Metabolites were classified by their Pubchem Compound Identification (CID) (https://pubchem.ncbi.nlm.nih.gov/) using the OMU package in R, which categorizes metabolites based on their chemical classification according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg).
"],"repository":["MetaboLights"],"study_status":["Public"],"ptm_modification":[""],"instrument_platform":["Liquid Chromatography MS - negative - reverse phase","Gas Chromatography MS -","Liquid Chromatography MS - positive - reverse phase"],"chromatography_protocol":["GC-MS:
-Data are acquired using the following chromatographic parameters, with more details to be found in Fiehn O. et al. Plant J. 53 (2008) 691–704.
-Instrument: Sciex TTOF 6600
-Column: Restek corporation Rtx-5Sil MS (30 m length x 0.25 mm internal diameter with 0.25 µm film made of 95% dimethyl/5%diphenylpolysiloxane)
-Mobile phase: Helium
-Column temperature: 50-330°C Flow rate: 1 mL min-1
-Injection volume: 0.5 µL
-Injection: 25 splitless time into a multi-baffled glass liner
-Injection temperature: 50°C ramped to 250°C by 12°C s-1
-Oven temperature program: 50°C for 1 min, then ramped at 20°C min-1to 330°C, held constant for 5 min.
LC-MS: Polyphenols and flavonoids were detected via tandem mass spectrometry (MS/MS) utilizing a Vanquish UHPLC system (Thermo Fisher Scientific) with an Acquity Primer BEH C18 column (Waters Corporation, Milford, MA, USA) coupled with a Q-Exactive HF Orbitrap mass spectrometer (Thermo Fisher) following an in-house protocol. Specifically, samples were extracted using 1 mL of 80:20 MeOH:H2O. Samples were vortexed and centrifuged and 450 uL of the supernatant was dried for analysis. Dried samples were resuspended with 100 uL of a solution 75:25 H2O:ACN containing internal standards (CUDA, D3-L-Carnitine, Val-Tyr-Val, D4-Daidzein, D9-Reserpine, and D5-Hippuric Acid). The samples were vortexed for 10 seconds, sonicated for 5 minutes at room temperature, and centrifuged for 2 min at 16,000 xg. A 60 uL supernatant from each sample was transferred into an LC-MS vial containing a glass microinsert and a 30 uL supernatant from each sample was transferred into a microcentrifuge tube and vortexed for use as a pool. Samples were injected onto a Waters Acquity Premier BEH C18 1.7 µm, 2.1 x 50 mm column. The gradient used was 0 min 1%, 0.50 min 1%, 7.50 min 99%, 9.00 min 99%, 9.20 min 1%, 10.00 min 1%, with a flow rate of 0.6 mL/min. Mobile phase A was 100% LC/MS grade water + 0.1% Formic Acid and mobile phase B was 100% ACN + 0.1% Formic Acid and injection volume into the Vanquish UHPLC system (ThermoFisher Scientific) varied between 0.1 uL and 5 uL.
"],"publication":["Exometabolomic-enabled discovery of compounds associated with Escherichia coli O157:H7 population dynamics in the lettuce phyllosphere. 10.1186/s12870-026-08917-9. PMID:42106610"],"submitter_affiliation":["University of California, Davis","UC Davis"],"submitter_name":["Maeli Melotto","David Bridges"],"organism_part":["pool","solvent","plant epidermis","leaf"],"technology_type":["mass spectrometry assay"],"disease":[""],"extraction_protocol":["Samples were filter sterilized and keep frozen at -80 C before analysis. Pooled samples were included for quality control purposes in all runs. Internal standards for the LC-MS included CUDA, D3-L-Carnitine, Val-Tyr-Val, D4-Daidzein, D9-Reserpine, and D5-Hippuric Acid.
"],"organism":["solvent blank","Lactuca sativa"],"full_dataset_link":["https://www.ebi.ac.uk/metabolights/MTBLS14653"],"author":["Maeli Melotto. Department of Plant Sciences, University of California, Davis. 104 MANN LAB, UC Davis, One Shields Ave, Davis CA 95616. melotto@ucdavis.edu.","David Bridges. UC Davis Department of Plant Sciences. One Shields Avenue, Davis, CA 95616, USA. dbridges@ucdavis.edu."],"data_transformation_protocol":["Raw data files are preprocessed directly after data acquisition and stored as ChromaTOF-specific *.peg files, as generic *.txt result files and additionally as generic ANDI MS *.cdf files. ChromaTOF vs. 2.32 is used for data preprocessing without smoothing, 3 s peak width, baseline subtraction just above the noise level, and automatic mass spectral deconvolution and peak detection at signal/noise levels of 5:1 throughout the chromatogram. Apex masses are reported for use in the BinBase algorithm. Result *.txt files are exported to a data server with absolute spectra intensities and further processed by a filtering algorithm implemented in the metabolomics BinBase database.
The BinBase algorithm (rtx5) used the settings: validity of chromatogram (<10 peaks with intensity >10^7 counts s-1), unbiased retention index marker detection (MS similarity>800, validity of intensity range for high m/z marker ions), retention index calculation by 5th order polynomial regression. Spectra are cut to 5% base peak abundance and matched to database entries from most to least abundant spectra using the following matching filters: retention index window ±2,000 units (equivalent to about ±2 s retention time), validation of unique ions and apex masses (unique ion must be included in apexing masses and present at >3% of base peak abundance), mass spectrum similarity must fit criteria dependent on peak purity and signal/noise ratios and a final isomer filter. Failed spectra are automatically entered as new database entries if s/n >25, purity <1.0 and presence in the biological study design class was >80%. All thresholds reflect settings for ChromaTOF v. 2.32. Quantification is reported as peak height using the unique ion as default, unless a different quantification ion is manually set in the BinBase administration software BinView.
Data were then further sum normalized, log10 transformed, and auto-scaled using MetaboAnalyst 5.0
"],"study_factor":["Treatment","Assay type","Day sampled"],"submitter_email":["melotto@ucdavis.edu","dbridges@ucdavis.edu"],"sample_collection_protocol":["Lettuce genotypes and growth conditions:
A set of 31 cultivated lettuce (Lactuca sativa L.) genotypes, representing seven different horticultural classes, were included in this study (Table S1). Genotypes were given an experimental code name to protect source information. Seeds of each genotype were sown on water-soaked paper towels in petri dishes and incubated for 2 days at 20 degrees C. After germination, three seedlings of the same genotype were transplanted into 11.36-liter pots (28 cm diameter) containing a commercial growing mix (Sun Gro® Sunshine® 1 Grower Mix with RESiLIENCE™, MA, USA) and grown at 20 degrees C under light conditions of 240 ± 10 µmol m-2 sec-1 with a 12-hour photoperiod. Seven genotypes were maintained in the environment room for 4 weeks under the same conditions described above (Table S1) and were fertilized with 0.05 g/plant Peters® Excel Multi-Purpose 19-11-21 fertilizer (ICL Group Ltd, Tev Aviv, Israel). At seven days post transplanting, the remaining 24 genotypes were transported to the field at the Crop Improvement and Protection Research Unit (United States Department of Agriculture, Agricultural Research Service) in Salinas, CA, where they were grown on outdoor open benches for three weeks throughout July-October 2021. Temperature conditions for these field conditions are provided in Figure S1. Plants were watered as needed and fertilized with Peters® Professional 20-20-20 (ICL Group Ltd, Tev Aviv, Isreal) once per week per manufacturer’s instructions. Two weeks after transplanting, Marathon® 1% Granular (OPH, Inc., NC, USA) was added to the topsoil at the manufacturer’s recommend dosage. Four weeks old plants were transported back to the UC Davis campus and were acclimated to the growing conditions described above for four days.
Preparation of O157:H7 inoculum:
O157:H7 strain 86-24 (National Center for Biotechnology Information accession number PRJNA1073941) was streaked from a frozen glycerol stock on Low Salt Luria-Bertani (LSLB) agar (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl, 15 g/l agar) medium supplemented with 50 µg/ml streptomycin (Thermo Fisher Scientific (Watham, MA) overnight at 28 degrees C. A single colony from these agar plates was inoculated into liquid LSLB broth and cultures were kept in an orbital shaker incubator set at 200 rpm and 28 degrees C until reaching an OD600 0.85-1 (approximately 12 h). Liquid cultures were centrifuged at 5000 xg at 4 degrees C for 5 min and the cell pellet was resuspended with sterile deionized water (SDW) The above centrifugation and resuspension step was repeated twice to ensure removal of leftover media, and the cell suspension was resuspended in SDW to create 500 ml of 6 log10 CFU/ml inoculum solution.
Plant inoculation and leaf sampling:
The third fully expanded leaf of 4-week-old plants was inoculated with either 6 log CFU/ml O157:H7 inoculum or SDW as a mock control. Inoculation of the leaf apoplast was performed using a needless syringe following the protocol described by Katagiri et al. (2002). In addition to apoplastic infiltration, the surface of each leaf was inoculated by dipping into 200 ml of inoculum or mock containing 0.03% Silwet L-77 (Lehle Seeds Co., Round Rock, TX, USA) for 5 sec., to ensure overall inoculation of the leaf. Plants were allowed to dry for 3 h before being placed back in the growth conditions described above. At 1- and 7-days post inoculation (DPI), inoculated leaves were removed from the plant using a sterile razor blade and placed in a sterile petri dish with their excision wound covered with parafilm. Images of each leaf were taken on a gridded background with a scale to estimate the surface area using ImageJ (https://imagej.net/ij/; Abràmoff et al. 2004). Three different plants (n = 3) were sampled per treatment at each time point.
Exudate sampling and bacterial enumeration:
To collect the leaf surface exudates, sampled leaves were individually rinsed with 10 ml of sterile phosphate buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4; 1.47 mM KH2PO4) by shaking at 125 rpm for 10 min. The rinse of each leaf was aspirated off, set aside, and labeled as surface wash fluid (SWF). Water soluble metabolites and O157:H7 within the apoplast of the same leaves were recovered by collecting apoplastic wash fluid (AWF) as outlined by O’Leary et al. (2014). Briefly, the parafilm covering the excision would of leaf was removed, and the sample was infiltrated with PBS, carefully blotted dry, and centrifuged at 500 xg for 8 min at 4 degrees C to collect the AWF.
"],"omics_type":["Metabolomics"],"study_design":["Pegasus IV","study reference material","untargeted analysis","solvent blank","pool","leaf","experimental sample","untargeted metabolite profiling","Escherichia coli O157","Vanquish","plant metabolite","TripleTOF 6600","Q-Exactive HF Orbitrap mass spectrometer (Thermo Fisher)","solvent","plant epidermis","Lactuca sativa"],"curator_keywords":["Pegasus IV","study reference material","untargeted analysis","solvent blank","pool","leaf","experimental sample","untargeted metabolite profiling","Escherichia coli O157","Vanquish","plant metabolite","Q-Exactive HF Orbitrap mass spectrometer (Thermo Fisher)","TripleTOF 6600","solvent","plant epidermis","Lactuca sativa"],"mass_spectrometry_protocol":["GC-MS: Mass spectrometry parameters are used as follows: a Leco Pegasus IV mass spectrometer is used with unit mass resolution at 17 spectra s-1from 80-500 Da at -70 eV ionization energy and 1800 V detector voltage with a 230°C transfer line and a 250°C ion source.
LC-MS: A Thermo Q-Exactive HF Orbitrap MS instrument was used in both positive and negative ESI modes to acquire LC-MS/MS data with the following parameters: mass range 80−1200 m/z; full scan MS1 mass resolving power 60,000, data-dependent MSMS (dd-MSMS) 2 scans per cycle (4 scans per cycle for pooled MSMS injections), normalized collision energy at 20%, 30%, and 40%, dd-MSMS mass resolving power
"],"metabolite_name":["xylose"],"pubmed_abstract":["Contamination of fresh produce with human pathogens remains a serious public health and economic concerns due to the absence of effective kill steps in the farm-to-fork chain. In particular, the pathogenic Escherichia coli O157:H7 has been implicated in multiple illness outbreaks linked to lettuce, and its survival in the phyllosphere is significantly affected by the plant genotype and associated metabolic traits. In this study, we examined the exometabolomic profile of 31 lettuce genotypes and identified substantial variation in the chemical composition of their leaf surface and leaf apoplast. Correlation analyses revealed that genotype-specific metabolomic patterns are associated with differential E. coli O157:H7 survival and allowed the identification of metabolites significantly associated, both positively and negatively, with the pathogen population net growth. Furthermore, inoculation with E. coli O157:H7 induced changes in the overall phyllosphere chemistry, with multiple differentially accumulated metabolites identified across genotypes. These shifts were dependent on lettuce genotype, leaf compartment, and compound class. This information guided the selection of metabolite cocktails for supplementation experiments, which significantly altered O157:H7 net growth and demonstrated the functional role of specific compounds in modulating pathogen dynamics on and in lettuce leaves. Overall, our results provide new chemical insights into how the phyllosphere exometabolome influences the survival of O157:H7 in lettuce leaves, offering potential targets to mitigate food safety concerns.
","dates":{"publication":"2026-06-11","submission":"2026-06-02"},"accession":"MTBLS14653","cross_references":{"pubmed":["42106610"]}}