#METABOLOMICS WORKBENCH OlafTyc_20230316_052558 DATATRACK_ID:3791 STUDY_ID:ST002511 ANALYSIS_ID:AN004135 PROJECT_ID:PR001621 VERSION 1 CREATED_ON March 21, 2023, 8:29 am #PROJECT PR:PROJECT_TITLE Enhanced niche colonization and competition during bacterial adaptation to a PR:PROJECT_TITLE fungus PR:PROJECT_TYPE Fungal / bacterial interaction PR:PROJECT_SUMMARY Bacterial-fungal interactions (BFIs) influence microbial community performance PR:PROJECT_SUMMARY of most ecosystems and elicit specific microbial behaviors, including the PR:PROJECT_SUMMARY stimulation of specialized metabolite production. Using a simple BFI system PR:PROJECT_SUMMARY encompassing the Gram-positive bacterium Bacillus subtilis and the black mold PR:PROJECT_SUMMARY fungus Aspergillus niger, we established a co-culture experimental evolution PR:PROJECT_SUMMARY method to investigate bacterial adaptation to the presence of a fungus. In the PR:PROJECT_SUMMARY evolving populations, B. subtilis was rapidly selected for enhanced production PR:PROJECT_SUMMARY of the lipopeptide surfactin and accelerated surface spreading that led to an PR:PROJECT_SUMMARY inhibition of fungal expansion and environment acidification. These phenotypes PR:PROJECT_SUMMARY were explained by specific mutations in the DegS-DegU two-component system. In PR:PROJECT_SUMMARY the presence of surfactin, the fungal hyphae exhibited bulged cells with PR:PROJECT_SUMMARY delocalized secretory vesicles and RlmA-dependent cell wall stress induction. PR:PROJECT_SUMMARY Increased surfactin production generally enhances the competitive success of the PR:PROJECT_SUMMARY bacterium against various Aspergillus species that likely explains the primary PR:PROJECT_SUMMARY adaption path in the presence of A. niger. PR:INSTITUTE Netherlands Institute of Ecology PR:DEPARTMENT Microbial Ecology PR:LABORATORY GCMS Lab PR:LAST_NAME Tyc PR:FIRST_NAME Olaf PR:ADDRESS Theodor-Stern-Kai 7, Building 11, 60590 Frankfurt Germany PR:EMAIL Olaf.Tyc@kgu.de PR:PHONE 016094726403 #STUDY ST:STUDY_TITLE Enhanced niche colonization and competition during bacterial adaptation to a ST:STUDY_TITLE fungus ST:STUDY_TYPE Fungal / bacterial interaction ST:STUDY_SUMMARY Enhanced niche 1 colonization and competition during bacterial adaptation to a ST:STUDY_SUMMARY fungus ST:INSTITUTE Netherlands Institute of Ecology ST:DEPARTMENT Microbial Ecology ST:LAST_NAME Tyc ST:FIRST_NAME Olaf ST:ADDRESS Droevendaalsesteeg 10, 6708 PB Wageningen, The Netherlands ST:EMAIL Olaf.Tyc@kgu.de ST:PHONE +496963018046 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Aspergillus niger/Bacillus subtilis SU:TAXONOMY_ID 5061/1423 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - A niger mono day 3.1 treatment:A. niger mono | timepoint:day 3 RAW_FILE_NAME=qtof-2017-0103 A niger mono 268208.mzML SUBJECT_SAMPLE_FACTORS - A niger mono day 3.2 treatment:A. niger mono | timepoint:day 3 RAW_FILE_NAME=qtof-2017-0105 A niger mono 268210.mzML SUBJECT_SAMPLE_FACTORS - A niger mono day 3.3 treatment:A. niger mono | timepoint:day 3 RAW_FILE_NAME=qtof-2017-0107 A niger mono 268213.mzML SUBJECT_SAMPLE_FACTORS - B subtilis mono day 3.1 treatment:B. subtilis mono | timepoint:day 3 RAW_FILE_NAME=qtof-2017-0109 B sp 3610 anc mono 268233.mzML SUBJECT_SAMPLE_FACTORS - B subtilis mono day 3.2 treatment:B. subtilis mono | timepoint:day 3 RAW_FILE_NAME=qtof-2017-0111 B sp 3610 anc mono 268237.mzML SUBJECT_SAMPLE_FACTORS - B subtilis mono day 3.3 treatment:B. subtilis mono | timepoint:day 3 RAW_FILE_NAME=qtof-2017-0113 B sp 3610 anc mono 288253.mzML SUBJECT_SAMPLE_FACTORS - A niger B subtilis interaction 3.1 treatment:A. niger B. subtilis interaction | timepoint:day 3 RAW_FILE_NAME=qtof-2017-0115 A niger B sp 3610 anc int 288255.mzML SUBJECT_SAMPLE_FACTORS - A niger B subtilis interaction 3.2 treatment:A. niger B. subtilis interaction | timepoint:day 3 RAW_FILE_NAME=qtof-2017-0119 A niger B sp 3610 anc int 288258.mzML SUBJECT_SAMPLE_FACTORS - Control day 3.1 treatment:control | timepoint:day 3 RAW_FILE_NAME=qtof-2017-0121 TSB media control 288266.mzML SUBJECT_SAMPLE_FACTORS - Control day 3.2 treatment:control | timepoint:day 3 RAW_FILE_NAME=qtof-2017-0123 TSB media control 288269.mzML SUBJECT_SAMPLE_FACTORS - Control day 3.3 treatment:control | timepoint:day 3 RAW_FILE_NAME=qtof-2017-0125 TSB media control 288279.mzML SUBJECT_SAMPLE_FACTORS - A niger mono day 5.1 treatment:A. niger mono | timepoint:day 5 RAW_FILE_NAME=qtof-2017-0127 A niger mono 263895.mzML SUBJECT_SAMPLE_FACTORS - A niger mono day 5.2 treatment:A. niger mono | timepoint:day 5 RAW_FILE_NAME=qtof-2017-0129 A niger mono 268204.mzML SUBJECT_SAMPLE_FACTORS - A niger mono day 5.3 treatment:A. niger mono | timepoint:day 5 RAW_FILE_NAME=qtof-2017-0131 A niger mono 268209.mzML SUBJECT_SAMPLE_FACTORS - B subtilis mono day 5.1 treatment:B. subtilis mono | timepoint:day 5 RAW_FILE_NAME=qtof-2017-0133 B sp 3610 anc mono 268221.mzML SUBJECT_SAMPLE_FACTORS - B subtilis mono day 5.2 treatment:B. subtilis mono | timepoint:day 5 RAW_FILE_NAME=qtof-2017-0135 B sp 3610 anc mono 268223.mzML SUBJECT_SAMPLE_FACTORS - B subtilis mono day 5.3 treatment:B. subtilis mono | timepoint:day 5 RAW_FILE_NAME=qtof-2017-0137 B sp 3610 anc mono 268234.mzML SUBJECT_SAMPLE_FACTORS - A niger B subtilis interaction 5.1 treatment:A. niger B. subtilis interaction | timepoint:day 5 RAW_FILE_NAME=qtof-2017-0139 A niger B sp 3610 anc int 268236.mzML SUBJECT_SAMPLE_FACTORS - A niger B subtilis interaction 5.2 treatment:A. niger B. subtilis interaction | timepoint:day 5 RAW_FILE_NAME=qtof-2017-0141 A niger B sp 3610 anc int 287345.mzML SUBJECT_SAMPLE_FACTORS - A niger B subtilis interaction 5.3 treatment:A. niger B. subtilis interaction | timepoint:day 5 RAW_FILE_NAME=qtof-2017-0143 A niger B sp 3610 anc int 288245.mzML SUBJECT_SAMPLE_FACTORS - Control day 5.1 treatment:control | timepoint:day 5 RAW_FILE_NAME=qtof-2017-0145 TSB media control 288282.mzML SUBJECT_SAMPLE_FACTORS - Control day 5.2 treatment:control | timepoint:day 5 RAW_FILE_NAME=qtof-2017-0147 TSB media control 288288.mzML SUBJECT_SAMPLE_FACTORS - Control day 5.3 treatment:control | timepoint:day 5 RAW_FILE_NAME=qtof-2017-0149 TSB media control 288289.mzML #COLLECTION CO:COLLECTION_SUMMARY For analysis and trapping of volatile organic compounds two-compartment glass CO:COLLECTION_SUMMARY Petri-dishes (Garbeva et al., 2014) containing 1% LB-A (Carl-Roth LB- Agar) were CO:COLLECTION_SUMMARY applied. The fungus Aspergillus niger was inoculated 24 hours before addition of CO:COLLECTION_SUMMARY the bacterial strain on the left side of the two compartment glass petri-dish CO:COLLECTION_SUMMARY and incubated overnight at 28 °C. The bacterial strain Bacillus subtilis 3610 CO:COLLECTION_SUMMARY was incubated overnight at 28 °C in LB- liquid medium (Carl-Roth LB- Medium). CO:COLLECTION_SUMMARY After overnight _incubation a volume of 2 µl of the grown Bacillus subtilis CO:COLLECTION_SUMMARY 3610 culture was spotted on the opposite side of the Aspergillus niger culture. CO:COLLECTION_SUMMARY The glass Petri-dishes were kept open for 25 min. for drying of the droplets. CO:COLLECTION_SUMMARY After the drying process plates were closed by a lid with an outlet connected to CO:COLLECTION_SUMMARY a steel trap containing 150 mg Tenax TA and 150 mg Carbopack B (Markes CO:COLLECTION_SUMMARY International Ltd) and incubated at 28 °C. The Tenax steel traps were collected CO:COLLECTION_SUMMARY after three and five days of incubation and stored at 4 °C until GC-Q-TOF CO:COLLECTION_SUMMARY analysis. As controls, glass Petri-dishes containing LB agar media without CO:COLLECTION_SUMMARY inoculated bacteria or fungi were used. CO:SAMPLE_TYPE Fungal/bacterial volatiles CO:COLLECTION_METHOD Tenax Trap CO:COLLECTION_LOCATION Wageningen, The Netherlands CO:COLLECTION_FREQUENCY 3 days and 5 days CO:COLLECTION_DURATION 5 min. CO:STORAGE_CONDITIONS 4℃ CO:COLLECTION_VIALS Tenax CO:STORAGE_VIALS Metal tubes #TREATMENT TR:TREATMENT_SUMMARY The fungus Aspergillus niger was inoculated 24 hours before addition of the TR:TREATMENT_SUMMARY bacterial strain on the left side of the two compartment glass petri-dish and TR:TREATMENT_SUMMARY incubated overnight at 28 °C. The bacterial strain Bacillus subtilis 3610 was TR:TREATMENT_SUMMARY incubated overnight at 28 °C in LB- liquid medium (Carl-Roth LB- Medium). After TR:TREATMENT_SUMMARY overnight _incubation a volume of 2 µl of the grown Bacillus subtilis 3610 TR:TREATMENT_SUMMARY culture was spotted on the opposite side of the Aspergillus niger culture. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The collected volatiles were desorbed from the traps using a desorption unit SP:SAMPLEPREP_SUMMARY (Unity TD-100, Markes International Ltd., Llantrisant, UK) at 210 °C for 12 min SP:SAMPLEPREP_SUMMARY (He flow 50 mL/min) and trapped on a cold trap at -10 °C. The volatiles were SP:SAMPLEPREP_SUMMARY introduced into the GC-Q-TOF (model Agilent 7890B GC and the Agilent 7200A QTOF, SP:SAMPLEPREP_SUMMARY Santa Clara, USA) by heating the cold trap for 12 min to 250 °C. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The volatiles were desorbed from the traps using an desorption unit (Unity CH:CHROMATOGRAPHY_SUMMARY TD-100, Markes International Ltd., Llantrisant, UK) at 210 °C for 12 min (He CH:CHROMATOGRAPHY_SUMMARY flow 50 mL/min) and trapped on a cold trap at -10 °C. The volatiles were CH:CHROMATOGRAPHY_SUMMARY introduced into the GC-Q-TOF (model Agilent 7890B GC and the Agilent 7200A QTOF, CH:CHROMATOGRAPHY_SUMMARY Santa Clara, USA) by heating the cold trap for 12 min to 250 °C. The used split CH:CHROMATOGRAPHY_SUMMARY ratio was 1:10 and the used column was a 30 × 0.25 mm ID RXI-5MS, film CH:CHROMATOGRAPHY_SUMMARY thickness 0.25 µm (Restek 13424-6850, Bellefonte, PA, USA). Temperature program CH:CHROMATOGRAPHY_SUMMARY was as follows: 39 °C for 2 min, from 39 °C to 95 °C at 3.5 °C/min, then to CH:CHROMATOGRAPHY_SUMMARY 165 °C at 4 °C/min, to 280 °C at 15 °C/min and finally to 320 °C at 30 CH:CHROMATOGRAPHY_SUMMARY °C/min, hold 7 min. The volatile organic compounds were ionized in EI mode at CH:CHROMATOGRAPHY_SUMMARY eV and mass spectra were acquired in full-scan-mode (30–400 U @ 5 scans/s). CH:CHROMATOGRAPHY_SUMMARY Mass-spectra were extracted with MassHunter Qualitative Analysis Software V CH:CHROMATOGRAPHY_SUMMARY B.06.00 Build 6.0.633.0 (Agilent Technologies, Santa Clara, USA). CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890B CH:COLUMN_NAME Restek Rtx-5Sil MS (30m x 0.25mm, 0.25um) CH:SOLVENT_A - CH:SOLVENT_B - CH:FLOW_GRADIENT - CH:FLOW_RATE 50 mL/min CH:COLUMN_TEMPERATURE 280°C #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Netherlands Insitute of Ecology, NIOO-KNAW, Wageningen AN:OPERATOR_NAME Hans Zweers #MS MS:INSTRUMENT_NAME Agilent 7200 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS The volatile organic compounds were ionized in EI mode at eV and mass spectra MS:MS_COMMENTS were acquired in full-scan-mode (30–400 U @ 5 scans/s). Mass-spectra were MS:MS_COMMENTS extracted with MassHunter Qualitative Analysis Software V B.06.00 Build MS:MS_COMMENTS 6.0.633.0 (Agilent Technologies, Santa Clara, USA). The obtained mass spectra MS:MS_COMMENTS were transformed to netCDF files and imported into MZmine V2.20 (Copyright © MS:MS_COMMENTS 2005-2012) MZmine Development Team) (Pluskal et al., 2010). Compounds were MS:MS_COMMENTS identified via their mass spectra using deconvolution function and local-minimum MS:MS_COMMENTS search algorithm in combination with two mass-spectral-libraries: NIST 2014 MS:MS_COMMENTS V2.20 (National Institute of Standards and Technology, USA http://www.nist.gov) MS:MS_COMMENTS and Wiley 7th edition spectral libraries and by their linear retention indexes MS:MS_COMMENTS (LRI). The LRI values were calculated by using AMDIS 2.72 (National Institute of MS:MS_COMMENTS Standards and Technology, USA). After deconvolution and mass identification peak MS:MS_COMMENTS lists containing the mass features of each treatment were exported as csv file MS:MS_COMMENTS format for further analysis. MS:MS_RESULTS_FILE ST002511_AN004135_Results.txt UNITS:m/z Has m/z:Yes Has RT:Yes RT units:Minutes #END