r3406 strain (Schering-Plough corporation)
86
Structured Review
Schering-Plough corporation
r3406 strain
R3406 Strain, supplied by Schering-Plough corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/r3406 strain/product/Schering-Plough corporation
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
R3406 Strain, supplied by Schering-Plough corporation, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/r3406 strain/product/Schering-Plough corporation
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
r3406 strain - by Bioz Stars,
2025-01
86/100 stars
Images
1) Product Images from "The RecA-directed recombination pathway of natural transformation initiates at chromosomal replication forks in Streptococcus pneumoniae"
Article Title: The RecA-directed recombination pathway of natural transformation initiates at chromosomal replication forks in Streptococcus pneumoniae
Journal: bioRxiv
doi: 10.1101/2022.08.04.502747
Figure Legend Snippet: (A) Schematic representation of the steps involved in pneumococcal transformation (1) DNA capture. DNA is captured by a long transformation pilus, formed from the ComG proteins, and transferred to the DNA receptor ComEA. (2) ssDNA uptake. ComEA transfers the DNA to EndA, which degrades one strand of DNA, with the remaining single strand pulled through the ComEC transformation pore by the ComFA ATPase. (3) ssDNA protection and DprA-mediated RecA filamentation. Once internalised, ssDNA interacts with SsbB and the RMP DprA, which loads RecA onto the DNA. Polymerization of RecA along the ssDNA generates the early HR intermediate known as the presynaptic filament. (4) Homology search and ssDNA pairing. The presynaptic filament interacts with the chromosome in an unknown manner, and RecA promotes homology search. Once homology is found, the homologous strand of the recipient chromosome is displaced, and RecA facilitates pairing between the transforming ssDNA and the complementary strand, forming the so-called displacement loop (D-loop). D-loop extension is facilitated by the helicase RadA which unwinds the recipient chromosome on either side of the D-loop. (5) Integrative resolution into daughter chromosomes. The D-loop structure is resolved by the passage of the replication machinery, generating one transformed and one untransformed daughter chromosome. (B) Sample fluorescence microscopy images of R3728 strain ( comC0, dprA-gfp ) producing DprA-GFP 15 minutes after competence induction and 5 minutes after DNA addition (250 ng µL -1 ). Scale bars, 1 µm. Black arrows, polar DprA-GFP foci; white arrows, midcell DprA-GFP foci. (C) Schematic representation of focus density maps with half cells represented as vertical lines in ascending size order and localisation of foci represented along the length axis of each half cell. (D) Addition of transforming DNA shifts the localisation profile of DprA-GFP foci towards midcell. Data represented as focus density maps plotted on the longitudinal axis of half cells ordered by cell length. Each spot represents the localisation of an individual focus, and spot colour represents focus density at a specific location on the half cell. Cells with >0 foci shown for each time point. In cells possessing >1 foci, foci were represented adjacently on cells of the same length. DNA-, 5,739 cells and 4,920 foci analysed; DNA+, 3,406 cells and 3,899 foci analysed. (E) localisation of DprA-GFP foci split into three categories on a half-cell of arbitrary length 1 where midcell is 0 and the pole is 1. Midcell, 0-0.3; betwixt, 0.3-0.7; polar, 0.7-1.
Techniques Used: Transformation Assay, Fluorescence, Microscopy
Figure Legend Snippet: (A) Sample fluorescence microscopy images of R4262 strain ( comC0, CEP lac -dprA-gfp , dprA::spc ) grown in 6 µM IPTG to produce ∼300 DprA-GFP dimers, 15 minutes after competence induction and 5 minutes after DNA addition (250 ng µL -1 ). Scale bars, 1 µm. White arrows, midcell DprA-GFP foci. (B) Low level DprA-GFP accumulates at midcell upon addition of transforming DNA. Representations as focus density maps as described in . DNA-, 21,055 cells and 1,512 foci analysed; Homologous DNA, 54,058 cells and 27,811 foci analysed; Heterologous DNA, 23,962 cells and 8,819 foci analysed. (C) Foci localisation on density heat maps with cells split into six cell cycle categories by sise and constriction status (see Materials and Methods). White lines represent the average cell contour of the sample set and each spot represents an individual focus, with colour representing density. Microscopy images represent sample images of each cell category showing preferential focus localisation. Scale bars, 1 µm. Homologous transforming DNA; Small cells, 17,888 cells and 6,272 foci analysed; medium cells, 16,744 cells and 8,862 foci analysed; large cells, 13,659 cells and 8,395 foci analysed; cons. start cells, 880 cells and 446 foci analysed; cons. middle cells, 3824 cells and 2,799 foci analysed; cons. end cells, 1063 cells and 1,037 foci analysed. Heterologous transforming DNA; Small cells, 7,557 cells and 1,902 foci analysed; medium cells, 7,562 cells and 2,742 foci analysed; large cells, 6,318 cells and 2,910 foci analysed; cons. start cells, 377 cells and 153 foci analysed; cons. middle cells, 1,708 cells and 861 foci analysed; cons. end cells, 440 cells and 251 foci analysed. (D) Sample fluorescence microscopy images of low level DprA-GFP foci within cells of R4415 ( comC0, CEP lac -dprA QNQ -gfp, dprA::spc ) and R4429 ( comC0, CEP lac -dprA-gfp, dprA::spc, recA::cat) strains 15 minutes after competence induction and 5 minutes after DNA addition (250 ng µL -1 ). Scale bars, 1 µm. (E) Low level DprA-GFP foci change localisation profile in the absence of recA of in a dprA QNQ -gfp mutant which cannot interact with RecA. Representations focus density maps as described in . R4415, 7,912 cells and 6,723 foci analysed, R4429, 35,318 cells and 10,974 foci analysed. (F) Foci localisation on density heat maps as described in panel C for R4415 ( comC0, CEP lac -dprA QNQ -gfp, dprA::spc ) and R4429 ( comC0, CEP lac -dprA-gfp, dprA::spc, recA::cat ) strains. Data used as in panel E . Microscopy images represent sample images of each cell category showing preferential focus localisation. Scale bars, 1 µm. R4415: Small cells, 2,729 cells and 1,233 foci analysed; medium cells, 2,180 cells and 1,809 foci analysed; large cells, 2,132 cells and 2,461 foci analysed; cons. start cells, 131 cells and 135 foci analysed; cons. middle cells, 559 cells and 768 foci analysed; cons. end cells, 181 cells and 317 foci analysed. R4429: Small cells, 14,338 cells and 2,988 foci analysed; medium cells, 9,534 cells and 3,294 foci analysed; large cells, 7,508 cells and 2,925 foci analysed; cons. start cells, 727 cells and 270 foci analysed; cons. middle cells, 2,534 cells and 1,079 foci analysed; cons. end cells, 740 cells and 418 foci analysed.
Techniques Used: Fluorescence, Microscopy, Mutagenesis
Figure Legend Snippet: (A) Time-course experiment of low-level DprA-GFP foci per cell in competent cells. 15 min, 2,355 cells and 1,023 foci analysed; 20 min, 2,260 cells and 1,129 foci analysed; 40 min, 5,988 cells and 2,001 foci analysed; 60 min, 5,244 cells and 1,216 foci analysed. (B) Low level DprA-GFP foci persist at midcell up to 60 minutes after competence induction. Representations as focus density maps as described in . Cells and foci analysed as in panel D . (C) Analysis of the repartition of low-level DprA-GFP foci within competent cells in a gradient of transforming DNA. 0 ng µL -1 DNA, 2,621 cells and 128 foci analysed; 0.25 ng µL -1 DNA, 2,222 cells and 425 foci analysed; 2.5 ng µL -1 DNA, 6,038 cells and 1,098 foci analysed; 25 ng µL -1 DNA, 4,215 cells and 1,194 foci analysed; 250 ng µL -1 DNA, 2,190 cells and 1,313 foci analysed. (D) Sample fluorescence microscopy images and analysis of the repartition of low level DprA-GFP foci within cells of R4262 ( comC0, CEP lac -dprA-gfp, dprA::spc ), R4401 ( comC0, CEP lac -dprA-gfp, dprA::spc, comEC::ery ) and R4413 ( comC0, CEP lac -dprA AR -gfp, dprA::spc ) strains 15 minutes after competence induction and 5 minutes after DNA addition (250 ng µL -1 ). Scale bars, 1 µm. (E) Distribution of low level DprA-GFP foci in cells of R4415 ( comC0, CEP lac -dprA QNQ -gfp, dprA::spc ) and R4429 ( comC0, CEP lac -dprA-gfp, dprA::spc, recA::cat) strains 15 minutes after competence induction and 5 minutes after DNA addition (250 ng µL -1 ). Data taken from and . Midcell, betwixt and polar localisations defined as in . (F) Sample fluorescence microscopy image of low level DprA-GFP foci within cells of R4618 ( comC0, CEP lac -dprA-gfp, dprA::spc, comEC::trim, recA::cat ). Conditions as in . (G) Analysis of the repartition of low level DprA-GFP foci within cells of R4618. 2,455 cells and 22 foci analysed.
Techniques Used: Fluorescence, Microscopy
Figure Legend Snippet: (A) Sample fluorescence microscopy image of strain R4400 ( comC0, CEP lac -dprA-gfp, dprA::spc, ssbB::cat) 15 minutes after competence induction and 5 minutes after DNA addition (250 ng µL -1 ). Scale bars, 1 µm. (B) Low cellular DprA-GFP accumulates at midcell upon addition of transforming DNA in the absence of SsbB. Representations as focus density maps as described in . 41,788 cells and 30,213 foci analysed. (C) DprA-GFP localisation in the absence of SsbB represented as density heat maps as in . Microscopy images represent sample images of each cell category showing preferential focus localisation. Scale bars, 1 µm. Small cells, 16,723 cells and 9,548 foci analysed; medium cells, 11,722 cells and 7,962 foci analysed; large cells, 9,029 cells and 7,619 foci analysed; cons. start cells, 948 cells and 1024 foci analysed; cons. middle cells, 2,689 cells and 3,217 foci analysed; cons. end cells, 677 cells and 843 foci analysed. (D) Sample fluorescence microscopy image of strain R4625 ( comC0, CEP lac -dprA-gfp, dprA::spc, radA::trim) 15 minutes after competence induction and 5 minutes after DNA addition (250 ng µL -1 ). Scale bars, 1 µm. (E) Low cellular DprA-GFP accumulates at midcell upon addition of transforming DNA in the absence of RadA. Representations as described in . 17,925 cells and 9,667 foci analysed. (F) DprA-GFP localisation in the absence of RadA represented as density heat maps as in . Microscopy images represent sample images of each cell category showing preferential focus localisation. Small cells, 7,544 cells and 2,638 foci analysed; medium cells, 6,678 cells and 2,982 foci analysed; large cells, 5,315 cells and 2,528 foci analysed; cons. start cells, 421 cells and 222 foci analysed; cons. middle cells, 1,528 cells and 984 foci analysed; cons. end cells, 439 cells and 313 foci analysed.
Techniques Used: Fluorescence, Microscopy
Figure Legend Snippet: (A) Western blot probed with α-RecA antibodies showing cellular levels of RecA and RecA-mTurquoise in competent (CSP+) and non-competent (CSP-) cells. Samples also probed with α-SsbB antibodies to confirm induction of competence. Samples taken 15 minutes after competence induction. Strains used; RecA, R1501 ( comC0 ); RecA-mTurquoise, R4712 ( comC0, recA- mturquoise ). (B) Western blot probed with α-RecA antibodies showing cellular levels of RecA and RecA-mTurquoise in a strain possessing recA and CEP lac -recA-mturquoise grown in varying concentrations of IPTG and in absence of CSP. Wildtype RecA strain included as a control in presence or absence of CSP. Strains used; RecA, R1501 ( comC0 ); RecA/RecA-mTurquoise, R4848 ( comC0, CEP lac -recA-mturquoise ). (C) Comparison of transformation efficiencies of various recA mutant strains to wild type. Saturating (300 ng mL -1 ) and non-saturating (0.03 ng mL -1 ) concentrations of rpsL41 PCR fragment, conferring streptomycin resistance via point mutation, used. DNA identity; 3,434 bp rpsL41 PCR fragment amplified from R304 strain using MB117-MB120 primer pair. Transformation pre-cultures prepared in 0 or 50 µM IPTG as noted. Strains used: wt , R1501 ( comC0 ); recA-mturquoise , R4712 ( comC0, recA-mturquoise ), CEP lac -recA-mturquoise , R4848 ( comC0, CEP lac -recA-mturquoise ). Data represented as mean ± s.e.m. of triplicate repeats. Asterisks represent significant difference between test samples and wildtype controls for a given DNA concentration (** = p < 0.01, n. s. = not significant). (D) Spot tests comparing growth of competent (CSP+) and non-competent (CSP-) recA mutants to wildtype in presence or absence of methanemethylsulfonate (MMS, 0.02 %). Strains used: wt , R1501 ( comC0 ); recA- , (R4857, comC0, recA::trim ), recA-mturquoise , R4712 ( comC0, recA- mturquoise ), CEP lac -recA-mturquoise , R4848 ( comC0, CEP lac -recA-mturquoise ); R4664 ( comC0, CEP lac -recA ). R4848 and R4664 grown and plated with 0 and 50 µM IPTG to compare presence and absence of CEP lac -recA-mturquoise induction.
Techniques Used: Western Blot, Transformation Assay, Mutagenesis, Amplification, Concentration Assay
Figure Legend Snippet: (A) Sample microscopy images of RecA-mTurquoise in non-competent cells and competent cells in presence or absence of homologous transforming DNA. Images taken 15 minutes after competence induction. Strain used, R4712 ( comC0, recA-mturquoise ). Black arrows, polar foci. (B) RecA-mTurquoise alone accumulates at the cell poles in a majority of transforming cells. Representations as focus density maps as described in . 8,125 cells and 5,858 foci analysed. (C) RecA-mTurquoise forms centrally localised bundles in a minority of non- competent cells. Representations as focus density maps as described in . 5,512 cells and 901 bundles analysed. (D) RecA-mTurquoise forms foci at the cell poles in competent cells in absence of transforming DNA. Representations as focus density maps as described in . 6,066 cells and 3,874 foci analysed. (E) The presence of transforming DNA does not alter the polar localisation of RecA-mTurquoise. Representations as focus density maps as described in . 8,125 cells and 5,858 foci analysed. (F) The absence of DprA does not alter RecA-mTurquoise localisation in competent cells. Representations as focus density maps as described in . 6,066 cells and 3,874 foci analysed. (G) RecA-mTurquoise forms polar foci in a strain expressing DprA-YFP in the presence of transforming DNA. Representations as focus density maps as described in . 9,337 cells, 10,367 RecA- mTurquoise foci analysed. Strain used, R4742, comC0 , P lac -dprA-yfp , recA-mturquoise , dprA :: spc . (H) DprA-YFP forms polar foci in a strain expressing RecA-mTurquoise in the presence of transforming DNA. Strain and images used as in panel E . Representations as focus density maps as described in . 9,337 cells, 3,721 DprA-YFP foci analysed. (I) RecA- mTurquoise forms polar foci in a strain expressing DprA-YFP in the presence of transforming DNA. Representations as focus density maps as described in . 7,074 cells, 8,493 RecA-mTurquoise foci analysed. Strain used as in panel E . (J) DprA-YFP forms polar foci in a strain expressing RecA-mTurquoise in the presence of transforming DNA. Images used as in panel G . Representations as focus density maps as described in . 7,074 cells, 6,120 DprA-YFP foci analysed. Strain used as in panel E . (K) Sample microscopy images of a strain expressing low level DprA-YFP and RecA-mTurquoise in competent, transforming cells and colocalisation of DprA-YFP with RecA-mTurquoise in these cells. Images taken 15 minutes after competence induction and 5 minutes after DNA addition (250 ng µL -1 ). Scale bars, 1 µm. Phase, phase contrast images of cells; overlay, overlay of all 3 other images. Strain used as in panels I and J .
Techniques Used: Microscopy, Expressing
Figure Legend Snippet: (A) Sample microscopy images of RecA/RecA-mTurquoise mixed filaments in non-competent cells and competent cells in presence or absence of homologous tDNA. Images taken 15 minutes after competence induction. Strain used, R4848 ( comC0, P lac -recA-mturquoise ). White arrows, midcell RecA-mTurquoise foci. (B) Mixed filaments of RecA/RecA-mTurquoise accumulate at midcell, reminiscent of DprA. Representations as focus density maps as described in . P lac -recA-mturquoise , 3,481 cells and 3,022 foci analysed. (C) RecA/RecA-mTurquoise mixed filaments accumulate at midcell in transforming cells. Representations on density heat maps as described in . Microscopy images represent sample images of each cell category showing preferential focus localisation. Small cells, 923 cells and 591 foci analysed; medium cells, 1,242 cells and 984 foci analysed; large cells, 958 cells and 948 foci analysed; cons. start cells, 45 cells and 46 foci analysed; cons. middle cells, 229 cells and 318 foci analysed; cons. end cells, 84 cells and 135 foci analysed. (D) Formation of RecA-mTurquoise foci expressed from P lac -recA-mturquoise ectopic expression platform in merodiploid cells is dependent on transforming DNA and DprA. Strains used, P lac -recA-mturquoise , R4848 ( comC0, CEP lac -recA-mturquoise ); P lac -recA- mturquoise , dprA - , R4851, ( comC0, CEP lac -recA-mturquoise, dprA::spc ). (E) Formation of RecA- mTurquoise foci expressed from P lac -recA-mturquoise ectopic expression platform in merodiploid cells varies depending on the length of tDNA fragments used. Strain used, P lac - recA-mturquoise , R4848 ( comC0, CEP lac -recA-mturquoise ).
Techniques Used: Microscopy, Expressing
Figure Legend Snippet: (A) Heatmaps of RecA-mTurquoise foci in competent, transforming RecA/RecA-mTurquoise cells grown in varying IPTG concentrations of IPTG (3-50 µM), as described in . Strain used: R4848, comC0, CEP lac -recA-mturquoise . 3 µM IPTG, Small cells, 7,056 cells and 680 foci analysed; medium cells, 3,594 cells and 784 foci analysed; large cells, 3,012 cells and 808 foci analysed; cons. start cells, 282 cells and 51 foci analysed; cons. middle cells, 964 cells and 367 foci analysed; cons. end cells, 321 cells and 154 foci analysed. 6 µM IPTG, Small cells, 8,533 cells and 710 foci analysed; medium cells, 4,617 cells and 1,059 foci analysed; large cells, 3,911 cells and 1,133 foci analysed; cons. start cells, 321 cells and 59 foci analysed; cons. middle cells, 1,238 cells and 508 foci analysed; cons. end cells, 362 cells and 192 foci analysed. 12 µM IPTG, Small cells, 6,037 cells and 1,112 foci analysed; medium cells, 3,424 cells and 1,786 foci analysed; large cells, 3,089 cells and 1,914 foci analysed; cons. start cells, 229 cells and 106 foci analysed; cons. middle cells, 854 cells and 793 foci analysed; cons. end cells, 271 cells and 322 foci analysed. 25 µM IPTG, Small cells, 1,187 cells and 379 foci analysed; medium cells, 1,002 cells and 584 foci analysed; large cells, 806 cells and 523 foci analysed; cons. start cells, 49 cells and 37 foci analysed; cons. middle cells, 186 cells and 198 foci analysed; cons. end cells, 66 cells and 81 foci analysed. 50 µM IPTG, Small cells, 923 cells and 591 foci analysed; medium cells, 1,242 cells and 984 foci analysed; large cells, 958 cells and 948 foci analysed; cons. start cells, 45 cells and 46 foci analysed; cons. middle cells, 229 cells and 318 foci analysed; cons. end cells, 84 cells and 135 foci analysed. (B) Focus density maps of RecA-mTurquoise foci in competent, transforming RecA/RecA-mTurquoise cells as described in . Strains, conditions and images used as in panel A . 3 µM IPTG, 15,229 cells and 2,844 foci analysed; 6 µM IPTG, 18,982 cells and 3,661 foci analysed; 12 µM IPTG, 13,940 cells and 6,033 foci analysed; 25 µM IPTG, 3,296 cells and 1,802 foci analysed; 50 µM IPTG, 3,481 cells and 3,022 foci analysed. (C) Percentage of RecA-mTurquoise foci in RecA/RecA-mTurquoise cells grown in varying IPTG concentrations. Strains, conditions and images used as in panel A . (D) Cellular localisation of RecA-mTurquoise foci in RecA/RecA-mTurquoise cells grown in varying IPTG concentrations. Strains, conditions and images used as in panel A . (E) RecA-mTurquoise foci per cell in transformation experiments using DNA fragments of varying sizes. Strain used, R4848 ( comC0, CEP lac -recA-mturquoise ).
Techniques Used: Transformation Assay
Figure Legend Snippet: (A) Comparison of transformation of rpsL41 PCR fragments containing either dTTP or dUTP. dUTP bases were either unlabelled, labelled with fluorescein, or labelled with amino-allyl and tagged with Dylight 550 or 650 fluorophores. Asterisks represent significant difference between test samples and dTTP control (*** = p < 0.005, **** = p < 0.001). dUTP, p = 0.0005; d-UTP-fluorescein, p < 0.0001; d-UTP-Dylight 550, p < 0.0001; d-UTP-Dylight 650, p = 0.0006. (B) Comparison of microscopy images of competence cells of R1501 ( comC0 ) transformed with DNA labelled with fluorescein, Dylight 550 or 650. Analysis of microscopy images of cells transformed with DNA labelled with various fluorophores, showing cells possessing detectable fluorescent foci. (C) Comparison of transformation efficiency of Dylight 550 transformed with PCR fragments of rpsL41 or radA::spc in a concentration gradient of transforming DNA. Values plotted represent ratios of transformation efficiency between labelled and labelled PCR fragments. Strain used as in panel A. Asterisks represent significant difference ratios for each PCR fragment (** = p < 0.01, *** = p < 0.005, n. s., not significant). ∼1 pg µL -1 DNA, p = 0.0069; ∼10 pg µL -1 DNA, p = 0.0007; ∼100 pg µL -1 DNA, p = 0.12; ∼1,000 pg µL -1 DNA, p = 0.056. (D) Comparison of transformation efficiency of Dylight 650 transformed with PCR fragments of rpsL41 or radA::spc in a concentration gradient of transforming DNA. Values plotted as in panel C . Strain used as in panel A. Asterisks represent significant difference ratios for each PCR fragment (* = p < 0.05, ** = p < 0.01, n. s., not significant). ∼1 pg µL -1 DNA, p = 0.017; ∼10 pg µL -1 DNA, p = 0.006; ∼100 pg µL -1 DNA, p = 0.025; ∼1,000 pg µL -1 DNA, p = 0.11. (E) Comparison of foci present in cells of various transformasome mutants after exposure to Dylight 650-labelled rpsL41 PCR fragments. Strains used: wt, R1501 ( comC0 ); comG - , R4655 ( comC0, comC-luc, comG::kan ); endA - , R2811 ( comC0, endA::cat ); comEC - , R2586 ( comC0, comEC::ery ); comEAC - , R4653 ( comC0, comC-luc, comEAC::spc ); dprA - , R2018 ( comC0, dprA::spc ). (F) Comparison of focus localisation in cells of various transformasome mutants after exposure to Dylight 650-labelled rpsL41 PCR fragments. Strains used as in panel E .
Techniques Used: Transformation Assay, Microscopy, Concentration Assay
Figure Legend Snippet: (A) Sample microscopy images of cells possessing YFP-DnaX in competence and non-competent cells. Strain used, R4840 ( comC0, ssbB::luc, CEP M -yfp-dnaX, CEPII-P lac -recA-mturquoise ). (B) Competence mediates a slight reduction in YFP-DnaX foci. Representations as focus density maps as described in . CSP-, 7,911 cells and 6,947 foci analysed. CSP+, 8,425 cells and 6,055 foci analysed. (C) RecA-mTurquoise and YFP-DnaX colocalise in competent cells in the presence of heterologous tDNA ( E. coli gDNA). Strain used, R4840 ( comC0, ssbB::luc, CEP M -yfp-dnaX, CEPII-P lac -recA-mturquoise ). 2,443 cells, 1,345 RecA- mTurquoise foci and 1,475 YFP-DnaX foci analysed. (D) Focus density maps of RecA- mTurquoise and YFP-DnaX, as described in . Strain, cell and foci details as in panel C . (E) Time-lapse images of RecA-mTurquoise and YFP-DnaX with time representing time after CSP addition (tDNA added at t = 10 min). Strain used as in panel C .
Techniques Used: Microscopy
Figure Legend Snippet: (A) Low level DprA-mTurquoise foci display a localisation profile similar to a YFP-DnaX fluorescent fusion of the replisome clamp loader expressed in the same R4631 cells ( comC0, CEP M -yfp-dnaX, CEPIIP lac -dprA-mTurquoise, dprA - ). Representations as focus density maps as described in . 29,942 cells analysed possessing 14,281 DprA- mTurquoise foci and 20,975 YFP-DnaX foci analysed. (B) DprA-mTurquoise and YFP-DnaX foci localisation on density heat maps as described in for R4631 strain. Data used as in panel A . Microscopy images represent sample images of each cell category showing preferential focus localisation. Small cells, 9,979 cells, 2,716 DprA-mTurquoise and 4,476 YFP- DnaX foci analysed; medium cells, 8,517 cells, 3,830 DprA-mTurquoise and 6,148 YFP-DnaX foci analysed; large cells, 7,952 cells, 4,855 DprA-mTurquoise and 6,352 YFP-DnaX foci analysed; cons. start cells, 508 cells, 309 DprA-mTurquoise and 426 YFP-DnaX foci analysed; cons. middle cells, 2,390 cells, 1,989 DprA-mTurquoise and 2,615 YFP-DnaX foci analysed; cons. end cells, 596 cells, 582 DprA-mTurquoise and 958 YFP-DnaX foci analysed. (C) Sample microscopy images of R4631 strain expressing low level DprA-mTurquoise and YFP-DnaX and colocalisation of DprA-mTurquoise with YFP-DnaX in these cells. Images taken 15 minutes after competence induction and 5 minutes after DNA addition (250 ng µL -1 ). Scale bars, 1 µm. Phase contrast, phase contrast images of cells; overlay, overlay of all 3 other images. (D) DnaX and DprA produce similarly dynamic foci in competent, transforming cells. DprA-mTurquoise and YFP-DnaX observed during time-lapse microscopy of strain R4631 ( comC0, CEP M -yfp-dnaX, CEPII-P lac -dprA-mturquoise, dprA::spc ) starting 10 min after competence induction and five min after DNA addition. Images taken every two min. Phase, phase contrast images of cells. Overlay, a merge of the three other images. Scale bars, 1 µm. (E) The replisome clamp loader DnaX interacts with transforming ssDNA in early HR intermediates, as shown by co-purification of heterologous transforming ssDNA with YFP-DnaX at levels comparable to DprA-GFP and DprA-YFP in ChIP-PCR experiments. In contrast, DprA AR -GFP co-purified at levels comparable to the GFP alone negative control (with an enrichment value of 1, to which all other samples were normalised). Strains used, R2546, comC0, CEP X -gfp ; R3406, comC0, CEPM-yfp-dnaX ; R3728, comC0, dprA-gfp ; R4046, comC0, dprA AR -gfp ; R4404, comC0, dprA-yfp . Asterisks represent significant difference between samples (** = p < 0.005, n. s. = not significant). DprA AR -GFP, p = 0.52; DprA-GFP, p = 0.0029; DprA-YFP, p = 0.0019; YFP-DnaX, p = 0.0012. (F) Split-luciferase assay comparing cellular proximity of DnaX and DprA in presence or absence of tDNA. Luminescence signal increases when tDNA is added to competent cells containing dprA-lgbit and dnaX-smbit (R4856), indicating an increased proximity of these fusion proteins in the presence of tDNA. When dprA-lgbit is replaced by the dimerization mutant dprA AR -lgbit (R4861), the increase in luminescence upon addition of tDNA is attenuated. A strain containing dprA-lgbit and P lac -dprA-smbit (R4858) was used as a positive control for interaction since DprA dimerises, and shows high luminescence irrespective of tDNA addition. Each point represents an individual replicate, with 9 replicates done for each condition. RLU, relative luminescence units. Asterisks represent significant difference between samples (**** = p < 0.001, ** = p < 0.01, n. s. = not significant). dnaX-smbit , dprA-lgbit, p = 0.00009; dnaX-smbit , dprA AR -lgbit , p = 0.009; CEP lac -dprA-smbit , dprA-lgbit, p = 0.0066.
Techniques Used: Microscopy, Expressing, Time-lapse Microscopy, Copurification, Purification, Negative Control, Luciferase, Mutagenesis, Positive Control
Figure Legend Snippet: (A) RecA-mTurquoise and YFP-DnaX colocalise in competent cells in the presence of tDNA. Strain used, R4840 ( comC0, ssbB::luc, CEP M -yfp-dnaX, CEPII-P lac - recA-mturquoise ). 5,866 cells, 4,923 RecA-mTurquoise foci and 5,081 YFP-DnaX foci analysed. (B) Focus density maps of RecA-mTurquoise and YFP-DnaX, as described in . Strain, cell and foci details as in panel A . (C) Heatmaps of RecA-mTurquoise and YFP-DnaX as described in . Small cells, 1,730 cells, 1,072 RecA-mTurquoise and 1,071 YFP-DnaX foci analysed; medium cells, 1,766 cells, 1,409 RecA-mTurquoise and 1,582 YFP-DnaX foci analysed; large cells, 1,603 cells, 1,449 RecA-mTurquoise and 1,582 YFP-DnaX foci analysed; cons. start cells, 93 cells, 88 RecA-mTurquoise and 92 YFP-DnaX foci analysed; cons. middle cells, 510 cells, 1,989 RecA-mTurquoise and 614 YFP-DnaX foci analysed; cons. end cells, 165 cells, 264 RecA-mTurquoise and 257 YFP-DnaX foci analysed. (D) Microscopy images showing filaments of RecA-mTurquoise emanating from YFP-DnaX foci. (E) Time-lapse images of RecA- mTurquoise and YFP-DnaX in microfluidics experiment with time representing time after tDNA addition. Strain used as in panel A . dynamic extension of filaments ( , Movie 4). In contrast to the RecA filaments formed in cells exposed to norfloxacin, those that emanate from replication forks in transformed cells were short, extending on average 0.22 (+/- 0.05) µm either side of a replisome colocalisation point. Similar short tDNA-dependent RecA filaments were observed with heterologous tDNA (genomic DNA from E. coli ) and, therefore, are not the result of pairing with a complementary sequence. Thus, these dynamic RecA polymers may represent presynaptic HR filaments assembled on tDNA and mediating homology search after having accessed the recipient chromosome via the replisome landing pad.
Techniques Used: Microscopy, Transformation Assay, Sequencing
Figure Legend Snippet: (A) Most RecA/RecA-mTurquoise cells do not display RecA- mTurquoise accumulation in absence of norfloxacin exposure. RecA-mTurquoise observed during time-lapse microscopy of strain R4848 ( comC0, CEPlac-recA-mturquoise ). Images taken every 1 min. (B) Most RecA/RecA-mTurquoise cells display RecA-mTurquoise accumulation into filaments in presence of norfloxacin. RecA-mTurquoise observed during time-lapse microscopy of strain R4848 ( comC0, CEP lac -recA-mturquoise ) after 20 min exposure to 100 ng µL -1 norfloxacin (MIC 3 ng µL -1 ). Images taken every 1 min. (C) Single image of cells showing lack of RecA-mTurquoise accumulation in absence of norfloxacin exposure. Scale bar, 1 µm. Strain used as in panel A. (D) Single image of cells showing RecA-mTurquoise filamentation in almost all cells after norfloxacin exposure. Scale bar, 1 µm. Strain used as in panel A .
Techniques Used: Time-lapse Microscopy
Figure Legend Snippet: (A) Growth of pneumococcal cells in presence of varying concentrations of HpUra. Strain used, R1501 ( comC0 ). (B) Comparison of RecA/RecA-mTurquoise and YFP-DnaX localisation in non-competent recO +/- cells exposed to HpUra (1,8 µg mL -1 ) or not. (C) Colocalisation of RecA-mTurquoise and YFP-DnaX foci in competent recO - cells transformed with homologous tDNA in presence or absence of HpUra. Strain used, R4892 ( comC0, ssbB::luc, CEP M -yfp-dnaX, CEPII-P lac -recA-mturquoise, recO::spc ). HpUra-, 9,391 cells, 3,996 RecA-mTurquoise foci and 9,072 YFP-DnaX foci analysed. HpUra+, 11,358 cells, 2,791 RecA- mTurquoise foci and 9,555 YFP-DnaX foci analysed. (D) Distribution of RecA-mTurquoise and YFP-DnaX foci per cell. Strain and data used as in panel C. (E) Focus density maps of RecA-mTurquoise and YFP-DnaX in transforming recO - cells in the absence of HpUra. Strain and data used as in panel C. (F) Focus density maps of RecA-mTurquoise and YFP-DnaX in transforming recO - cells in the presence of HpUra. Strain and data used as in panel C .
Techniques Used: Transformation Assay