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: Frontiers in Public Health

Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

doi: 10.3389/fpubh.2016.00131

Figure Lengend Snippet: Primers, sequence, and amplicons size used in epigenetic influence of Dam studies .

Article Snippet: In addition, the laboratory E. coli strain MG1655 and JM109 [genotype K-12sp rec A − , end A − , F′] (Promega, WI, USA) were utilized as dam- positive control and competency for cloning, respectively.

Techniques: Sequencing

Journal: Frontiers in Public Health

Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

doi: 10.3389/fpubh.2016.00131

Figure Lengend Snippet: Genotypic and growth characteristics displayed by parental and dam- mutant strains of UPEC . (A) Schematic diagram of gene disruption strategy for chromosomal insertion of chloramphenicol resistance gene from pKD3 into dam gene within UPEC chromosome subsequent to λ red recombineering with pKM208. (B) Amplified dam fragment from wild type UPEC strains CFT073 (lane 1) and cured parental strains C119 (lane 2) to produce 1071 bp amplicon. MW is 1 kb DNA ladder (Bioneer Corporation, Republic of Korea) and −ve is negative control. (C) PCR screening of UPEC candidates for dam mutation observed as 1323 bp products using primers UR427 and UR428. MW is a 1 kb Plus DNA ladder (Invitrogen, USA). (D) Dam methylation pattern in UPEC CFT073 wild type (lanes 1, 2, 8, 9, 14, 15), C119 wild type (lanes 3, 4, 10, 11, 16, 17), and E. coli K-12 substrain MG1655 (5, 12, 18) strains subsequent to digestion with Mbo I, Sau 3AI, and Dpn I. The negative control (7, 13, 19) and 1 kb Plus DNA ladder (MW) are also shown. (E) Dam methylation pattern in UPEC dam mutants CFT073 (lanes 1, 2, 3, 8, 9, 10, 15, 16, 17) and C119 wild-type (lanes 4, 5, 6, 11, 12, 13, 18, 19) subsequent to digestion with Sau 3AI, Mbo I, and Dpn I. The negative control (lanes 7, 14) and 1 kb Plus DNA ladder (MW) are also shown. (F) Growth curve (CFU/milliliter versus time) for UPEC strains CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam .

Article Snippet: In addition, the laboratory E. coli strain MG1655 and JM109 [genotype K-12sp rec A − , end A − , F′] (Promega, WI, USA) were utilized as dam- positive control and competency for cloning, respectively.

Techniques: Mutagenesis, Disruption, Amplification, Negative Control, Methylation

Journal: Frontiers in Public Health

Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

doi: 10.3389/fpubh.2016.00131

Figure Lengend Snippet: Antimicrobial susceptibility responses of E. coli wild type (wt), cured (c), dam mutant, and pGEMQA-bearing strains subjected to the sensititre substrate-in-well GNUR2F Gram-negative MIC plate test .

Article Snippet: In addition, the laboratory E. coli strain MG1655 and JM109 [genotype K-12sp rec A − , end A − , F′] (Promega, WI, USA) were utilized as dam- positive control and competency for cloning, respectively.

Techniques: Mutagenesis

Journal: Frontiers in Public Health

Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

doi: 10.3389/fpubh.2016.00131

Figure Lengend Snippet: Phenotypic influence of Dam on P fimbriae . (A) PCR screening for pap EF in UPEC strains cC119 (lane 4), CFT073 (lane 5), and cU155 (lane 6). The 100-bp molecular weight marker (Invitrogen), negative control and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 1 and 2, respectively. (B) PCR screening for pap I– pap B intergenic regulatory region in UPEC strains from UPEC strains cC119 (lane 2), CFT073 (lane 3), and cU155 (lane 4). The 1-kb plus molecular marker (Invitrogen, CA, USA), negative control, and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 2 and 5, respectively. (C) Schematic representation of pSAMS1 recombinant plasmid containing cloned pap IB insert within pCRII–TOPOII vector. (D) Dam methylation patterns for pap I-B regulatory region. Sau 3AI (lane 2), Mbo I (lane 3), and Dpn I (lane 4) digests of pSAMS2 isolated from cC119 are shown. MW represents the 1 kb Plus molecular marker (Invitrogen). An undigested pap IB fragment (lane 5) is also represented. (E) Semi-quantitative (sq) RT-PCR for pap I expression in cC119 (lane 1), cC119 Δ dam (lane 2), CFT073 wild-type (lane 3) and CFT073 Δ dam (lane 4). The 1 kb Plus molecular marker (Invitrogen) and amplified chromosomal DNA for UPEC strains cC119 and CFT073 are shown in lanes MW, 5 and 6, respectively.

Article Snippet: In addition, the laboratory E. coli strain MG1655 and JM109 [genotype K-12sp rec A − , end A − , F′] (Promega, WI, USA) were utilized as dam- positive control and competency for cloning, respectively.

Techniques: Molecular Weight, Marker, Negative Control, Positive Control, Recombinant, Plasmid Preparation, Clone Assay, Methylation, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification

Journal: Frontiers in Public Health

Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

doi: 10.3389/fpubh.2016.00131

Figure Lengend Snippet: Data representing mean ± SD of separate experiments for attachment assay of CFT073 wt, CFT073 Δ dam mutant, cC119, and cC119 Δ dam mutant to HEK-293 kidney and HTB-9 bladder cells .

Article Snippet: In addition, the laboratory E. coli strain MG1655 and JM109 [genotype K-12sp rec A − , end A − , F′] (Promega, WI, USA) were utilized as dam- positive control and competency for cloning, respectively.

Techniques: Mutagenesis