Extractions from the culture Selleckchem RG7420 supernatant were performed as described by Vallet-Gely et al. . Briefly, 200 ml of bacterial culture in PMS minimal medium was pelleted by centrifugation after 7 days of growth. The supernatants were passed through a 0.2-μm filter (Millipore Corporation, Bedford, MA); the pH was
adjusted to 5.0 with HCl or NaOH, and the preparation was extracted three times with dichloromethane. Initially, the preparations were extracted with 100 ml of solvent, then again with 70 ml of solvent and finally with 50 ml of solvent. The extracts were pooled, dried with anhydrous Na2SO4, filtered through Whatman paper, evaporated to dryness EVP4593 cost and dissolved in 1 ml of methanol. To supplement the growth medium with extract, 150 μl of methanolic extract was added to a 15-ml PMS culture, which was subsequently
allowed to grow for 24 h. The mangotoxin production was analysed as previously described, and cell-free filtrates of UMAF0158 and UMAF0158ΔmgoA supplemented with extracts from UMAF0158 and UMAF0158ΔmgoA were tested. Cell-free filtrates from P. syringae pv. syringae UMAF0158 Dorsomorphin mouse and UMAF0158ΔmgoA grown in PMS supplemented with 150 μl of methanol were used as controls, as were cell-free filtrates of UMAF0158 and UMAF0158ΔmgoA that were grown in PMS under standard conditions. Bioinformatics Database searches were performed using the website of the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov). Homology searches and the analysis of conserved protein domains were performed using the NCBI Specialized BLAST programme, the protein tools (InterProScan) of the EMBL European Bioinformatics Institute (http://www.ebi.ac.uk) PR-171 purchase and the Pfam database (http://pfam.sanger.ac.uk). The restriction maps were constructed and analysed using the JustBio website (http://www.justbio.com). The primers were designed using Primer3 online software (http://primer3.sourceforge.net). The annotation and general manipulation of sequences was performed using Artemis
software (Sanger Institute, Cambridge, U.K.). The plasmid maps were constructed using the programme Plasmid Map Enhancer 3.1 (Scientific & Educational Software). The promoter prediction was performed by SoftBerry online software http://linux1.softberry.com/berry.phtml. Acknowledgements This study was supported by funding from Consejería de Innovación, Ciencia y Empresa, Secretaría General de Universidades, Investigación y Tecnología, Junta de Andalucía, Spain (Proyecto de Excelencia P07-AGR-2471), cofinanced by FEDER funds (EU). This work was developed during my hired by the CSIC in the program mode JAEDoc “”Junta para la Ampliación de Estudios”" cofinanced by ESF. Electronic supplementary material Additional file 1: Figure S1. Analysis of the plasmid integration in UMAF0158::mgoB.