Purification and characterization of recombinant pea-seed ferritins expressed in Escherichia coli: influence of N-terminus deletions on protein solubility and core formation in vitro

Plant ferritin subunits are synthesized as precursor molecules; the transit peptide (TP) in their NH2 extremity, responsible for plastid targeting, is cleaved during translocation to this compartment. In addition, the N-terminus of the mature subunit contains a plant-specific sequence named extension peptide (EP) [Ragland, Briat, Gagnon, Laulhère, Massenet, and Theil, E.C. (1990) J. Biol. Chem. 265, 18339-18344], the function of which is unknown. A novel pea-seed ferritin cDNA, with a consensus ferroxidase centre conserved within H-type animal ferritins has been characterized. This pea-seed ferritin cDNA has been engineered using oligonucleotide-directed mutagenesis to produce DNA fragments (1) corresponding to the wild-type (WT) ferritin precursor, (2) with the TP deleted, (3) with both the TP and the plant specific EP sequences deleted and (4) containing the TP but with the EP deleted. These four DNA fragments have been cloned in an Escherichia coli expression vector to produce the corresponding recombinant pea-seed ferritins. Expression at 37 degrees C led to the accumulation of recombinant pea-seed ferritins in inclusion bodies, whatever the construct introduced in E. coli. Expression at 25 degrees C in the presence of sorbitol and betaine allowed soluble proteins to accumulate when constructs with the TP deleted were used; under this condition, E. coli cells transformed with constructs containing the TP were unable to accumulate recombinant protein. Recombinant ferritins purified from inclusion bodies were found to be assembled only when the TP was deleted; however assembled ferritin under this condition had a ferroxidase activity undetectable at acid pH. On the other hand, soluble recombinant ferritins with the TP deleted and expressed at 25 degrees C were purified as 24-mers containing an average of 40-50 iron atoms per molecule. Despite the conservation in the plant ferritin subunit of a consensus ferroxidase centre, the iron uptake activity in vitro at pH 6.8 was found to be lower than that of the recombinant human H-ferritin, though it was much more active than the recombinant human L-ferritin. The recombinant ferritin with both the TP and the EP deleted (r delta TP/EP) assembled correctly as a 24-mer; it has slightly higher ferroxidase activity and decreased solubility compared with the wild-type protein with the TP deleted (r delta TP). In addition, on denaturation by urea followed by renaturation by dialysis the r delta TP/EP protein showed a 25% increase in core-formation in vitro compared with the r delta TP protein.(ABSTRACT TRUNCATED AT 400 WORDS)

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Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.3468-3474.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 3468-3474 ◽

Cited By ~ 19

Author(s):

Gyeong Tae Eom ◽

Jae Kwang Song ◽

Jung Hoon Ahn ◽

Yeon Soo Seo ◽

Joon Shick Rhee

Keyword(s):

Escherichia Coli ◽

Directed Evolution ◽

Abc Transporter ◽

Microtiter Plate ◽

Single Amino Acid ◽

Wild Type ◽

E Coli ◽

Single Amino Acid Change ◽

Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

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In Vivo Titration of Mitomycin C Action by Four Escherichia coli Genomic Regions on Multicopy Plasmids

Journal of Bacteriology ◽

10.1128/jb.183.7.2259-2264.2001 ◽

2001 ◽

Vol 183 (7) ◽

pp. 2259-2264 ◽

Cited By ~ 26

Author(s):

Yan Wei ◽

Amy C. Vollmer ◽

Robert A. LaRossa

Keyword(s):

Escherichia Coli ◽

Mitomycin C ◽

Transcriptional Activation ◽

Efflux Pump ◽

Wild Type ◽

Potent Inducer ◽

E Coli ◽

Genomic Regions

ABSTRACT Mitomycin C (MMC), a DNA-damaging agent, is a potent inducer of the bacterial SOS response; surprisingly, it has not been used to select resistant mutants from wild-type Escherichia coli. MMC resistance is caused by the presence of any of four distinctE. coli genes (mdfA, gyrl, rob, andsdiA) on high-copy-number vectors. mdfAencodes a membrane efflux pump whose overexpression results in broad-spectrum chemical resistance. The gyrI (also called sbmC) gene product inhibits DNA gyrase activity in vitro, while the rob protein appears to function in transcriptional activation of efflux pumps. SdiA is a transcriptional activator of ftsQAZ genes involved in cell division.

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Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine

Infection and Immunity ◽

10.1128/iai.00052-07 ◽

2007 ◽

Vol 75 (7) ◽

pp. 3315-3324 ◽

Cited By ~ 47

Author(s):

Eric J. Gauger ◽

Mary P. Leatham ◽

Regino Mercado-Lubo ◽

David C. Laux ◽

Tyrrell Conway ◽

...

Keyword(s):

Escherichia Coli ◽

Regulatory Region ◽

Flagellar Motor ◽

Wild Type ◽

E Coli ◽

Mouse Intestine ◽

Flagellum Biosynthesis ◽

Colonizing Ability

ABSTRACT Previously, we reported that the mouse intestine selected mutants of Escherichia coli MG1655 that have improved colonizing ability (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). These mutants grew 10 to 20% faster than their parent in mouse cecal mucus in vitro and 15 to 30% faster on several sugars found in the mouse intestine. The mutants were nonmotile and had deletions of various lengths beginning immediately downstream of an IS1 element located within the regulatory region of the flhDC operon, which encodes the master regulator of flagellum biosynthesis, FlhD4C2. Here we show that during intestinal colonization by wild-type E. coli strain MG1655, 45 to 50% of the cells became nonmotile by day 3 after feeding of the strain to mice and between 80 and 90% of the cells were nonmotile by day 15 after feeding. Ten nonmotile mutants isolated from mice were sequenced, and all were found to have flhDC deletions of various lengths. Despite this strong selection, 10 to 20% of the E. coli MG1655 cells remained motile over a 15-day period, suggesting that there is an as-yet-undefined intestinal niche in which motility is an advantage. The deletions appear to be selected in the intestine for two reasons. First, genes unrelated to motility that are normally either directly or indirectly repressed by FlhD4C2 but can contribute to maximum colonizing ability are released from repression. Second, energy normally used to synthesize flagella and turn the flagellar motor is redirected to growth.

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Enterohemorrhagic Escherichia coli O157:H7 gal Mutants Are Sensitive to Bacteriophage P1 and Defective in Intestinal Colonization

Infection and Immunity ◽

10.1128/iai.01342-06 ◽

2006 ◽

Vol 75 (4) ◽

pp. 1661-1666 ◽

Cited By ~ 36

Author(s):

Theresa Deland Ho ◽

Matthew K. Waldor

Keyword(s):

Escherichia Coli ◽

Genetic Manipulation ◽

Enterohemorrhagic Escherichia Coli ◽

Deletion Strain ◽

Growth Defect ◽

Intestinal Colonization ◽

Wild Type ◽

E Coli ◽

O Antigen

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC), especially E. coli O157:H7, is an emerging cause of food-borne illness. Unfortunately, E. coli O157 cannot be genetically manipulated using the generalized transducing phage P1, presumably because its extensive O antigen obscures the P1 receptor, the lipopolysaccharide (LPS) core subunit. The GalE, GalT, GalK, and GalU proteins are necessary for modifying galactose before it can be assembled into the repeating subunit of the O antigen. Here, we constructed E. coli O157:H7 gal mutants which presumably have little or no O antigen. These strains were able to adsorb P1. P1 lysates grown on the gal mutant strains could be used to move chromosomal markers between EHEC strains, thereby facilitating genetic manipulation of E. coli O157:H7. The gal mutants could easily be reverted to a wild-type Gal+ strain using P1 transduction. We found that the O157:H7 galETKM::aad-7 deletion strain was 500-fold less able to colonize the infant rabbit intestine than the isogenic Gal+ parent, although it displayed no growth defect in vitro. Furthermore, in vivo a Gal+ revertant of this mutant outcompeted the galETKM deletion strain to an extent similar to that of the wild type. This suggests that the O157 O antigen is an important intestinal colonization factor. Compared to the wild type, EHEC gal mutants were 100-fold more sensitive to a peptide derived from bactericidal permeability-increasing protein, a bactericidal protein found on the surface of intestinal epithelial cells. Thus, one way in which the O157 O antigen may contribute to EHEC intestinal colonization is to promote resistance to host-derived antimicrobial polypeptides.

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Positive control of sulphate reduction in Escherichia coli. The nature of the pleiotropic cysteineless mutants of E. coli K 12

Biochemical Journal ◽

10.1042/bj1100597 ◽

1968 ◽

Vol 110 (3) ◽

pp. 597-602 ◽

Cited By ~ 21

Author(s):

M. C. Jones-Mortimer

Keyword(s):

Escherichia Coli ◽

Mutant Allele ◽

Positive Control ◽

Wild Type Allele ◽

Wild Type ◽

E Coli ◽

Type Allele ◽

K 12 ◽

Sulphite Reductase

1. The function of the wild-type alleles of the pleiotropic mutants cysB and cysE of Escherichia coli was investigated. 2. The wild-type allele cysB+ is dominant to the mutant allele cysB in stable and transient heterozygotes. 3. The wild-type allele cysE+ is dominant to the mutant allele cysE, as predicted. 4. Sulphur-starved cultures of cysB or cysE strains contain less than 0·2nmole of free cysteine/mg. dry wt. 5. Complementation in vitro is not observed between extracts of cysB mutants and mutants lacking sulphite reductase only. 6. A scheme, involving positive control of the enzymes of sulphate activation and reduction, is suggested to account for the control of cysteine biosynthesis.

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ucFabV Requires Functional Reductase Activity to Confer Reduced Triclosan Susceptibility in Escherichia coli

Journal of Molecular Microbiology and Biotechnology ◽

10.1159/000441640 ◽

2015 ◽

Vol 25 (6) ◽

pp. 394-402 ◽

Cited By ~ 1

Author(s):

Taylor L. Fischer ◽

Robert J. White ◽

Katherine F.K. Mares ◽

Devin E. Molnau ◽

Justin J. Donato

Keyword(s):

Escherichia Coli ◽

Reductase Activity ◽

Acid Synthesis ◽

Temperature Sensitive ◽

Wild Type ◽

E Coli ◽

Enoyl Acp Reductase ◽

Selection For

Background/Aims: We previously identified the Triclo1 fosmid in a functional metagenomic selection for clones that increased triclosan tolerance in Escherichia coli. The active enzyme encoded by Triclo1 is ucFabV. Although ucFabV is homologous to FabV from other organisms, ucFabV contains substitutions at key positions that would predict differences in substrate binding. Therefore, a detailed characterization of ucFabV was conducted to link its biochemical activity to its ability to confer reduced triclosan sensitivity. Methods: ucFabV and a catalytic mutant were purified and used to reduce crotonoyl-CoA in vitro. The mutant and wild-type enzymes were introduced into E. coli, and their ability to confer triclosan tolerance as well as suppress a temperature-sensitive mutant of FabI were measured. Results: Purified ucFabV, but not the mutant, reduced crotonoyl-CoA in vitro. The wild-type enzyme confers increased triclosan tolerance when introduced into E. coli, whereas the mutant remained susceptible to triclosan. Additionally, wild-type ucFabV, but not the mutant, functionally replaced FabI within living cells. Conclusion: ucFabV confers increased tolerance through its function as an enoyl-ACP reductase. Furthermore, ucFabV is capable of restoring viability in the presence of compromised FabI, suggesting ucFabV is likely facilitating an alternate step within fatty acid synthesis, bypassing FabI inhibition.

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Effects of nucleotide substitutions within the T-loop of precursor tRNAs on interaction with ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and yeast

Biochemical Journal ◽

10.1042/bj3140049 ◽

1996 ◽

Vol 314 (1) ◽

pp. 49-53 ◽

Cited By ~ 13

Author(s):

Zengji LI ◽

Kimberly A. GILLIS ◽

Lisa A. HEGG ◽

Jianchao ZHANG ◽

David L. THURLOW

Keyword(s):

Escherichia Coli ◽

Wild Type ◽

Inhibitory Effects ◽

Nucleotide Substitutions ◽

E Coli ◽

Naturally Occurring ◽

Base Substitutions

Recognition of tRNA and tRNA-like substrates by the enzyme ATP/CTP:tRNA nucleotidyltransferase requires chemically intact nucleotides within the T-loop, especially at positions 57 and 58, which are invariant purines among naturally occurring tRNAs. To test the effects of base substitutions at these positions, which are distant from the site of catalysis, we synthesized mutant tRNAGlu molecules. These in vitro-synthesized RNAs also contained an extra 33 bases at the 5´ end and lacked post-transcriptionally modified bases. The precursor tRNAs were used as substrates for nucleotidyltransferases from Escherichia coli and yeast. Substitution of cytidines at either position 57 or 58 had dramatic inhibitory effects on recognition by both enzymes, including raising the apparent Km and lowering the apparent Vmax.; substitution of an adenosine at position 57 or a uridine at position 58 inhibited the reaction only slightly by comparison. Our results demonstrate that the identities of nucleotides at positions 57 and 58 are relevant to recognition by nucleotidyltransferase, and that a purine is required at position 57. The extra bases at the 5´ end and the lack of post-transcriptionally modified bases did not substantially inhibit interaction with the enzyme, as judged by the wild-type precursor tRNAGlu acting as an effective substrate for both enzymes in the presence of equal concentrations of appropriate tRNA substrates isolated from E. coli.

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Antimicrobial Activity Evaluation of Tebipenem (SPR859), an Orally Available Carbapenem, against a Global Set of Enterobacteriaceae Isolates, Including a Challenge Set of Organisms

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02618-18 ◽

2019 ◽

Vol 63 (6) ◽

Cited By ~ 3

Author(s):

S. J. Ryan Arends ◽

Paul R. Rhomberg ◽

Nicole Cotroneo ◽

Aileen Rubio ◽

Robert K. Flamm ◽

...

Keyword(s):

Escherichia Coli ◽

Antimicrobial Activity ◽

Urinary Tract Infection ◽

Urinary Tract ◽

Tract Infection ◽

Broth Microdilution ◽

Wild Type ◽

Content Type ◽

E Coli

ABSTRACT The antimicrobial activity of tebipenem and other carbapenem agents were tested in vitro against a set of recent clinical isolates responsible for urinary tract infection (UTI), as well as against a challenge set. Isolates were tested by reference broth microdilution and included Escherichia coli (101 isolates), Klebsiella pneumoniae (208 isolates), and Proteus mirabilis (103 isolates) species. Within each species tested, tebipenem showed equivalent MIC50/90 values to those of meropenem (E. coli MIC50/90, ≤0.015/0.03 mg/liter; K. pneumoniae MIC50/90, 0.03/0.06 mg/liter; and P. mirabilis MIC50/90, 0.06/0.12 mg/liter) and consistently displayed MIC90 values 8-fold lower than imipenem. Tebipenem and meropenem (MIC50, 0.03 mg/liter) showed equivalent MIC50 results against wild-type, AmpC-, and/or extended-spectrum β-lactamase (ESBL)-producing isolates. Tebipenem also displayed MIC50/90 values 4- to 8-fold lower than imipenem against the challenge set. All carbapenem agents were less active (MIC50, ≥8 mg/liter) against isolates carrying carbapenemase genes. These data confirm the in vitro activity of the orally available agent tebipenem against prevalent UTI Enterobacteriaceae species, including those producing ESBLs and/or plasmid AmpC enzymes.

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ISKpn40-Mediated Mobilization of the Colistin Resistance Gene mcr-3.11 in Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00851-20 ◽

2020 ◽

Vol 64 (10) ◽

Author(s):

Yu-Zhang He ◽

Teng-Fei Long ◽

Cai-Ping Chen ◽

Bing He ◽

Xing-Ping Li ◽

...

Keyword(s):

Escherichia Coli ◽

Resistance Gene ◽

Wild Type ◽

Colistin Resistance ◽

Content Type ◽

First Report ◽

E Coli ◽

Target Sites

ABSTRACT The mobile colistin resistance gene mcr-3 has globally disseminated since it was first reported in 2017 in Escherichia coli. In vitro mobilization assays in this study demonstrate the functionality of the composite transposon structure ISKpn40-mcr-3.11-dgkA-ISKpn40 in wild-type and recA− E. coli strains. These transpositions generated 4-bp duplications at the target sites. This is the first report demonstrating the mobility of the mcr-3.11 gene by transposition.

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Recombinant soluble human tissue factor secreted by Saccharomyces cerevisiae and refolded from Escherichia coli inclusion bodies: glycosylation of mutants, activity and physical characterization

Biochemical Journal ◽

10.1042/bj3100605 ◽

1995 ◽

Vol 310 (2) ◽

pp. 605-614 ◽

Cited By ~ 56

Author(s):

M J Stone ◽

W Ruf ◽

D J Miles ◽

T S Edgington ◽

P E Wright

Keyword(s):

Escherichia Coli ◽

Saccharomyces Cerevisiae ◽

Fusion Protein ◽

Tissue Factor ◽

Inclusion Bodies ◽

Factor Viia ◽

Factor X ◽

Wild Type ◽

Proteolytic Activation ◽

E Coli

Tissue factor (TF) is the cell-surface transmembrane receptor that initiates both the extrinsic and intrinsic blood coagulation cascades. The abilities of TF to associate with Factor VIIa and Factor X in a ternary complex and to enable proteolytic activation of Factor X by Factor VIIa reside in the extracellular domain of TF. We describe the expression of the surface domain of TF (truncated TF, tTF) in both Saccharomyces cerevisiae and Escherichia coli and the biochemical and physical characterization of the recombinant proteins. Wild-type tTF and several glycosylation-site mutants were secreted efficiently by S. cerevisiae under the control of the yeast prepro-alpha-signal sequence; the T13A,N137D double mutant was the most homogeneous variant expressed in milligram quantities. Wild-type tTF was expressed in a non-native state in E. coli inclusion bodies as a fusion protein with a poly(His) leader. The fusion protein could be fully renatured and the leader removed by proteolysis with thrombin; the correct molecular mass (24,729 Da) of the purified protein was confirmed by electrospray mass spectrometry. Recombinant tTFs from yeast, E. coli and Chinese hamster ovary cells were identical in their abilities to bind Factor VIIa, to enhance the catalytic activity of Factor VIIa and to enhance the proteolytic activation of Factor X by Factor VIIa. Furthermore, CD, fluorescence emission and NMR spectra of the yeast and E. coli proteins indicated that these proteins are essentially identical structurally.

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