scholarly journals Mammalian antioxidant protein complements alkylhydroperoxide reductase (ahpC) mutation in Escherichia coli

1995 ◽  
Vol 307 (2) ◽  
pp. 377-381 ◽  
Author(s):  
K Tsuji ◽  
N G Copeland ◽  
N A Jenkins ◽  
M Obinata
Keyword(s):  
Escherichia Coli ◽  
Cellular Proliferation ◽  
Sequence Similarity ◽  
Human Chromosomes ◽  
Subunit Gene ◽  
Sequence Comparisons ◽  
E Coli ◽  
Antioxidant Protein ◽  
Gene Coding ◽  
Human And Mouse

The MER5 [now called the Aop1 (antioxidant protein 1) gene] was cloned as a transiently expressed gene of murine erythroleukaemia (MEL) cell differentiation and its antisense expression inhibited differentiation of MEL cells. We found that the Aop1 gene shows significant nucleotide sequence similarity to the gene coding for the C22 subunit of Salmonella typhimurium alkylhydroperoxide reductase, which is also found in other bacteria, suggesting it functions as an antioxidant protein. Expression of the Aop1 gene product in E. coli deficient in the C22-subunit gene rescued resistance of the bacteria to alkylhydroperoxide. The human and mouse Aop1 genes are highly conserved, and they mapped to the regions syntenic between mouse and human chromosomes. Sequence comparisons with recently cloned mammalian Aop1 homologues suggest that these genes consist of a family that is responsible for regulation of cellular proliferation, differentiation and antioxidant functions.

scholarly journals Identification of a novel NADH-specific aldo-keto reductase using sequence and structural homologies

2006 ◽  
Vol 400 (1) ◽  
pp. 105-114 ◽  
Author(s):  
Eric Di Luccio ◽  
Robert A. Elling ◽  
David K. Wilson
Keyword(s):  
Escherichia Coli ◽  
Sinorhizobium Meliloti ◽  
Thermotoga Maritima ◽  
Xylose Reductase ◽  
Sulfolobus Solfataricus ◽  
Sequence Comparisons ◽  
E Coli ◽  
Fluorescence Measurements ◽  
Dual Specificity ◽  
Substrate Dependence

The AKRs (aldo-keto reductases) are a superfamily of enzymes which mainly rely on NADPH to reversibly reduce various carbonyl-containing compounds to the corresponding alcohols. A small number have been found with dual NADPH/NADH specificity, usually preferring NADPH, but none are exclusive for NADH. Crystal structures of the dual-specificity enzyme xylose reductase (AKR2B5) indicate that NAD+ is bound via a key interaction with a glutamate that is able to change conformations to accommodate the 2′-phosphate of NADP+. Sequence comparisons suggest that analogous glutamate or aspartate residues may function in other AKRs to allow NADH utilization. Based on this, nine putative enzymes with potential NADH specificity were identified and seven genes were successfully expressed and purified from Drosophila melanogaster, Escherichia coli, Schizosaccharomyces pombe, Sulfolobus solfataricus, Sinorhizobium meliloti and Thermotoga maritima. Each was assayed for co-substrate dependence with conventional AKR substrates. Three were exclusive for NADPH (AKR2E3, AKR3F2 and AKR3F3), two were dual-specific (AKR3C2 and AKR3F1) and one was specific for NADH (AKR11B2), the first such activity in an AKR. Fluorescence measurements of the seventh protein indicated that it bound both NADPH and NADH but had no activity. Mutation of the aspartate into an alanine residue or a more mobile glutamate in the NADH-specific E. coli protein converted it into an enzyme with dual specificity. These results show that the presence of this carboxylate is an indication of NADH dependence. This should allow improved prediction of co-substrate specificity and provide a basis for engineering enzymes with altered co-substrate utilization for this class of enzymes.

scholarly journals Optimization of the apolipoprotein B mRNA editing enzyme catalytic polypeptidelike-3G (APOBEC3G) gene to enhance its expression in Escherichia coli

2020 ◽  
Vol 29 (2) ◽  
pp. 120-8
Author(s):  
Rizkyana Avissa ◽  
Silvia Tri Widyaningtyas ◽  
Budiman Bela
Keyword(s):  
Escherichia Coli ◽  
Apolipoprotein B ◽  
Plasmid Vector ◽  
Nitrilotriacetic Acid ◽  
Mrna Editing ◽  
Specific Domain ◽  
Iptg Induction ◽  
E Coli ◽  
Gene Coding ◽  
Editing Enzyme

BACKGROUND Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like-3G (APOBEC3G) can abolish HIV infection by inducing lethal mutations in the HIV genome. The HIV protein virion infectivity factor (Vif) can interact with APOBEC3G protein and cause its degradation. Development of a method that can screen substances inhibiting the APOBEC3G-Vif interaction is necessary for identification of substances that potentially used in anti-HIV drug development. In order to increase expression of recombinant APOBEC3G protein that will be used in APOBEC3G-Vif interaction assay, we developed an optimized APOBEC3G gene for expression in Escherichia coli.  METHODS The gene coding APOBEC3G was codon-optimized in accordance with prokaryotic codon using DNA 2.0 software to avoid bias codons that could inhibit its expression. The APOBEC3G gene was synthesized and sub-cloned into pQE80L plasmid vector. pQE80L containing APOBEC3G was screened by polymerase chain reaction, enzyme restriction, and sequencing to verify its DNA sequence. The recombinant APOBEC3G was expressed in E. coli under isopropyl-β-D-thiogalactoside (IPTG) induction and purified by using nickel-nitrilotriacetic acid (Ni-NTA) resin.  RESULTS The synthetic gene coding APOBEC3G was successfully cloned into the pQE80L vector and could be expressed abundantly in E. coli BL21 in the presence of IPTG.  CONCLUSIONS Recombinant APOBEC3G is robustly expressed in E. coli BL21, and the APOBEC3G protein could be purified by using Ni-NTA. The molecular weight of the recombinant APOBEC3G produced is smaller than the expected value. However, the protein is predicted to be able to interact with Vif because this interaction is determined by a specific domain located on the N-terminal of APOBEC3G. 

scholarly journals Characterization of human and mouse granulocyte-macrophage-colony-stimulating factors derived from Escherichia coli

1987 ◽  
Vol 247 (1) ◽  
pp. 195-199 ◽  
Author(s):  
J L Schrimsher ◽  
K Rose ◽  
M G Simona ◽  
P Wingfield
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Natural Material ◽  
Animal Cells ◽  
Colony Stimulating Factors ◽  
E Coli ◽  
Macrophage Colony ◽  
The One ◽  
Human And Mouse

Human and mouse granulocyte-macrophage-colony-stimulating factors (hGM-CSF and mGM-CSF, respectively), isolated from Escherichia coli cells expressing the corresponding human and mouse genes, have been characterized. The observed properties of the proteins have been compared with those properties which can be deduced from the DNA sequence alone and the published properties of natural GM-CSFs. The purified E. coli-derived proteins were found to have the expected molecular masses, amino acid compositions and N- and C-terminal amino acid sequences. The finding of 70-90% unprocessed N-terminal methionine for both proteins is discussed. The four Cys residues were found to be involved in two intramolecular disulphide bonds, linking the first and third, and second and fourth Cys residues. This disulphide bond arrangement is probably the one existing in natural material, since, although not glycosylated, both E. coli-derived proteins showed biological activity (colony stimulating assay for hGM-CSF, and cell proliferation assay for mGM-CSF) comparable with that reported for the respective proteins purified from animal cells.

scholarly journals Evolution of Chi motifs in Proteobacteria

2020 ◽  
Author(s):  
Angélique Buton ◽  
Louis-Marie Bobay
Keyword(s):  
Escherichia Coli ◽  
Sequence Similarity ◽  
Bacterial Species ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Large Dataset ◽  
High Sequence Similarity ◽  
Strand Breaks ◽  
E Coli ◽  
Dna Motif

AbstractHomologous recombination is a key pathway found in nearly all bacterial taxa. The recombination complex allows bacteria to repair DNA double strand breaks but also promotes adaption through the exchange of DNA between cells. In Proteobacteria, this process is mediated by the RecBCD complex, which relies on the recognition of a DNA motif named Chi to initiate recombination. The Chi motif has been characterized in Escherichia coli and analogous sequences have been found in several other species from diverse families, suggesting that this mode of action is widespread across bacteria. However, the sequences of Chi-like motifs are known for only five bacterial species: E. coli, Haemophilus influenzae, Bacillus subtilis, Lactococcus lactis and Staphylococcus aureus. In this study we detected putative Chi motifs in a large dataset of Proteobacteria and we identified four additional motifs sharing high sequence similarity and similar properties to the Chi motif of E. coli in 85 species of Proteobacteria. Most Chi motifs were detected in Enterobacteriaceae and this motif appears well conserved in this family. However, we did not detect Chi motifs for the majority of Proteobacteria, suggesting that different motifs are used in these species. Altogether these results substantially expand our knowledge on the evolution of Chi motifs and on the recombination process in bacteria.

scholarly journals Characterization of the hca Cluster Encoding the Dioxygenolytic Pathway for Initial Catabolism of 3-Phenylpropionic Acid in Escherichia coliK-12

1998 ◽  
Vol 180 (11) ◽  
pp. 2915-2923 ◽  
Author(s):  
Eduardo Díaz ◽  
Abel Ferrández ◽  
José L. García
Keyword(s):  
Sequence Similarity ◽  
Recombinant Bacteria ◽  
Sequence Comparisons ◽  
Phenylpropionic Acid ◽  
E Coli ◽  
Catabolic Genes ◽  
Dihydrodiol Dehydrogenase ◽  
Class Iib ◽  
K 12

ABSTRACT We have identified, cloned, and sequenced the hcacluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic acid (PP) in Escherichia coli K-12. This cluster maps at min 57.5 of the chromosome and is composed of five catabolic genes arranged as a putative operon (hcaA1A2CBD) and two additional genes transcribed in the opposite direction that encode a potential permease (hcaT) and a regulator (hcaR). Sequence comparisons revealed that whilehcaA1A2CD genes encode the four subunits of the 3-phenylpropionate dioxygenase, the hcaB gene codes for the corresponding cis-dihydrodiol dehydrogenase. This type of catabolic module is homologous to those encoding class IIB dioxygenases and becomes the first example of such a catabolic cluster in E. coli. The inducible expression of the hca genes requires the presence of the hcaR gene product, which acts as a transcriptional activator and shows significant sequence similarity to members of the LysR family of regulators. Interestingly, the HcaA1A2CD and HcaB enzymes are able to oxidize not only PP to 3-(2,3-dihydroxyphenyl)propionate (DHPP) but also cinnamic acid (CI) to its corresponding 2,3-dihydroxy derivative. Further catabolism of DHPP requires the mhp-encoded meta fission pathway for the mineralization of 3-hydroxyphenylpropionate (3HPP) (A. Ferrández, J. L. Garcı́a, and E. Dı́az, J. Bacteriol. 179:2573–2581, 1997). Expression in Salmonella typhimurium of the mhp genes alone or in combination with the hca cluster allowed the growth of the recombinant bacteria in 3-hydroxycinnamic acid (3HCI) and CI, respectively. Thus, the convergent mhp- and hca-encoded pathways are also functional in S. typhimurium, and they are responsible for the catabolism of different phenylpropanoid compounds (3HPP, 3HCI, PP, and CI) widely available in nature.

scholarly journals AtzC Is a New Member of the Amidohydrolase Protein Superfamily and Is Homologous to Other Atrazine-Metabolizing Enzymes

1998 ◽  
Vol 180 (1) ◽  
pp. 152-158 ◽  
Author(s):  
Michael J. Sadowsky ◽  
Zhaokun Tong ◽  
Mervyn de Souza ◽  
Lawrence P. Wackett
Keyword(s):  
Amino Acids ◽  
Cyanuric Acid ◽  
Sequence Similarity ◽  
Cytosine Deaminase ◽  
Cosmid Library ◽  
Sequence Comparisons ◽  
Protein Superfamily ◽  
E Coli ◽  
Sequence Identity ◽  
Dna Fragment

ABSTRACT Pseudomonas sp. strain ADP metabolizes atrazine to cyanuric acid via three plasmid-encoded enzymes, AtzA, AtzB, and AtzC. The first enzyme, AtzA, catalyzes the hydrolytic dechlorination of atrazine, yielding hydroxyatrazine. The second enzyme, AtzB, catalyzes hydroxyatrazine deamidation, yielding N-isopropylammelide. In this study, the third gene in the atrazine catabolic pathway,atzC, was cloned from a Pseudomonas sp. strain ADP cosmid library as a 25-kb EcoRI DNA fragment inEscherichia coli. The atzC gene was further delimited by functional analysis following transposon Tn5mutagenesis and subcloned as a 2.0-kbEcoRI-AvaI fragment. An E. colistrain containing this DNA fragment expressedN-isopropylammelide isopropylamino hydrolase activity, metabolizing N-isopropylammelide stoichiometrically to cyanuric acid and N-isopropylamine. The 2.0-kb DNA fragment was sequenced and found to contain a single open reading frame of 1,209 nucleotides, encoding a protein of 403 amino acids. AtzC showed modest sequence identity of 29 and 25%, respectively, to cytosine deaminase and dihydroorotase, both members of an amidohydrolase protein superfamily. The sequence of AtzC was compared to that of E. coli cytosine deaminase in the regions containing the five ligands to the catalytically important metal for the protein. Pairwise comparison of the 35 amino acids showed 61% sequence identity and 85% sequence similarity. AtzC is thus assigned to the amidohydrolase protein family that includes cytosine deaminase, urease, adenine deaminase, and phosphotriester hydrolase. Similar sequence comparisons of the most highly conserved regions indicated that the AtzA and AtzB proteins also belong to the same amidohydrolase family. Overall, the data suggest that AtzA, AtzB, and AtzC diverged from a common ancestor and, by random events, have been reconstituted onto an atrazine catabolic plasmid.

scholarly journals Synthetic promoter design in Escherichia coli based on a deep generative network

2020 ◽  
Vol 48 (12) ◽  
pp. 6403-6412 ◽  
Author(s):  
Ye Wang ◽  
Haochen Wang ◽  
Lei Wei ◽  
Shuailin Li ◽  
Liyang Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Predictive Model ◽  
De Novo ◽  
Sequence Similarity ◽  
Small Subset ◽  
Vast Number ◽  
Significant Sequence Similarity ◽  
E Coli ◽  
Synthetic Promoters ◽  
Naturally Occurring

Abstract Promoter design remains one of the most important considerations in metabolic engineering and synthetic biology applications. Theoretically, there are 450 possible sequences for a 50-nt promoter, of which naturally occurring promoters make up only a small subset. To explore the vast number of potential sequences, we report a novel AI-based framework for de novo promoter design in Escherichia coli. The model, which was guided by sequence features learned from natural promoters, could capture interactions between nucleotides at different positions and design novel synthetic promoters in silico. We combined a deep generative model that guides the search for artificial sequences with a predictive model to preselect the most promising promoters. The AI-designed promoters were optimized based on the promoter activity in E. coli and the predictive model. After two rounds of optimization, up to 70.8% of the AI-designed promoters were experimentally demonstrated to be functional, and few of them shared significant sequence similarity with the E. coli genome. Our work provided an end-to-end approach to the de novo design of novel promoter elements, indicating the potential to apply deep learning methods to de novo genetic element design.

scholarly journals Acquisition of mcr-1 and Cocarriage of Virulence Genes in Avian Pathogenic Escherichia coli Isolates from Municipal Wastewater Influents in Japan

2019 ◽  
Vol 85 (22) ◽  
Author(s):  
Wataru Hayashi ◽  
Hayato Tanaka ◽  
Yui Taniguchi ◽  
Masaki Iimura ◽  
Eiji Soga ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Virulence Genes ◽  
Sequence Similarity ◽  
Multidrug Resistant ◽  
Common Source ◽  
High Sequence Similarity ◽  
Content Type ◽  
E Coli ◽  
Query Coverage ◽  
Food Animals

ABSTRACT This study focused on the detection of the plasmid-mediated mcr colistin resistance gene in Escherichia coli isolates from wastewater treatment plants (WWTPs). Seven influent samples were collected from three WWTPs in Nagano Prefecture, Japan, during August and December 2018. Colistin-resistant E. coli isolates were selected on colistin-supplemented CHROMagar ECC plates. mcr-1-positive isolates were subjected to whole-genome sequencing (WGS) analysis. From six influent samples, seven mcr-1-positive but extended-spectrum β-lactamase (ESBL)-negative isolates belonging to different genetic lineages, namely, B2-O25:H4-ST131-fimH22, B2-O2:H1-ST135-fimH2, B1-O8:H9-ST764-fimH32, B1-O23:H16-ST453-fimH31, A-O81:H27-ST10-fimH54, A-O16:H5-ST871-fimH25, and F-O11:H6-ST457-fimH145, were detected. The MICs of colistin for these isolates ranged from 4 to 16 mg/liter. The mcr-1 genes were located on plasmids belonging to IncX4 and IncI2 in five and two isolates, respectively. Four IncX4 plasmids with the same size (33,309 bp) showed high sequence similarity (4 single-nucleotide variations). The remaining one IncX4 plasmid, with a size of 33,858 bp, carried the mcr-1 gene with the single synonymous nucleic substitution T27C. Two IncI2 plasmids with sizes of 60,710 bp and 60,733 bp had high sequence similarity (99.9% identity; 100% query coverage). Two of five isolates carrying IncX4 plasmids and both of the isolates carrying IncI2 plasmids harbored ColV plasmids carrying virulence-associated genes of avian pathogenic E. coli (APEC). In addition, another isolate of the B2-O25:H4-ST131-fimH22 lineage had those APEC-associated virulence genes on its chromosome. In conclusion, mcr-1-positive E. coli environmental isolates were mostly characterized as positive for APEC-associated virulence genes. The copresence of those genes may suggest the existence of a common source in animals and/or their associated environments. IMPORTANCE Colistin is considered a last-line therapeutic option in severe infections due to multidrug-resistant Gram-negative bacteria, in particular carbapenemase-producing Enterobacteriaceae and multidrug-resistant Acinetobacter baumannii. An increasing prevalence of mcr genes in diverse Enterobacteriaceae species, mainly Escherichia coli and Klebsiella pneumoniae from humans and food animals, has become a significant concern to public health all over the world. In Japan, mcr genes have so far been detected in food animals, raw meat, wastewater, and human clinical samples. This study reports the copresence of mcr-1 and avian pathogenic E. coli (APEC)-associated virulence genes in five of seven E. coli isolates recovered from aquatic environments in Japan. Our study highlights the importance and urgency of action to reduce environmental contamination by mcr genes that may likely occur due to exposure to untreated wastewater through combined sewer overflow by recent unusual weather.

scholarly journals A Novel Pathogenicity Island Integrated Adjacent to the thrW tRNA Gene of Avian Pathogenic Escherichia coli Encodes a Vacuolating Autotransporter Toxin

2003 ◽  
Vol 71 (9) ◽  
pp. 5087-5096 ◽  
Author(s):  
V. R. Parreira ◽  
C. L. Gyles
Keyword(s):  
Escherichia Coli ◽  
Serine Protease ◽  
Trna Gene ◽  
Signal Sequence ◽  
Sequence Similarity ◽  
Pathogenicity Island ◽  
Open Reading Frames ◽  
Integrase Gene ◽  
E Coli ◽  
Reading Frames

ABSTRACT We report the complete nucleotide sequence and genetic organization of the Vat-encoding pathogenicity island (PAI) of avian pathogenic Escherichia coli strain Ec222. The 22,139-bp PAI is situated adjacent to the 3′ terminus of the thrW tRNA gene, has a G+C content of 41.2%, and includes a bacteriophage SfII integrase gene, mobile genetic elements, two open reading frames with products exhibiting sequence similarity to known proteins, and several other open reading frames of unknown function. The PAI encodes an autotransporter protein, Vat (vacuolating autotransporter toxin), which induces the formation of intracellular vacuoles resulting in cytotoxic effects similar to those caused by the VacA toxin from Helicobacter pylori. The predicted 148.3-kDa protein product possesses the three domains that are typical of serine protease autotransporters of Enterobacteriaceae: an N-terminal signal sequence of 55 amino acids, a 111.8-kDa passenger domain containing a modified serine protease site (ATSGSG), and a C-terminal outer membrane translocator of 30.5 kDa. Vat has 75% protein homology with the hemagglutinin Tsh, an autotransporter of avian pathogenic E. coli. A vat deletion mutant of Ec222 showed no virulence in respiratory and cellulitis infection models of disease in broiler chickens. We conclude that the newly described PAI and Vat may be involved in the pathogenicity of avian septicemic E. coli strain Ec222 and other avian pathogenic E. coli strains.

scholarly journals A Mutation within the β Subunit of Escherichia coli RNA Polymerase Impairs Transcription from Bacteriophage T4 Middle Promoters

2010 ◽  
Vol 192 (21) ◽  
pp. 5580-5587 ◽  
Author(s):  
Tamara D. James ◽  
Michael Cashel ◽  
Deborah M. Hinton
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Bacteriophage T4 ◽  
Β Subunit ◽  
Subunit Gene ◽  
Wild Type ◽  
Promoter Activation ◽  
E Coli ◽  
Growth Phenotype

ABSTRACT During infection of Escherichia coli, bacteriophage T4 usurps the host transcriptional machinery, redirecting it to the expression of early, middle, and late phage genes. Middle genes, whose expression begins about 1 min postinfection, are transcribed both from the extension of early RNA into middle genes and by the activation of T4 middle promoters. Middle-promoter activation requires the T4 transcriptional activator MotA and coactivator AsiA, which are known to interact with σ70, the specificity subunit of RNA polymerase. T4 motA amber [motA(Am)] or asiA(Am) phage grows poorly in wild-type E. coli. However, previous work has found that T4 motA(Am)does not grow in the E. coli mutant strain TabG. We show here that the RNA polymerase in TabG contains two mutations within its β-subunit gene: rpoB(E835K) and rpoB(G1249D). We find that the G1249D mutation is responsible for restricting the growth of either T4 motA(Am)or asiA(Am) and for impairing transcription from MotA/AsiA-activated middle promoters in vivo. With one exception, transcription from tested T4 early promoters is either unaffected or, in some cases, even increases, and there is no significant growth phenotype for the rpoB(E835K G1249D) strain in the absence of T4 infection. In reported structures of thermophilic RNA polymerase, the G1249 residue is located immediately adjacent to a hydrophobic pocket, called the switch 3 loop. This loop is thought to aid in the separation of the RNA from the DNA-RNA hybrid as RNA enters the RNA exit channel. Our results suggest that the presence of MotA and AsiA may impair the function of this loop or that this portion of the β subunit may influence interactions among MotA, AsiA, and RNA polymerase.

Sign in / Sign up

Export Citation Format

Share Document