Journal: Nucleic Acids Research

Article Title: A-to-I mRNA editing in bacteria can affect protein sequence, disulfide bond formation, and function

doi: 10.1093/nar/gkaf584

Figure Lengend Snippet: A-to-I mRNA editing can affect protein sequence and function in bacteria as evidenced by the case of HokB. ( A ) Adenosine is deaminated to inosine, which is similar to guanosine in its base-pair properties. ( B ) A-to-I mRNA editing by TadA in E. coli occurs in 80%–90% of endogenously expressed hokB transcripts at the logarithmic phase, and is assumed to recode a tyrosine to a cysteine codon at position 29 of HokB. ( C ) Growth analysis of E. coli (Top10-DH10B) co-expressing mCherry (control, black) or mCherry-HokB (green), with either GFP-TadA (left panel) or GFP (right panel). The mean and standard error of three biological replicates conducted on different days ( N = 3), each with 21 technical replicates, are shown. The expression of mCherry and HokB was induced from the beginning of the experiment (time point “0”) with 0.2% arabinose from a pBAD vector. Expression of GFP-TadA or GFP was induced with 1 mM IPTG from a pME6032 vector. ( D ) A-to-I mRNA editing identification by next-generation sequencing (NGS; Illumina; amplicon-seq) of plasmid-borne hokB RNA (cDNA) co-overexpressed with GFP-TadA or with GFP alone. Minimum observed reads coverage per sample that passed our quality filters ≥17 515 391. Statistical analysis was conducted using Student’s t -test; P -value <.0001 (****). ( E ) A-to-I mRNA editing validation by Sanger sequencing of plasmid-borne DNA and RNA (cDNA) of hokB when co-overexpressed with GFP-TadA or with GFP. The sequence above the chromatograms represents the gene (DNA) sequence. A black arrow marks the double peak of A and G(I) that was observed only in the cDNA (complementary DNA) samples. Note that the G(I) peak is higher than the A peak when overexpressing GFP-TadA and vice versa when overexpressing only GFP. ( F ) MS/MS spectrum of non-edited (Y29; top) and edited (C29; bottom) HokB peptides found in strains co-overexpressing HokB with GFP (top) or GFP-TadA (bottom). Black arrows mark identified peptides and their mass in the MS/MS spectra that show a mass shift corresponding to tyrosine or cysteine at the edited site. The gray arrow marks an example of a peptide and its mass in the MS/MS spectra that does not include the edited site (same mass). All peptides were discovered with false discovery rate (FDR) ≤ 0.01. The peaks weight, font size, and axis were adjusted from the original figure for better visualization and comparison. A comprehensive mass distribution and the original MS/MS spectra and data can be found in – .

Article Snippet: All experiments in this work used the E. coli Top10 strains (DH10B-WT, DH10B-Δ dsbA , and DH10B-Δ dsbC , a generous gift from the lab of Professor Jan Michiels, KU Leuven), except for the pathogenic bacterial species [enterohemorrhagic E. coli (O157:H7 EDL933), enteropathogenic E. coli (O127:H6 strain E2348/69), uropathogenic E. coli that was isolated from a patient suffering from urinary tract infection, and Shigella sonnei (ATCC 25931)].

Techniques: Sequencing, Bacteria, Expressing, Control, Plasmid Preparation, Next-Generation Sequencing, Amplification, Biomarker Discovery, Tandem Mass Spectroscopy, Comparison

Journal: Nucleic Acids Research

Article Title: A-to-I mRNA editing in bacteria can affect protein sequence, disulfide bond formation, and function

doi: 10.1093/nar/gkaf584

Figure Lengend Snippet: DNA-encoded cysteine residues are essential for the toxicity of edited HokB. ( A ) The protein sequence of non-edited and edited HokB according to their respective transcript. DNA-coded cysteines are shown in bold. ( B ) A description of the different plasmids containing different versions of HokB used in the growth assay is presented. ( C ) Growth analysis of E. coli (Top10-DH10B) WT strain expressing the HokB (Y29#, green), non-edited HokB (Y29, blue), and edited HokB (C29, red) fused to mCherry reporter protein (N-terminus) from the plasmid shown in panel (B). As a reference control, a plasmid harboring only mCherry was used (black). As previously reported , when highly expressed, edited HokB (C29) induces the highest level of toxicity. ( D ) Growth analysis as in panel (C), with all three versions of HokB having the C9S substitution. ( E ) Growth analysis as in panel (C), with all three versions of HokB having the C14S substitution. ( F ) Growth analysis as in panel (C), with all three versions of HokB having the C46S substitution. In all growth experiments, protein expression was induced from the beginning of the experiment (time point “0”) with 0.2% arabinose from a pBAD vector. The mean and standard error of three biological replicates conducted on different days ( N = 3), each with 21 technical replicates, are shown.

Article Snippet: All experiments in this work used the E. coli Top10 strains (DH10B-WT, DH10B-Δ dsbA , and DH10B-Δ dsbC , a generous gift from the lab of Professor Jan Michiels, KU Leuven), except for the pathogenic bacterial species [enterohemorrhagic E. coli (O157:H7 EDL933), enteropathogenic E. coli (O127:H6 strain E2348/69), uropathogenic E. coli that was isolated from a patient suffering from urinary tract infection, and Shigella sonnei (ATCC 25931)].

Techniques: Sequencing, Growth Assay, Expressing, Plasmid Preparation, Control

Journal: Nucleic Acids Research

Article Title: A-to-I mRNA editing in bacteria can affect protein sequence, disulfide bond formation, and function

doi: 10.1093/nar/gkaf584

Figure Lengend Snippet: In vivo disulfide bond formation is essential for the toxicity of the edited HokB. ( A ) Growth analysis of an E. coli Δ dsbA strain that expresses one of three versions of HokB, fused to mCherry from an inducible plasmid. As a reference control, we used a plasmid encoding only mCherry. ( B ) Growth analysis as in panel (A), but with overexpressing DsbA from a second plasmid (pME6032). ( C ) Growth analysis, as in panel (B), using an empty plasmid (pME6032 with no dsbA insert).

Article Snippet: All experiments in this work used the E. coli Top10 strains (DH10B-WT, DH10B-Δ dsbA , and DH10B-Δ dsbC , a generous gift from the lab of Professor Jan Michiels, KU Leuven), except for the pathogenic bacterial species [enterohemorrhagic E. coli (O157:H7 EDL933), enteropathogenic E. coli (O127:H6 strain E2348/69), uropathogenic E. coli that was isolated from a patient suffering from urinary tract infection, and Shigella sonnei (ATCC 25931)].

Techniques: In Vivo, Plasmid Preparation, Control

Journal: Nucleic Acids Research

Article Title: A-to-I mRNA editing in bacteria can affect protein sequence, disulfide bond formation, and function

doi: 10.1093/nar/gkaf584

Figure Lengend Snippet: Western blot analysis supports that A-to-I mRNA editing mediates an intramolecular disulfide bond between C29 and C46 in HokB. ( A ) Western blot of membrane enriched protein fraction of E. coli (Top10-DH10B) WT strain expressing either mCherry only (control; black) or the HokB (Y29#, green), non-edited HokB (Y29, blue), and edited HokB (C29, red) fused to mCherry (N-terminus) from the plasmid shown in Fig. . ( B ) Same as panel (A) but with the C9S substitution in the different expressed HokB versions. ( C ) Same as panel (A) but with the C14S substitution in the different expressed HokB versions. ( D ) Same as panel (A) but with the C46S substitution in the different expressed HokB versions.

Article Snippet: All experiments in this work used the E. coli Top10 strains (DH10B-WT, DH10B-Δ dsbA , and DH10B-Δ dsbC , a generous gift from the lab of Professor Jan Michiels, KU Leuven), except for the pathogenic bacterial species [enterohemorrhagic E. coli (O157:H7 EDL933), enteropathogenic E. coli (O127:H6 strain E2348/69), uropathogenic E. coli that was isolated from a patient suffering from urinary tract infection, and Shigella sonnei (ATCC 25931)].

Techniques: Western Blot, Membrane, Expressing, Control, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: A-to-I mRNA editing in bacteria can affect protein sequence, disulfide bond formation, and function

doi: 10.1093/nar/gkaf584

Figure Lengend Snippet: Lower levels of edited HokB induce early entrance to the stationary phase. ( A ) Growth analysis of WT E. coli as described in Fig. with 1:1000 lower arabinose concentration. The expression of mCherry and HokB was induced from the beginning of the experiment (time point “0”) with 0.0002% arabinose from a pBAD vector. Black and white triangles correspond to sampling times for panel (B). ( B ) CFU counts at 5 and 6 h of the beginning of growth. Notice that there are fewer CFUs when edited HokB is expressed, with similar numbers at 5 and 6 h after growth. The mean and standard error of four biological replicates conducted on different days ( N = 4) are shown. Statistical analysis was conducted using Student’s paired t -test followed by Benjamini–Hochberg FDR correction: P -value ≤.05 (*).

Article Snippet: All experiments in this work used the E. coli Top10 strains (DH10B-WT, DH10B-Δ dsbA , and DH10B-Δ dsbC , a generous gift from the lab of Professor Jan Michiels, KU Leuven), except for the pathogenic bacterial species [enterohemorrhagic E. coli (O157:H7 EDL933), enteropathogenic E. coli (O127:H6 strain E2348/69), uropathogenic E. coli that was isolated from a patient suffering from urinary tract infection, and Shigella sonnei (ATCC 25931)].

Techniques: Concentration Assay, Expressing, Plasmid Preparation, Sampling

Journal: Nucleic Acids Research

Article Title: A-to-I mRNA editing in bacteria can affect protein sequence, disulfide bond formation, and function

doi: 10.1093/nar/gkaf584

Figure Lengend Snippet: A-to-I mRNA editing of hokB is conserved in pathogenic E. coli and Shigella strains. Sanger sequencing of the endogenous hokB gene and its mRNA from the same sample of non-pathogenic E. coli (used throughout this work), enterohemorrhagic E. coli , enteropathogenic E. coli , uropathogenic E. coli , and Shigella sonnei . A black arrow marks the double peak of A and G(I) observed only in the cDNA samples. Note that the G(I) peak (black) is higher than the A peak (green) in most samples. Sequences were aligned to the E. coli reference genome ( NC_000913.3 ) and positions 1491982–1491990 are shown. See for exact genomic coordinates of the full-length hokB gene in each species.

Article Snippet: All experiments in this work used the E. coli Top10 strains (DH10B-WT, DH10B-Δ dsbA , and DH10B-Δ dsbC , a generous gift from the lab of Professor Jan Michiels, KU Leuven), except for the pathogenic bacterial species [enterohemorrhagic E. coli (O157:H7 EDL933), enteropathogenic E. coli (O127:H6 strain E2348/69), uropathogenic E. coli that was isolated from a patient suffering from urinary tract infection, and Shigella sonnei (ATCC 25931)].

Techniques: Sequencing

Journal: Journal of Virology

Article Title: Molecular cloning and host range analysis of three cytomegaloviruses from Mastomys natalensis

doi: 10.1128/jvi.02147-24

Figure Lengend Snippet: STAR cloning of MnatCMV2. ( A ) Linearized cloning vector and vDNA from MnatCMV2 virions were used to transform yeast spheroplasts. Homology hooks (60 bp, blue and green) for recombination were homologous to regions 110 bp upstream and downstream from the vDNA termini. Circular DNA consisting of the vector and the long unique region from vDNA was isolated from yeast and transferred to E. coli . ( B ) BAC DNA originating from two independent yeast clones isolated from E. coli clones was digested with NotI or HindIII and analyzed by gel electrophoresis with vDNA as a reference. M, DNA size markers. Vector-vDNA junction fragments (arrowheads) and genome terminal fragments (asterisks) absent from the circular BAC clones are indicated.

Article Snippet: A 5–10 μL yeast-derived DNA solution was used to transform E. coli strain DH10B by electroporation using a BioRad Gene Pulser II.

Techniques: Cloning, Plasmid Preparation, Isolation, Clone Assay, Nucleic Acid Electrophoresis

Journal: bioRxiv

Article Title: Homology-Based Enzymatic Assembly of Modular T7 Phage Genome

doi: 10.1101/2025.04.24.650493

Figure Lengend Snippet: Assembled phage containing landing pad insert rebooted in DH10B. (A) The linear genome phage containing the Sapl and EcoRl containing landing pad transformed in DH10B. (B) PCR assessment of in vitro assembly. Gibson Assembly reactions were amplified via PCR using primers spanning the assembly junctions. (C) Genome map of linear assembled phage with location of respective junctions.

Article Snippet: Escherichia coli strain DH10B was obtained from New England Biolabs (C3019I, obtained from New England Biolabs) and used to reboot modified phages.

Techniques: Transformation Assay, In Vitro, Amplification

Journal: bioRxiv

Article Title: Homology-Based Enzymatic Assembly of Modular T7 Phage Genome

doi: 10.1101/2025.04.24.650493

Figure Lengend Snippet: Circular assembly of phage containing landing pad insert. (A) The WT phage genome assembled as a circular DNA transformed into DH10B. (B) PCR assessment of in vitro circular assembly. Gibson Assembly were amplified via PCR using primers spanning the assembly junctions. (C) Genome map of circular assembled phage with location of respective junctions.

Article Snippet: Escherichia coli strain DH10B was obtained from New England Biolabs (C3019I, obtained from New England Biolabs) and used to reboot modified phages.

Techniques: Transformation Assay, In Vitro, Amplification

Journal: bioRxiv

Article Title: Homology-Based Enzymatic Assembly of Modular T7 Phage Genome

doi: 10.1101/2025.04.24.650493

Figure Lengend Snippet: Dual Sapl containing landing pad alters mRNA structure and phage growth. The red and green box indicate the location of the recognition sequence of Sapl and respectively with respect to the landing pad insert. (A) Predicted hairpin formation of the WT sequence of gene 2.5 mRNA (B) Predicted hairpin formation of the mutated gene 2.5 mRNA following genome assembly and rebooting in E. coli does not contain Sapl sites. (C) Predicted hairpin formation of gene 2.5 mRNA containing inverted Sapl sites. (D) Predicted hairpin formation of gene 2.5 mRNA containing Sapl and (E) Table with predicted thermodynamic parameters for mRNA structures

Article Snippet: Escherichia coli strain DH10B was obtained from New England Biolabs (C3019I, obtained from New England Biolabs) and used to reboot modified phages.

Techniques: Sequencing