Article Figures & Data
- FIG 1
Role of the distinct pSVH1 proteins on conjugative plasmid transfer. (A) Arrangement of genes in pEB211. The regulatory gene traR is drawn in red, spd genes in orange, traB in blue, putative spd genes in light grey, rep and dso-sso regions in dark grey. The E. coli part of pEB211 is highlighted in black. (B) Transfer frequencies of pEB211 derivatives. Approximately 107 spores of S. lividans TK54 containing pEB211 (Kanr) and its mutated derivatives were plated with equal amounts of plasmid-free TK64 (Strr). After 7 days of incubation, spores were harvested, and the transfer frequencies (ratio of transconjugants [Strr, Kanr] and recipients [Strr]) were determined. Transfer frequencies are the mean values from three mating experiments (see Table S5). Error bars indicate standard deviations. (C) Pock formation, indicating intramycelial plasmid spreading. Approximately 102 spores of S. lividans TK54 containing derivatives of plasmid pEB211 (Kanr) were streaked onto a lawn (~105 spores) of plasmid-free TK64 (Strr). After 7 days of incubation at 30°C, pocks associated with the conjugative plasmid transfer were visible, and the fully sporulated plates were replica plated onto antibiotic-containing LB agar to select for transconjugants. Sizes of the pocks and the corresponding transconjugant patches indicate efficiency of plasmid transfer and intramycelial spreading.
- FIG 2
TraR specifically binds to the traR-spdB3 intergenic region. (A) Electrophoretic mobility shift assays demonstrate binding of TraR-His to DNA fragments comprising the traR-spdB3 intergenic region. With increasing TraR-His concentration (0 to 3.75 pmol), two retarded bands appeared (arrows). The negative-control fragment, marked by an asterisk, is not bound by TraR-His. (B) A schematic drawing of the DNA fragments (a to d) analyzed for TraR binding is given. The positions of the 14-bp TTTGGTACACAACT repeats (black arrows) and the 12-bp inverted repeat (grey arrows) are indicated. (C) Nucleotide sequence of the traR-spdB3 intergenic region. The start codons of traR and spdB3, the 14-bp perfect repeats (black arrows), the imperfect repeat (dotted arrow), and the 12-bp inverted repeat (grey arrows) are marked. The KpnI site is underlined. DNA fragment “c,” which is bound by TraR, is highlighted by shading.
- FIG 3
Peptidoglycan binding activity of pSVH1-encoded proteins. Purified soluble proteins were incubated with S. lividans PG sacculi (+) or buffer (−). Following precipitation of the PG sacculi by centrifugation, the supernatant (S) and the pellet (P) fractions were analyzed by immunoblotting with anti-His tag-specific antibodies for the presence of the respective proteins. Detection of the protein in the pellet fraction indicated PG binding activity.
- FIG 4
SpdB2-His promotes spheroplast formation by T7 lysozyme and forms unstable pores in planar lipid bilayers. (A) When expression of SpdB2-His was induced in BL21/pLysS, spheroplast (arrows) were formed. In contrast, spheroplasts were not observed in the absence of spdB2 (B) or when expressing spdB2 in BL21 lacking the T7 amidase (C). (D) When purified SpdB2-His was added to the cis side of a lipid bilayer, SpdB2-His inserted into the planar lipid bilayer, showing flickering pore structures. (E) The corresponding all-points conductance level histogram shows that there is no discrete conductance state of the open pore indicated by the long tail in the histogram.
- FIG 5
Complex interaction network of the pSVH1-encoded proteins. (A) Summary of the interactions of the pSVH1 proteins revealed by bacterial two-hybrid analyses. (B) Subcellular localization and the interaction pattern of pSVH1 proteins involved in conjugative DNA transfer and intramycelial plasmid spreading. The complex interaction network of TraB and various Spd proteins suggests a membrane-localized multiprotein DNA translocation apparatus involved in intramycelial plasmid spreading. Lines mark protein-protein interactions. Self-interactions are indicated by double ellipses. Transmembrane regions and protein orientations were predicted with TMpred (39). The dashed line indicates that Orf108 has to be secreted by an unknown route to be able to interact with PG.
Supplemental Material
Table S5
Transfer frequencies of different pEB211 derivatives. Table S5, DOCX file, 0.02 MB.
Copyright © 2015 Thoma et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S1
Purification of pSVH1-encoded proteins. (A) pSVH1-encoded proteins were purified from E. coli or S. lividans (SpdB2) via Ni-NTA affinity chromatography and analyzed by SDS-PAGE. 1, TraR-His (28.2 kDa); 2, SpdB2-His (45.4 kDa); 3, Orf108-His (12.7 kDa); 4, Orf140-His (16.5 kDa). (B) For single-channel recordings, SpdB2-His was purified from S. lividans membranes by affinity chromatography (Ni-NTA) and ion exchange chromatography (SP-FF). The purity of the protein was determined by Coomassie blue and silver staining. Download Figure S1, TIF file, 1.4 MB.
Copyright © 2015 Thoma et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S4
Primer sequences used for gene expression and EMSA. Table S4, DOCX file, 0.02 MB.
Copyright © 2015 Thoma et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S2
Oligomerization of TraR, Orf140, and SpdB2. (A and B) Chemical cross-linking of purified TraR-His (A) and Orf140-His (B). Purified proteins were incubated with increasing amounts of glutaraldehyde (indicated above the gel). Following electrophoretic separation on a 12.5% SDS-polyacrylamide gel, the protein complexes were visualized by immunoblotting with anti-His tag-specific antibodies. The thick arrow marks the monomeric protein, while thin arrows indicate dimeric and oligomeric forms. (C) Blue native gel electrophoresis of SpdB2-His. SpdB2-His was extracted from the membrane fraction of S. lividans TK23/pYT90 with 1% Triton X-100 and purified by Ni-NTA affinity chromatography. Blue native PAGE was performed according to Fiala et al. (G. J. Fiala, W. W. A. Schamel, B. Blumenthal, J Vis Exp 48:e2164, 2011, http://dx.doi.org/10.3791/2164). The purified protein was dialyzed against BN dialysis buffer supplemented with 1% Triton overnight and loaded on a 4 to 15% BN-PAGE gel. As a marker protein, BSA was used. Download Figure S2, TIF file, 2.3 MB.
Copyright © 2015 Thoma et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S6
Bacterial two-hybrid analyses of pSVH1-encoded proteins. Table S6, DOCX file, 0.02 MB.
Copyright © 2015 Thoma et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S1
Strains and plasmids. Table S1, DOCX file, 0.02 MB.
Copyright © 2015 Thoma et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S2
Primer sequences used for gene replacement. Table S2, DOCX file, 0.02 MB.
Copyright © 2015 Thoma et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S3
Primer sequences used for bacterial two-hybrid analyses. Table S3, DOCX file, 0.02 MB.
Copyright © 2015 Thoma et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S3
Example plate showing the interactions of Cya-T25-TraB1–772 and T25-TraB274–772 with Cya-T18 fusions to pSVH1-encoded proteins. Overnight cultures of BTH101 carrying the indicated plasmids (A) were grown in LB, washed with PBS, and spotted on MM supplemented with lactose, X-Gal, ampicillin, and kanamycin. As a positive control (+) served pUT18c-Zip/pKT25-Zip, and the negative control (−) was pUT18c/pKT25. (B) Plates were incubated for 48 h at 37°C. Blue circles mark positive interactions. The black circle indicates the negative control. Experiments were repeated three times with independent cultures. Only positive reactions in all three experiments were considered positive interactions. (1, pUT18c-spdB3; 2, pUT18c-spdB79; 3, pUT18c-spdB2; 4, pUT18c-orf108; 5, pUT18c-spd198; 6, pUT18c-spd40; 7, pUT18c-orf140; 8, pUT18c-spdA; 9, pUT18-spdB3; 10, pUT18-spdB79; 11, pUT18-spdB2; 12, pUT18-orf108; 13, pUT18-spd198; 14, pUT18-spd40; 15, pUT18-TraB1-772; 16, pUT18-TraB274-772; 17, pUT18-TraB1-130; 18, pUT18-TraB1-77,699-772; 19, pUT18-SpdB21-99; 20, pUT18-SpdB2206-409; 21, pUT18-spdA; 22, pUT18-orf140; −, pUT18/pKT25; +, pUT18zip/pKT25zip. Download Figure S3, EPS file, 0.7 MB.
Copyright © 2015 Thoma et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Additional Files
Supplementary Data
Supplementary Data
- Figure sf1, TIF - Figure sf1, TIF
- Figure sf2, TIF - Figure sf2, TIF
- Figure sf3, EPS - Figure sf3, EPS
- Table st1, DOCX - Table st1, DOCX
- Table st2, DOCX - Table st2, DOCX
- Table st3, DOCX - Table st3, DOCX
- Table st4, DOCX - Table st4, DOCX
- Table st5, DOCX - Table st5, DOCX
- Table st6, DOCX - Table st6, DOCX
