Functionality of the Dimeric Form of NhaA Na+/H+ Antiporter

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Abraham Rimon ◽  
Ramakanta Mondal ◽  
Assaf Friedler ◽  
Etana Padan
Keyword(s):  
Binding Site ◽  
The Other ◽  
Phosphatidyl Glycerol ◽  
Dimer Interface ◽  
Future Studies ◽  
E Coli ◽  
Lipid Interactions ◽  
Dimeric Form

AbstractCardiolipin (CL) was shown to bound to the dimer interface of NhaA Na+/H+ antiporter. Here, we explore the cardiolipin-NhaA interaction both in vitro and in vivo. Using a novel and straightforward in-vitro assay in which n-dodecyl β-D maltoside (DDM) detergent is used to delipidate the dimer interface and to split the dimers into monomers; the monomers are subsequently exposed to cardiolipin or the other E. coli phospholipids. Most efficient reconstitution of dimers is observed by cardiolipin. This assay is likely to be applicable to future studies of protein–lipid interactions. In-vivo experiments further reveal that cardiolipin is necessary for NhaA survival. Although less efficient phosphatidyl-glycerol (PG) can also reconstitute NhaA monomers to dimers. We also identify a putative cardiolipin binding site. Our observations may contribute to drug design, as human NhaA homologues, which are involved in severe pathologies, might also require specific phospholipids.

scholarly journals Convergent In Vivo and In Vitro Selection of Ceftazidime Resistance Mutations at Position 167 of CTX-M-3 β-Lactamase in Hypermutable Escherichia coli Strains

2008 ◽  
Vol 52 (4) ◽  
pp. 1297-1301 ◽  
Author(s):  
Marina N. Stepanova ◽  
Maxim Pimkin ◽  
Anatoly A. Nikulin ◽  
Varvara K. Kozyreva ◽  
Elena D. Agapova ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Typing ◽  
In Vitro Selection ◽  
Resistance Mutations ◽  
Natural Evolution ◽  
E Coli ◽  
Low Activity ◽  
Selection Of

ABSTRACT We report on a novel CTX-M extended-spectrum β-lactamase (ESBL), designated CTX-M-42, with enhanced activity toward ceftazidime. CTX-M-42 was identified in a hypermutable Escherichia coli nosocomial isolate (isolate Irk2320) and is a Pro167Thr amino acid substitution variant of CTX-M-3. By molecular typing of ESBL-producing E. coli strains previously isolated in the same hospital ward, we were able to identify a putative progenitor (strain Irk1224) of Irk2320, which had a mutator phenotype and harbored the CTX-M-3 β-lactamase. To reproduce the natural evolution of CTX-M-3, we selected for ceftazidime resistance mutations in bla CTX-M-3 gene in vitro both in clinical isolate Irk1224 and in laboratory-derived hypermutable (mutD5) strain GM2995. These experiments yielded CTX-M-3Pro167Ser and CTX-M-3Asn136Lys mutants which conferred higher levels of resistance to ceftazidime than to cefotaxime. CTX-M-3Asn136Lys had a level of low activity toward ampicillin, which may explain its absence from clinical isolates. We conclude that the selection of CTX-M-42 could have occurred in vivo following treatment with ceftazidime and was likely facilitated by the hypermutable background.

scholarly journals In Vivo Transduction with Shiga Toxin 1-Encoding Phage

1998 ◽  
Vol 66 (9) ◽  
pp. 4496-4498 ◽  
Author(s):  
David W. K. Acheson ◽  
Joachim Reidl ◽  
Xiaoping Zhang ◽  
Gerald T. Keusch ◽  
John J. Mekalanos ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gastrointestinal Tract ◽  
Shiga Toxin ◽  
Recipient Strain ◽  
Gene Encoding ◽  
E Coli ◽  
A Subunit

ABSTRACT To facilitate the study of intestinal transmission of the Shiga toxin 1 (Stx1)-converting phage H-19B, Tn10d-blamutagenesis of an Escherichia coli H-19B lysogen was undertaken. Two mutants containing insertions in the gene encoding the A subunit of Stx1 were isolated. The resultant ampicillin-resistantE. coli strains lysogenic for these phages produced infectious H-19B particles but not active toxin. These lysogens were capable of transducing an E. coli recipient strain in the murine gastrointestinal tract, thereby demonstrating that lysogens of Shiga toxin-converting phages give rise to infectious virions within the host gastrointestinal tract.

scholarly journals Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis

2015 ◽  
Vol 197 (11) ◽  
pp. 1952-1962 ◽  
Author(s):  
Katherine A. Black ◽  
Patricia C. Dos Santos
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cysteine Desulfurase ◽  
Content Type ◽  
E Coli ◽  
Wobble Position ◽  
Lysine Trna ◽  
Domains Of Life

ABSTRACTThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s2U inEscherichia colirequires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). TheE. coliMnmA adenylates and subsequently thiolates tRNA to form the s2U modification.Bacillus subtilislacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene,yrvO, directly adjacent tomnmA. The genomic synteny ofyrvOandmnmAcombined with the absence of the Tus proteins indicated a potential functionality of these proteins in s2U formation. Here, we provide evidence that theB. subtilisYrvO and MnmA are sufficient for s2U biosynthesis. A conditionalB. subtilisknockout strain showed that s2U abundance correlates with MnmA expression, andin vivocomplementation studies inE. coliIscS- or MnmA-deficient strains revealed the competency of these proteins in s2U biosynthesis.In vitroexperiments demonstrated s2U formation by YrvO and MnmA, and kinetic analysis established a partnership between theB. subtilisproteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype ofE. coliΔiscSand ΔmnmAstrains associated with s2U depletion is recovered byB. subtilis yrvOandmnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s2U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA.IMPORTANCEThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s2U inEscherichia colirequires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE).Bacillus subtilisand most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, themnmAcoding sequence is located adjacent toyrvO, encoding a cysteine desulfurase. In this work, we provide evidence that theB. subtilisYrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s2U biosynthesis in a pathway independent of the one used inE. coli.

scholarly journals Awakening a latent carbon fixation cycle in Escherichia coli

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Ari Satanowski ◽  
Beau Dronsella ◽  
Elad Noor ◽  
Bastian Vögeli ◽  
Hai He ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Carbon Fixation ◽  
Pentose Phosphate ◽  
Biochemical Processes ◽  
E Coli ◽  
Phosphogluconate Dehydrogenase ◽  
Thermodynamic Driving Force ◽  
Natural Carbon ◽  
Pentose Sugars

AbstractCarbon fixation is one of the most important biochemical processes. Most natural carbon fixation pathways are thought to have emerged from enzymes that originally performed other metabolic tasks. Can we recreate the emergence of a carbon fixation pathway in a heterotrophic host by recruiting only endogenous enzymes? In this study, we address this question by systematically analyzing possible carbon fixation pathways composed only of Escherichia coli native enzymes. We identify the GED (Gnd–Entner–Doudoroff) cycle as the simplest pathway that can operate with high thermodynamic driving force. This autocatalytic route is based on reductive carboxylation of ribulose 5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (Gnd), followed by reactions of the Entner–Doudoroff pathway, gluconeogenesis, and the pentose phosphate pathway. We demonstrate the in vivo feasibility of this new-to-nature pathway by constructing E. coli gene deletion strains whose growth on pentose sugars depends on the GED shunt, a linear variant of the GED cycle which does not require the regeneration of Ru5P. Several metabolic adaptations, most importantly the increased production of NADPH, assist in establishing sufficiently high flux to sustain this growth. Our study exemplifies a trajectory for the emergence of carbon fixation in a heterotrophic organism and demonstrates a synthetic pathway of biotechnological interest.

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

2000 ◽  
Vol 182 (10) ◽  
pp. 2879-2885 ◽  
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.

scholarly journals Awakening a latent carbon fixation cycle in Escherichia coli

2020 ◽  
Author(s):  
Ari Satanowski ◽  
Beau Dronsella ◽  
Elad Noor ◽  
Bastian Vögeli ◽  
Hai He ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Carbon Fixation ◽  
Pentose Phosphate ◽  
Biochemical Processes ◽  
E Coli ◽  
Phosphogluconate Dehydrogenase ◽  
Thermodynamic Driving Force ◽  
Natural Carbon ◽  
Pentose Sugars

AbstractCarbon fixation is one of the most important biochemical processes. Most natural carbon fixation pathways are thought to have emerged from enzymes that originally performed other metabolic tasks. Can we recreate the emergence of a carbon fixation pathway in a heterotrophic host by recruiting only endogenous enzymes? In this study, we address this question by systematically analyzing possible carbon fixation pathways composed only of Escherichia coli native enzymes. We identify the GED (Gnd-Entner-Doudoroff) cycle as the simplest pathway that can operate with high thermodynamic driving force. This autocatalytic route is based on reductive carboxylation of ribulose 5-phosphate (Ru5P) by 6-phosphogluconate dehydrogenase (Gnd), followed by reactions of the Entner-Doudoroff pathway, gluconeogenesis, and the pentose phosphate pathway. We demonstrate the in vivo feasibility of this new-to-nature pathway by constructing E. coli gene deletion strains whose growth on pentose sugars depends on the GED shunt, a linear variant of the GED cycle which does not require the regeneration of Ru5P. Several metabolic adaptations, most importantly the increased production of NADPH, assist in establishing sufficiently high flux to sustain this growth. Our study exemplifies a trajectory for the emergence of carbon fixation in a heterotrophic organism and demonstrates a synthetic pathway of biotechnological interest.

scholarly journals Molecular analysis of lambda bio transducing phage produced by oxolinic acid-induced illegitimate recombination in vivo.

Genetics ◽  
1995 ◽  
Vol 140 (3) ◽  
pp. 889-896 ◽  
Author(s):  
H Shimizu ◽  
H Yamaguchi ◽  
H Ikeda
Keyword(s):  
Dna Gyrase ◽  
Oxolinic Acid ◽  
Illegitimate Recombination ◽  
Prophage Induction ◽  
Minority Class ◽  
E Coli ◽  
Gyrase A ◽  
A Subunit ◽  
Dependent Mechanism

Abstract To study the mechanism of DNA gyrase-mediated illegitimate recombination in Escherichia coli, we examined the formation of lambda Spi- phage during prophage induction. The frequency of Spi- phage was two to three orders of magnitude higher in the presence of oxolinic acid, an inhibitor of DNA gyrase A subunit, than in the absence of the drug, while it was very low in nalAr bacteria with the drug. RecA function is not required for the formation of these phages, indicating that this enhancement is not caused by the expression of SOS-controlled genes. Analyses of att region and recombination junctions of Spi- phages revealed that they have essentially the same structures as lambda bio transducing phages but are classified into two groups with respect to recombination sites. In the majority class of the transducing phages, there were not more than 3-bp homologies between the parental E. coli bio and lambda recombination sites. In the minority class of the transducing phages, on the other hand, 9-10-bp homologies were found between the parental recombination sites. These results suggested that oxolinic acid-induced illegitimate recombination takes place by two variants of a DNA gyrase-dependent mechanism.

scholarly journals The bacterial actin-like cell division protein FtsA forms curved antiparallel double filaments upon binding of FtsN

2021 ◽  
Author(s):  
Tim Nierhaus ◽  
Stephen H McLaughlin ◽  
Frank Bürmann ◽  
Danguole Kureisaite-Ciziene ◽  
Sarah Maslen ◽  
...  
Keyword(s):  
Activation Mechanism ◽  
Lipid Monolayers ◽  
Site Specific ◽  
Division Plane ◽  
Curvature Sensing ◽  
E Coli ◽  
Z Ring ◽  
Cell Division Protein ◽  
Concentrate Cell

Cell growth and division of walled bacteria depend on the synthesis and remodelling of peptidoglycan (PG). These activities are carried out by two multiprotein complexes, the elongasome and the divisome during cell elongation and division, respectively. Filaments of tubulin-like FtsZ form the cytoplasmic scaffold for divisome assembly, the Z-ring. In E. coli, the actin homologue FtsA anchors the Z-ring to the membrane and recruits downstream divisome components, including bitopic FtsN. FtsN is recruited late and activates the periplasmic PG synthase FtsWI. To start unravelling the activation mechanism involving FtsA and FtsN, we showed that E. coli FtsA forms antiparallel double filaments on lipid monolayers when also binding FtsN's cytoplasmic tail, and that Vibrio maritimus FtsA crystallised as an equivalent double filament. We structurally located the FtsA-FtsN interaction site in FtsA's IA-IC interdomain cleft and confirmed FtsA double filament formation in vivo using site-specific cysteine cross-linking. FtsA-FtsN double filaments reconstituted on and in liposomes preferred negative Gaussian curvature, as was previously shown for the elongasome's actin, MreB. MreB filaments serve as curvature-sensing "rudders", orienting insertion of PG around the cell's circumference. We propose that curved antiparallel FtsA double filaments function similarly in the divisome: FtsA filaments, together with dynamic FtsZ filaments orient and concentrate cell-constricting septal PG synthesis in the division plane.

A Comparison of the in Vitro and in Vivo Thrombogenic Activity of Factor IX Concentrates Using Stasis (Wessler) and Non-Stasis Rabbit Models

1980 ◽  
Vol 44 (02) ◽  
pp. 081-086 ◽  
Author(s):  
C V Prowse ◽  
A E Williams
Keyword(s):  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Factor Ix ◽  
Coagulation System ◽  
In Vitro Tests ◽  
Clinical Use ◽  
Low Activity

SummaryThe thrombogenic effects of selected factor IX concentrates were evaluated in two rabbit models; the Wessler stasis model and a novel non-stasis model. Concentrates active in either the NAPTT or TGt50 in vitro tests of potential thrombogenicity, or both, caused thrombus formation in the Wessler technique and activation of the coagulation system in the non-stasis model. A concentrate with low activity in both in vitro tests did not have thrombogenic effects in vivo, at the chosen dose. Results in the non-stasis model suggested that the thrombogenic effects of factor IX concentrates may occur by at least two mechanisms. A concentrate prepared from platelet-rich plasma and a pyrogenic concentrate were also tested and found to have no thrombogenic effect in vivo.These studies justify the use of the NAPTT and TGt50 in vitro tests for the screening of factor IX concentrates prior to clinical use.

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