biostudies-arrayexpressXylella fastidiosahttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14493Xylella fastidiosa is a Gram-negative plant pathogen responsible for severe diseases in a variety of economically important crops. A critical aspect of its pathogenicity is the production of outer membrane vesicles (OMVs). While some knowledge exists on the molecular cargo of X. fastidiosa (Xf)-OMVs, nucleic acid cargoes remain unknown. Since OMVs protect DNA from degradation during transfer, vesiduction increases the efficiency of HGT, aiding bacterial evolution and adaptation. In order to understand if this mechanism is relevant for Xf, we sequenced DNA extracted from OMVs and compared it to DNA extracted from whole bacterial cells.biostudies-arrayexpressGrowth Protocol - Xylella fastidiosa fastidiosa subsp. Temecula 1 was routinely grown for 5-10 days on Pierce’s Disease 3 (PD3) plates (Davis, 1980). For liquid cultures, 200 mL PD2 (Davis, 1980) was inoculated with resuspended Xf inoculum in PBS and cultures were grown for ca. 4-7 days in PD2 (+ 50 µg/mL for Temecula 1-GFP) at 28 °C and 140 rpm reaching an OD600 > 0.2.Sequencing - Sequencing on a MiSeq sequencer (Illumina; 2 x 300 bp paired-end sequencing, v3 chemistry) was performed in the Genomics Service Unit (LMU Biocenter, Martinsried, Germany).Nucleic Acid Extraction - DNA isolation was performed using phenol/chloroform extraction as published in (Spada et al., 2020). Samples were incubated with 100 µL of iZon concentrator beads at RT for 1 h and pelleted at 16’800 x g for 10 min. OMV pellets were resuspended in DNA extraction buffer (SDS 0.5%, Tris-HCL 50 mM pH8, EDTA 0.1 M) and 0.1 mg/mL of ProtK and incubated at 56 °C overnight. Then, exosomal DNA was isolated using phenol/chloroform. DNA was pelleted for 2 h at -80 °C and precipitation was facilitated by addition of 1 µL glycogen (Thermo Scientific, FERR0051). DNA of untreated Xff-OMVs was then used to perform DNA sequencing.Sample Collection - For WCL samples, 2 mL cultures were centrifuged for 15 min, 4’000 x g. Supernatants were removed and pellets were flash-frozen and kept at -80 °C until further use. For Xf-OMV isolation, the remaining of 200 mL cultures were centrifuged at 4’000 x g for 15 min, supernatants were filtered through 0.22 µm filters (Milipore, SEGTPT0045). Filtered supernatants were centrifuged at 38’000 x g for 1 h to remove cellular debris and then further centrifuged at 150’000 x g for 4 h. Then, pellets were resuspended in 2 mL filtered 1x PBS (pH 7.4) and further purified using qEV2 iZON SEC-columns (iZon qEV2 columns, 70 nm series, IC2-70, France). Xff-OMVs were isolated from 1x 200 mL cultures and concentrated using iZon concentrator beads.Library Construction - Size and concentration of DNA was analyzed using DNA High sensitivity bioanalyzer chip. Sequencing libraries were constructed from 1 ng of DNA with the Nextera XT DNA Sample Preparation Kit (Illumina, Germany) according to the manufacturer’s protocol. The library was quality controlled by analysis on an Agilent 2000 Bioanalyzer with the Agilent High Sensitivity DNA Kit (Agilent Technologies, Germany) for fragment sizes of ca. 500-800 bp.MINSEQE ScoreAssays and DataProcessed DataorganisationMAGE-TAB FilesData Transformation - Conversion, sorting, and filtering (unmapped reads) of SAM/BAM files was done with samtools v.1.19. Unmapped and discordantly aligned reads were removed.Sequence Alignment - The DNA sequences were adapter and quality trimmed with fastp v.0.23.4 using default settings without length filtering ('--disable_length_filtering'). Due to a larger region of badly aligned reads in rRNA regions, DNA reads of rRNAs were filtered out with SortMeRNA v.4.3.6 using \\"smr_v4.3_default_db.fasta\\" from https://github.com/biocore/sortmerna/releases/download/v4.3.4/database.tar.gz. Afterwards, the reads were aligned to NCBI accession GCF_000007245.1 with bowtie2 v.2.5.3 in '--very-sensitive' mode and a maximal insert size of 1,000bp (-X 1000). Conversion, sorting, and filtering (unmapped reads) of SAM/BAM files was done with samtools v.1.19.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina MiSeqDNA-seqXylella fastidiosaERP164697Patrick BlumenkampAlessa RufSilke RobatzekfalseDNA-seq of Xylella fastidiosa OMVs and WCLXylella fastidiosa is a Gram-negative plant pathogen responsible for severe diseases in a variety of economically important crops. A critical aspect of its pathogenicity is the production of outer membrane vesicles (OMVs). While some knowledge exists on the molecular cargo of X. fastidiosa (Xf)-OMVs, nucleic acid cargoes remain unknown. Since OMVs protect DNA from degradation during transfer, vesiduction increases the efficiency of HGT, aiding bacterial evolution and adaptation. In order to understand if this mechanism is relevant for Xf, we sequenced DNA extracted from OMVs and compared it to DNA extracted from whole bacterial cells.2025-05-30T00:00:00Z2026-05-27T13:14:32.653Z2024-10-02T15:10:07.18ZE-MTAB-14493ERP164697EFO_0002944EFO_0004170EFO_0003789EFO_0002693EFO_0004917EFO_0005518EFO_0003816EFO_0004184