ER 3.20.9 (Robinson et al., 2010). Damaging binomial GLMs have been fitted to model read counts for every single gene in each sample and a dispersion parameter which accounts for variability in between biological replicates was calculated (Lun et al., 2016). For DE analysis, nine comparisons (contrasts) had been defined (SIP vs. C, M vs. C, R vs. C, SIP + M vs. SIP, SIP + R vs. SIP, SIP + R vs. R, SIP + M vs. M, SIP + M vs. SIP + R, see Figure 1 for experimental setup). A gene was regarded as differentially expressed (DE) when the false discovery price (FDR) adjusted p-values had been beneath 0.01 plus the absolute log2 fold alter (LFC) was equal or greater than 1. To confirm GTP specificity in the 2-Hydroxyisobutyric acid Autophagy putativeRguanylate cyclases (GC), a a number of sequence alignment was carried out in MEGA 7 (Kumar et al., 2016) to check the presence of guanylate cyclase-specific motifs ( Winger et al., 2008). For genes DE in one particular certain contrast, Gene Ontology enrichment for single comparisons was determined employing a gene set enrichment approach (GSEA) as implemented in CAMERA (Wu and Smyth, 2012), integrated inside the R package limma v.three.34.9 (Ritchie et al., 2015). Redundant GO terms had been removed utilizing REVIGO4 (Supek et al., 2011) utilizing a low similarity value of 0.5. GO enrichment of genes that were DE in various contrasts was performed using Fisher’s exact test as well as the “weight” algorithm for GO group scoring as implemented in TopGO (Alexa and Rahnenf rer, 2009). Venn diagrams were generated with the R package VennDiagram v. 1.6.20 and with the web-based application Venny v. 2.1 (Oliveros, 20070155 ).Exometabolome ExtractionA total of 150 mL of filtered medium from each culture flask was transferred to sterile and cleaned 250 mL Erlenmeyer flasks, which had been covered immediately with aluminum foil and cooled down to four C just before solid phase extraction. Roseovarius sp. and Maribacter sp. exudates (n = 4, diluted to an equivalent OD600 = 0.05 with minimal medium) had been prepared and stored in the exact same way. Just before extraction, 15 nmol of caffeine dissolved in methanol [HPLC grade, Sigma ldrich, Chromasolv Plus (99.9 )] was added to each sample as an internal regular. The medium was extracted on 60 mg Oasis HLB-SPE cartridges (Waters, Eschborn, Germany), following the manufacturer’s guidelines. Gentle vacuum was applied for the cartridges having a VisiprepTM SPE Vacuum Manifold (Sigma ldrich) to possess a flow-through of ca. 1 drop per second. The cartridges have been eluted three instances with 1 mL of methanol. The 3 mL of eluate was stored in four mL vial glass at -80 C until further analysis. Medium blanks (n = 3) were prepare within the exact same way by extracting sterile F2 medium. 1.five mL on the eluate from each and every sample was transferred to a clean vial, evaporated beneath a stream of nitrogen, and dissolved in 50 of methanol. Two excellent handle (QC) samples were prepared by pooling five from every single sample in one particular clean vial.R RUHPLC-MS MeasurementsAfter randomizing the measuring order list in the samples and including QC every single 7 samples, five of each and every sample had been analyzed by UHPLC Dionex UltiMate 3000 (Thermo Fisher Scientific, Dreieich, Germany), coupled to an ESI-Orbitrap MS Q-Exactive Plus (Thermo Fisher Scientific, Dreieich, Germany). Liquid chromatography was performed on an Accucore C18 column (2.1 100mm, 2.six particle size; Thermo Scientific, Dreieich, Germany). The composition on the mobile phase was set to 100 A (0.1 HCOOH and two ACN in H2 O) for 0.2 min and ramped to one hundred B (0.1 HCOOH in ACN) in a linear gradi.
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