Journal: Cell host & microbe
Article Title: Separating Functions of the Phage-Encoded Quorum-Sensing-Activated Antirepressor Qtip
doi: 10.1016/j.chom.2020.01.024
Figure Lengend Snippet: (A) Schematic depicting the phage VP882-encoded QS pathway and the reporter system used in this work. Top: VqmAPhage, when bound to DPO (white hexagon), activates expression of the qtip gene encoding the Qtip antirepressor. Qtip inactivates the cIVP882 repressor, enabling expression of Pq and subsequent Q-mediated host cell lysis. Bottom: The reporter system used in this work to monitor Qtip and cIVP882 activity. Pq is fused to the luciferase operon (lux). Light production is low when the cIVP882 repressor is functional and light production is high when Qtip is active and/or the cIVP882 repressor is non-functional. (B) Upper panel: EMSA analysis of Pq DNA retarded by cIVP882-HALO protein purified alone or in complex with HIS-Qtip. The relative amount of cIVP882-HALO in each lane is indicated (1x = ~12 nM). Lower panel: The reactions from the upper panel were subjected to SDS-PAGE analysis and imaged using a Cy5 filter set to visualize HALO-Alexa660, to which the cIVP882-HALO protein had been conjugated. The molecular weight marker is designated M. (C) Light production from E. coli harboring the Pq-lux reporter (no plasmid) or the Pq-lux reporter and a plasmid encoding WT cIVP882 or the cIVP882DBD*-HALO allele. Relative light units (RLU) were calculated by dividing bioluminescence by OD600. Data represented as mean ± SD with n = 3 biological replicates. (D) Upper panel: Average individual cell images of recombinant E. coli producing Qtip and either, cIVP882-HALO, cIVP882DBD*-HALO, or the HALO tag. HALO-TMR fluorescence intensity is displayed as a red heat map (black and white reflect the lowest and highest intensity, respectively). Dashed lines denote the average cell outlines. Scale bar = 1 μm. Upper left inset: Schematic showing aligned individual cells averaged to produce composite images in the upper panel (n = 20–25 cells per condition, see Methods). In the schematic, the HALO-TMR fluorescence intensity from three representative individual cells harboring Qtip and cIVP882DBD*-HALO is shown in inverted greyscale. Lower panel: Line plots of HALO-TMR fluorescence intensity extracted from individual cell images used to generate the composite images displayed in the upper panel. The distance along the x-axis is relative to the left most edge of each cell. Shaded regions represent ± 1 SD from the mean. See also Figure S1.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Virus Strains V. parahaemolyticus VP882 lysogen BCRC BCRC 80155 E. coli T7Express lysY/I q NEB Cat# C3013I E. coli TOP10 Invitrogen Cat# C404010 E. coli cI857 lysogen (Sussman and Jacob, 1962); gift of Tom Silhavy N/A Chemicals, Peptides, and Recombinant Proteins SNAP-JF 503 Lavis Lab N/A SNAP-Cell 647-SiR NEB Cat# S9102S HALO-TMR Promega Cat# G8251 HALO-Alexa660 Promega Cat# G8471 RecA and RecA Buffer NEB Cat# M0249L ATP-γ-S Sigma Cat# A1388–5MG Novex 6% DNA Retardation Gel Thermo Cat# EC63655BOX SYBR Green I Nucleic Acid Gel Stain Thermo Cat# S7567 4–20% Mini-PROTEAN TGX Protein Gels Bio-Rad Cat# 4561096 GeneMorph II EZClone Domain Mutagenesis Kit Agilent Cat# 200552 Anhydrotetracycline Takara Cat# 631310 Isopropyl β-D-1-thiogalactopyranoside Fisher Scientific Cat# BP1755–10 Q5 High Fidelity Polymerase NEB Cat# M0491L HiFi DNA assembly Mix NEB Cat# E2621L Ni-NTA Superflow Resin Qiagen Cat# 30430 Critical Commercial Assays Synergy Neo2 Multi-Mode Reader BioTek N/A BioSpa8 Automated Incubator BioTek N/A ImageQuant LAS4000 GE N/A Leica White Light Laser Confocal Microscope TCS SP8 Leica Microsystems N/A Superdex 200 Increase 10/300 GL GE Healthcare Cat# 28990944 HiTrap 5 ml Heparin HP GE Healthcare Cat# 17040701 AKTA FPLC System GE Healthcare N/A Deposited Data Raw data This paper, Zenodo (10.5281/zenodo.3576389) https://doi.org/10.5281/zenodo.3576389 Oligonucleotides Primers used for plasmid construction This study, IDT Table S2 Primers used for construction of EMSA probe This study, IDT Table S2 dsDNA fragments (gBlocks) This study, IDT Table S2 Recombinant DNA Reporter and cI expression constructs This study Table S3 cI VP882 R11x -HALO-HIS mutants This study Table S3 SNAP-Qtip mutants and Ant constructs This study Table S3 Software and Algorithms Prism v6 GraphPad https://www.graphpad.com/scientific-software/prism/ FIJI v1.52p FIJI http://fiji.sc/ LASX v3.5.5.19976 Leica Microsystems https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/ RStudio Desktop 1.2.5033 RStudio / The R Foundation https://rstudio.com Open in a separate window KEY RESOURCES TABLE The phage antirepressor Qtip sequesters its partner cI repressor to the cell poles Qtip bound repressor is inhibited for DNA-binding and autoproteolysis Qtip recognizes the N-terminal domain of the cI repressor Repressor recognition and polar localization are separable properties of Qtip
Techniques: Expressing, Lysis, Activity Assay, Luciferase, Functional Assay, Purification, SDS Page, Molecular Weight, Marker, Plasmid Preparation, Recombinant, Fluorescence
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