Ferric Enterochelin Transport in Yersinia enterocolitica: Molecular and Evolutionary Aspects

ABSTRACT Yersinia enterocolitica is well equipped for siderophore piracy, encompassing the utilization of siderophores such as ferrioxamine, ferrichrome, and ferrienterochelin. In this study, we report on the molecular and functional characterization of theYersinia fep-fes gene cluster orthologous to theEscherichia coli ferrienterochelin transport genes (fepA, fepDGC, and fepB) and the esterase gene fes. In vitro transcription-translation analysis identified polypeptides of 30 and 35 kDa encoded byfepC and fes, respectively. A frameshift mutation within the fepA gene led to expression of a truncated polypeptide of 40 kDa. The fepD,fepG, and fes genes of Y. enterocolitica were shown to complement corresponding E. coli mutants. Insertional mutagenesis of fepD orfes genes abrogates enterochelin-supported growth ofY. enterocolitica on iron-chelated media. In contrast toE. coli, the fep-fes gene cluster inY. enterocolitica consists solely of genes required for uptake and utilization of enterochelin (fep) and not of enterochelin synthesis genes such as entF. By Southern hybridization, fepDGC and fes sequences could be detected in Y. enterocolitica biotypes IB, IA, and II but not in biotype IV strains, Yersinia pestis, andYersinia pseudotuberculosis strains. According to sequence alignment data and the coherent structure of the Yersinia fep-fes gene cluster, we suggest early genetic divergence of ferrienterochelin uptake determinants among species of the familyEnterobacteriaceae.

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Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽

10.1099/mic.0.28753-0 ◽

2006 ◽

Vol 152 (7) ◽

pp. 2129-2135 ◽

Cited By ~ 17

Author(s):

Taku Oshima ◽

Francis Biville

Keyword(s):

Functional Characterization ◽

Anaerobic Growth ◽

Functional Identification ◽

New Members ◽

E Coli ◽

Inner Membrane Protein ◽

Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

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Prevalence of the “High-Pathogenicity Island” of Yersinia Species among Escherichia coliStrains That Are Pathogenic to Humans

Infection and Immunity ◽

10.1128/iai.66.2.480-485.1998 ◽

1998 ◽

Vol 66 (2) ◽

pp. 480-485 ◽

Cited By ~ 257

Author(s):

S. Schubert ◽

A. Rakin ◽

H. Karch ◽

E. Carniel ◽

J. Heesemann

Keyword(s):

Gene Cluster ◽

Yersinia Pseudotuberculosis ◽

Pathogenicity Island ◽

Uptake System ◽

Highly Pathogenic ◽

E Coli ◽

Yersinia Species ◽

The Family ◽

High Pathogenicity ◽

Dna Sequence Comparison

ABSTRACT The fyuA-irp gene cluster contributes to the virulence of highly pathogenic Yersinia (Yersinia pestis,Yersinia pseudotuberculosis, and Yersinia enterocolitica 1B). The cluster encodes an iron uptake system mediated by the siderophore yersiniabactin and reveals features of a pathogenicity island. Two evolutionary lineages of this “high pathogenicity island” (HPI) can be distinguished on the basis of DNA sequence comparison: a Y. pestis group and a Y. enterocolitica group. In this study we demonstrate that the HPI of the Y. pestis evolutionary group is disseminated among species of the family Enterobacteriaceae which are pathogenic to humans. It prevails in enteroaggregativeEscherichia coli and in E. coli blood culture isolates (93 and 80%, respectively), but is rarely found in enteropathogenic E. coli, enteroinvasive E. coli, and enterotoxigenic E. coli isolates. In contrast, the HPI was absent from enterohemorrhagic E. coli, Shigella, and Salmonella entericastrains investigated. Polypeptides encoded by the fyuA,irp1, and irp2 genes located on the HPI could be detected in E. coli strains pathogenic to humans. However, these E. coli strains showed a reduced sensitivity to the bacteriocin pesticin, whose uptake is mediated by the FyuA receptor. Escherichia strains do not possess thehms gene locus thought to be a part of the HPI of Y. pestis. Deletions of the fyuA-irp gene cluster affecting solely the fyuA part of the HPI were identified in 3% of the E. coli strains tested. These results suggest horizontal transfer of the HPI between Y. pestis and some pathogenic E. coli strains.

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Fluorescence-free quantification of protein/nucleic-acid binding through single-molecule kinetic locking

10.1101/2020.09.30.321232 ◽

2020 ◽

Author(s):

Martin Rieu ◽

Jessica Valle-Orero ◽

Bertrand Ducos ◽

Jean-François Allemand ◽

Vincent Croquette

Keyword(s):

Single Molecule ◽

Functional Characterization ◽

Nucleic Acid Binding ◽

Single Molecule Force Spectroscopy ◽

E Coli ◽

Natural Function ◽

Protein Nucleic Acid ◽

Free Quantification

ABSTRACTFluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a new method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein’s natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5,6,7 bases) and use it to measure the dynamical interactions of E. coli RecQ helicase with its DNA substrate.

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Single-molecule kinetic locking allows fluorescence-free quantification of protein/nucleic-acid binding

Communications Biology ◽

10.1038/s42003-021-02606-z ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Martin Rieu ◽

Jessica Valle-Orero ◽

Bertrand Ducos ◽

Jean-François Allemand ◽

Vincent Croquette

Keyword(s):

Single Molecule ◽

Functional Characterization ◽

Nucleic Acid Binding ◽

Single Molecule Force Spectroscopy ◽

E Coli ◽

Natural Function ◽

Protein Nucleic Acid ◽

Free Quantification

AbstractFluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein’s natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5, 6, 7 bases) and use it to measure the dynamical interactions of Escherichia coli/E. coli RecQ helicase with its DNA substrate.

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cfrB, cfrC, and a potential new cfr-like gene in Clostridium difficile strains recovered across Latin America

10.1101/649020 ◽

2019 ◽

Author(s):

Vanja Stojković ◽

María Fernanda Ulate ◽

Fanny Hidalgo-Villeda ◽

Emmanuel Aguilar ◽

Camilo Monge-Cascante ◽

...

Keyword(s):

Latin America ◽

Ribosomal Proteins ◽

Functional Characterization ◽

Ribosomal Subunit ◽

23S Rrna ◽

Cross Resistance ◽

Protection Factor ◽

E Coli

ABSTRACTCfr is a radical S-adenosyl-L-methionine (SAM) enzyme that confers cross-resistance to all antibiotics targeting the large ribosomal subunit through hypermethylation of nucleotide A2503 of 23S rRNA. Of the four known cfr genes known to date, cfr(B) and cfr(C) have been sporadically found in C. difficile, yet functional characterization of cfr(C) is still lacking. We identified genes for putative Cfr-like enzymes among clinical C. difficile strains from Mexico, Honduras, Costa Rica, and Chile. To confirm their identity and activity, we obtained minimum inhibitory concentrations for ribosome-targeting antibiotics, annotated whole genome sequences, and performed a functional characterization of Cfr(C). The seven representative isolates analyzed displayed different levels of resistance to PhLOPSA antibiotics in the absence of the ribosome protection factor OptrA, and mutations in genes for 23S rRNAs or the ribosomal proteins L3 and L4. cfr(B) was detected in four isolates as part of a Tn6218-like transposon or an un-described mobile genetic element. In turn, cfr(C) was found integrated into an ICE-element. One isolate harbored a putative cfr-like gene that shows only 51-58% of sequence identity to Cfr and known Cfr-like enzymes. Moreover, our in vitro assays confirmed that Cfr(C) methylates E. coli and C. difficile 23S rRNA fragments. These results indicate selection of cfr-like genes in C. difficile from Latin America, suggest that the diversity of cfr-like resistance genes is larger than anticipated, and provide the first assessment of the methylation activity of Cfr(C).

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Structural and Functional Characterization of PC2 and RNA Polymerase II-Associated Subpopulations of Metazoan Mediator

Molecular and Cellular Biology ◽

10.1128/mcb.25.6.2117-2129.2005 ◽

2005 ◽

Vol 25 (6) ◽

pp. 2117-2129 ◽

Cited By ~ 35

Author(s):

Sohail Malik ◽

Hwa Jin Baek ◽

Weizhen Wu ◽

Robert G. Roeder

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Specific Activity ◽

Functional Characterization ◽

In Vitro Transcription ◽

Dynamic Nature ◽

Pol Ii ◽

General Transcription Factors

ABSTRACT The coactivator complexes TRAP/SMCC and PC2 represent two forms of Mediator. To further understand the implications of the heterogeneity of the cellular Mediator populations for regulation of RNA polymerase II (Pol II) transcription, we used a combination of affinity and conventional chromatographic methods. Our analysis revealed a spectrum of complexes, including some containing significant proportions of Pol II. Interestingly, the subunit composition of the Pol II-associated Mediator population resembled that of PC2 more closely than that of the larger TRAP/SMCC complex. In in vitro transcription assays reconstituted from homogeneous preparations of general transcription factors, Mediator-associated Pol II displayed a greater specific activity (relative to that of standard Pol II) in activator-independent (basal) transcription in addition to the previously described effects of Mediator on activator-dependent transcription. Purified PC2 complex also stimulated basal activity under these conditions. Immobilized template assays in which activator-recruited preinitiation complexes were allowed to undergo one cycle of transcription revealed partial disruption of Mediator that resulted in a PC2-like complex being retained in the scaffold. This result implies that PC2 could originate as a result of a normal cellular process. Our results are thus consistent with a dynamic nature of the Mediator complex and further extend the functional similarities between Saccharomyces cerevisiae and metazoan Mediator complexes.

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Functional Characterization of POFUT1 Variants Associated with Colorectal Cancer

Cancers ◽

10.3390/cancers12061430 ◽

2020 ◽

Vol 12 (6) ◽

pp. 1430 ◽

Cited By ~ 1

Author(s):

Marlène Deschuyter ◽

Florian Pennarubia ◽

Emilie Pinault ◽

Sébastien Legardinier ◽

Abderrahman Maftah

Keyword(s):

Colorectal Cancer ◽

Notch Signaling ◽

Functional Characterization ◽

Missense Mutations ◽

Colorectal Tumors ◽

Protein Targets ◽

E Coli ◽

Notch Receptors

Background: Protein O-fucosyltransferase 1 (POFUT1) overexpression, which is observed in many cancers such as colorectal cancer (CRC), leads to a NOTCH signaling dysregulation associated with the tumoral process. In rare CRC cases, with no POFUT1 overexpression, seven missense mutations were found in human POFUT1. Methods: Recombinant secreted forms of human WT POFUT1 and its seven mutated counterparts were produced and purified. Their O-fucosyltransferase activities were assayed in vitro using a chemo-enzymatic approach with azido-labeled GDP-fucose as a donor substrate and NOTCH1 EGF-LD26, produced in E. coli periplasm, as a relevant acceptor substrate. Targeted mass spectrometry (MS) was carried out to quantify the O-fucosyltransferase ability of all POFUT1 proteins. Findings: MS analyses showed a significantly higher O-fucosyltransferase activity of six POFUT1 variants (R43H, Y73C, T115A, I343V, D348N, and R364W) compared to WT POFUT1. Interpretation: This study provides insights on the possible involvement of these seven missense mutations in colorectal tumors. The hyperactive forms could lead to an increased O-fucosylation of POFUT1 protein targets such as NOTCH receptors in CRC patients, thereby leading to a NOTCH signaling dysregulation. It is the first demonstration of gain-of-function mutations for this crucial glycosyltransferase, modulating NOTCH activity, as well as that of other potential glycoproteins.

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Sweet Basil Has Distinct Synthases for Eugenol Biosynthesis in Glandular Trichomes and Roots with Different Regulatory Mechanisms

International Journal of Molecular Sciences ◽

10.3390/ijms22020681 ◽

2021 ◽

Vol 22 (2) ◽

pp. 681

Author(s):

Vaishnavi Amarr Reddy ◽

Chunhong Li ◽

Kumar Nadimuthu ◽

Jessica Gambino Tjhang ◽

In-Cheol Jang ◽

...

Keyword(s):

Glandular Trichomes ◽

Functional Characterization ◽

Rna Seq ◽

Post Translational Modifications ◽

E Coli ◽

Sweet Basil ◽

Specific Regulation

Production of a volatile phenylpropene; eugenol in sweet basil is mostly associated with peltate glandular trichomes (PGTs) found aerially. Currently only one eugenol synthase (EGS), ObEGS1 which belongs to PIP family is identified from sweet basil PGTs. Reports of the presence of eugenol in roots led us to analyse other EGSs in roots. We screened for all the PIP family reductase transcripts from the RNA-Seq data. In vivo functional characterization of all the genes in E. coli showed their ability to produce eugenol and were termed as ObEGS2-8. Among all, ObEGS1 displayed highest expression in PGTs and ObEGS4 in roots. Further, eugenol was produced only in the roots of soil-grown plants, but not in roots of aseptically-grown plants. Interestingly, eugenol production could be induced in roots of aseptically-grown plants under elicitation suggesting that eugenol production might occur as a result of environmental cues in roots. The presence of ObEGS4 transcript and protein in aseptically-grown plants indicated towards post-translational modifications (PTMs) of ObEGS4. Bioinformatics analysis showed possibility of phosphorylation in ObEGS4 which was further confirmed by in vitro experiment. Our study reveals the presence of multiple eugenol synthases in sweet basil and provides new insights into their diversity and tissue specific regulation.

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Identification and Characterization of Sporulation-Dependent Promoters Upstream of the Enterotoxin Gene (cpe) of Clostridium perfringens

Journal of Bacteriology ◽

10.1128/jb.180.1.136-142.1998 ◽

1998 ◽

Vol 180 (1) ◽

pp. 136-142 ◽

Cited By ~ 103

Author(s):

Yuling Zhao ◽

Stephen B. Melville

Keyword(s):

Clostridium Perfringens ◽

Dna Sequences ◽

Promoter Region ◽

Food Poisoning ◽

In Vitro Transcription ◽

Shuttle Vectors ◽

E Coli ◽

Dna Template

ABSTRACT Three promoter sites (P1, P2, and P3) responsible for the sporulation-associated synthesis of Clostridium perfringensenterotoxin, a common cause of food poisoning in humans and animals, were identified. Nested and internal deletions of the cpepromoter region were made to narrow down the location of promoter elements. To measure the effects of the deletions on the expression ofcpe, translational fusions containing the promoter deletions were made with the gusA gene of Escherichia coli, which codes for β-glucuronidase; E. coli-C. perfringens shuttle vectors carrying the fusions were introduced into C. perfringens by electroporation. In addition, in vitro transcription assays were performed with the cpepromoter region as the DNA template for extracts made from sporulating cells. DNA sequences upstream of P1 were similar to consensus SigK-dependent promoters, while P2 and P3 were similar to consensus SigE-dependent promoters. SigE and SigK are sporulation-associated sigma factors known to be active in the mother cell compartment of sporulating cells of Bacillus subtilis, the same compartment in which enterotoxin is synthesized in C. perfringens.

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