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conditions/protocols/BacterialCultureConditions.md new file mode 100644 index 0000000..11aa508 --- /dev/null +++ b/studies/Bacterial culture conditions/protocols/BacterialCultureConditions.md @@ -0,0 +1,3 @@ +## Bacterial culture conditions + +Bacteria were streaked from glycerol stocks (25% glycerol) on TSA plates (15 g/l Tryptic Soy Broth, Sigma Aldrich; with 10 g/l Bacto Agar, Duchefa Biochemie) and grown at 25 °C. Single colonies were inoculated into liquid 50% TSB (15 g/l Tryptic Soy broth, Sigma Aldrich) and grown until dense at 25 °C with 180 rpm agitation. Dense cultures were then stored at 4 °C and diluted 1 to 10 in TSB the day before the experiment and cultured at 25 °C with 180 rpm agitation overnight to ensure sufficient cell densities for slow- and rapidly growing bacteria. Glycerol stocks were stored at -80 °C and kept on dry ice when transported. \ No newline at end of file diff --git a/studies/Bacterial culture conditions/protocols/BacterialStrains.md b/studies/Bacterial culture conditions/protocols/BacterialStrains.md new file mode 100644 index 0000000..6a7fb1f --- /dev/null +++ b/studies/Bacterial culture conditions/protocols/BacterialStrains.md @@ -0,0 +1,3 @@ +## Bacterial Strains + +The bacterial strains used in this study have been initially isolated from unplanted soil, *A. thaliana* roots or shoots and are summarized in **Table S1**. *Ralstonia solanacearum* GMI1000 and GMI1600 have also been reported previously. 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Seeds were submerged in 70% ethanol and left shaking at 40 rpm for 14 minutes. Ethanol was removed before the seeds were submerged in 8.3% sodium hypochlorite (Roth) containing 1 μl of Tween 20 (Sigma-Aldrich) and left shaking at 40 rpm for 4 minutes. Under sterile conditions, the seeds were washed 7x times and finally taken up with sterile 10 mM MgC12. Seeds were left for stratification at 4 °C for 3 days. 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Using a forceps, uniform seedlings were then transferred to freshly prepared 1â„2 MS plates without sucrose. \ No newline at end of file diff --git a/studies/Plant Growth Conditions/protocols/Flowpot.md b/studies/Plant Growth Conditions/protocols/Flowpot.md new file mode 100644 index 0000000..4fb1763 --- /dev/null +++ b/studies/Plant Growth Conditions/protocols/Flowpot.md @@ -0,0 +1,3 @@ +## Flowpot + +Flowpots were assembled and inoculated as described below. Each Flowpot was first flushed with 50 ml sterile MiliQ water and then 50 ml half strength Murashige and Skoog medium with vitamins (1â„2 MS; 2.2 g/l, Duchefa Biochemie, 0.5 g/l MES, pH 5.7) containing the bacterial inoculum. Per Flowpot, five surface-sterilized and stratified *A. thaliana* Col-0 seeds were pipetted. Microboxes were then incubated in a light cabinet under short day conditions (10 h light at 21 °C, 14 h dark at 19 °C) for 14 days and randomized every 2–3 days. \ No newline at end of file -- GitLab From 803b4703e4002679530041cfc51d33c9ef905d2a Mon Sep 17 00:00:00 2001 From: Mekhrona Mirzoeva <memir101@hhu.de> Date: Wed, 13 Nov 2024 13:38:29 +0100 Subject: [PATCH 08/35] new assay Screen for antagonistic interbacterial interactions --- .gitattributes | 2 ++ .../README.md | 0 .../dataset/.gitkeep | 0 .../dataset/Table S2.xlsx | 3 +++ .../dataset/pnas.2221508120fig01.jpg | 3 +++ .../isa.assay.xlsx | Bin 0 -> 7499 bytes .../protocols/.gitkeep | 0 .../AntagonisticInterbacterialInteractions.md | 3 +++ isa.investigation.xlsx | Bin 11349 -> 7876 bytes .../isa.study.xlsx | Bin 12306 -> 12305 bytes .../Plant Growth Conditions/isa.study.xlsx | Bin 15939 -> 15939 bytes 11 files changed, 11 insertions(+) create mode 100644 assays/Screen for antagonistic interbacterial interactions/README.md create 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interactions/protocols/.gitkeep b/assays/Screen for antagonistic interbacterial interactions/protocols/.gitkeep new file mode 100644 index 0000000..e69de29 diff --git a/assays/Screen for antagonistic interbacterial interactions/protocols/AntagonisticInterbacterialInteractions.md b/assays/Screen for antagonistic interbacterial interactions/protocols/AntagonisticInterbacterialInteractions.md new file mode 100644 index 0000000..22b90fd --- /dev/null +++ b/assays/Screen for antagonistic interbacterial interactions/protocols/AntagonisticInterbacterialInteractions.md @@ -0,0 +1,3 @@ +## Screen for antagonistic interbacterial interactions + +For the initial mBA experiment (**Fig. 1**), bacterial strains were cultured for seven days in 25% TSB (7.5 g/l Tryptic Soy Broth, Sigma-Aldrich). Briefly, 100 μl of a bacterial solution were resuspended in 50 ml cooled (~38 oC), but still molten, 25% TSA (15 g/l Bacto-Agar, Duchefa Biochemie) and poured into a square petri dish (120x120 mm). After medium solidification, 24 bacterial isolates were spotted on top of the medium using a multi-stamp replicator. The replicator was sterilized by dipping in 70% EtOH (v/v) followed by flaming and cooling. The screen comprising 39,204 binary interactions was conducted once and validated by randomly re-screening 7,470 interaction pairs as described above. All bacterial strains that showed antagonistic activity were rescreened two more times. For the *Ralstonia* inhibition screen, *R. solanacearum* GMI1000 was pre-cultured for two days in CPG medium (1% peptone, 0.5% glucose and 0.1% casamino acids; pH7.0). Before spotting the bacterial cultures, each CPG agar plate (1% Peptone, 0.5% D-glucose, 0.1% casamino acids and 1.5% agar; pH 7.0) was overlaid with 5 ml of *R. solanacearum* suspension (50 μl of pre-cultured *R. solanacearum* in pure sterile water). Excess *R. solanacearum* suspension was removed and the plates were briefly dried, then 5 μl of the bacterial culture were spotted onto the *R. solanacearum*-overlaid plates. All isolates were tested three times. For all subsequent halo assays, strains were cultivated in 50% TSB until turbidity, stored at 4 °C and diluted 1:10 in 50% TSB one day before the experiment. Bacterial cultures were pelleted at 4,000 rpm for 15 min. The resulting bacterial pellets were subsequently washed 3 times and resuspended in 1 ml 10 mM MgCl2. OD600 were measured and set depending on the strain. One hundred microliters bacterial culture were inoculated per 50 ml 25% TSA. After drying, up to nine different 3-μl droplets of bacterial suspensions with 0.4 OD600 were applied with equal distances. For all experiments, plates were incubated at 25 °C for up to 96 hours and photographs were taken thereafter for quantitative image analysis. The size of the halo of inhibition was measured using ImageJ with up to five separate measurements, which were subsequently averaged to reduce variation. 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acetate and methanol, to capture greater small molecule diversity. Each bacterial strain was grown separately on 25% TSA plates (25% BBLTM TrypticaseTM Soy, BD with 1.8% Bacto-Agar; BD, Germany). After seven days of incubation at 25 °C, three to four agar plugs were taken from the periphery and inside of the bacterial colony. Agar plugs were crushed and washed with 500 μl water followed by extraction in 500 μl ethyl acetate and methanol. Between each extraction step, samples were vortexed for 30–45 s. After each extraction, the solvents were evaporated, and the residue was redissolved in 500 μl LC-MS-grade methanol and filtered through a 0.2-μm membrane into HPLC vials. Solvents for blanks (uninoculated medium) were extracted according to the same protocol. The extraction protocol was also used to analyse the inhibition zones upon inter-bacterial interactions. To this end, a bacterial lawn of a sensitive target strain, either R472D3, R480 or R553, was prepared as top agar and the antibiotic producer strains (R63, R68, R71, R342, R401, R562, R569, R690, R920 and R1310, respectively) were inoculated on top. +The agar plugs were taken from the zone of inhibition and inside of the antibiotic-producing colony. In total, 20 interactions were analysed. All samples were analysed by HPLC-MS/MS on a micrOTOF-Q mass spectrometer (Bruker) with ESI-source coupled with a HPLC Dionex Ultimate 3000 (Thermo Scientific, Germany) using a Zorbax Eclipse Plus C18 1.8 μm column, 2.1×50 mm (Agilent). The column temperature was 45 °C. MS data were acquired over a range 100–3000 m/z in positive mode. Auto MS/MS fragmentation was achieved with rising collision energy (35–50 keV over a gradient from 500–2000 m/z) with a frequency of 4 Hz for all ions over a threshold of 100. uHPLC began with 90% H2O containing 0.1% acetic acid. The gradient started after 0.5 min to 100% acetonitrile (0.1% acetic acid) in 4 min. Two microliters sample solution were injected to a flow of 0.8 ml/min. All MS/MS data were converted to ‘.mzxml’ format and transferred to the GNPS server (gnps.ucsd.edu) (4). Molecular networking was performed based on the GNPS data analysis workflow using the spectral clustering algorithm (5). The data was filtered by removing all MS/MS peaks within +/- 17 Da of the precursor m/z. MS/MS spectra were window-filtered by choosing only the top 6 peaks in the +/- 50 Da window throughout the spectrum. The data was then clustered with MS-Cluster with a parent mass tolerance of 0.02 Da and a MS/MS fragment ion tolerance of 0.02 Da to create consensus spectra. Consensus spectra that contained less than two spectra were discarded. Networks were then created from the single cultivation and from the competition experiments. Edges were filtered to have a cosine score above 0.5 (0.6 for interaction network) and more than four matched peaks. Further edges between two nodes were kept in the network only if each of the nodes appeared in each other's respective top ten most similar nodes. Sample attributes were assigned to the data files (strain, genus, family, order, class phylum extraction solvent). For the network analysis, all nodes that contained ions from the blank medium were removed. 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a/assays/DetectionOfR401DAPGandPyoverdine/protocols/.gitkeep b/assays/DetectionOfR401DAPGandPyoverdine/protocols/.gitkeep new file mode 100644 index 0000000..e69de29 diff --git a/assays/DetectionOfR401DAPGandPyoverdine/protocols/DAPGandPyoverdineDetection.md b/assays/DetectionOfR401DAPGandPyoverdine/protocols/DAPGandPyoverdineDetection.md new file mode 100644 index 0000000..b81ba3a --- /dev/null +++ b/assays/DetectionOfR401DAPGandPyoverdine/protocols/DAPGandPyoverdineDetection.md @@ -0,0 +1,3 @@ +## Detection of R401 DAPG and pyoverdine + +For the detection of iron-chelating compounds, a R401 preculture was grown over night in 5 ml LB medium at 30 °C and 200 rpm. Before inoculation of the main culture, cells were washed twice with the main culture medium to remove potential traces of iron from the medium. Erlenmeyer flasks containing 20 ml of modified MM63 (KH2PO4 13.61 g/L, KOH 4.21 g/L, (NH4)SO4 1.98 g/L, MgSO4*7 H2O 0.25 g/L, NaCl 0.5g/L, Glucose * H2O 5,00 g/L, pH 7.1 KOH/HCl) with or without addition of 0.0011 g/L FeSO4 * 7 H2O were inoculated with 100 μl of preculture and cultivated for 6 days at 30 °C and 200 rpm. Every second day, 0.5 ml sample were taken, cells were removed by centrifugation and 5 μl of supernatant were analysed on a Bruker microTOFq-II high-resolution mass spectrometer coupled to an Agilent 1290 UPLC system with an Acquity UPLC BEH C-18 reverse phase column, run in a gradient of MeCN/H2O + 0.1% formic acid. 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Subject: [PATCH 15/35] renamed Metabolomics assay, antiSMASH v6.0 assay added --- .gitattributes | 1 + .../README.md | 0 .../dataset/.gitkeep | 0 .../dataset/Table S2.xlsx | 3 +++ .../isa.assay.xlsx | Bin 0 -> 9258 bytes .../protocols/.gitkeep | 0 .../protocols/BGCAntiSMASH.md | 3 +++ .../isa.assay.xlsx | Bin 11885 -> 11885 bytes assays/Metabolomics Analysis/README.md | 0 assays/Metabolomics Analysis/dataset/.gitkeep | 0 .../dataset/Metabolomics.md | 0 .../isa.assay.xlsx | Bin 10742 -> 10746 bytes .../Metabolomics Analysis/protocols/.gitkeep | 0 .../protocols/MetabolomicAnalysis.md | 0 .../isa.assay.xlsx | Bin 11666 -> 11666 bytes isa.investigation.xlsx | Bin 7876 -> 7876 bytes .../isa.study.xlsx | Bin 12308 -> 12308 bytes .../Plant Growth Conditions/isa.study.xlsx | Bin 15939 -> 15939 bytes 18 files changed, 7 insertions(+) rename assays/{MetabolomicAnalysis => BGC prediction using antiSMASH}/README.md (100%) rename assays/{MetabolomicAnalysis => BGC prediction using antiSMASH}/dataset/.gitkeep (100%) create mode 100644 assays/BGC prediction using antiSMASH/dataset/Table S2.xlsx create mode 100644 assays/BGC prediction using antiSMASH/isa.assay.xlsx rename assays/{MetabolomicAnalysis => BGC prediction using antiSMASH}/protocols/.gitkeep (100%) create mode 100644 assays/BGC prediction using antiSMASH/protocols/BGCAntiSMASH.md create mode 100644 assays/Metabolomics Analysis/README.md create mode 100644 assays/Metabolomics Analysis/dataset/.gitkeep rename assays/{MetabolomicAnalysis => Metabolomics Analysis}/dataset/Metabolomics.md (100%) rename assays/{MetabolomicAnalysis => Metabolomics Analysis}/isa.assay.xlsx (63%) create mode 100644 assays/Metabolomics Analysis/protocols/.gitkeep rename assays/{MetabolomicAnalysis => Metabolomics Analysis}/protocols/MetabolomicAnalysis.md (100%) diff --git a/.gitattributes b/.gitattributes index fdeb834..6608dce 100644 --- a/.gitattributes +++ b/.gitattributes @@ -3,3 +3,4 @@ _publication/pnas.2221508120.sapp.pdf 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+Bacterial genomes were downloaded from “www.at-sphere.com†or NCBI and https://antismash.secondarymetabolites.org/ version 6.0. Output data from antiSMASH analysis are listed as BGC classes and predicted BGCs for each genome in **Table S2**. Only high-quality genomes, as assessed by CheckM with ≥90% completeness and ≤5% contamination ratio were used for the analysis. 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Generation of pK18mobsaB-derived plasmid containing flanking regions of the gene of interest. + +Primers were designed to amplify a 750-bp DNA sequence (i.e., flanking region) directly upstream and downstream of the target region, sharing terminal sequence overlaps to the linearized pK18mobsacB vector and the other respective flanking region using Geneious Prime. R401 genomic DNA was isolated from 6 μl dense R401 culture in 10 μl of buffer I (pH 12) containing 25 mM NaOH, 0.2 mM EDTA at 95 °C for 30 min, before the pH was readjusted using 10 μl of buffer II (pH 7.5) containing 40 mM Tris-HCl. The R401 genomic DNA was used for amplification of the flanking regions through PCR using the respective flanking region-specific primer combinations **(Table S4)**. PCR was conducted with 0.2 μl Phusion Hot Start High-Fidelity DNA polymerase (New England Biolabs) in 20-μl reactions containing 4 μl 5x Phusion HF buffer (New England Biolabs), 0.4 μl 10 mM dNTPs, 1 μl of 10 μM forward primer, 1 μl of 10 μM reverse primer, 2 μl of R401 genomic DNA as template, filled up to 20 μ1 with nuclease-free water. The tubes were placed into a preheated (98 °C) thermal cycler set at the following program: 98 °C for 30 s, 35 cycles of 98°C for 7 s, 60°C for 20 s, 72°C for 15 s, then a final extension at 72°C for 7 min. +Five microliters of the PCR product were combined with 1 μl Orange DNA Loading Dye (6x; New England Biolabs), loaded on 1% agarose gels containing 0.05% EtBr, and run at 110 mV. After confirmation of successful amplification, the PCR product was purified using AMPure XP (Beckman-Coulter) and subsequently quantified using Nanodrop (Thermo Fisher Scientific). Plasmid purification was performed on an *E. coli* culture containing plasmid pK18mobsacB using the QIAprep Spin Miniprep Kit for plasmid DNA purification (QIAGEN) following the manufacturer's instructions. The pkl8mobsacB vector was then amplified and linearized through PCR using the PKSF and PKSR primers +361 **(Table S4)**. PCR was conducted with 0.2 μl Phusion Hot Start High-Fidelity DNA polymerase (New England Biolabs) in 20-μl reactions, largely as described above with 1 μl 0.1 ng/μl pkl8mobsac as a template. Annealing temperature was decreased to 55 °C and extension time increased to 150 s for each cycle. Template DNA was digested by DpnI (New England Biolabs) in 50-μl reactions containing 1 μl DpnI, 1 μg DNA, 5 μl Cutsmart buffer (New England Biolabs) and filled up to 50 μl with nuclease-free water. The tubes were then incubated at 37 °C for 15 min followed by heat inactivation at 80 °C for 20 min. Five microliters of the DpnI-digested plasmid were combined with 1 μl Orange DNA Loading Dye and analysed by DNA agarose electrophoresis. Upon successful verification of amplification and digestion, the remaining sample was purified using AMPure XP and subsequently quantified using Nanodrop. Linearized pK18mobsacB and both flanking regions were mixed in a molar ratio of 1:3:3 into a 10-μl total volume, added to 10 μl 2X Gibson Assembly® Master Mix (New England Biolabs) and incubated at 50 °C for 1 h. diff --git a/assays/Mutant Generation/protocols/MutantGeneration.md b/assays/Mutant Generation/protocols/MutantGeneration.md new file mode 100644 index 0000000..6a3d7f4 --- /dev/null +++ b/assays/Mutant Generation/protocols/MutantGeneration.md @@ -0,0 +1,6 @@ +## Mutant Generation + +Marker-free knockouts in R401 were generated through homologous recombination using the cloning vector pK18mobsacB (GenBank accession: FJ437239), which encodes the *kanR* and *sacB* genes conferring resistance to kanamycin and susceptibility to sucrose, respectively. In this method, upstream and downstream sequences of the gene to be deleted are integrated into the pKl8mobsacB suicide plasmid by Gibson assembly. The resulting plasmid is transformed into BW29427 *E. coli* cells and subsequently conjugated into R401. The plasmid is then integrated into the chromosome by homologous recombination and deletion mutants are generated by a second sucrose counter-selection-mediated homologous recombination event. The protocol is adapted from (14). + + +14. B. H. Kvitko, A. 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cells according to the following heat shock protocol: 2 μl of the vector were gently mixed with 50 μl of competent cells, and the resulting mixture was incubated on ice for 30 min. The mixture was transferred to a water bath at 42 °C for 1 min and put back on ice for 2 min. Then, 1 ml of 50% TSB with 50 μg/ml diaminopimelic acid (DAP; Sigma-Aldrich) was added to the heat-shocked cells, the mixture was left to regenerate at 37 °C for 1 h and then plated on 50% TSA containing 25 μg/ml Kanamycin (Kan) and 50 μg/ml DAP. The plates were incubated at 37 °C overnight. Resulting colonies were validated by colony PCR using the M13F and M13R primers. Colony PCR was performed on at least four separate colonies with 0.4 μl DFS-Taq polymerase (BIORON) in 25 μl reactions containing 2.5 μl 10x incomplete buffer (BIORON), 0.5 10 mM MgC12, 0.5 μl 10 mM dNTPs, 0.75 μl 10 μM forward primer, 0.75 μl 10 μM reverse primer, a small fraction of a colony and filled up to 25 μl with nuclease-free water. The tubes were placed in a thermocycler set at the following program: 94°C for 2 min, 35 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 2 min, then a final extension at 72 °C for 10 min. Five microliters of the PCR product were combined with 1 μl Orange DNA Loading Dye and analysed by DNA agarose electrophoresis. 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-0,0 +1,3 @@ +## Conjugation of E. coli and R401 and selection for first homologous recombination event + +*E. coli* BW29427 cells containing the plasmid and R401 were inoculated into 4 ml of 50% TSB containing 25 μg/ml Kan and 50 μg/ml DAP or 50% TSB and incubated overnight at 37 °C with 180 rpm agitation or 25 °C with 180 rpm agitation, respectively. Cells were harvested by centrifugation at 8,000 rpm for 2 min at room temperature, then washed 3x and subsequently resuspended in 1 ml of 50% TSB followed by centrifugation, after which the supernatant was discarded. After quantifying OD600, both cultures were mixed to equal parts and approx. 10x concentrated by centrifugation. The bacterial suspension was plated on 50% TSA plates containing 50 μg/ml DAP and incubated at 25 °C overnight to allow for conjugation events. The mating patches were scraped of the plate and resuspended in 1 ml 50% TSB. Then, 100 μl were spread on 50% TSA plates containing 25 μg/ml Kan and 50 μg/ml Nitrofurantoin (Nitro; Sigma-Aldrich; to counter-select *E. coli*) and incubated at 25 °C. Colonies were validated for successful genomic insertion of the plasmid via colony PCR using a primer specific to the genomic DNA approx. 150 bp upstream of the upward flanking region (upup) and the plasmid specific M13F primer. Colony PCR was performed on at least 15 separate colonies and a WT control with 0.4 μl DFS-Taq polymerase in 25-μl reactions as described previously, but with an annealing temperature of 60 °C. Five microliters of the PCR product were combined with 1 ml Orange DNA Loading Dye and analysed by DNA agarose electrophoresis followed by Sanger sequencing following the manufacturer's protocol. \ No newline at end of file diff --git a/assays/Mutant Generation/protocols/SucroseSecondHomologousRecombination.md b/assays/Mutant Generation/protocols/SucroseSecondHomologousRecombination.md new file mode 100644 index 0000000..dcfd4b1 --- /dev/null +++ b/assays/Mutant Generation/protocols/SucroseSecondHomologousRecombination.md @@ -0,0 +1,3 @@ +## Sucrose counter-selection to induce the second homologous recombination event + +A R401 colony with a successful genomic insertion of the plasmid was resuspended from a plate into 1 ml of 50% TSB. The cell density in the medium was then measured using the Multisizer 4e Coulter Counter (Beckman Coulter) following the manufacturer's protocol. One hundred microliters of 500 cells/μl, 5,000 cells/μl and 50,000 cells/μl dilutions were spread on three separate 50% TSA plates containing 300 mM sucrose. The plates were incubated at 25 °C for approx. 48 h. At least 30 colonies were examined by colony PCR using the respective upup and dwdw primers. Colony PCR was performed with 0.4 μl DFS-Taq polymerase in 25-μl reactions as described previously with an annealing temperature of 60 °C. Five microliters of the PCR product were combined with 1 μl Orange DNA Loading Dye and analysed by DNA agarose electrophoresis. Positive colonies were purified by streaking on new 50% TSA plates and further verified by Sanger sequencing (Eurofins Scientific) following the manufacturer's protocol. They were also streaked on 50% TSA containing 25 μg/ml Kan to verify loss of the plasmid. A second colony PCR was performed on positive colonies and a wt control to validate the absence of the GOI, using a forward (inF) and reverse (inR) primer inside the GOI. Colony PCR was performed with 0.4 μl DFS-Taq polymerase in 25 μl reactions as described previously. Five microliters of the PCR product were combined with 1 ml Orange DNA Loading Dye and analysed by DNA agarose electrophoresis. Upon successful verification, 4 ml of 50% TSB were inoculated with a positive colony and grown overnight at 25 °C at 180 rpm. 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HcmV?d00001 diff --git a/assays/Mini-Tn5TransposonMutantInR401andR569/protocols/.gitkeep b/assays/Mini-Tn5TransposonMutantInR401andR569/protocols/.gitkeep new file mode 100644 index 0000000..e69de29 diff --git a/assays/Mini-Tn5TransposonMutantInR401andR569/protocols/Mini-Tn5TransposonMutantInR401andR569.md b/assays/Mini-Tn5TransposonMutantInR401andR569/protocols/Mini-Tn5TransposonMutantInR401andR569.md new file mode 100644 index 0000000..b4a2866 --- /dev/null +++ b/assays/Mini-Tn5TransposonMutantInR401andR569/protocols/Mini-Tn5TransposonMutantInR401andR569.md @@ -0,0 +1,3 @@ +## Establishment of mini-Tn5 transposon mutant collections in R401 and R569 + +Mini Tn5-mutant collections of R401 and R569 were established similarly with only minor changes as described below. Liquid cultures of R401 or R569 and *E. coli* strain BW29427 carrying plasmid pUTmTn5Km2 (15) were grown overnight in no antibiotics or 25 μg/ml Kan and 50 μg/ml DAP at 25 °C or 37 °C, respectively. Conjugation was carried out as described above in *“Conjugation of E. coli and R401 and selection for first homologous recombination eventâ€*. For R401, the mating patch was taken up in 1 ml 50% TSB liquid medium and subsequently plated on 50% TSA plates containing 25 μg/ml Kan and 50 μg/ml Zeocin in four different dilutions (undiluted, 1:3, 1:4, and 1:5) and left to grow at 25 °C for 48 h. Individual colonies were picked in 100 μl sterile 50% TSB in 96-well culture plates, sealed and left to grow at 25 °C and 180 rpm for 24 h. Subsequently, 100 μl 50% glycerol were added to each well and plates were frozen until further processing. The outer rows and columns were left uninoculated as to avoid positional effects in the subsequent forward genetic screen. For R569, resuspended mating patches were stocked at -80 °C in 700-μl aliquots using a final concentration of 25% Glycerol. 1:4 dilutions were plated onto 50% TSA plates supplemented with 120 μg/ml Kan, 50 μg/ml Rifampicin and 50 μg/ml Zeocin and incubated at 25 °C for 48h. Individual colonies were inoculated in 100 μl 50% TSB supplemented with the same antibiotics at the same concentrations in 96-well plates and incubated at 25 °C and 180 rpm for 48 h. Then, 100 μl 50% glycerol were added to each culture and plates were frozen at -80 °C. \ No newline at end of file -- GitLab From fad359aedaa9c851e05c2d3b6b623ab23263c3e7 Mon Sep 17 00:00:00 2001 From: Mekhrona Mirzoeva <memir101@hhu.de> Date: Tue, 4 Mar 2025 17:04:14 +0100 Subject: [PATCH 21/35] added protocol and dataset for loss of R401s growth inhibition of Rs GMI1600 --- .gitattributes | 1 + .../README.md | 0 .../dataset/.gitkeep | 0 .../dataset/Table S5.xlsx | 3 +++ .../isa.assay.xlsx | Bin 0 -> 6926 bytes .../protocols/.gitkeep | 0 ...n5ScreenLossR401sGrowthInhibitionRsGMI1600.md | 5 +++++ 7 files changed, 9 insertions(+) create mode 100644 assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/README.md create mode 100644 assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/.gitkeep create mode 100644 assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/Table S5.xlsx create mode 100644 assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/isa.assay.xlsx create mode 100644 assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/protocols/.gitkeep create mode 100644 assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/protocols/Mini-Tn5ScreenLossR401sGrowthInhibitionRsGMI1600.md diff --git a/.gitattributes b/.gitattributes index 7ca77f5..51fdd51 100644 --- a/.gitattributes +++ b/.gitattributes @@ -5,3 +5,4 @@ _publication/dataverse_files/Table[[:space:]]S2.xlsx filter=lfs diff=lfs merge=l assays/Screen[[:space:]]for[[:space:]]antagonistic[[:space:]]interbacterial[[:space:]]interactions/dataset/Table[[:space:]]S2.xlsx filter=lfs diff=lfs merge=lfs -text assays/BGC[[:space:]]prediction[[:space:]]using[[:space:]]antiSMASH/dataset/Table[[:space:]]S2.xlsx filter=lfs diff=lfs merge=lfs -text assays/Mini-Tn5TransposonMutantInR401andR569/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text +assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text diff --git a/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/README.md b/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/README.md new file mode 100644 index 0000000..e69de29 diff --git a/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/.gitkeep b/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/.gitkeep new file mode 100644 index 0000000..e69de29 diff --git a/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/Table S5.xlsx b/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/Table S5.xlsx new file mode 100644 index 0000000..9d758ba --- /dev/null +++ b/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/Table S5.xlsx @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:ee62110bf04b25262ecbaa77d8c9a5cd8d87a34fd4d2d7ab6657d1086f3884ce +size 13477 diff --git a/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/isa.assay.xlsx 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b/assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/protocols/Mini-Tn5ScreenLossR401sGrowthInhibitionRsGMI1600.md @@ -0,0 +1,5 @@ +## Mini-Tn5 transposon mutant screen for loss of R401s growth inhibition of Rs GMI1600 + +Each R401 mini-Tn5 transposon mutant was screened individually for loss of inhibitory activity against GFP-expressing *Rs* GMI1600 and for wild type-like growth, as we observed in first trials that mutants that are impaired in growth are also more likely to have reduced inhibitory activity against Rs GMI1600. This screen was conducted in a 96-well plate format with GFP expression of *Rs* GMI1600 in response to individual R401 mutants and Abs~600~ of axenically grown, individual R401 mutants as readouts. Therefore, for each mutant, two wells were inoculated in parallel, one in the presence of *Rs* GMI1600 and one grown axenically. Rs GMI1600 was streaked on 50% TSA plates and left to grow at 25 °C for 96 h. The same day, R401 mutants were each inoculated into 150 μl Artificial Root Exudates (ARE; **Table S5**) in 96-well culture plates (‘R401 preculture plates’) from glycerol stocks using a multi-stamp replicator and left to grow at 25 °C and 150 rpm for 96 h until saturation. Then, a *Rs* GMI1600 preculture was inoculated into 10 ml ARE and grown overnight at 25 °C and 150 rpm. Approximately 24 h later, the preculture was 1:10 diluted with 90 ml ARE and left to grow under the same conditions. Approximately 24 h later, the 100 ml *Rs* GMI1600 culture was concentrated by centrifugation at 4,000 rpm for 15 min, washed 3x and resuspended in ARE. Subsequently, OD600 was quantified and set to 0.2 in ARE. Then, 75 μl of this *Rs* GMI1600 suspension were transferred to each well of a sterile 96-well bacterial culture plate (Greiner-CELLSTAR-96-Well plate, transparent, flatbottom; Sigma-Aldrich) per ‘R401 preculture plate’, referred to as the ‘R401-*Rs* interaction plate’. Per ‘R401 preculture plate’ one sterile 96-well bacterial culture plate (Greiner-CELLSTAR-96-Well plate, transparent, flatbottom; Sigma-Aldrich) was filled with 150 μl ARE per well and R401 mutants were inoculated from the ‘R401 preculture plate’ using a multi-stamp replicator; this plate is referred to as the ‘axenic R401 plate’. After mixing by pipetting, 75 μl R401 mutant suspension were transferred from the ‘axenic R401 plate’ to the ‘R401-Rs interaction plate’. Finally, 75 μl ARE were added to each well of the ‘axenic R401 plate’. The ‘axenic R401 plate’ and ‘R401-Rs interaction plate’ thereby contain the same volume and concentration of R401 mutants, while the latter also contains a final concentration of OD~600~ 0.1 *Rs* GMI1600 per well. Both plates were closed with a lid and incubated at 25 °C and 150 rpm for 48 h. Subsequently, Abs~600~ was measured for the ‘axenic R401 plate’ and GFP fluorescence was quantified at ðœ†~excitation~=475 nm and ðœ†~emission~=510 nm for the ‘R401-Rs interaction plate’. Both measurements were taken using a microplate reader (Infinite M200 PRO, Tecan). Subsequently, candidate R401 mutants were selected based on two criteria: (I) loss of *Rs* GMI1600 inhibition; a mutant was considered a candidate if the GFP fluorescence in the ‘R401-Rs interaction plate’ was lower than 3-fold the absolute deviation around the median (MAD) (16) compared to the respective plates median, and (II) wild-type-like growth; a mutant was considered a candidate if the Abs~600~ in the ‘axenic R401 plate’ was not lower than 3x MAD compared to the respective plate’s median. Candidate R401 mutants were freshly picked from glycerol stocks and validated twice more independently using the same assay. Finally, 38 mutants that showed wild-type-like growth and robustly reduced inhibitory activity against *Rs* GMI1600 were subsequently tested in an orthogonal mBA experiment with Rs as target bacterium, as described before. + +(16) C. Leys, C. Ley, O. Klein, P. Bernard, L. 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--git a/assays/Mini-Tn5ForPyoverdineLackofR569/protocols/.gitkeep b/assays/Mini-Tn5ForPyoverdineLackofR569/protocols/.gitkeep new file mode 100644 index 0000000..e69de29 diff --git a/assays/Mini-Tn5ForPyoverdineLackofR569/protocols/Mini-Tn5TransposonScreenPyoverdineLackofR569.md b/assays/Mini-Tn5ForPyoverdineLackofR569/protocols/Mini-Tn5TransposonScreenPyoverdineLackofR569.md new file mode 100644 index 0000000..9aba88c --- /dev/null +++ b/assays/Mini-Tn5ForPyoverdineLackofR569/protocols/Mini-Tn5TransposonScreenPyoverdineLackofR569.md @@ -0,0 +1,3 @@ +## Mini-Tn5 transposon mutant screen for lack of pyoverdine fluorescence of R569 + +R569 mini-Tn5 mutants were cultured in 96 well plates at 25 °C and 180 rpm for five days in 50% TSB medium. For the initial mutant screen, fluorescence was acquired at ðœ†~excitation~=395 nm and ðœ†~emission~=470 nm in a microplate reader (Infinite M200 PRO, Tecan). Out of ~2,000 mutants analysed, the fluorescence-based screening identified twelve R569 mutants that showed severely reduced pyoverdine-specific fluorescence but retained median-like growth behaviour (Thresholds: -6x MAD fluorescence, >-1x MAD Abs~600~). For validation, cultures were pre-grown for five days in 50% TSB before sub-culturing into fresh siderophore medium (see **Table S5**) and growth for five additional days. Finally, bacterial culture density (Abs~600~) was determined, and pyoverdine-specific fluorescence of bacterial culture supernatants was captured at ðœ†~excitation~=410 nm and ðœ†~emission~=500 nm. 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b/assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/protocols/Mini-Tn5IdentificationofIntegrationSitesR401andR569.md @@ -0,0 +1,6 @@ +## Identification of mini-Tn5 transposon integration sites in the genomes of R401 and R569 + +The chromosomal mini-Tn5 transposon integration sites in the R401 or R569 genomes were determined similarly as described before (15). Briefly, a colony from each strain was resuspended in 20 μl of sterile deionized water. Subsequently, a two-step PCR (TAIL-PCR) was performed starting with an arbitrarily primed PCR (PCR1), followed by a nested PCR (PCR2) on the generated product using the primers as described (**Table S4**). PCR reactions were conducted using the BIORON DFS-Taq polymerase reaction kit according to the manufacturer’s instructions. One microliter of a resuspended bacterial colony was used as template for PCR1 in a total PCR reaction volume of 25 μl. PCR1 was conducted using primers JO4 and JO28 and the following steps were applied: 95 °C for 5 min, six +cycles of 95 °C for 15 sec, 30 °C for 30 sec and 1 min elongation at 72 °C, followed by 30 cycles with 95 °C for 15 seconds, 45 °C for 30 seconds and 1 min elongation at 72 °C. Final elongation was for 5 min at 72°C. PCR2 was conducted using 0.3 μl of the PCR1 product and the primers JO1 and JO5. The following steps were applied: 95 °C for 5 min followed by 30 cycles of 95 °C for 15 sec, 57 °C for 30 sec and 1 min elongation at 72 °C. +Final elongation was for 5 min at 72°C. R401 or R569 wild type was included to account for unspecific amplifications. PCR products were separated by agarose gel electrophoresis. One of the most prominent bands from each sample was extracted using the Nucleospin Gel and PCR clean-up kit (Macherey-Nagel). The PCR product was eluted into a final buffer volume of 20 μl; DNA concentration was determined with a spectrophotometer (NanoDrop One, Thermo Scientific), and 75 ng of each product were sent for Sanger + sequencing (Eurofins genomics). Finally, chromosomal Tn5 integration sites were assessed by alignment of the obtained sequences flanked by the integrated Tn5 transposon in the R401 or R569 genome using the Geneious Prime or CLC Genomics Workbench software, respectively (Qiagen Digital Insights). Matching open reading frames (ORFs) were further aligned to the NCBI BLAST (17) nucleotide and protein data base (using BLASTn and BLASTp algorithm). \ No newline at end of file -- GitLab From 95ca08ee29431d6f8a30fbaeec3ef633cd2a6ebf Mon Sep 17 00:00:00 2001 From: Mekhrona Mirzoeva <memir101@hhu.de> Date: Thu, 13 Mar 2025 15:47:59 +0100 Subject: [PATCH 25/35] R401pvdY Complementation assay and protocol --- .gitattributes | 1 + assays/ComplementationR401pvdY/README.md | 0 assays/ComplementationR401pvdY/dataset/.gitkeep | 0 assays/ComplementationR401pvdY/isa.assay.xlsx | Bin 0 -> 9425 bytes .../ComplementationR401pvdY/protocols/.gitkeep | 0 .../ComplementationR401\342\210\206pvdy.md" | 5 +++++ 6 files changed, 6 insertions(+) create mode 100644 assays/ComplementationR401pvdY/README.md create mode 100644 assays/ComplementationR401pvdY/dataset/.gitkeep create mode 100644 assays/ComplementationR401pvdY/isa.assay.xlsx create mode 100644 assays/ComplementationR401pvdY/protocols/.gitkeep create mode 100644 "assays/ComplementationR401pvdY/protocols/ComplementationR401\342\210\206pvdy.md" diff --git a/.gitattributes b/.gitattributes index d99cf23..fb6ffed 100644 --- a/.gitattributes +++ b/.gitattributes @@ -8,3 +8,4 @@ assays/Mini-Tn5TransposonMutantInR401andR569/dataset/.gitkeep filter=lfs diff=lf assays/Mini-Tn5ScreenLossOfR401sGrowthInhibitionRsGMI1600/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text assays/Mini-Tn5ForPyoverdineLackofR569/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text +assays/ComplementationR401pvdY/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text diff --git a/assays/ComplementationR401pvdY/README.md b/assays/ComplementationR401pvdY/README.md new file mode 100644 index 0000000..e69de29 diff --git a/assays/ComplementationR401pvdY/dataset/.gitkeep b/assays/ComplementationR401pvdY/dataset/.gitkeep new file mode 100644 index 0000000..e69de29 diff --git a/assays/ComplementationR401pvdY/isa.assay.xlsx b/assays/ComplementationR401pvdY/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..981f97cfbe1278262d6f3099df057f033f4c56d6 GIT binary patch literal 9425 zcmahvbzGEL*GPkOcQ*q_BZ8!KcXtg4gEUgo-Q6H1NOyxE(hVXdNOwv|euJ#5tGn-a z|CpKIGbipneb0Fmq@kb@As`^&A@&`uwUCF_sVpBrK%B!sKwyHOYKwqu987H-^i^DK zP3?7=U96vXsr1`)JVI+eW2DemG+I<p#r75fMlvaF!T}BgG<a%FQ>PZ3jp>RQ$b?0= zsNa67Vz07t6OYv`i9zON2_Kp!+pdCkH1wP3PxBaD7^6)%htPV(MqpnnK1{P5IO+}> zrHw207%D>BVP9{3Qx2PnADZGMwQLFhbvt2BxWb3}$gu6U=V1=iR#ZTMZ@9QJz_3L4 zs4-{N7d^1AYGoDTd9@wvM+Qy7ERWXFrAVolDm`@UM()%l`{=I@s}Ki=Oudai!#RGC z^zKE{aS2ROa4$IjSU=g3r0SHu5+<#jP$_5_;wVd>T!$ibj>tN}5X$;iXiKlj6WWcW zGs~Eg_J+^{>9$uq%PlZ)|GFfIgURRV4)#T9q(Dq9z51epJFGZrwLMf@!>ks{LpC>f zUHvwRm#dpLdn!3sg}zLxu7!d=LunNmV)31`Z!eRd$vRmE!|O&(MV{tLOgPtJrs^SJ znOD0K%`Jlz{!$IZch!Kk@%MrS|I)WJeQwY41Lj_RN12i{o8bSXAt4|h{td%?2U!yH zPOkG2lHk7kvz5yf8wvRAye6{Tc^ZzV?Nl~|HQ_n$efRc*qXdz{fp8HocGk`Nw+H#c zx5AZ9iI4zq39w-LhXRM%`)`0RP#GS=22#SY+2$gpu9;oSP)Zf{wnS=TOZ33Lj6TnD ztmT(YVvNA*lkW!#(+^K<2067yd@U;7cev<%wdPk?J0v*MNd4xTb&T`5jW%dAde`tZ znYq?gADxK|aj>`R#C}?U`dT)F!pOR=+EmjOe?f-CNOo3jngzOYq1;lxR+}TytS-X( zaG11k-E9L=ioXw~vnGP^726ms_lP^h4~hIKH7Kw|T%P|evOfV3?g9QyvNx^KF^Ay4 z#lS=eeg$&|*;yHZKvs9I@m<g@F@u(!kI?+~JrFC-v}!L!=%elF{dKg@9{APN$~>2} zs1xwKEJC&>g3pH=k4?gUa?CnrcFBsegiLwznVzna2)gk@>Ssqpy{+#4Cur|y@cCT= z5oxGCso|#55^)p|S9*{|%Mj6*4}8&+vIIn=u#t}BrGD&Iw2!HX72<ZA&juV{seNdG zQ=!c;GRJzKPA@p2+}`GLS-VT<elWNl6frC+u>_R3di}9NM9p?Y^XP%=(^eh!jB#d? 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Plasmid construction was conducted using Gibson assembly as described before. In brief, the coding region of R401 *pvdY* was co amplified with a 1.5 kb region upstream of the gene and ligated into a linearized pSEVA22l (18) vector backbone. This vector was integrated into *E. coli* BW29427 cells and subsequently integrated into R401 *∆pvdy via* bi-parental conjugation. + +(18) E. MartÃnez-GarcÃa, *et al.*, SEVA 3.0: an update of the Standard European Vector Architecture for enabling portability of genetic constructs among diverse bacterial hosts. *Nucleic Acids Res* **48**, D1164–D1170 (2020). \ No newline at end of file -- GitLab From 97f288cc0d6bfafb0bf617a9bcd1ee845945aa18 Mon Sep 17 00:00:00 2001 From: Mekhrona Mirzoeva <memir101@hhu.de> Date: Thu, 13 Mar 2025 16:07:15 +0100 Subject: [PATCH 26/35] iron mobilization protocol --- .gitattributes | 1 + assays/InvitroIronMobilizationAssay/README.md | 0 .../dataset/.gitkeep | 0 .../InvitroIronMobilizationAssay/isa.assay.xlsx | Bin 0 -> 6907 bytes .../protocols/.gitkeep | 0 .../protocols/InvitroIronMobilizationAssay.md | 11 +++++++++++ 6 files changed, 12 insertions(+) create mode 100644 assays/InvitroIronMobilizationAssay/README.md create mode 100644 assays/InvitroIronMobilizationAssay/dataset/.gitkeep create mode 100644 assays/InvitroIronMobilizationAssay/isa.assay.xlsx create mode 100644 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In vitro iron mobilization assay + +The capability of R401 and R569 mutants of solubilizing inaccessible ferric iron was tested using a previously described photometric assay (19). Bacteria were cultured at 25 °C and 180 rpm for five days in 50% TSB and subsequently subcultured and 1:50 diluted in siderophore medium (**Table S5**). Diluted cultures were grown for an additional five days under the same conditions. At five days post inoculation (dpi), bacterial load was quantified by OD600 determination. Subsequently, cells were pelleted by centrifugation for 15 min at 4,000 rpm. The cell-free supernatant was diluted 5-fold with 10 mM MgCl2 and 25 μl were mixed with 100 μl of CAS assay solution (20) in three technical replicates and incubated for approx. 40 min at room temperature in the dark. Using an Infinite 200 PRO plate reader (TECAN), Abs636 was measured as an indication for the transition from complexed iron (blue complex) to solubilized or siderophore-bound iron (yellow). Finally, bacterial iron mobilizing capacity was computed according to the following formula: + + +Fe-mobilizing capacity [μM Fe / OD₆₀₀] = +((Abs₆₃₆(Medium) − Abs₆₃₆(Sample)) / +Abs₆₃₆(Sample)) × 2 nmol ───────────────────────────── +(Volume(Supernatant) × OD₆₀₀) + + -- GitLab From b2243d57928debda0c3c90acf1910af520430d47 Mon Sep 17 00:00:00 2001 From: Mekhrona Mirzoeva <memir101@hhu.de> Date: Fri, 14 Mar 2025 12:05:47 +0100 Subject: [PATCH 27/35] Iron Mobilization assay --- .../isa.assay.xlsx | Bin 6907 -> 10874 bytes 1 file changed, 0 insertions(+), 0 deletions(-) diff --git a/assays/InvitroIronMobilizationAssay/isa.assay.xlsx b/assays/InvitroIronMobilizationAssay/isa.assay.xlsx index 914bb893093a07c87af3c9eb215778a134aa91bb..a624fe563a8f60b198c14347210335914fda4cc5 100644 GIT binary patch delta 6859 zcmaJ`1z6NwvtK%-Wu>J%loD`7K)O-7yE~<q20=h}L4*Zq=~_~136buQ5|9=|1S!e8 z;QM}DeeQS9KKq>cpZ}aWzcX`o&dlto;E4uUO%V+p7XScY1Il%r^zaAQne0#ifMW~* 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Date: Fri, 14 Mar 2025 12:35:53 +0100 Subject: [PATCH 28/35] Bacterial Grwoth rate assay and protocol, some changes in tables column names --- .gitattributes | 1 + assays/BacterialGrowthRates/README.md | 0 assays/BacterialGrowthRates/dataset/.gitkeep | 0 assays/BacterialGrowthRates/isa.assay.xlsx | Bin 0 -> 10078 bytes .../BacterialGrowthRates/protocols/.gitkeep | 0 .../BacterialGrowthRatesValidation.md | 6 ++++++ assays/ComplementationR401pvdY/isa.assay.xlsx | Bin 9425 -> 9445 bytes .../isa.assay.xlsx | Bin 10874 -> 10874 bytes .../isa.assay.xlsx | Bin 10297 -> 10298 bytes .../isa.assay.xlsx | Bin 15561 -> 15566 bytes 10 files changed, 7 insertions(+) create mode 100644 assays/BacterialGrowthRates/README.md create mode 100644 assays/BacterialGrowthRates/dataset/.gitkeep create mode 100644 assays/BacterialGrowthRates/isa.assay.xlsx create mode 100644 assays/BacterialGrowthRates/protocols/.gitkeep create mode 100644 assays/BacterialGrowthRates/protocols/BacterialGrowthRatesValidation.md diff --git a/.gitattributes b/.gitattributes index 9c182c3..a008537 100644 --- a/.gitattributes +++ b/.gitattributes @@ -10,3 +10,4 @@ assays/Mini-Tn5ForPyoverdineLackofR569/dataset/.gitkeep filter=lfs diff=lfs merg assays/Mini-Tn5IdentificationofIntegrationSitesR401andR569/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text assays/ComplementationR401pvdY/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text assays/InvitroIronMobilizationAssay/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text +assays/BacterialGrowthRates/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text diff --git a/assays/BacterialGrowthRates/README.md b/assays/BacterialGrowthRates/README.md new file mode 100644 index 0000000..e69de29 diff --git a/assays/BacterialGrowthRates/dataset/.gitkeep b/assays/BacterialGrowthRates/dataset/.gitkeep new file mode 100644 index 0000000..e69de29 diff --git a/assays/BacterialGrowthRates/isa.assay.xlsx b/assays/BacterialGrowthRates/isa.assay.xlsx new file mode 100644 index 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b/assays/BacterialGrowthRates/protocols/.gitkeep new file mode 100644 index 0000000..e69de29 diff --git a/assays/BacterialGrowthRates/protocols/BacterialGrowthRatesValidation.md b/assays/BacterialGrowthRates/protocols/BacterialGrowthRatesValidation.md new file mode 100644 index 0000000..74bb5be --- /dev/null +++ b/assays/BacterialGrowthRates/protocols/BacterialGrowthRatesValidation.md @@ -0,0 +1,6 @@ +## Validation of bacterial growth rates + +The growth of individual R401 mutants and wild type was assessed by continuously measuring OD~600~ of actively growing bacterial cultures in an Infinite 200 PRO plate reader (TECAN) over 48 h at 25 °C and approx. 300 rpm. Overnight cultures in 50% TSB were pelleted and washed as described before. Subsequently, OD~600~ was measured and set to 0.02 in either artificial root exudates (ARE) or ARE lacking FeCl~3~. Composition of ARE +was adopted from (21) and can be found in **Table S5**. + +(21) E. Baudoin, E. Benizri, A. 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b/assays/MicrobiotaReconstitutionFlowpot/protocols/MicrobiotaReconstitutionFlowpot.md new file mode 100644 index 0000000..cdf67a0 --- /dev/null +++ b/assays/MicrobiotaReconstitutionFlowpot/protocols/MicrobiotaReconstitutionFlowpot.md @@ -0,0 +1,5 @@ +## Microbiota reconstitution in the gnotobiotic Flowpot system + +Flowpot assembly was performed according to (24) with minor adjustments. A 2:1 mixture of peat potting mix and vermiculite was used as a matrix. The matrix was sterilized two times (25 min liquid cycle (121 °C) and 45 min solid cycle (134 °C)) and stored at 60 °C until completely dry. Prior to Flowpot assembly, the matrix was rehydrated with sterile MiliQ water. Flowpots were assembled by adding a layer of glass beads to the conical end of a truncated syringe, followed by a layer of the rehydrated, sterile substrate, subsequently covered with a sterile mesh secured by a cable tie. Assembled Flowpots were sterilized on a 25 min liquid cycle, stored at 60 °C overnight and sterilized twice on a 45 min solid cycle. Bacterial strains, cultivated as described before, were harvested, 3x washed and pooled in equal ratios. Then, 1.25 ml bacterial pool (OD~600~ 1.0) were added to 500 ml of 1â„2 MS medium for a final bacterial OD~600~ of 0.0025. Flowpots were first flushed with sterile MiliQ water, then inoculated with 50 ml of 1â„2 MS. Eight inoculated Flowpots were placed into each sterile microbox (TP1200, Sac O2) and stored at room temperature overnight. Exactly five sterilized seeds were sown per Flowpot and left to grow under the previously described conditions. At 21 dpi, shoot fresh weight was measured individually for each plant. Roots from a single Flowpot were thoroughly cleaned from soil particles in sterile water using tweezers. Six representative Flowpots were selected for harvesting root and matrix samples. Cleaned roots from each Flowpot were pooled in 2 ml lysing matrix E tubes (MP Biomedicals), snap-frozen and stored at -80 °C until further use. Additionally, <100 mg of soil were taken from each Flowpot, snap-frozen and stored in weighed 2 ml lysing matrix E tube (MP Biomedicals) at -80 °C until further use. + +(24) J. M. Kremer, et al., Peat-based gnotobiotic plant growth systems for Arabidopsis microbiome research. *Nature Protocols 2021 16:5* **16**, 2450–2470 (2021). \ No newline at end of file -- GitLab From 094fd9794ff62bda03e9c44e3410d6f5e0adf015 Mon Sep 17 00:00:00 2001 From: Mekhrona Mirzoeva <memir101@hhu.de> Date: Fri, 14 Mar 2025 16:06:05 +0100 Subject: [PATCH 30/35] Athaliana seedlings --- .gitattributes | 1 + assays/BacterialGrowthRates/isa.assay.xlsx | Bin 10078 -> 10079 bytes .../README.md | 0 .../dataset/.gitkeep | 0 .../isa.assay.xlsx | Bin 0 -> 14425 bytes .../protocols/.gitkeep | 0 .../R401onAthalianaSeedlingsAgarPlates.md | 3 +++ 7 files changed, 4 insertions(+) create mode 100644 assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/README.md create mode 100644 assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/dataset/.gitkeep create mode 100644 assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/isa.assay.xlsx create mode 100644 assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/.gitkeep create mode 100644 assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/R401onAthalianaSeedlingsAgarPlates.md diff --git a/.gitattributes b/.gitattributes index 50a354a..e09f562 100644 --- a/.gitattributes +++ b/.gitattributes @@ -12,3 +12,4 @@ assays/ComplementationR401pvdY/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -t assays/InvitroIronMobilizationAssay/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text assays/BacterialGrowthRates/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text assays/MicrobiotaReconstitutionFlowpot/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text +assays/Mono-association[[:space:]]experiment[[:space:]]of[[:space:]]R401[[:space:]]on[[:space:]]Athaliana[[:space:]]seedlings[[:space:]]in[[:space:]]agar[[:space:]]plates/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text diff 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Athaliana seedlings in agar plates/protocols/.gitkeep new file mode 100644 index 0000000..e69de29 diff --git a/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/R401onAthalianaSeedlingsAgarPlates.md b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/R401onAthalianaSeedlingsAgarPlates.md new file mode 100644 index 0000000..c7878ba --- /dev/null +++ b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/protocols/R401onAthalianaSeedlingsAgarPlates.md @@ -0,0 +1,3 @@ +## Mono-association experiment of R401 on A. thaliana seedlings in agar plates + +This protocol is adapted from (25). In brief, *A. thaliana* seeds were sterilized, germinated, and transferred to 1â„2 MS agar plates without sucrose, as described before. After transfer of seedlings, plants were grown for another 14 days under the same conditions. R401 wild-type and mutants were grown in 50% TSB overnight as described before. Bacterial cells were pelleted by centrifugation, washed 3x in 10 mM MgCl2 and OD~600~ was measured and adjusted to 0.0001. Agar plates were flushed with 15 ml of bacterial suspension for 5 min. The bacterial suspension were removed, and plants were carefully transferred to new 1â„2 MS agar plates. After 24 h, roots were cut using a sterile scalpel and collected in pre-weighed, sterile 2 ml tubes containing 1 steel bead (3 mm diameter). Tubes were weighed again to assess the root fresh weight. Subsequently, roots were ground in a Precellys 24 TissueLyser (Bertin Technologies) for 2 x 30 s at 6,200 rpm at 15 s intervals. Then, 250 μl of sterile 10 mM MgCl2 were added to each tube and roots were ground again under the same conditions. Each sample was subsequently 5x 1:10 diluted in sterile 10 mM MgCl2. Undiluted samples and each dilution were plated on 50% TSA square plates, dried and left to grow at 25 °C until single colonies appeared. 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Isolation/dataset/.gitkeep | 0 assays/DNA Isolation/isa.assay.xlsx | Bin 0 -> 9419 bytes assays/DNA Isolation/protocols/.gitkeep | 0 .../DNA Isolation/protocols/DNAIsolation.md | 4 ++++ .../isa.assay.xlsx | Bin 13480 -> 10656 bytes 7 files changed, 5 insertions(+) create mode 100644 assays/DNA Isolation/README.md create mode 100644 assays/DNA Isolation/dataset/.gitkeep create mode 100644 assays/DNA Isolation/isa.assay.xlsx create mode 100644 assays/DNA Isolation/protocols/.gitkeep create mode 100644 assays/DNA Isolation/protocols/DNAIsolation.md diff --git a/.gitattributes b/.gitattributes index e09f562..eac0854 100644 --- a/.gitattributes +++ b/.gitattributes @@ -13,3 +13,4 @@ assays/InvitroIronMobilizationAssay/dataset/.gitkeep filter=lfs diff=lfs merge=l assays/BacterialGrowthRates/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text assays/MicrobiotaReconstitutionFlowpot/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text 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b/assays/DNA Isolation/protocols/DNAIsolation.md new file mode 100644 index 0000000..abb6f07 --- /dev/null +++ b/assays/DNA Isolation/protocols/DNAIsolation.md @@ -0,0 +1,4 @@ +## DNA isolation + +For DNA extractions, root and soil samples were homogenized in a Precellys 24 TissueLyser (Bertin Technologies) for 2 x 30 s at 6,200 rpm with 15 s intervals. DNA was extracted with a modified, high-throughput version of the FastDNA SPIN kit for Soil (MP Biomedicals). In brief, samples were taken up in sodium phosphate buffer (MP Biomedicals) and MT buffer (MP Biomedicals), then homogenized as described before. +After centrifugation for 15 min at 13,000 rpm, 150 μl of the supernatant were transferred to a 96-well Acroprep Advance filter plate (with 0.2 μm Supor filter; Pall). Once full, the filter plate was positioned on a PCR plate filled with 50 μl Binding Matrix (MP Biomedicals) per well and centrifuged for 15 min to remove residual soil particles. This and all subsequent centrifugation steps were carried out in a swing out centrifuge at 1,500 rpm. The PCR plate was sealed and shaken for 3 min to allow binding of the DNA to the Binding Matrix. The suspension was pipetted onto a second filter plate of the same kind, positioned on a collection plate, and centrifuged for 15 min. The flowthrough was discarded. Then, 200 μl SEWS-M washing buffer (MP Biomedicals) were pipetted into each well of the filter plate and centrifuged for 5 min. This washing step was carried out a second time. The flowthrough was discarded and followed by centrifugation for 5 min to remove residual SEWS-M buffer. Finally, 30 μl nuclease-free water were added to each well and left to incubate at room temperature for 3 min. Subsequent centrifugation for 5 min eluted the DNA into a clean PCR plate. 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bacterial 16S rRNA gene profiling/protocols/LibraryPreparation16SrRNAProfiling.md b/assays/Library preparation for bacterial 16S rRNA gene profiling/protocols/LibraryPreparation16SrRNAProfiling.md new file mode 100644 index 0000000..17be9b0 --- /dev/null +++ b/assays/Library preparation for bacterial 16S rRNA gene profiling/protocols/LibraryPreparation16SrRNAProfiling.md @@ -0,0 +1,3 @@ +## Library preparation for bacterial 16S rRNA gene profiling + +The v5v7 variable regions of the bacterial *16S* rRNA gene were amplified in 96-wells plates through PCR using 799F and 1192R primers (PCR I; **Table S4**). PCR I was performed with 0.4 μl DFS-Taq polymerase (BIORON) in 25-μl reactions containing 2.5 μl 10x incomplete buffer (BIORON), 0.5 μl 10 mM MgC12, 2.5 μl 3% BSA, 0.5 μl 10 mM dNTPs, 0.75 μl 10 μM 799F, 0.75 μl 10 μM 1192R, 1 μl of isolated DNA and adjusted to 25 μl with nuclease-free water. Samples were placed in a thermocycler set at the following program: 94°C for 2 min, 25 cycles of 94°C for 30 s, 55°C for 30 s,72°C for 1 min, then a final extension at 72 °C for 10 min. Remaining primers and dNTPs were digested by Antarctic phosphatase and Exonuclease I. To each 25 μl PCR reaction, 1 μl of Antarctic phosphatase (New-England BioLabs), 1 μl of Exonuclease I (New-England BioLabs) and 3 μl of Antarctic phosphatase buffer (New-England BioLabs) were added; the resulting mixture was incubated at 37 °C for 30 min followed by heat inactivation of the enzymes at 85 °C for 15 min. The digested PCR I product was centrifuged at 3,000 rpm and 4 °C for 10 minutes. Then, 3 μl of the supernatant were used as a template in a second PCR, using a unique combination of uniquely barcoded 799F- and 1192-based primers containing Illumina adaptors for each sample (PCR II; **Table S4**). PCR II was performed with 0.4 μl DFS-Taq polymerase in 25-μl reactions containing 2.5 μl 10x incomplete buffer, 0.5 μl 10 mM MgC12, 2.5 μl 3% BSA, 0.5 μl 10 mM dNTPs, 0.75 μl 10 μM unique forward primer, 0.75 μl 10 μM unique reverse primer, 3 μl cleaned PCR I-product and adjusted to 25 μl with nuclease-free water. Samples were placed in a thermocycler set at the following program: 94°C for 2 min, 10 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, then a final extension at 72 °C for 10 min. Subsequently, 5 μl of the PCR product were combined with 1 ml Orange DNA Loading Dye, loaded on a 1% agarose gel containing 0.05% EtBr, and run at 110 mV. The expected PCR-product was an approx. 500 bp band containing the variable v5v7 regions, barcodes, and Illumina adaptors. After verification of successful amplification, samples were purified by AMPure XP (Beckman-Coulter) according to the manufacturer’s protocol. The DNA concentrations of each sample were quantified using the Quant-iT dsDNA Assay-Kit (Invitrogen) and pooled per full factorial replicate in equimolar values. All pools were purified twice using AMPure XP and fluorescently quantified using the Quant-iT dsDNA Assay-Kit. All pooled samples were combined into a single pool based on equimolarity and subsequently purified three times using AMPure XP and fluorescently quantified using the Qubit dsDNA HS Assay Kit (Invitrogen). The final concentration was set to 10 ng/μl. Paired-end Illumina sequencing was performed in-house using the MiSeq sequencer and custom sequencing primers (**Table S4**). diff --git a/assays/MicrobiotaReconstitutionFlowpot/README.md b/assays/MicrobiotaReconstitutionFlowpot/README.md index e69de29..0519ecb 100644 --- a/assays/MicrobiotaReconstitutionFlowpot/README.md +++ b/assays/MicrobiotaReconstitutionFlowpot/README.md @@ -0,0 +1 @@ + \ No newline at end of file diff --git a/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/README.md b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/README.md index e69de29..0519ecb 100644 --- a/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/README.md +++ b/assays/Mono-association experiment of R401 on Athaliana seedlings in agar plates/README.md @@ -0,0 +1 @@ + \ No newline at end of file -- GitLab From 7be4314fe65e651562401f735942613dfdc367e7 Mon Sep 17 00:00:00 2001 From: Mekhrona Mirzoeva <memir101@hhu.de> Date: Thu, 20 Mar 2025 16:20:48 +0100 Subject: [PATCH 34/35] 16S profiling analysis --- .gitattributes | 2 ++ assays/Analysis of 16S profiling/README.md | 0 .../Analysis of 16S profiling/dataset/.gitkeep | 0 .../dataset/ComProfiling.jpg | 3 +++ assays/Analysis of 16S profiling/isa.assay.xlsx | Bin 0 -> 11322 bytes .../protocols/.gitkeep | 0 .../protocols/16SProfilingAnalysis.md | 12 ++++++++++++ 7 files changed, 17 insertions(+) create mode 100644 assays/Analysis of 16S profiling/README.md create mode 100644 assays/Analysis of 16S profiling/dataset/.gitkeep create mode 100644 assays/Analysis of 16S profiling/dataset/ComProfiling.jpg create mode 100644 assays/Analysis of 16S profiling/isa.assay.xlsx create mode 100644 assays/Analysis of 16S profiling/protocols/.gitkeep create mode 100644 assays/Analysis of 16S profiling/protocols/16SProfilingAnalysis.md diff --git a/.gitattributes b/.gitattributes index 4b96006..7bc447f 100644 --- a/.gitattributes +++ b/.gitattributes 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z`Cp~U0|7mvA6oyXP<cT8e~Ao6`de=JfcAsq-$~>HN9y0vex{UvWB;AEJYXM@{l@fH n7V|gy-#6_8x);U$c7G2{3ewQ`aTymhk#__Q2F6D99sK_QxJDHM literal 0 HcmV?d00001 diff --git a/assays/Analysis of 16S profiling/protocols/.gitkeep b/assays/Analysis of 16S profiling/protocols/.gitkeep new file mode 100644 index 0000000..e69de29 diff --git a/assays/Analysis of 16S profiling/protocols/16SProfilingAnalysis.md b/assays/Analysis of 16S profiling/protocols/16SProfilingAnalysis.md new file mode 100644 index 0000000..19f9193 --- /dev/null +++ b/assays/Analysis of 16S profiling/protocols/16SProfilingAnalysis.md @@ -0,0 +1,12 @@ +## Analysis of 16S profiling data + +### ASV table generation + +Amplicon sequencing reads from *A. thaliana* roots and Flowpot soil were quality filtered and demultiplexed according to their barcode sequence using QIIME (26) and unique amplicon sequencing variants (ASVs) were inferred from error-corrected reads, followed by chimera filtering. ASVs were mapped to the reference 16S rRNA sequences (downloaded from “www.at-sphere.comâ€) to generate an ASV count table. All steps were carried out using the Rbec R package (27). Analysis was performed on samples with a sequencing depth of at least 500 high-quality reads. + + +### Alpha- and beta-diversity + +Analyses and visualization were performed in the R statistical environment (Version 4.1.2). +Alpha and beta diversity were calculated on non-rarefied ASV count tables (28). Alpha-diversity (Shannon index) was calculated with the “plot_richness†function in phyloseq package (29). +Beta-diversity (Bray-Curtis dissimilarities) was calculated using the “ordinate†function in phyloseq package and used for unconstrained ordination by Principal Coordinate Analysis (PCoA). Statistical significances were assessed using permutational multi-variate analysis of variance (PERMANOVA) using the adonis function in the vegan package. -- GitLab From 8912854314db1ba65cb4cbbb79e7810113a4b479 Mon Sep 17 00:00:00 2001 From: Mekhrona Mirzoeva <memir101@hhu.de> Date: Thu, 20 Mar 2025 16:30:52 +0100 Subject: [PATCH 35/35] figure descr --- .gitattributes | 1 + assays/Analysis of 16S profiling/dataset/ComProfiling.md | 3 +++ 2 files changed, 4 insertions(+) create mode 100644 assays/Analysis of 16S profiling/dataset/ComProfiling.md diff --git a/.gitattributes b/.gitattributes index 7bc447f..d194a34 100644 --- a/.gitattributes +++ b/.gitattributes @@ -17,3 +17,4 @@ assays/DNA[[:space:]]Isolation/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -t assays/Library[[:space:]]preparation[[:space:]]for[[:space:]]bacterial[[:space:]]16S[[:space:]]rRNA[[:space:]]gene[[:space:]]profiling/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text assays/Analysis[[:space:]]of[[:space:]]16S[[:space:]]profiling/dataset/.gitkeep filter=lfs diff=lfs merge=lfs -text assays/Analysis[[:space:]]of[[:space:]]16S[[:space:]]profiling/dataset/ComProfiling.jpg filter=lfs diff=lfs merge=lfs -text +assays/Analysis[[:space:]]of[[:space:]]16S[[:space:]]profiling/dataset/ComProfiling.md filter=lfs diff=lfs merge=lfs -text diff --git a/assays/Analysis of 16S profiling/dataset/ComProfiling.md b/assays/Analysis of 16S profiling/dataset/ComProfiling.md new file mode 100644 index 0000000..b7be10a --- /dev/null +++ b/assays/Analysis of 16S profiling/dataset/ComProfiling.md @@ -0,0 +1,3 @@ +version https://git-lfs.github.com/spec/v1 +oid sha256:922853bf9051d5889e00ee8abecfb4a2f9d775bdc2260eb08105c68b6a75cfe9 +size 2429 -- GitLab