Microbial cationic peptides as a natural defense mechanism against insect

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1 Microbial cationic peptides as a natural defense mechanism against insect antimicrobial peptides Tien Duy Vo 1*, Christoph Spahn 2*, Mike eilemann 2, elge B. Bode 1,3,4,5 1 Fachbereich Biowissenschaften, Molekulare Biotechnologie, Goethe-Universität Frankfurt, Frankfurt am Main 60438, Germany 2 Single Molecule Biophysics, Institute of Physical and Theoretical Chemistry, Goethe-Universität Frankfurt, Frankfurt am Main 60438, Germany 3 Buchmann Institute for Life Sciences (BMLS), Goethe-Universität Frankfurt, Frankfurt am Main 60438, Germany 4 Senckenberg Gesellschaft für aturforschung, Frankfurt 5 Max-Planck-Institute for Terrestrial Microbiology, Marburg 35043, Germany * These authors contributed equally correspondence: Supporting Information 1

2 Supplementary methods PAX peptides used in this study R 2 R 1 Table S1: PAX derivatives used in this study. PAX derivatives 1 and 2 represent the naturally occurring PAX 1 and PAX 3 (1), respectively. Derivatives 3 and 4 were synthesized as described in detail below. 1 4 R 1 R2 Chemical formula Calculated m/z [M+2 + ] 8 measured m/z [M+2 + ] C C C C The following amino acids were used as building blocks and were either acquired from Sigma Aldrich, Bachem, Carbolution or Iris Biotech: Fmoc-Glycine, Fmoc-Boc-L-Lys, Fmoc-Boc-D-Lys, Fmoc-Lys(Alloc)-, and Fmoc-L-Lys( 3 )-. Synthesis route of PAX derivatives 2

3 Peptide elongation Loading 2-CTC-Resin Cl 5 R 1 Boc Boc Alloc deprotection Boc 6 R 1 Boc Boc n-resin cyclisation Boc 7 Fmoc R 1 Fatty acid coupling 8 R 1 3, 4 2 Peptides were created with Fmoc solid-phase synthesis. First, the process started with a Fmoc-L-Lys-All, which was linked to the resin via an amine group (5). ext, the Fmoc group is removed and the peptide was successively built to the fifth lysine. ere, 3

4 a Fmoc-Lys(Alloc) is used and the peptide was continually built to the amino acid glycine (6). After that, the Alloc protective groups were removed (7) to perform an onresin cyclization between the lysine at positions one and five (8). In the last step, the final product was obtained by coupling the peptide with the fatty acid (3,4). Loading of 2-CTC-Resin with Fmoc-L-Lys-All*Cl Cl mg 2-CTC resin (100 μmol, 1.0 eq) was suspended in 1.5 ml dry DCM in a polyethylene syringe under nitrogen and incubated for 30 min at room temperature. In addition, 22 μl thionyl chloride (300 μmol, 3.0 eq) was added and incubated for another 1h. In a separate flask, 45 mg Fmoc-L-Lys-All*Cl (100 μmol, 1.0 eq) was dissolved in dry DCM with 7 μl DIPEA and incubated for 1h at room temperature. After 1h thionyl chloride was discarded and the syringe was washed five times with dry DCM. Finally, the mixture with Fmoc-L-Lys-All*Cl was mounted into the syringe and incubated overnight at room temperature. The Fmoc group was deprotected by 20% piperidine and the product yield was elucidated by UV/VIS (Genesys 10S, Thermo Fisher Scientific). The loading efficiency was ~ 80%. 4

5 Peptide elongation Boc Boc R 1 Boc 6 The peptide was elongated by the peptide synthesizer (Syro Wave, Biotage). The common workflow comprised the following steps: washing (three times with MP, DMF, and DCM), deprotection with piperidine, washing, amino acid activation, coupling, and washing. The products were confirmed by PLC-MS. The yield was determined by UV-Measurement during Fmoc deprotection and was around 90% for all samples. Alloc deprotection Boc Boc R 1 Boc 7 In a Schlenk tube, 29.0 mg (25 μmol, 1 eq) TPP palladium(0) and 31 μl (250 μmol, 10 eq) phenylsilane was added to 2 ml dry DCM under nitrogen atmosphere. The syringe was flushed with nitrogen and the mixture from the Schlenk tube was mounted. The syringe was incubated for 4h at room temperature. The resin was washed three times with the following solvents: DCM, 1 M pyridine*cl in Me, Me, 25 mm Sodium diethyldithiocarbamate. In addition, the resin was washed excessively with 5

6 DCM until the resin showed a faint yellow color. The yield was determined by PLC-MS measurement of the deprotected product and was around 90%. n-resin cyclisation Fmoc R mg (250 μmol, 10 eq) xyma Pure was mixed with 48.0 mg (250 μmol, 10 eq) EDC*Cl and dissolved in 1 ml DMF. The reaction mixture was drawn up into the syringe and was incubated overnight at room temperature. ext, the Fmoc group was removed and the yield was elucidated by UV/VIS. Complete cyclization was achieved. The yield was determined by PLC-MS measurement of the cyclized product. 6

7 Fatty acid coupling R 1 2 3, mg (143 μmol, 5.7 eq) (R)-3-hydroxydodecanoic acid, 48.0 mg (125 μmol, 5 eq) ATU, 17.0 mg (125 μmol, 5 eq) AT, 43 μl (250 μmol, 10 eq) DIPEA was dissolved in 1.5 ml DMF. In the next step, the syringe was filled with the reaction mixture and incubated overnight at 37 C. The peptide was cleaved with a TFA, TIS, and Water solution (v/v 95:2.5:2.5). The final products were purified by reverse-phase PLC-MS (20-80%). The product was dissolved in 2 and freeze-dried. The overall yield was reported at around 50%. R-MS Analysis A trace amount of the synthesized peptides was dissolved in 1 ml methanol. 10 µl was injected and measured by the Bruker Impact II ESI -TF using a C18 column and a 5-95% AC/ 2 gradient. MR Analysis The peptides were dissolved in D 2 and then measured by using a 500 Mz Bruker MR spectrometer. Spectra were analyzed using MestReova (MestreLab). 7

8 Cell culture and sample preparation Bacterial strains and growth conditions The bacterial strains used in this study are listed in Table S4. An X. doucetiae pax - strain was used for experiments in which the synthetic PAX derivative was added to the culture medium (2). X. doucetiae strains were grown in LB Lennox (Carl Roth) at 30 C and E. coli strains in LB Miller (Carl Roth) at 37 C. Single colonies were picked for overnight () cultures, which were diluted 1:100 (X. doucetiae) or 1:200 (E. coli) for the working day cultures used in the experiments. Culture viability was assessed by monitoring the D 600. Treatment with synthetic azido-pax Cells were grown to D 600 ~ 0.25 as described above and azido-pax (3) (in dd 2 ) was added for 11 min at various concentrations (10, 30, and 100 µg ml -1 ). For 10 µg ml -1 and 30 µg ml -1 final concentration, 3 was pre-diluted 1:10 or 3:10 in dd 2 to keep the volume of added water constant. As a control, 10% dd 2 (v/v) was added to one culture aliquot. During PAX treatment, 70 μl of PAX control solution were added to 630 μl cell suspension in 2 ml reaction tubes (Eppendorf). Cell fixation and immobilization Cells were chemically fixed for 15 min at room temperature by directly adding fixation solution to the culture aliquots, resulting in a final concentration of 2% methanol-free formaldehyde (Thermo Fisher Scientific, catalogue number 28908), 0.1% EM-grade glutaraldehyde (Electron Microscopy Sciences, catalogue number 16200) and 33 mm sodium phosphate buffer (p 7.5). After incubation, cells were pelleted (3 min at 5,000 g) and resuspended in freshly prepared PBS containing 0.2% ab 4 (w/v) to quench excess aldehydes. After 3 min incubation, cells were washed thrice in PBS and immobilized on K-cleaned, poly-l-lysine coated chamber slides (Sarstedt, catalogue number ) as described elsewhere (3). 8

9 Click-labelling Immobilized cells were permeabilized using 0.5% TX-100 in PBS for min and subsequently washed twice with PBS. 250 μl Click-labelling solution containing 100 mm tris p 8.0 (Thermo Fisher Scientific), 1 mm CuS 4 (Sigma Aldrich), 100 nm Sulfo-Cy5 alkyne (Lumiprobe) and 100 mm L-ascorbic acid (Sigma Aldrich) was added to each chamber and samples were incubated 60 min in the dark. Samples were then rinsed thrice and washed thrice (5 min) with PBS to remove unbound fluorophores. A scheme of the click reaction is shown in Fig. S13. Quantification utilizing PLC-MS Cell cultures of X. doucetiae wild type, pax - and pax + were grown in triplicate for 24 h at 30 C and 220 rpm. pax + cultures hereby represent the pax - strain induced with 0.2% arabinose (2). 1 ml of each sample was pelleted at 5000 rpm for 4 min at 20 C. The cell pellet and the supernatant fraction were separated. In addition, the pellet was sonicated for 15 min. Both fractions were freeze-dried (Alpha 1-2 LDplus, Christ) and resuspended in a 400 μl methanol/water mix (v/v 1:1) and acidified with 1% formic acid. The cell pellet fraction was sonicated again for 5 min. Both fractions were pelleted at 5000 rpm for 4 min at 20 C. The insoluble fractions were then separated by centrifugation (5000 rpm, 4 min at 20 C). All samples were analyzed by reverse-phase PLC using a semi-preparative C18 column (AmaZon, Bruker, linear-gradient acetonitrile 5%-95%, 0,1% TFA). This protocol allowed us to map the peaks of two naturally occurring PAX derivatives with high resolution and signal-to-noise ratio. The integrated area of the respective peaks was determined in the extracted chromatograms and normalized to a standard curve of known concentrations of compound 4 (Fig. 1, Fig. S1), yielding the concentration of PAX derivatives 1 and 2. Confocal microscopy Confocal images were acquired on a commercial Leica SP8 system (Leica Microsystems), equipped with a 63x 1.40 A oil immersion objective (Leica C PL AP CS2). 633 nm diode laser was used for Cy5 excitation and the fluorescence emission was collected in the window nm. 9

10 Super-resolution microscopy Image acquisition dstrm and PAIT imaging were performed on a custom-built microscope for singlemolecule detection (described elsewhere (3)). For multi-color imaging, 1-3 nm JF oechst (kindly provided by Luke Lavis, Janelia Farm Research Campus) and pm ile Red (Sigma Aldrich) were diluted in imaging buffer containing 100 mm tris p 8.0, 100 mm cysteamine hydrochloride (Sigma Aldrich), 10 mm acl (Sigma Aldrich) and 50% D 2 (Eurisotop). The different channels were recorded sequentially using 647 nm laser excitation for Cy5, 488 nm for JF 503 -oechst, and 568 nm for ile Red at appropriate excitation powers (typically 2-5 kw cm - ²), with 10,000 15,000 frames acquired at 33 z frame rate for each channel. Single-molecule fitting and filtering PAIT and dstrm movies were analyzed using the open-source software Picasso (4). Single-molecule signals were identified using the Picasso module Filter, adjusting the box size (5 or 7 pixels) and gradient with respect to the emission wavelength and signal brightness. After fitting (least square fitting algorithm), sample drift was corrected using the redundant cross-correlation (RCC) routine implemented in the Picasso module Render (5). Fluorescence emission over consecutive frames was spatiotemporally linked within 80 nm distance while allowing for 1 dark frame. Finally, the localization list was filtered (Picasso module Filter ) for the PSF standard deviation (both x and y), maintaining localizations with 80 nm < < 190 nm for ile Red and JF 503 -oechst or 95 nm < < 205 nm for Cy5. Correction of chromatic aberrations and image registration Multi-color images were corrected for chromatic aberrations using the linear alignment tool implemented in rapidstrm (6). Image sequences of glass-immobilized tetraspecks were recorded in each channel and used to create the linear alignment matrices. Picasso localization lists were converted into rapidstrm format and both JF 503 -oechst and Cy5 data were aligned to the ile Red channel. The resulting images were registered using the open-source image processing platform Fiji (7). Brightness and contrast in each channel were manually adjusted. 10

11 Bioactivity tests Details about the compounds used for bioactivity tests a broth microdilution assays are listed in Table S5. Broth microdilution assays of X. doucetiae WT, X. doucetiae pax - and E. coli MG1655 strains X. doucetiae WT, the pax - mutant and E. coli were grown in LB overnight at 30 C and 220 rpm. The cultures were harvested by centrifugation at 5000 rpm, 4 min at 20 C. The pellet was dissolved in 1 ml 0.9% acl, adjusted to McFarland standard 0.5, and diluted 1:10 ( 1x10 7 CFU ml -1 ). The assays were performed in 96 well plates and triplicates. 100 μl cation-adjusted Mueller inton II Broth (Sigma Aldrich) was added to each well. 100 μl of the standard AMP concentration was added in the first row while 100 μl of water was added to the negative control. Polymyxin B (Carl Roth) was used as positive control and the pax - mutant was induced by 0.2% arabinose. After addition, the fluids were mixed and 100 μl from the top row was mixed with the next row resulting in a serial dilution with a factor of two. Finally, 5 μl of the tested cultures were added to each well (~ CFU). The optical density for each well was measured using a plate reader (Tecan Spark 150 M, Tecan Group) shaking at 37 C over a time course of 20 h (20 min interval). Statistical methods The amount of PAX found in the cell pellet and supernatant of X. doucetiae WT and pax + cultures was tested for significance using the Welch s t-test. 11

12 Supplementary Tables Table S2: PAX amounts in different fractions for different X. doucetiae cultures as quantified using PLC-MS. P = pellet fraction, S = supernatant fraction. umbers were obtained from 1 ml culture and adjusted D 600 = 1. Bold numbers indicate the total amount of 1 and 2 produced by the respective strains. PAX PAX amount [µg ml -1 ] (WT) PAX amount [µg ml -1 ] (pax + ) derivative P S P + S P S P + S ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.8 Table S3: m/z and mass errors of b- and y-ions generated by CID of 3 and 4. This table refers to Fig. S6 and S CID-Ion m/z Δ ppm m/z Δ ppm y y y y y y b b b b b Table S4: Bacterial strains used in this study Strain Genotype Source Reference X. doucetiae DSM17909 Wild type DSMZ (8) X. doucetiae pax - P BAD PAX Bode lab (2) E. coli MG1655 F- lambda- ilvg- rfb-50 rph-1 CGSC (#6300) (9) 12

13 Table S5: Compounds used for MIC and bioactivity tests performed in this study, /A = not applicable. Drosocin represents the peptide lacking the naturally occurring disaccharide. Compound Sequence Supplier Cat. # Cecropin A KWKLFKKIEKVGQIRDGIIKAGPAVAVVGQATQIAK Sigma C MG Drosocin GKPRPYSPRPTSPRPIRV Anaspec AS Melittin GIGAVLKVLTTGLPALISWIKRKRQQ Sigma M2272-1MG Polymyxin B /A Carl Roth

14 Supplementary Figures Figure S1: PLC-MS standard curve of PAX B174 (4) for quantification of PAX derivative 1 and 2. Cell pellet fraction Supernatant fraction Figure S2: PLC-MS chromatograms for the quantification of PAX derivatives naturally produced by the X. doucetiae WT strain. Base peak chromatograms (BPC) of the cell pellet (top panel) and supernatant fractions (middle panel) are shown including the peaks assigned to derivatives 1 and 2. The bottom panel compares the extracted ion chromatograms (EIC) of both derivatives for the respective fractions. 14

15 Cell pellet fraction Supernatant fraction Figure S3: PLC-MS chromatograms for the quantification of PAX derivatives produced by the X. doucetiae pax + strain after induction with 0.2% arabinose. BPC of the cell pellet (top panel) and supernatant fractions (middle panel) are shown including the peaks assigned to derivative 1 and 2. The bottom panel compares the extracted ion chromatograms (EIC) of both derivatives for the respective fractions. Figure S4: PLC-MS chromatograms for the quantification of PAX derivatives produced by the X. doucetiae pax - strain. BPC from pax - cell pellet (red, top), overlaid with the EICs of 1 (blue) and 2 (black). In the bottom panel, a magnified view of the EICs of 1 and 2 alone is shown (Intensity EIC/BPC ratio = 0.01). nly noise can be detected for both derivatives. 15

16 Figure S5: PLC-MS chromatograms for the quantification of PAX derivatives naturally produced by the X. doucetiae pax - strain. BPC from pax - cell supernatant (golden, top), overlaid with the EICs of 1 (blue) and 2 (black). In the bottom panel, a magnified view of the EICs of 1 and 2 alone are shown (Intensity EIC/BPC ratio = 0.01). nly noise can be detected for both derivatives. Compound 3 relative intensity [%] A y1 y2 y3 y4 y5 y6 b6 b y6 y5 y4 y3 y2 y1 B FA-G-K-K-K- 3 -K-K mass [m/z] b6 b7 Figure S6: (A) CID-ion (Collision-induced dissociation) spectrum of 3 ([M+ + ] = ) recorded using the Bruker Impact II ESI -TF. b-ions are labeled with dashed lines and y- ions with dotted lines. Intensities are relative to [M+ + ]. (B) Primary structure of 3 with detected b- and y-ions. 16

17 Compound 4 A relative intensity [%] 8 4 y1 y2 y3 b3 y4 b4 y5 b5 y6 b6 b y6 y5 y4 y3 y2 y1 B FA-G-K-K-K-K-K-K mass [m/z] b3 b4 b5 b6 b7 Figure S7: (A) CID-ion spectrum of 4 ([M+ + ] = ) recorded using the Bruker Impact II ESI -TF. b-ions are labeled with dashed lines and y-ions with dotted lines. Intensities are relative to [M+ + ]. (B) Primary structure of 4 with detected b- and y-ions f1 (ppm) Figure S8: 1 MR (500 Mz, D 2 ) of compound 3. δ 4.29 (m, 4), 4.19 (t, 1), 4.06 (dt, J = 11.7, 6.1 z, 1), 3.99 (dd, 2), 3.65 (s, 1), 3.46 (m, 1), 3.37 (t, 2), 3.01 (m, 8), 2.55 (dt, 2), (m, 58), 0.88 (t, 3). 17

18 Azido-Modifikation C-Atom connected to the azide group f1 (ppm) Figure S9: 13 C MR (125 Mz, D 2 ) of compound f1 (ppm) Figure S10: 1 MR (500 Mz, D 2 ) of compound 4. δ 4.4 (dd, J = 9.4, 5.8 z, 1), 4.32 (dt, J = 8.8, 6.0 z, 2), (m, 2), 4.05 (m, 1), 3.49 (m, 1), 3.02 (q, J = 6.8 z, 10), (dd, 2), (m, 56), 0.88 (t, 3). 18

19 f1 (ppm) Figure S11: 13 C MR (125 Mz, D 2 ) of compound MIC [μg ml -1 ] Figure S12: MIC tests (triplicates) of different PAX derivatives on E. coli MG1655 (blue bar), X. doucetiae WT (dark grey bar), and pax - mutant (orange bar) cultures. umbers indicate the PAX constructs 3 and 4 as shown in the section PAX peptide synthesis. 19

20 K 3 S S mm tris p mm CuS nm Sulfo-Cy5 alkyne 100 mm L-ascorbic acid 60 min (dark) - 3 S K 3 S Figure S13: Scheme of the CuAAC reaction. 20

21 Figure S14: Titration of synthetic PAX 3 to X. doucetiae pax - and E. coli MG1655 cultures. (A) Representative images of X. doucetiae pax - cells treated with various concentrations of compound 3. (B) Representative images E. coli MG1655 cells treated with various concentrations of compound 3. Brightness and contrast were kept identical in all PAX-Cy5 images except for E. coli cells treated with 100 µg ml -1 (4-fold reduction, lower right image corner). Scale bars are 5 µm. (C) Binding analysis. The intensity of the fluorescence signal 21

22 was measured for single cells and normalized to the cell cross-section area. For E. coli cells treated with 100 µg ml -1, the dataset was split into cells with membrane-localized signals and cells with cytosolic signals (asterisk). (D) Average cell length vs. PAX concentration. PAX treatment has no apparent influence on the average cell length both for E. coli (filled black circles) and X. doucetiae (filled red circles). Figure S15: Localization of PAX on X. doucetiae pax - cells. Compound 3 (click-labeled with Cy5, red) colocalizes with the bacterial membrane (labeled with ile Red, cyan) independent of the concentration applied. o change in nucleoid morphology (labeled with JF 503 -oechst, yellow hot) was observed, indicating that X. doucetiae cells maintain membrane integrity. Scale bars are 2 µm. 22

23 Figure S16: Localization of PAX on E. coli MG1655 cells. Compound 3 (click-labeled with Cy5, red) mainly colocalizes with the bacterial membrane (labeled with ile Red, cyan) at 10 and 30 µg ml -1 PAX concentration. A fraction of cells shows the strong azido-pax signal in the cytosol, only omitting regions where chromosomal DA (labeled with JF 503 -oechst, yellow hot) is localized. Flooding of the cytosol by PAX is accompanied by a strong reorganization of the nucleoid. Scale bars are 1 µm. 23

24 Figure S17: Growth checkpoint analysis of the data shown in Fig. 3. Shown are the times when the cultures reach an absorption value of 0.2. Lacking data points (e.g. t = 100 min for drosocin treatment) indicate that cultures did not reach this value over the entire experiment time of 25 h. Data points represent mean values and shaded areas of the respective standard deviation. Figure S18: Growth inhibition assay of E. coli MG1655 cultures treated with different insect AMPs. E. coli MG1655 cultures were grown in the absence (control, black dots and shaded areas) and presence of (A) drosocin, (B) melittin and (C) cecropin alone (blue dots and shaded areas) or together with PAX (compound 4, red dots and shaded areas). Since 2.5 µg ml -1 cecropin showed no antimicrobial effect, the experiment was repeated with a higher cecropin and PAX concentrations. Data points represent mean values and shaded areas of the respective standard deviation. 24

25 References 1. Fuchs, S. W., Proschak, A., Jaskolla, T. W., Karas, M., and Bode,. B. (2011) Structure elucidation and biosynthesis of lysine-rich cyclic peptides in Xenorhabdus nematophila, rg. Biomol. Chem. 9, Bode, E., einrich, A. K., irschmann, M., Abebew, D., Shi, Y.-., Vo, T. D., Wesche, F., Shi, Y.-M., Grün, P., Simonyi, S., Keller,., Engel, Y., Wenski, S., Bennet, R., Beyer, S., Bischoff, I., Buaya, A., Brandt, S., Cakmak, I., Çimen,., Eckstein, S., Frank, D., Fürst, R., Gand, M., Geisslinger, G., azir, S., enke, M., eermann, R., Lecaudey, V., Schäfer, W., Schiffmann, S., Schüffler, A., Schwenk, R., Skaljac, M., Thines, E., Thines, M., Ulshöfer, T., Vilcinskas, A., Wichelhaus, T. A., and Bode,. B. (2019) Promoter Activation in Δhfq Mutants as an Efficient Tool for Specialized Metabolite Production Enabling Direct Bioactivity Testing, Angew. Chem. Int. Ed. 58, Spahn, C. K., Glaesmann, M., Grimm, J. B., Ayala, A. X., Lavis, L. D., and eilemann, M. (2018) A toolbox for multiplexed super-resolution imaging of the E. coli nucleoid and membrane using novel PAIT labels, Scientific reports 8, Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F., and Jungmann, R. (2017) Superresolution microscopy with DA-PAIT, at. Protoc. 12, Wang, Y., Schnitzbauer, J., u, Z., Li, X., Cheng, Y., uang, Z.-L., and uang, B. (2014) Localization events-based sample drift correction for localization microscopy with redundant cross-correlation algorithm, pt. Express 22, Wolter, S., Schüttpelz, M., Tscherepanow, M., van de Linde, S., eilemann, M., and Sauer, M. (2010) Real-time computation of subdiffraction-resolution fluorescence images, Journal of microscopy 237, Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.-Y., White, D. J., artenstein, V., Eliceiri, K., Tomancak, P., and Cardona, A. (2012) Fiji: an open-source platform for biological-image analysis, at. Methods 9, Bode, E., e, Y., Vo, T. D., Schultz, R., Kaiser, M., and Bode,. B. (2017) Biosynthesis and function of simple amides in Xenorhabdus doucetiae, Environ. Microbiol. 19, Guyer, M. S., Reed, R. R., Steitz, J. A., and Low, K. B. (1981) Identification of a sex-factor-affinity site in E. coli as gamma delta, Cold Spring arb Symp. Quant. Biol. 45 Pt 1,

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