Journal: Nature Communications

Article Title: Interaction between transcribing RNA polymerase and topoisomerase I prevents R-loop formation in E. coli

doi: 10.1038/s41467-022-32106-5

Figure Lengend Snippet: Circular maps show fold enrichment profiles of EcTopoI (ChIP-Seq, light orange; Topo-Seq, cyan and dark orange for two separate DNA strands), RNAP (ChIP-Seq, green), and DNA gyrase (Topo-Seq, dark-red). Additionally, GC-content (%, purple) and mean expression levels (FPKM, RNA-Seq, gray) for annotated TUs (inner blue segments) are also shown. Blue asterisks indicate positions of rRNA operons on the innermost orange ring representing E. coli DY330 genome. The numbers on the outside of the orange ring indicate genome coordinates in megabase pairs (Mbs). Three gaps around ~0.3, ~0.8, and ~1.2 Mb correspond to deletions in the E. coli DY330 genome relative to the E. coli W3110 reference genome. Insets provide a zoom-in view of representative regions with high EcTopoI signals. Coordinates in kb are indicated on top of each inset. For ChIP-Seq, fold enrichment is given relative to the input sample in all figures. The maps were constructed with the Circos tool , and the insets were prepared using IGV .

Article Snippet: DNA fragments of dps , potF , or nuoN were PCR-amplified from E. coli DY330 genomic DNA (for primers, see Supplementary Table ) and purified by GeneJET Gel Extraction and DNA cleanup micro kit (PCR cleanup protocol, Thermo Fisher).

Techniques: ChIP-sequencing, Expressing, RNA Sequencing, Construct

Journal: Nature Communications

Article Title: Interaction between transcribing RNA polymerase and topoisomerase I prevents R-loop formation in E. coli

doi: 10.1038/s41467-022-32106-5

Figure Lengend Snippet: a Representative regions of the E. coli chromosome with EcTopoI ChIP-Seq peaks matching the EcTopoI Topo-Seq TCSs ( dps , potF ) and a region lacking EcTopoI-binding and activity ( nuoN ). EcTopoI cleavage activity is shown strand-specifically. A control, non-induced culture, is shown. Positions of regions used for ChIP-qPCR and affinity measurements are indicated by gray rectangles. b Affinity of purified EcTopoI to three amplified genomic regions from the panel a measured by EMSA. Red asterisks mark the lowest concentration of EcTopoI at which a gel-shift was detected. c Metagene plot of EcTopoI ChIP-Seq enrichment (untreated condition, black curve) and EcTopoI Topo-Seq cleavage signal (blue and red curves for coding and template strands, respectively). Confidence bands around the mean metagene signal are represented by ±SEM. Analysis was performed for the HETU set. Regions used for further quantification of enrichment in panel d are shown by colored areas on the plot. d Mean EcTopoI cleavage signal in different regions relative to HETUs. Means were compared by a two-sided Welch t -test. P values <4e-3 are indicated by asterisks and Bonferroni correction for multiple testing was applied. Bars represent mean values ±SEM, n = 200 TUs. e Metagene plot of EcTopoI Topo-Seq cleavage activity for all TUs, LETU, and HETU sets. Cleavage is shown strand-specifically. f Logo of EcTopoI-binding motif identified in sequences under the ChIP-Seq peaks. A motif is shown in both orientations. g EcTopoI cleavage motif identified by alignment of TCSs. The cleavage site between nucleotides −1 and 1 is indicated by a dashed line. The cleavage signal (N3E) is plotted below. h Affinity of purified EcTopoI to oligonucleotides measured with EMSA. The binding of forward (left) and reverse-complement (right) oligonucleotides is shown. Red asterisks mark the lowest concentration of EcTopoI at which a gel-shift was detected (Supplementary Table ). i Affinity of purified EcTopoI to oligonucleotides measured with MST. Data were represented as mean values ± SEM, minimum of three independent MST experiments were performed. For source data, see Table in the Source Data file. j Cleavage of oligonucleotides by purified EcTopoI. A control with EcTopoI inactivated by a high temperature is indicated with a boxed + sign. Cleavage products are marked with a blue arrow. For ChIP-Seq, fold enrichment is given relative to the input sample.

Article Snippet: DNA fragments of dps , potF , or nuoN were PCR-amplified from E. coli DY330 genomic DNA (for primers, see Supplementary Table ) and purified by GeneJET Gel Extraction and DNA cleanup micro kit (PCR cleanup protocol, Thermo Fisher).

Techniques: ChIP-sequencing, Binding Assay, Activity Assay, Control, ChIP-qPCR, Affinity Purification, Amplification, Concentration Assay, Gel Shift, Purification

Journal: Nature Communications

Article Title: Interaction between transcribing RNA polymerase and topoisomerase I prevents R-loop formation in E. coli

doi: 10.1038/s41467-022-32106-5

Figure Lengend Snippet: a Growth curves for E. coli DY330 topA -SPA harboring pCA24 GFP (i), pCA24 14 kDa CTD (ii), or pCA24 topA (iii) plasmids. Data for induced (+IPTG 1 mM) and non-induced (−IPTG) cultures are shown. Shade represents a 0.95 confidential interval of the mean of three biological replicates. Gray lines mark aliquots collection for plasmid extraction. b Quantification of cell length in CTD or GFP producing cultures (left). Cells >10 µm are collected into an overflowing bin. The number of cells is indicated in parentheses. Representative fields are shown on the right. c Graphical representation of truncated versions of a topA gene constructed by recombineering in E. coli BW25113. d Growth curves of E. coli BW25113 strains with truncated versions of topA and the wild-type control (left). Shade represents 0.95 confidential intervals of the mean. Quantification of doubling time for exponential regions of growth curves (right). e Quantification of cell length for E. coli BW25113 strains with truncated versions of topA and the wild-type (left). Vertical dashed line marks 2*mean cell length for wild-type. Representative fields are shown on the right. f Mutations in gyrase genes ( gyrA , gyrB ) found in E. coli BW25113 topAΔ30 clones. An asterisk indicates amplification of a chromosomal region containing TopoIV genes; clones lacking compensatory mutations are highlighted in green. g Supercoiling of pCA24 GFP (i), pCA24 topA (ii), and pCA24 14 kDa CTD (iii) plasmids extracted from exponentially growing E. coli DY330 topA -SPA. Time-points correspond to panel a . Supercoiling of pCA24 GFP (iv) plasmid extracted from exponentially growing E. coli BW25113 topAΔ 30 (time-course, on the left) or E. coli BW25113 wt (two rightmost lanes). (v) Supercoiling level of the pCA24 GFP plasmid extracted from overnight cultures of different clones of E. coli BW25113 topA mutants and from the wild-type control. Clone numbers correspond to panel f . Nic - nicked plasmid, L - linear plasmid, −sc - negatively supercoiled plasmid, HCF - hypercompacted plasmid. h Metagene plots of normalized strand-specific read coverage depth obtained in DRIP-Seq experiments for E. coli DY330 topA -SPA for HETU (upper panel, rRNA operons were excluded) and LETU (lower panel) sets. Schematic TUs are shown below. Data for CTD-/Rif- condition are shown with a dashed line, coverage depths for the coding and template strands are indicated by dark-red and dark-blue fillings, respectively. Data for CTD+/Rif− condition are shown with a solid line, coverage depths for the coding, and template strands are indicated by light-red and light-blue fillings, respectively. i DRIP-Seq data for pCA24 14 kDa CTD for CTD+/Rif− and CTD+/Rif+ conditions and corresponding RNase HI-treated controls. Coverage depths for “−” and “+” strands are shown in light-blue and light-red, respectively. A linearized map of the plasmid is shown below. Source data are provided as a Source Data file.

Article Snippet: DNA fragments of dps , potF , or nuoN were PCR-amplified from E. coli DY330 genomic DNA (for primers, see Supplementary Table ) and purified by GeneJET Gel Extraction and DNA cleanup micro kit (PCR cleanup protocol, Thermo Fisher).

Techniques: Plasmid Preparation, Extraction, Construct, Control, Clone Assay, Amplification

Journal: Nature Communications

Article Title: Interaction between transcribing RNA polymerase and topoisomerase I prevents R-loop formation in E. coli

doi: 10.1038/s41467-022-32106-5

Figure Lengend Snippet: Average normalized enrichment of TopoI, DNA gyrase, and RNAP over transcription units of E. coli ( a , “open” model) and Mycobacterium ( b , “closed” model). Graphical representations of twin-domain sub-models are shown below. ChIP-Seq data for M. tuberculosis MtbRNAP, MtbGyrase, and M. smegmatis MsmTopoI was taken from publicly available datasets , , . c Other “semi-open” hypothetical variations of the twin-domain model, based on the interaction of key topoisomerases (TopoI, DNA gyrase) with RNAP and their activity within a complex. For ChIP-Seq, fold enrichment is given relative to the input sample.

Article Snippet: DNA fragments of dps , potF , or nuoN were PCR-amplified from E. coli DY330 genomic DNA (for primers, see Supplementary Table ) and purified by GeneJET Gel Extraction and DNA cleanup micro kit (PCR cleanup protocol, Thermo Fisher).

Techniques: ChIP-sequencing, Activity Assay

Journal: Nature Communications

Article Title: RNA-guided RNA silencing by an Asgard archaeal Argonaute

doi: 10.1038/s41467-024-49452-1

Figure Lengend Snippet: a HrAgo1 associates with 5’ phosphorylated (5’ P) small RNAs in vivo from E. coli . Nucleic acids that co-purified with HrAgo1 were [γ- 32 P] labeled, treated with RNase A or DNase I, and resolved on a denaturing gel (15% polyacrylamide 7 M urea). nt: nucleotides. b Length distribution of small RNAs associated with HrAgo1 as determined by small RNA sequencing. c Small RNAs associated with HrAgo1 have a bias for uracil bases at the 5’ end. d Sequences of guide and target oligonucleotides used in in vitro cleavage assays. e HrAgo1 cleaves ssRNA (but not ssDNA) targets with ssRNA guides, and ssDNA guides at lower efficiency, in the presence of Mg 2+ . HrAgo1 was incubated with ssDNA or ssRNA guides and Cy5-labeled ssDNA or RNA targets. Cy5-labeled cleavage products were resolved through denaturing (7 M urea) polyacrylamide gel electrophoresis and visualized by fluorescence imaging. Both ssRNA and ssDNA targets are 45nt. The HrAgo1-bound RNA extraction and digestion was carried out once; the results of the cleavage assays were confirmed by at least three repetitions.

Article Snippet: A plasmid suitable for expression of a HrAgo1 catalytic double-mutant (D585A & E623A; HrAgo1 DM ) was generated by Quikchange Site-Directed Mutagenesis using primers oPB199 and oPB201 for D585A and oPB200 and oPB198 for E623A, using E. coli strain NEB 5-alpha (New England Biolabs) (Table ). pX-sfGFP vector was a kind gift from Prof. Jae-Sung Woo (Korea University, South Korea).

Techniques: In Vivo, Purification, Labeling, RNA Sequencing Assay, In Vitro, Incubation, Polyacrylamide Gel Electrophoresis, Fluorescence, Imaging, RNA Extraction

Journal:

Article Title: Characterization of the 4-Carboxy-4-Hydroxy-2-Oxoadipate Aldolase Gene and Operon Structure of the Protocatechuate 4,5-Cleavage Pathway Genes in Sphingomonas paucimobilis SYK-6

doi: 10.1128/JB.185.1.41-50.2003

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: To determine the N-terminal amino acid sequence, the cell extract of E. coli BL21(DE3) harboring pETK was subjected to SDS-15% PAGE and electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, Calif.).

Techniques: Plasmid Preparation, Mutagenesis, Clone Assay, Expressing

Journal: eLife

Article Title: Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome

doi: 10.7554/eLife.42549

Figure Lengend Snippet:

Article Snippet: 5x KCM buffer (0.5M KCl, 0.15M CaCl 2 , 0.25M MgCl 2 ), Escherichia coli strain K12 CJ236 (NEB, E4141), SOC outgrowth medium (ThermoFisher Scientific, 15544034), LB-agar plates supplemented with 100 µg/ml ampicillin (Roth, K029.2).

Techniques: Recombinant, Gel Extraction, Plasmid Preparation, Electroporation, Software