Journal: Nucleic Acids Research

Article Title: Bacteria sense the antibiotic rifampicin through a widespread dual-promoter based alarm system

doi: 10.1093/nar/gkaf1407

Figure Lengend Snippet: Effect of HelD on RIF resistance of B. subtilis . (A) Quantitative mass spectrometry analysis of B. subtilis ( Bsu ) wt strain (LK2711) in the presence vs absence of sub-MIC concentration (0.03 μg/ml) of RIF. The analysis was done from six biological replicates. The abundance of individual proteins was compared by a two-tailed Student’s t -test. The permutation-based FDR was used as an adjustment of the p -value. The enrichment is shown with a volcano plot (−log 10 p -value > 2 on the y -axis, protein enrichments > 1.5 on the x -axis). Significantly enriched (upregulated) proteins are shown on the right hand side as light orange dots, significantly downregulated proteins on the left hand side as black dots. The identity of the two most enriched proteins is indicated (HelD, Pps); for the full list, see . (B), (C) Growth of strains in liquid LB medium in the absence (-RIF) or presence (+RIF) of sub-MIC concentration (0.03 μg/ml) of RIF. The strains were inoculated in a 24-well plate, and the OD 600 was measured every 30 min for ∼16 h. A representative result is shown from a total of three independent growth experiments. Black dots, wt strain (LK2711); grey triangles, Δ helD strain (LK2840); orange squares, helD overexpression strain (LK2934); light orange diamonds, helD complementation strain (LK2935). (D), (E) Growth of strains on solid LB medium in the absence (-RIF) or presence (+RIF) of sub-MIC concentration (0.03 μg/ml) of RIF. A representative result is shown; the experiment was repeated three times with identical results. (F) A representative SDS-PAGE of pull-down of FLAG-tagged RNAP (LK3132) in the absence (-RIF) or presence (+RIF) of sub-MIC concentration (0.03 μg/ml) of RIF. The identity of HelD was determined by mass spectrometry. The experiment was performed in three biological replicates.

Article Snippet: RM RNA was a fragment of 16S rRNA from M. smegmatis prepared by in vitro T7 RNAP (NEB)-dependent transcription from a DNA fragment prepared by PCR with primers #1281 and #1282 and M. smegmatis mc 2 155 chromosomal DNA purified with Mini Bacterial Kit (Invitrogen) from LK2980.

Techniques: Mass Spectrometry, Concentration Assay, Two Tailed Test, Over Expression, SDS Page

Journal: Nucleic Acids Research

Article Title: Bacteria sense the antibiotic rifampicin through a widespread dual-promoter based alarm system

doi: 10.1093/nar/gkaf1407

Figure Lengend Snippet: Role of Bsu_ HelD N-terminal domain in RIF resistance. (A) A scheme of Bsu_ HelD domain organization with acidic aa in the N-terminal domain is indicated. The N-terminal domain [also known as secondary channel arm (SCA)] is indicated and colored ruby-brown. Amino acid residues at the domain borders are indicated. (B) A structural analysis of B. subtilis RNAP showing deformation of the expected RIF binding pocket in the presence of HelD. Teal, RNAP β-E521 (+HelD); dark blue, RNAP β-E521 (-HelD); ruby brown, semitransparent surface of the HelD N-terminal domain with the peptide backbone indicated within; orange, RIF; magenta, catalytic Mg 2+ .The black arrow shows repositioning of β-E521 forced by the HelD N-terminal domain. The model is based on the superposition of structures aligned by the β subunit: Bsu RNAP elongation complex (PDB ID: 6WVJ,), Bsu RNAP with HelD (PDB ID: 6ZFB,[ – ]). The expected position of rifampicin is adopted from the Mycobacterium tuberculosis ( Mtb ) RNAP complex (PDB ID: 5UHC,). (C) A close-up of the wt Bsu_ HelD N-terminal domain tip interacting with Bsu RNAP (PDB ID: 6WVK,) with the three acidic residues and their interactions with RNAP indicated with blue dashed lines. RNAP is represented as a semitransparent grey surface. The Mg 2+ ion in the RNAP active site is shown as the magenta sphere. The N-terminal domain of HelD is shown in ruby brown, secondary structure representation with relevant aa residues shown as sticks. RNAP is shown as a grey surface. Their interaction partners (in the case of wt) from the β’ domain are shown as grey sticks and marked. The approximate position of RIF (carbon in orange, represented as sticks and marked) in the active site is adopted from the complex with Mtb RNAP (PDB ID: 5UHC,). The graphics were created using PyMOL. (D) Growth of indicated strains in liquid LB medium in the absence (-RIF) or presence (+RIF) of sub-MIC concentration (0.03 μg/ml) of RIF. The strains were inoculated in a 24-well plate, and the OD 600 was measured every 30 min for ∼ 16 h. A representative result from three growth experiments is shown. Black dots, wt (LK2711); grey triangles, Δ helD (LK2840); brown squares, HelDΔN (HelD lacking the N-terminal domain; LK3772); red diamonds, HelDmutTIP (D56A, D57A, E60A; LK3784). (E) Multiple round transcriptions from the P veg (LK1177) promoter were performed in the absence or presence of HelD or HelDΔN with increasing amounts of RIF. The 1:16 RNAPΔHelD:HelD or RNAPΔHelD:HelDΔN ratio was used in protein reconstitution. Transcription at zero RIF was set as 1 for both -/+ HelD/HelDΔN to facilitate visualization of the changes. The bars show averages of three independent experiments, the dots are individual experimental data, and the error bars show \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $ \pm $\end{document} SD. p -values were calculated using a two-tailed, unpaired t -test and indicated in the graph – this is also used in subsequent figures. Primary data are shown below the graph.

Article Snippet: RM RNA was a fragment of 16S rRNA from M. smegmatis prepared by in vitro T7 RNAP (NEB)-dependent transcription from a DNA fragment prepared by PCR with primers #1281 and #1282 and M. smegmatis mc 2 155 chromosomal DNA purified with Mini Bacterial Kit (Invitrogen) from LK2980.

Techniques: Binding Assay, Concentration Assay, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Bacteria sense the antibiotic rifampicin through a widespread dual-promoter based alarm system

doi: 10.1093/nar/gkaf1407

Figure Lengend Snippet: Model of regulation of helD expression by RIF and bioinformatically identified dual promoter arrangements (2-promoter systems) across bacteria. (A) Model of regulation of helD expression by RIF. In the absence of RIF (left), transcription predominantly occurs from the stronger P anti-helD promoter, which interferes with the weaker P helD promoter, allowing only occasional transcription from P helD . When RIF at sub-MIC levels is present, it primarily arrests RNAP at P anti-helD and stops the interference. RNAP (without RIF) from P helD has then sufficient time to bind and escape from the promoter. Once initiated, the elongating RNAP upon collision dislodges the arrested RNAP from P anti-helD (middle) and transcribes the helD gene (right). For more details, see the text. (B) Activity and inducibility (activity +RIF/-RIF) of P helD FULL and P helD CORE promoter- lacZ constructs in wt [P helD FULL (LK3005), P helD CORE (LK2970)] and RIF-resistant [RIF R , rpoB H482Y; P helD FULL (LK4439), PhelD CORE (LK4437)] genetic backgrounds in the exponential phase. Activity of P helD FULL wt in the absence of RIF was set as 1. The bars are averages from two independent experiments, the dots are individual experimental data, and the error bars show the range. (C) Activity and inducibility (activity +RIF/-RIF) of P helD FULL (LK3005), P helD CORE (LK2970), and P helD del17 (LK3114) promoter- lacZ constructs in the exponential phase. Activity of P helD FULL wt in the absence of RIF was set as 1. The bars are averages from three independent experiments, the dots are individual experimental data, and the error bars show ± SD. (D) Taxonomy tree overview of bacterial families in which two-promoter combinations, sequentially similar to those identified upstream of B. subtilis helD and pps , were identified. The NCBI taxonomy tree was trimmed to include only families that were present in the used RefSeq dataset (see Zenodo repository). The bars associated with individual leaves show the number of genome assemblies (log 2 ) in a particular leaf (family) in which the two-promoter system was found (outward part of the bar, light green sector) or not (inward part of the bar, light red sector). The larger the bars, the more assemblies within that taxonomic unit. The tree leaf labels are colored if they occur in the top seven most occurring taxonomic classes by genome assembly count in our dataset. (E) The pie-chart shows the top four GO slim molecular function terms by the sum of per-protein weighted occurrence of the term in the proteins found downstream of candidate two-promoter system sites. Two additional fractions are also shown: (i) GO terms that were not mapped to any GO slim term used (labeled “not mapped”) and (ii) GO slim terms different from the top four terms (labeled “other”).

Article Snippet: RM RNA was a fragment of 16S rRNA from M. smegmatis prepared by in vitro T7 RNAP (NEB)-dependent transcription from a DNA fragment prepared by PCR with primers #1281 and #1282 and M. smegmatis mc 2 155 chromosomal DNA purified with Mini Bacterial Kit (Invitrogen) from LK2980.

Techniques: Expressing, Bacteria, Activity Assay, Construct, Labeling