diff --git a/.gitattributes b/.gitattributes index 23b3b4ce418bd15fb5c389f48c5bd3e7a9c5956f..fdeb8343e32a54bf04b32f6d77dd278404e7ab38 100644 --- a/.gitattributes +++ b/.gitattributes @@ -3,4 +3,3 @@ _publication/pnas.2221508120.sapp.pdf filter=lfs diff=lfs merge=lfs -text _publication/dataverse_files/Table[[:space:]]S2.xlsx filter=lfs diff=lfs merge=lfs -text assays/Screen[[:space:]]for[[:space:]]antagonistic[[:space:]]interbacterial[[:space:]]interactions/dataset/Table[[:space:]]S2.xlsx filter=lfs diff=lfs merge=lfs -text -assays/Screen[[:space:]]for[[:space:]]antagonistic[[:space:]]interbacterial[[:space:]]interactions/dataset/pnas.2221508120fig01.jpg filter=lfs diff=lfs merge=lfs -text diff --git a/assays/MetabolomicAnalysis/README.md b/assays/MetabolomicAnalysis/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/MetabolomicAnalysis/dataset/.gitkeep b/assays/MetabolomicAnalysis/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/MetabolomicAnalysis/dataset/Metabolomics.md b/assays/MetabolomicAnalysis/dataset/Metabolomics.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/MetabolomicAnalysis/isa.assay.xlsx b/assays/MetabolomicAnalysis/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..073f8b3335111f128aafe70baec92a15396f762c Binary files /dev/null and b/assays/MetabolomicAnalysis/isa.assay.xlsx differ diff --git a/assays/MetabolomicAnalysis/protocols/.gitkeep b/assays/MetabolomicAnalysis/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/MetabolomicAnalysis/protocols/MetabolomicAnalysis.md b/assays/MetabolomicAnalysis/protocols/MetabolomicAnalysis.md new file mode 100644 index 0000000000000000000000000000000000000000..0ac5db92ccd3863c848026367fac9b7cedbf7f8e --- /dev/null +++ b/assays/MetabolomicAnalysis/protocols/MetabolomicAnalysis.md @@ -0,0 +1,4 @@ +## Metabolomic Analysis + +Metabolites were extracted from individual isolates (n = 198) grown on the same agar medium used in mBA experiments with two organic solvents with different polarity, ethyl 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. The network was visualized using Cytoscape 3.5.1. \ No newline at end of file diff --git a/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx b/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx index 58eee5275fac4699973d6210751fb92f534d8ae6..312ee8adba84ba51e4d90f15983ad73fff565f15 100644 Binary files a/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx and b/assays/Screen for antagonistic interbacterial interactions/isa.assay.xlsx differ diff --git a/isa.investigation.xlsx b/isa.investigation.xlsx index 7cb4cae0c5cf802a64f2339b1fdc28538899b8b3..305ebbc405a57b8d78bfeb275ecfb564e85eafc6 100644 Binary files a/isa.investigation.xlsx and b/isa.investigation.xlsx differ diff --git a/studies/Bacterial culture conditions/isa.study.xlsx b/studies/Bacterial culture conditions/isa.study.xlsx index 4494a085727022919adf6ba46033d9b119d9bc05..3ecab80be028f406a045cbf75e94115fbf46e6e1 100644 Binary files a/studies/Bacterial culture conditions/isa.study.xlsx and b/studies/Bacterial culture conditions/isa.study.xlsx differ diff --git a/studies/Plant Growth Conditions/isa.study.xlsx b/studies/Plant Growth Conditions/isa.study.xlsx index 5b4b101864c087d37fe3cb043be158415b1c48ff..e9fd0dca9c6296a9789b665e64ea1622c299587b 100644 Binary files a/studies/Plant Growth Conditions/isa.study.xlsx and b/studies/Plant Growth Conditions/isa.study.xlsx differ