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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/m_MTBLS15113_LC-MS_negative_normal-phase_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/s_MTBLS15113.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/a_MTBLS15113_LC-MS_negative_normal-phase.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_012_cabP.cdsA__80x_____GPL_r1_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_009_S2_Stat_80x_____GPL_r1_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_023_S2_Stat_80x_____GPL_r3_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_029_S2_G_80x_____GPL_r4_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_030_S2_Stat_80x_____GPL_r4_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_026_cabP.cdsA__80x_____GPL_r3_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_010_cabP_G_80x_____GPL_r1_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_022_S2_G_80x_____GPL_r3_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_024_cabP_G_80x_____GPL_r3_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_019_cabP.cdsA__80x_____GPL_r2_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_008_S2_G_80x_____GPL_r1_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_025_cabP_Stat_80x_____GPL_r3_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_017_cabP_G_80x_____GPL_r2_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_031_cabP_G_80x_____GPL_r4_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_015_S2_G_80x_____GPL_r2_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_032_cabP_Stat_80x_____GPL_r4_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_033_cabP.cdsA__80x_____GPL_r4_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_011_cabP_Stat_80x_____GPL_r1_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_016_S2_Stat_80x_____GPL_r2_t1_b1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113/FILES/CS006_018_cabP_Stat_80x_____GPL_r2_t1_b1.mzMLprimary200OKftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15113 The acquired dataset was extracted using mass spectrometry-data independent analysis software (MS-DIAL), version 4.8 (PMID: 32541957). MetaboLightsPublicLiquid Chromatography MS - negative - normal-phaseAgilent 1290 liquid chromatography system. Lipid class-specific separation was performed using normal-phase chromatography (Intersil SIL column, 3 μm, 2.1 × 150 mm, GL Sciences). Genome-wide mapping of genetic suppression events in Streptococcus pneumoniae.Novo Nordisk Foundation Centre for Metabolic Research, University of CopenhagenXueli GuanMicrobial Liquid Culturemass spectrometry assayLipids were analyzed as previously described (PMID: 40404014). Briefly, lipids were extracted using a modified Bligh-Dyer method (PMID: 13671378). Five OD units of cells (OD600 x mL of culture) in mid-log phase or from overnight culture were centrifuged at 3,000 × g for 5 min at 4°C, and washed twice in 1X PBS. Chloroform:methanol 1:2 (v/v) was added to the bacterial pellets, and the samples were incubated at 6°C for 4 h. After incubation, water and chloroform were added, and phase separation was achieved using centrifugation. The lower organic phase was collected, and the upper phase was re-extracted with chloroform. The organic extracts were pooled and dried using a nitrogen stream. Lipid extracts were stored at −80°C until further analysis.Streptococcus pneumoniaehttps://www.ebi.ac.uk/metabolights/MTBLS15113Xueli Guan. Novo Nordisk Foundation Centre for Metabolic Research, University of Copenhagen. xueliguan21@gmail.com.Lok-To Sham. National University of Singapore. lsham@nus.edu.sg. The acquired dataset was extracted using mass spectrometry-data independent analysis software (MS-DIAL), version 4.8 (PMID: 32541957). The relative peak areas of each sample were calculated by dividing the peak area of individual phospholipids by the sum of all identified phospholipids.Growth phaseStrainxueliguan21@gmail.com Five OD units of cells (OD600 x mL of culture) in mid-log phase or from overnight culture were collected and snap frozen. Metabolomicsuntargeted analysisGenome wide mappingTripleTOF 6600LipidomicsAgilent 1290 Infinity HPLCSup-seqMicrobial Liquid Cultureexperimental sampleStreptococcus pneumoniaeuntargeted analysisGenome wide mappingTripleTOF 6600LipidomicsAgilent 1290 Infinity HPLCSup-seqMicrobial Liquid Cultureexperimental sampleStreptococcus pneumoniaeMass spectrometry (MS) was performed using a SCIEX TripleTOF 6600 quadrupole time-of-flight mass spectrometer. Lipids were analyzed in the negative electrospray ionization mode, with collision-induced dissociation in the information-dependent acquisition mode for structural confirmation. falseGenome-wide mapping of genetic suppression events in Streptococcus pneumoniaeHistorically, suppressor analysis has been a powerful tool for uncovering the functions of essential genes. In this study, we report Sup-seq, a transposon-sequencing technique with random DNA barcodes that enables genome-wide detection of genetic suppression. When applied to seven essential genes in Streptococcus pneumoniae, Sup-seq revealed 53 suppressors across various cellular pathways. These suppressors function through diverse mechanisms, such as gene inactivation, overexpression, or downregulation, depending on the locations and orientations of the transposon insertions. Our proof-of-concept experiments identified a novel transcriptional regulator that controls chaperone and ribosomal protein expression, as well as a mechanism that bypasses the requirement for acetate kinase for growth. Furthermore, we found that phosphatidylglycerol is conditionally essential in pneumococcus, revealing an unexpected link between phospholipid synthesis and potassium transport. These findings demonstrate that Sup-seq is a facile approach for providing mechanistic insights into the functions of essential genes. Additionally, Sup-seq can be adapted to other bacteria, aiding in the identification of new antimicrobial targets.2026-09-012026-07-21MTBLS15113