Journal: Molecular Cell

Article Title: A cooperative PNPase-Hfq-RNA carrier complex facilitates bacterial riboregulation

doi: 10.1016/j.molcel.2021.05.032

Figure Lengend Snippet: The KH-S1 portal is crucial for PNPase-sRNA-Hfq complex formation (A–C) Electrophoretic mobility shift assays (EMSAs) of wild-type PNPase and KH-S1 mutants with 400 nM RyhB (A), 3ʹETS leuZ (B), and CyaR (C) in the absence and presence of 400 nM Hfq hexamer. Ternary complexes are highlighted with a red dot. Two different PNPase concentrations were used for every PNPase construct (1:1 and 1:3 RNA:PNPase trimer molar ratio), represented by a concentration bar. (D and E) RNA half-life experiments to determine RyhB and CyaR sRNA stabilities in an E. coli strain expressing a 3X-FLAG tagged construct of PNPase WT and mutants. RyhB and CyaR signal intensities were quantified using northern blots and normalized to their corresponding loading controls (SsrA). sRNA decay curves were generated by fitting the normalized signal intensities for each time point. Points and error bars in the curves represent the means and the standard errors (SEM) of at least three independent experiments. Northern blots for RyhB and CyaR half-life measurements corresponding to RNA stability curves are shown and values tabulated in . (F and G) Cell extracts prepared from late exponential phase cultures of E. coli strains expressing WT PNPase, or FLAG-tagged PNPase WT and mutants were used to assess coprecipitation of sRNAs, which were analyzed using northern blot. (G) Fold enrichment of a given RNA upon immunoprecipitation was determined by first calculating the signal intensity per microgram of RNA for the input and the elution from the northern blots in (F). The normalized elution signal was then divided by the input signal. An untagged wild-type strain (WT) was used as a control for data presented in (F) and (G). S1x2: PNPase K657A, R658A; KHx2: PNPase K566A, K571A; S1x4: PNPase R681A, Q682A, R684A, R686A.

Article Snippet: All strains used in this study are derivatives of E. coli K12 strain MG1655 (RRID:Addgene_61440) or BL21DE3.

Techniques: Electrophoretic Mobility Shift Assay, Construct, Concentration Assay, Expressing, Northern Blot, Generated, Immunoprecipitation, Control

Journal: Molecular Cell

Article Title: A cooperative PNPase-Hfq-RNA carrier complex facilitates bacterial riboregulation

doi: 10.1016/j.molcel.2021.05.032

Figure Lengend Snippet:

Article Snippet: All strains used in this study are derivatives of E. coli K12 strain MG1655 (RRID:Addgene_61440) or BL21DE3.

Techniques: Virus, Recombinant, Software

Journal: Applied and Environmental Microbiology

Article Title: Chromosome Engineering To Generate Plasmid-Free Phenylalanine- and Tyrosine-Overproducing Escherichia coli Strains That Can Be Applied in the Generation of Aromatic-Compound-Producing Bacteria

doi: 10.1128/AEM.00525-20

Figure Lengend Snippet: Constructed plasmids and generated strains in this study

Article Snippet: E. coli MG1655 (ME7986) was obtained from the National Institute of Genetics of Japan (National BioResource Project), and its λDE3 lysogen, MG1655(DE3), was used as the host strain for generating Phe- and Tyr-producing strains.

Techniques: Construct, Generated, Variant Assay, Derivative Assay, Transduction

Journal: Applied and Environmental Microbiology

Article Title: Chromosome Engineering To Generate Plasmid-Free Phenylalanine- and Tyrosine-Overproducing Escherichia coli Strains That Can Be Applied in the Generation of Aromatic-Compound-Producing Bacteria

doi: 10.1128/AEM.00525-20

Figure Lengend Snippet: Relationship between the chromosomal locus of PT7lac-lacZ and the β-galactosidase activity (reporter assay). Strain AR-G65 is a BW25113 derived strain harboring PT7lac-lacZ at the tyrR locus of the chromosome (41). (A) The β-galactosidase activities of the respective strains are shown as relative values when the value of strain AR-G65 was 100%. The data were obtained from three independent cultures, and error bars indicate standard deviations. (B) SDS-PAGE analysis of strains harboring T7-controlled shikimate pathway genes integrated into the respective loci of the chromosome. Cont., control strain MG1655(DE3). Combinations of integrated genes and loci are indicated as follows: M-ARG10, PT7lac-ppsA at the adhE locus; M-ARG3, PT7lac-aroGfbr at the tyrR locus; M-ARG4, PT7lac-pheAfbr at the ldhA locus; M-ARG14, PT7lac-tktA at the pflDC locus; M-ARG7, PT7lac-aroA at the pykF locus; and M-ARG99, PT7lac-aroB at the ascF locus.

Article Snippet: E. coli MG1655 (ME7986) was obtained from the National Institute of Genetics of Japan (National BioResource Project), and its λDE3 lysogen, MG1655(DE3), was used as the host strain for generating Phe- and Tyr-producing strains.

Techniques: Activity Assay, Reporter Assay, Derivative Assay, SDS Page, Control

Journal: Applied and Environmental Microbiology

Article Title: Chromosome Engineering To Generate Plasmid-Free Phenylalanine- and Tyrosine-Overproducing Escherichia coli Strains That Can Be Applied in the Generation of Aromatic-Compound-Producing Bacteria

doi: 10.1128/AEM.00525-20

Figure Lengend Snippet: Comparison of recent Phe- and Tyr-producing E. coli strains

Article Snippet: E. coli MG1655 (ME7986) was obtained from the National Institute of Genetics of Japan (National BioResource Project), and its λDE3 lysogen, MG1655(DE3), was used as the host strain for generating Phe- and Tyr-producing strains.

Techniques: Comparison, Plasmid Preparation

Journal: Applied and Environmental Microbiology

Article Title: Chromosome Engineering To Generate Plasmid-Free Phenylalanine- and Tyrosine-Overproducing Escherichia coli Strains That Can Be Applied in the Generation of Aromatic-Compound-Producing Bacteria

doi: 10.1128/AEM.00525-20

Figure Lengend Snippet: Synthetic routes for aromatic compounds. The genes used for modification of M-PAR-120 were ldhA(re) from Cupriavidus necator (synonym, Ralstonia eutropha), tdc(lb) from Lactobacillus brevis, ppd(ab) from Azospirillum brasilense, and yahK from Escherichia coli.

Article Snippet: E. coli MG1655 (ME7986) was obtained from the National Institute of Genetics of Japan (National BioResource Project), and its λDE3 lysogen, MG1655(DE3), was used as the host strain for generating Phe- and Tyr-producing strains.

Techniques: Modification, Full Display Name

Journal: Nature Communications

Article Title: Decorating bacteria with self-assembled synthetic receptors

doi: 10.1038/s41467-020-14336-7

Figure Lengend Snippet: a One way to decorate E. coli with artificial receptors, which are appended with a specific functionality ( X ), involves the binding of X -ODN-1 to a hexa-histidine tag (His-tag) fused to OmpC (I → II). This process can be reversed by subjecting the bacteria to EDTA (II → I). Another way to introduce an unnatural recognition motif ( Y ) to the bacterial surface is by adding to the bacteria decorated with ODN-1 a complementary strand modified with the desired functionality ( Y -ODN-2, II → III). Y-ODN-2 can be selectively removed by adding a complementary strand, ODN-3 (III → II). b Structure of X -ODN-1.

Article Snippet: E. coli K-12 strain KRX (Promega) was used for protein expression.

Techniques: Binding Assay, Bacteria, Introduce, Modification

Journal: Nature Communications

Article Title: Decorating bacteria with self-assembled synthetic receptors

doi: 10.1038/s41467-020-14336-7

Figure Lengend Snippet: a Merged bright-field and fluorescence images of the following: (Top left) E. coli expressing His-OmpC incubated with 500 nM of Cy5-ODN-1 and Ni (II). (Top right) Bacteria lacking His-tag incubated with 500 nM of Cy5-ODN-1 and Ni (II). (Bottom left) His-tagged bacteria incubated with 500 nM of Cy5-ODN-1 in the absence of Ni (II). (Bottom right) His-tagged bacteria incubated with 500 nM of Cy5-ODN (that lacks the NTA group) and Ni (II). b Flow cytometry analysis of His-tagged bacteria (yellow) and bacteria lacking His-tag (gray) incubated with TAMRA-ODN-1. c Images of E. coli expressing His-OmpC decorated with Cy5-ODN-1 in the presence of increasing concentrations of EDTA (0, 5, and 10 mM) (left), and following the subsequent addition of Cy5-ODN-1 in the presence of Ni (II) (right). d Growth curve of E. coli expressing His-OmpC (black) and the growth of the same bacteria decorated with TAMRA-ODN-1 (red). e Bright-field (top) and fluorescence images (bottom) of bacteria decorated with TAMRA-ODN-1 monitored at 0, 12, and 24 h. Source data are available in the Source Data file.

Article Snippet: E. coli K-12 strain KRX (Promega) was used for protein expression.

Techniques: Fluorescence, Expressing, Incubation, Bacteria, Flow Cytometry

Journal: Molecular microbiology

Article Title: Bactericidal peptidoglycan recognition protein induces oxidative stress in Escherichia coli through a block in respiratory chain and increase in central carbon catabolism

doi: 10.1111/mmi.13733

Figure Lengend Snippet: (A) Mutants with increased resistance to PGRP killing were identified by three-stage screening of the entire Keio collection of single gene deletion mutants (Fig. S1 and Table S1) and here the survival of the parental strain (BW25113) and mutants following 3-hr incubation with 200 µg/ml of BSA (as a control) or PGRP is shown. Gene products, their functions, and numerical data are shown in Table S1. (B) Mutants for the key genes for the respiratory chain and TCA cycle, and their regulators (cyaA and crp) were constructed in MG1655 and their sensitivity to killing by 100 µg/ml of PGRP was similarly tested. The results are means of 3 experiments, expressed as percent of initial inoculum (100%) + SEM; ^ P≤0.05, ^^ P<0.001, numbers of viable bacteria (colony forming units) of Δ mutants versus parental strain (t-test).

Article Snippet: The entire Keio collection of 3884 E. coli K-12 in-frame single-gene knockout mutants was obtained from the National BioResource Project, National Institute of Genetics, Japan ( Baba et al ., 2006 ).

Techniques: Incubation, Control, Construct, Bacteria

Journal: Molecular microbiology

Article Title: Bactericidal peptidoglycan recognition protein induces oxidative stress in Escherichia coli through a block in respiratory chain and increase in central carbon catabolism

doi: 10.1111/mmi.13733

Figure Lengend Snippet: (A) Time kinetics of changes of H2O2 in E. coli MG1655 treated with 100 µg/ml BSA or PGRP, or with 100 µM paraquat. (B) H2O2 in E. coli BW25113 and in the indicated deletion mutants from Keio collection treated with 100 µg/ml BSA or PGRP for 15 min. (C) H2O2 in E. coli MG1655 and in the indicated deletion mutants constructed in our laboratory treated with 100 µg/ml BSA or PGRP for 15 min. The results are means of 3–4 experiments ± SEM (SEM were within symbols if not visible); * P<0.05, ** P<0.001, PGRP vs BSA; +P<0.05, ++P<0.001, paraquat vs BSA; ^ P<0.05, ^^ P<0.001, PGRP-treated mutant vs parental strain.

Article Snippet: The entire Keio collection of 3884 E. coli K-12 in-frame single-gene knockout mutants was obtained from the National BioResource Project, National Institute of Genetics, Japan ( Baba et al ., 2006 ).

Techniques: Construct, Mutagenesis

Journal: Molecular microbiology

Article Title: Bactericidal peptidoglycan recognition protein induces oxidative stress in Escherichia coli through a block in respiratory chain and increase in central carbon catabolism

doi: 10.1111/mmi.13733

Figure Lengend Snippet: Parental E. coli or the indicated deletion mutants from Keio collection (BW25113) or from our laboratory (MG1655) were treated with 100 µg/ml BSA or PGRP for 15 min (A) or 5 min (B and C). The results are means of 3–4 experiments + SEM; * P<0.05, ** P<0.001, PGRP vs BSA; ^ P<0.05, ^^ P<0.001, mutant vs parental strain.

Article Snippet: The entire Keio collection of 3884 E. coli K-12 in-frame single-gene knockout mutants was obtained from the National BioResource Project, National Institute of Genetics, Japan ( Baba et al ., 2006 ).

Techniques: Mutagenesis

Journal: Molecular microbiology

Article Title: Bactericidal peptidoglycan recognition protein induces oxidative stress in Escherichia coli through a block in respiratory chain and increase in central carbon catabolism

doi: 10.1111/mmi.13733

Figure Lengend Snippet: E. coli and the indicated deletion mutants from Keio collection (BW25113) or from our laboratory (MG1655) were treated with 100 µg/ml BSA or PGRP for 30 min. The results are means of 3 experiments + SEM; * P<0.05, ** P<0.001, PGRP vs BSA; ^ P<0.05, mutant vs parental strain.

Article Snippet: The entire Keio collection of 3884 E. coli K-12 in-frame single-gene knockout mutants was obtained from the National BioResource Project, National Institute of Genetics, Japan ( Baba et al ., 2006 ).

Techniques: Mutagenesis