Journal: bioRxiv

Article Title: Thioproline formation as a driver of formaldehyde toxicity in Escherichia coli

doi: 10.1101/2020.03.19.981027

Figure Lengend Snippet: (A) E. coli BW25113 wild type (WT) and Δ pepP strains harboring the pBAD24 vector alone (V) or containing pepP (P) were cultured on M9 minimal medium containing 0.4% glucose and 0.02% arabinose, minus or plus 400 μM HCHO. Overnight liquid cultures of each strain were ten-fold serially diluted and 3.5-μl aliquots were spotted on the plates. Images were captured after incubation at 37°C for 1. d. (B) Wild type and Δ pepP strains were cultured in 150 μl of M9 liquid medium containing 10 mM glucose and the indicated concentrations of HCHO. Data are means ± s.e. ( n = 3).

Article Snippet: For Δ pepP mutant complementation, the E. coli PepP coding sequence was PCR-amplified (primers 1 and 2) from BW25113 genomic DNA prepared using a GeneJET Genomic DNA Purification Kit (Thermo Scientific, Cat No. K0721) according to the manufacturer’s instructions.

Techniques: Plasmid Preparation, Cell Culture, Incubation

Journal: bioRxiv

Article Title: Thioproline formation as a driver of formaldehyde toxicity in Escherichia coli

doi: 10.1101/2020.03.19.981027

Figure Lengend Snippet: E. coli BW25113 wild type (WT) and Δ pepP strains were grown in M9 minimal medium containing 0.4% glucose until OD 600 reached 0.4. Control (-HCHO) cells were then harvested; HCHO-treated (+ HCHO) cells were cultured for another 2 h after adding HCHO (final concentration 1 mM) and then harvested. Extracted proteins were hydrolyzed with S. griseus protease; the hydrolysate was analyzed by LC-MS for N 6 -formyl lysine. Data are means ± s.e. ( n = 6). Significance was determined by Student’s t -test. * P <0.05; ns, non-significant.

Article Snippet: For Δ pepP mutant complementation, the E. coli PepP coding sequence was PCR-amplified (primers 1 and 2) from BW25113 genomic DNA prepared using a GeneJET Genomic DNA Purification Kit (Thermo Scientific, Cat No. K0721) according to the manufacturer’s instructions.

Techniques: Control, Cell Culture, Concentration Assay, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Thioproline formation as a driver of formaldehyde toxicity in Escherichia coli

doi: 10.1101/2020.03.19.981027

Figure Lengend Snippet: (A) Schematic representation of the hypothesis. Compounds inferred or known to be toxic are in red font. The core parts of the hypothesis – the toxicity of thioproline-containing peptides and their cleavage by PepP – are boxed in gray. ThioPro, thioproline; X and Y represent any amino acid residue. (B) Growth of lawns of BW25113 wild type (WT) and Δ pepP cells on plates of M9 minimal medium without N, plus 0.4% glucose. The central disc contained the additions indicated; alanine was included as a positive control. Arrowheads mark growth halos around the discs. (A) Growth of wild type and Δ pepP cells in liquid M9 minimal medium without N, plus 10 mM glucose and the additions indicated. Data are means ± s.e. ( n = 3).

Article Snippet: For Δ pepP mutant complementation, the E. coli PepP coding sequence was PCR-amplified (primers 1 and 2) from BW25113 genomic DNA prepared using a GeneJET Genomic DNA Purification Kit (Thermo Scientific, Cat No. K0721) according to the manufacturer’s instructions.

Techniques: Residue, Positive Control

Journal: bioRxiv

Article Title: Thioproline formation as a driver of formaldehyde toxicity in Escherichia coli

doi: 10.1101/2020.03.19.981027

Figure Lengend Snippet: BW25113 wild type (WT) and Δ pepP strains were cultured in 150 μl of M9 liquid medium containing 10 mM glucose and the indicated concentrations of thioproline. Data are means ± s.e. ( n = 3).

Article Snippet: For Δ pepP mutant complementation, the E. coli PepP coding sequence was PCR-amplified (primers 1 and 2) from BW25113 genomic DNA prepared using a GeneJET Genomic DNA Purification Kit (Thermo Scientific, Cat No. K0721) according to the manufacturer’s instructions.

Techniques: Cell Culture

Journal: Microbiology Spectrum

Article Title: Comparison of the mechanism of antimicrobial action of the gold(I) compound auranofin in Gram-positive and Gram-negative bacteria

doi: 10.1128/spectrum.00138-24

Figure Lengend Snippet: Bacterial strains and plasmids used in this study ^a

Article Snippet: E. coli JW5156 , E. coli BW25113 Δ trxA , The National BioResource Project (National Institute of Genomics, Japan).

Techniques: Cell Culture, Control, Cloning, Expressing, Plasmid Preparation

Journal: Microbiology Spectrum

Article Title: Comparison of the mechanism of antimicrobial action of the gold(I) compound auranofin in Gram-positive and Gram-negative bacteria

doi: 10.1128/spectrum.00138-24

Figure Lengend Snippet: MICs of auranofin against various Gram-negative and Gram-positive bacterial species

Article Snippet: E. coli JW5156 , E. coli BW25113 Δ trxA , The National BioResource Project (National Institute of Genomics, Japan).

Techniques: Bacteria

Journal: Microbiology Spectrum

Article Title: Comparison of the mechanism of antimicrobial action of the gold(I) compound auranofin in Gram-positive and Gram-negative bacteria

doi: 10.1128/spectrum.00138-24

Figure Lengend Snippet: Proteomic response of E. coli MG1655 stressed with 120 µg/mL auranofin. ( a ) Growth curves of E. coli MG1655 grown in MOPS media at 37°C and 200 rpm without auranofin (◼︎) and after the addition of 120 µg/mL auranofin (◻︎). An arrow indicates the timepoint of auranofin addition. ( b ) Incorporation of L-[ 35 S]-methionine during a 5-min pulse starting 10 min after treatment with 120 µg/mL auranofin. ( c ) 2D-PAGE analysis of the pulse-labeled proteomics response profile of E. coli MG1655 treated with 120 µg/mL auranofin. False-color overlay of treated and untreated radioactive gels shows proteins synthesized after pulse labeling for 5 min after treatment with auranofin (red, Au) or in the untreated control (green, Co). Upregulated proteins with a regulation factor ≥2 were considered marker proteins and were labeled by arrows and the Spot ID Au_X. Marker proteins identified by mass spectrometry are additionally labeled with the protein’s name. For Spot ID, identified proteins, and functions, see . ( a and c ) A representative example from three ( a ) or two ( c ) biological replicates. ( b ) The average and standard deviation of three biological replicates.

Article Snippet: E. coli JW5156 , E. coli BW25113 Δ trxA , The National BioResource Project (National Institute of Genomics, Japan).

Techniques: Labeling, Synthesized, Control, Marker, Mass Spectrometry, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Comparison of the mechanism of antimicrobial action of the gold(I) compound auranofin in Gram-positive and Gram-negative bacteria

doi: 10.1128/spectrum.00138-24

Figure Lengend Snippet: Identified marker proteins of auranofin-treated E. coli MG1655 ^a

Article Snippet: E. coli JW5156 , E. coli BW25113 Δ trxA , The National BioResource Project (National Institute of Genomics, Japan).

Techniques: Marker, Binding Assay

Journal: Microbiology Spectrum

Article Title: Comparison of the mechanism of antimicrobial action of the gold(I) compound auranofin in Gram-positive and Gram-negative bacteria

doi: 10.1128/spectrum.00138-24

Figure Lengend Snippet: Auranofin inhibits E. coli ( ec TrxB) and B. subtilis TrxR ( bs TrxB) in a dose-dependent manner. Purified ec TrxB and bs TrxB (1 µM) were preincubated with 200 µM NADPH in the absence (0 µM auranofin) or presence of various auranofin concentrations (0.1–2 µM) for 5 min. The addition of 2 mM DTNB initiated the Reaction. Reaction progress was monitored by measuring the absorbance of the liberated TNB chromophore at 412 nm over time. Experiments were performed in triplicates. Mean (bars), individual values (dots), and standard deviation are shown (error bars).

Article Snippet: E. coli JW5156 , E. coli BW25113 Δ trxA , The National BioResource Project (National Institute of Genomics, Japan).

Techniques: Purification, Standard Deviation

Journal: Microbiology Spectrum

Article Title: Comparison of the mechanism of antimicrobial action of the gold(I) compound auranofin in Gram-positive and Gram-negative bacteria

doi: 10.1128/spectrum.00138-24

Figure Lengend Snippet: Loss of a functional Trx system and depletion of the glutathione pool increase E. coli’s susceptibility to auranofin. ( a ) Growth curves of E. coli BW25113 (wild type) and single-gene knockout mutants of the KEIO collection lacking a functional gor (Δ gor; E. coli JW3467) , gshA (Δ gshA; E. coli JW2663), trxB (Δ trxB; E. coli JW0871) , or trxA gene (Δ trxA; E. coli JW5156), respectively, in the absence of auranofin. ( b and c ) MICs of auranofin against E. coli wild-type and the various mutant strains. Cells were grown in an MOPS minimal medium until an OD 600 of 0.5 was reached. The various cultures were then used to inoculate MOPS minimal medium containing the indicated amounts of auranofin with 10 5 cells. After overnight incubation, bacterial growth was analyzed ( b ) visually and ( c ) photometrically by measuring the OD 600 of the cultures. ( b ) A representative MIC assay is shown. ( c ) Means and standard deviations of three independent experiments are shown.

Article Snippet: E. coli JW5156 , E. coli BW25113 Δ trxA , The National BioResource Project (National Institute of Genomics, Japan).

Techniques: Functional Assay, Gene Knockout, Mutagenesis, Incubation

Journal: Microbiology Spectrum

Article Title: Comparison of the mechanism of antimicrobial action of the gold(I) compound auranofin in Gram-positive and Gram-negative bacteria

doi: 10.1128/spectrum.00138-24

Figure Lengend Snippet: Auranofin exposure leads to roGFP2 oxidation in vivo . To evaluate the effects of auranofin on protein thiols in bacteria in situ , B. subtilis ( a ) and E. coli ( b ) expressing roGFP2 were treated with 0.5, 1, or 5 µM auranofin. Aldrithiol-2 and dithiothreitol were used to fully oxidize and fully reduce the probe within cells, respectively, and these baselines (not shown) were used to calculate the oxidation degree (OxD) of roGFP2. OxD normalizes the ratiometrically determined redox state and accounts for differences in probe synthesis and measurement settings. roGFP2 fluorescence was measured at an emission wavelength of 510 nm, and the excitation wavelength was scanned from 350–500 nm. roGFP2 oxidation was monitored for 60 min after adding auranofin, and then DTT was added at a concentration of 2 mM (indicated by an arrow). Means and standard deviations are shown.

Article Snippet: E. coli JW5156 , E. coli BW25113 Δ trxA , The National BioResource Project (National Institute of Genomics, Japan).

Techniques: In Vivo, Bacteria, In Situ, Expressing, Fluorescence, Concentration Assay

Journal: Microbiology Spectrum

Article Title: Comparison of the mechanism of antimicrobial action of the gold(I) compound auranofin in Gram-positive and Gram-negative bacteria

doi: 10.1128/spectrum.00138-24

Figure Lengend Snippet: List of marker protein spots induced after treatment with auranofin in E. coli MG1655 ^a

Article Snippet: E. coli JW5156 , E. coli BW25113 Δ trxA , The National BioResource Project (National Institute of Genomics, Japan).

Techniques: Marker, Binding Assay

Journal: Chembiochem

Article Title: Ribosomal Target‐Binding Sites of Antimicrobial Peptides Api137 and Onc112 Are Conserved among Pathogens Indicating New Lead Structures To Develop Novel Broad‐Spectrum Antibiotics

doi: 10.1002/cbic.202000109

Figure Lengend Snippet: Sequence alignment of 23S rRNA at the oncocin binding site. The residues depicted in bold are within 5 Å of oncocin in the crystal structure of oncocin bound to the T. thermophilus ribosome. Residues that interact with oncocin via the base are underlined. Four residues differ in the human 28S rRNA (boxed in red). T.t, S.a, E.c., K. p., P.a., A.b., and H.s. denote T. thermophilus, Staphylococcus aureus, E. coli , K. pneumoniae , P. aeruginosa , A. baumannii , and Homo sapiens , respectively. See Tables S2 and S3 for alignment statistics.

Article Snippet: The following bacteria were used: Escherichia coli BW25113 (lacI q rrnB3 ΔlacZ4787 hsdR514 DE(araBAD)567 DE(rhaBAD)568 rph‐1, Keio collection), Pseudomonas aeruginosa DSM 1117 / ATCC® 27853TM, Klebsiella pneumoniae DSM 681 / ATCC® 10031TM, Acinetobacter baumannii DSM 30008 / ATCC® 15308TM, Staphylococcus aureus DSM 6247 (DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany).

Techniques: Sequencing, Binding Assay

Journal: Microbial biotechnology

Article Title: Bacterial persistence increases as environmental fitness decreases

doi: 10.1111/j.1751-7915.2011.00327.x

Figure Lengend Snippet: Growth, stability and colony morphology of MqsR variants. A. Growth curves in LB medium with 1 mM IPTG induction at 37°C for BW25113 Δ mqsR strains containing pBS(Kan) (empty plasmid control), pBS(Kan)‐ mqsR (MqsR), pBS(Kan)‐ mqsR 2‐1 (MqsR 2‐1) and pBS(Kan)‐ mqsR 20‐14 (MqsR 20‐14). Error bars indicate standard deviation ( n = 3). B. Structure of MqsR in ribbon representation with MqsA in surface representation (based on Protein Data Bank accession code 3HI2). Cyan indicates MqsR active site residues (K56, Q68, Y81 and K96), yellow indicates the residues (K3 and N31) where the substitutions occur in MqsR 2‐1 (K3N and N31Y), and orange indicates the residues (R9, L35 and V70) where the substitutions occur in MqsR 20‐14 (R9C, L35F and V70I). C. Protein stability of native MqsR and the MqsR variants (MqsR K3N, MqsR N31Y and MqsR 2‐1). Western blot (upper panel) and SDS‐PAGE show the protein levels of His‐MqsR and variants detected by a His‐tagged antibody. MqsR and the variants were induced from pET28a‐based plasmids in E. coli BL21 (DE3) via 1 mM IPTG. Arrows indicate the MqsR proteins. D. Colony morphology of BW25113 Δ mqsR strains containing pBS(Kan) (empty plasmid), pBS(Kan)‐ mqsR and pBS(Kan)‐ mqsR 2‐1 grown on LB agar plates at 37°C after 24 h. Scale bars indicate 1 cm. Representative images are shown. E. Observation of BW25113 Δ mqsR strains containing pBS(Kan) (empty plasmid control), pBS(Kan)‐ mqsR and pBS(Kan)‐ mqsR 2‐1. IPTG was added to LB for 2 h to produce native MqsR and MqsR 2‐1 (middle row), while glucose (0.2%) was added to LB to repress MqsR production (upper row). Lower row shows the result of Live/Dead staining after producing MqsR and MqsR 2‐1. Live cells are stained in green, and dead cells are stained in red. Scale bars indicate 10 µm. Representative images are shown.

Article Snippet: For the whole‐transcriptome study of BW25113 Δ mqsR /pBS(Kan)‐ mqsR 2‐1 versus BW25113 Δ mqsR /pBS(Kan)‐ mqsR , planktonic cells were grown to a turbidity of 0.5 at 600 nm in LB medium with 1 mM IPTG at 37°C, adjusted to a turbidity to 1, and exposed to 20 μg ml −1 ampicillin with 1 mM IPTG for 1 h. Cells were isolated by centrifuging at 0°C, and RNA Later ® buffer (Applied Biosystems, Foster City, CA, USA) was added to stabilize RNA during the RNA preparation steps.

Techniques: Plasmid Preparation, Control, Standard Deviation, Western Blot, SDS Page, Staining

Journal: Microbial biotechnology

Article Title: Bacterial persistence increases as environmental fitness decreases

doi: 10.1111/j.1751-7915.2011.00327.x

Figure Lengend Snippet: Persister cell formation of MqsR variants. BW25113 Δ mqsR strains containing pBS(Kan) (empty plasmid control), pBS(Kan)‐ mqsR , pBS(Kan)‐ mqsR 2‐1 and pBS(Kan)‐ mqsR 20‐14 were grown to a turbidity of 0.5 at 600 nm in LB with 1 mM IPTG at 37°C, adjusted to a turbidity of 1, and exposed to 26 µg ml −1 ampicillin with 1 mM IPTG for 24 h. Error bars indicate the standard deviation ( n = 3).

Article Snippet: For the whole‐transcriptome study of BW25113 Δ mqsR /pBS(Kan)‐ mqsR 2‐1 versus BW25113 Δ mqsR /pBS(Kan)‐ mqsR , planktonic cells were grown to a turbidity of 0.5 at 600 nm in LB medium with 1 mM IPTG at 37°C, adjusted to a turbidity to 1, and exposed to 20 μg ml −1 ampicillin with 1 mM IPTG for 1 h. Cells were isolated by centrifuging at 0°C, and RNA Later ® buffer (Applied Biosystems, Foster City, CA, USA) was added to stabilize RNA during the RNA preparation steps.

Techniques: Plasmid Preparation, Control, Standard Deviation

Journal: Microbial biotechnology

Article Title: Bacterial persistence increases as environmental fitness decreases

doi: 10.1111/j.1751-7915.2011.00327.x

Figure Lengend Snippet: Effect of isogenic mutations on persister cell formation. BW25113 and its isogenic mutants were grown to a turbidity of 1 in LB at 37°C and exposed to 20 µg ml −1 ampicillin for 5 h (A). Time‐course of persister formation of BW25113 Δ rpoS exposed to 20 µg ml −1 ampicillin for 6 h (B). Persister formation of BW25113 Δ rpoS exposed to 1 µg ml −1 ciprofloxacin (Cipro) for 5 h (C). Persister formation of PA14 Δ rpoS exposed to 1 µg ml −1 Cipro for 5 h (D). Error bars indicate the standard deviation ( n = 3).

Article Snippet: For the whole‐transcriptome study of BW25113 Δ mqsR /pBS(Kan)‐ mqsR 2‐1 versus BW25113 Δ mqsR /pBS(Kan)‐ mqsR , planktonic cells were grown to a turbidity of 0.5 at 600 nm in LB medium with 1 mM IPTG at 37°C, adjusted to a turbidity to 1, and exposed to 20 μg ml −1 ampicillin with 1 mM IPTG for 1 h. Cells were isolated by centrifuging at 0°C, and RNA Later ® buffer (Applied Biosystems, Foster City, CA, USA) was added to stabilize RNA during the RNA preparation steps.

Techniques: Standard Deviation

Journal: Microbial biotechnology

Article Title: Bacterial persistence increases as environmental fitness decreases

doi: 10.1111/j.1751-7915.2011.00327.x

Figure Lengend Snippet: Persister formation after oxidative and acid stresses. Cell survival (%) with oxidative stress (20 mM H 2 O 2 ) for 10 min (A) and with acid stress (pH 2.5) for 2 min (B). BW25113 and its isogenic mutants were grown to a turbidity of 1 in LB at 37°C and exposed to H 2 O 2 or pH 2.5. C. Persister cell formation of BW25113 and its isogenic mutants exposed to 20 µg ml −1 ampicillin for 5 h after oxidative or acid stress. Error bars indicate the standard deviation ( n = 3).

Article Snippet: For the whole‐transcriptome study of BW25113 Δ mqsR /pBS(Kan)‐ mqsR 2‐1 versus BW25113 Δ mqsR /pBS(Kan)‐ mqsR , planktonic cells were grown to a turbidity of 0.5 at 600 nm in LB medium with 1 mM IPTG at 37°C, adjusted to a turbidity to 1, and exposed to 20 μg ml −1 ampicillin with 1 mM IPTG for 1 h. Cells were isolated by centrifuging at 0°C, and RNA Later ® buffer (Applied Biosystems, Foster City, CA, USA) was added to stabilize RNA during the RNA preparation steps.

Techniques: Standard Deviation

Journal: Microbial biotechnology

Article Title: Bacterial persistence increases as environmental fitness decreases

doi: 10.1111/j.1751-7915.2011.00327.x

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: For the whole‐transcriptome study of BW25113 Δ mqsR /pBS(Kan)‐ mqsR 2‐1 versus BW25113 Δ mqsR /pBS(Kan)‐ mqsR , planktonic cells were grown to a turbidity of 0.5 at 600 nm in LB medium with 1 mM IPTG at 37°C, adjusted to a turbidity to 1, and exposed to 20 μg ml −1 ampicillin with 1 mM IPTG for 1 h. Cells were isolated by centrifuging at 0°C, and RNA Later ® buffer (Applied Biosystems, Foster City, CA, USA) was added to stabilize RNA during the RNA preparation steps.

Techniques: