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ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075985/Q2508_SB_250804_DIA_Report_BGS_Factory_Report.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075985/Experimental.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075985/Q2508_SB_250804_DIA_Report.csvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075985/Q2508_SB_250804_Sulfit3_057.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075985/Q2508_SB_250804_DHPS2_049.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075985/Q2508_SB_250804_DHPS3_051.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075985/Q2508_SB_250804_DHPS1_047.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075985/Q2508_SB_250804_Sulfit1_053.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075985/Q2508_SB_250804_Sulfit2_055.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/09/PXD075985/database_Bilophila_wadsworthia_ATCC.faaprimaryOK200david.schleheck@uni-konstanz.deSabrina BorusakMass SpectrometryData-independent acquisitionDhpsBilophilaOrganosulfonatehttps://www.ebi.ac.uk/pride/archive/projects/PXD075985Bilophila wadsworthia 3.1.6 was grown with either 10 mM racemic 2,3-dihydroxypropane-1-sulfonate (DHPS) or 10 mM sulfite, each with 20 mM L-lactate as electron donor and additional C-source in carbonate-buffered salt medium (Widdel, 1981). The medium contained per L: 1.0 g NaCl, 0.4 g MgCl2 x 6 H2O, 0.2 g KH2PO4, 0.25 g NH4Cl, 0.5 g KCl, 0.15 g CaCl2 x 2 H2O, 1 ml of a 0.4 mg/ml Na-resazurine solution (redox indicator), 50 mM bicarbonate buffer (final concentration), 1 mM titan-(III)-nitrilotriacetate (final concentration, [Moench, 1983]), 200 µM Na2S (final concentration), 1 ml trace element solution SL10 (DSMZ medium 141), 1 ml selenite-tungstate solution (DSMZ medium 385), and 0.5 ml 7-vitamin solution (Widdel, 1981), 200 µg L-1 of 1,4-naphthoquinone (Laue, 1997) in double distilled H2O. The culture volume was 5 mL. Triplicate growth per growth condition. After two days the cultures were harvested by centrifugation (13,000 x g, 10 min, 4 °C) and the resulting pellet was washed with 1 mL 10 mM Tris, pH 8.0 before resuspending the pellet in the same buffer. Resuspended pellets were stored at -20 °C until further processed. 200 µl of sterilized zirconia/silica beads (0.1 mm diameter, ROTH) were added to the suspended cells in screw cap tubes and lysed by bead beating with a FastPrep F120 (Savant Bio 101). Cells underwent four rounds of 45 sec of beating at force 6.5 followed by 2 min on ice, to cool the sample. Sufficient disruption of the cells was checked by microscopy. Intact cells and cell debris were removed by centrifugation (10,000 x g, 10 min, 4 °C). The supernatant was transferred into new sterile tubes and submitted to the Proteomics core facility at the University of Konstanz (www.biologie.uni-konstanz.de/proteomics-centre/) for analysis of the total proteome. Before submission protein concentrations were measured with a Qubit 4 Fluorometer (Invitrogen) using the Qubit Protein BR assay kit (Invitrogen). Protein concentrations were adjusted by dilution with 10 mM Tris, pH8.0 to achieve the same protein concentration in each sample (210 µg mL-1). Proteomic analyses were performed by the Proteomics Core Facility at the University of Konstanz (www.biologie.uni-konstanz.de/proteomics-centre/) to assess the total proteome. For sample preparation, proteins were reduced with 10 mM dithiothreitol (DTT) for 30 min at 56 °C and alkylated with 50 mM chloroacetamide for 60 min at room temperature. Proteolytic digestion was carried out using trypsin at 0.01 µg µl-1 trypsin for 18 h at 33 °C. Peptide digests were analysed on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to an Easy-nLC 1200 nanoflow liquid chromatography system (Thermo Fisher Scientific, Bremen, Germany). Samples were reconstituted in 0.1% formic acid and loaded directly onto a heated analytical column (75 μm × 50 cm, 50 °C). Peptides were separated at a flow rate of 150 nL min-1 using a linear gradient of 6−48% solvent B (0.1% formic acid in 80% acetonitrile) over 108 min.PrideNot availableData-independent acquisition (DIA) was performed with full MS scans acquired over an m/z range of 300−1650 at a resolution of 120,000. The mass range was segmented into 24 variable isolation windows for fragmentation (window widths between 27 and 470 m/z), and MS/MS spectra were acquired at a resolution of 30,000. Fragmentation was achieved by higher-energy collision dissociation (HCD) with a normalized collision energy of 28%. Raw data were searched against the Bilophila wadsworthia 3.1.6 proteome (UniProt: UP000006034) using Spectronaut (Biognosys).ProteomicsDavid SchleheckDepartment of Biology, University of Konstanz, GermanyPARTIALBilophila Wadsworthia 3_1_6sabrina.borusak@uni-konstanz.de42441157 Barber H, Borusak S, Stewart AWE, Tahir H, Scott NE, Schleheck D, Lee M, Williams SJ. Chiral recognition of 2,3-dihydroxypropanesulfonate by bacterial transport proteins adapted to distinct ecological niches. Chem Sci. 2026 17(32):15566-15579 10.1039/d6sc02372jUniversity of Konstanz, Microbial Ecology and Limnic MicrobiologyGermanyBacterial catabolism of 2,3-dihydroxypropanesulfonate (DHPS) links algal production to marine degradation and connects sulfosugar metabolism to sulfide production in the gut. In surface seawater, DHPS occurs as a dilute, mixed R/S pool, whereas in the anaerobic gut it is produced predominantly as S-DHPS through bacterial sulfoglycolysis pathways. Uptake is achieved via tripartite ATP-independent periplasmic (TRAP) transporters that employ periplasmic substrate-binding proteins (HpsK), but the molecular basis of enantiomer recognition has not been defined. Here, we compare HpsK proteins from the marine bacterium Ruegeria pomeroyi and the gut anaerobe Bilophila wadsworthia using proteomics, biophysical analysis, X-ray crystallography, and bioinformatics. RpHpsK binds both R- and S-DHPS with low-nanomolar affinity (K D 5-9 nM), whereas BwHpsK binds selectively to S-DHPS (K D 530 nM), representing an ∼100-fold difference in affinity and strict stereoselectivity. Crystal structures reveal two contrasting strategies for chiral recognition: RpHpsK accommodates both enantiomers through subtle side-chain "toggling" within an otherwise conserved binding pocket, whereas BwHpsK achieves stereoselectivity through a distinct hydrogen-bonding network and a binding site that sterically excludes R-DHPS. Sequence similarity and genome neighbourhood analyses place these proteins in separate clusters associated with oxidative (HpsNOP) or glycyl radical enzyme-linked (HpsGH/HpfGH) pathways. These findings show how changes in binding-site architecture tune ligand stereoselectivity and illustrate the adaptation of TRAP-associated substrate binding proteins to distinct ecological and metabolic niches.Chiral recognition of 2,3-dihydroxypropanesulfonate by bacterial transport proteins adapted to distinct ecological niches.Barber Helen H, Borusak Sabrina S, Stewart Adam W E AWE, Tahir Hamza H, Scott Nichollas E NE, Schleheck David D, Lee Mihwa M, Williams Spencer J SJfalseComparative Proteomics of Bilophila wadsworthia 3.1.6 grown on DHPS or sulfiteBilophila wadsworthia 3.1.6 was cultivated with 2,3-dihydroxypropanesulfonate, or inorganic sulfite, each provided with L-lactate as the electron donor. Distinct enzymatic pathways are required for the respective respiratory processes, i.e., sulfite respiration versus organosulfonate respiration. Comparative proteomic analysis of B. wadsworthia 3.1.6 grown under these different conditions allows the identification and assessment of enzymes specifically involved in each type of respiration.2026-09-072026-03-22PXD075985NEWT:6945NEWT:184922NEWT:6703NEWT:3555NEWT:2NEWT:157546NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:544496NEWT:2102NEWT:2042546NEWT:45351NEWT:43179NEWT:4513NEWT:5722NEWT:1247NEWT:376741NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:309800NEWT:281395NEWT:10360NEWT:1211601NEWT:876138NEWT:47664NEWT:557436NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:272563NEWT:79220NEWT:224326NCBITaxon:79857NEWT:1096976NEWT:82688NEWT:95648NEWT:3885NEWT:3888NEWT:5821NEWT:1589NEWT:135622NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NEWT:272559NEWT:28909NEWT:515849NEWT:3641NEWT:383379NEWT:466585NEWT:10029NEWT:913645NEWT:1000589NEWT:85963NEWT:85962NEWT:143361NEWT:317447NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:398580NEWT:4565NEWT:1264690NEWT:515619NEWT:31155NEWT:192875NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:84645NEWT:626528NEWT:3347NEWT:139927NEWT:4558NEWT:209285NEWT:5888NEWT:211586NEWT:747078NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:294381NEWT:197NEWT:1390363NEWT:77133NEWT:288705NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:1270NEWT:374990NEWT:498217NEWT:156471NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:156476NEWT:286NEWT:391619NEWT:360104NEWT:287NEWT:246197NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:1148NEWT:580240NEWT:5508NEWT:294128NEWT:11676NEWT:55571NEWT:35500NEWT:1140NEWT:100226NEWT:4530NEWT:1143NEWT:4896NEWT:75058NEWT:13616NEWT:1390NEWT:1094343NEWT:1336795NEWT:172NEWT:296543NEWT:316435NEWT:1773NEWT:1895NEWT:1182590NEWT:3712NEWT:82380NEWT:105023NEWT:866628NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:375146NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NEWT:8175NCBITaxon:1313NEWT:43330NEWT:980415NEWT:1603293NEWT:408169NEWT:44544NEWT:47946NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:243277NEWT:990119NEWT:2850NEWT:408172NEWT:408170NEWT:493760NEWT:106590NEWT:260710NEWT:257313NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:106592NEWT:536231NEWT:1436733NEWT:460519NEWT:1187947NEWT:1432138NEWT:269796NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:9598NEWT:8030NEWT:9483NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:432359NEWT:46835NEWT:1182263NEWT:109757NEWT:943146NEWT:2711NEWT:300852NEWT:1502NEWT:376686NEWT:95486NEWT:9103NEWT:1883446NEWT:29159NEWT:253NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:89920NEWT:3635NEWT:5811NEWT:235443NEWT:180923NEWT:108458NEWT:272623NEWT:272624NEWT:2719099NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:118499NEWT:169963NEWT:32644NEWT:527796NEWT:225117NEWT:499175NEWT:109779NEWT:476272NEWT:3747NEWT:195051NEWT:367830NEWT:1255228NEWT:178616NEWT:649908NEWT:410289NEWT:373153NEWT:375451NEWT:352472NEWT:357NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:411469NEWT:84023NEWT:559292NEWT:39491NCBITaxon:5811NEWT:411464NEWT:411460NEWT:2014887NEWT:2762NEWT:1174673NEWT:1328426NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:571256NEWT:28038NEWT:1663NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:38293NEWT:327159NEWT:178876NEWT:1660NEWT:327160NEWT:573NEWT:9031NEWT:1872122NEWT:7091NEWT:108931NEWT:241368NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:303NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:7237NEWT:5180NEWT:256737NEWT:9541NEWT:115104NEWT:1121114NEWT:663NEWT:1081927NEWT:1238993NEWT:67825NEWT:185579NEWT:941442NEWT:220668NEWT:13076NEWT:1821314NEWT:1249668NEWT:7108NEWT:317NEWT:1808362NEWT:5286NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:9644NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:334542NEWT:554NEWT:27592NEWT:98334NEWT:426428NEWT:63459NEWT:1276815NEWT:36185NEWT:588858NEWT:9639NCBITaxon:11071NEWT:242231NEWT:7574NEWT:1715256NEWT:7215NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:2172103NEWT:6494NEWT:7460NEWT:6491NEWT:507601NEWT:643680NCBITaxon:6157NEWT:42752NEWT:746360NEWT:6239NEWT:470150NEWT:162425NEWT:216257NEWT:102169NEWT:543734NEWT:9986NEWT:4054NEWT:73239NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:118503NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NEWT:13164NEWT:216129NCBITaxon:2NEWT:985076NEWT:1215323NEWT:623NEWT:986NEWT:52641NEWT:7038NEWT:6192NEWT:28532NCBITaxon:38727NEWT:198618NEWT:353152NEWT:2829NEWT:366581NEWT:216599NEWT:216595NEWT:1194669NEWT:51329NEWT:243230NEWT:8355NEWT:633NEWT:9685NEWT:7029NEWT:1080772NEWT:8479NEWT:1283300NEWT:632NEWT:6183NEWT:6063NEWT:630NEWT:334747NEWT:61235NEWT:15368NEWT:6289NEWT:436486NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:170187NEWT:469008NEWT:260707NEWT:256318NCBITaxon:6191NEWT:1836NEWT:185431NEWT:2925NEWT:29760NEWT:260704NEWT:1719NEWT:703612NEWT:260705NEWT:80863NEWT:2697049NEWT:105231NEWT:1216981NEWT:52638NEWT:4097NCBITaxon:6073NEWT:884204NEWT:6279NEWT:1123869NEWT:9544NEWT:1329349NEWT:7370NEWT:83906NEWT:607699NCBITaxon:780NCBITaxon:1502NEWT:6282NEWT:426688NEWT:208963NEWT:208964NEWT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