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Subsequently, cysteine thiols were reduced and carbamidomethyl-ated in one step for 30 min at 70°C by the addition of 2 µL of 500 mM TCEP (tris(2-carboxyethyl)phosphine) and 625 mM 2-chloroacetamide (CAA) per 100 µg of total protein in 100 µl. Proteins were precipitated by MeOH/Chloroform/H2O and resolubilized in 100 mM TEAB in 5:95 trifluoroethanol/H2O (v/v). Proteins were digested for 18 h at 37°C after the addition of Tryp-sin/Lys-C mix at a protease-to-protein ratio of 1:25. Tryptic peptides were dried in a vacuum con-centrator and resolubilized in 30 µL of 0.05% TFA in H2O/ACN 98/2 (v/v), filtered through 10 kDa MWCO PES membrane spin filters.  Each sample was measured in triplicate. LC-MS/MS analysis was performed on an Ultimate 3000 nano RSLC system connected to a QExactive HF mass spectrometer (both Thermo Fisher Scientific). Peptide trapping for 5 min on an Acclaim Pep Map 100 column (2 cm x 75 µm, 3 µm) at 5 µL/min was followed by separation on an analytical Acclaim Pep Map RSLC nano column (50 cm x 75 µm, 2µm). Mobile phase gradient elution of eluent A (0.1% (v/v) formic acid in water) mixed with eluent B (0.1% (v/v) formic acid in 90/10 acetonitrile/water) was performed using the following gradient: 0-5 min at 4% B, 30 min at 7% B, 60 min at 10% B, 100 min at 15% B, 140 min at 25% B, 180 min at 45% B, 200 min at 65% B, 210-215 min at 96% B, 215.1-240 min at 4% B. Positively charged ions were generated at spray voltage of 2.2 kV using a stainless steel emitter attached to the Nanospray Flex Ion Source. The instrument was operated in DDA mode. Precursor ions were monitored at m/z 300-1500 at a resolution of 120,000 FWHM using a maximum injection time (ITmax) of 120 ms and an AGC target of 3×10^6. Precursor ions with a charge state of z=2-5 were filtered at an isolation width of m/z 4.0 amu for further fragmentation at 28% HCD collision energy. MS2 ions were scanned at 15,000 FWHM (ITmax=100 ms, AGC= 2×10^5)."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["The protein database search was done by searching the tandem mass spectra against the UniProt database (2024/01/31, YYYY/MM/DD) of Clostridium ljungdahlii strain ATCC 55383 (https://www.uniprot.org/proteomes/UP000001656) using Proteome Discoverer (PD) 3.1 (Thermo) and the database search algorithms (threshold search engine scores in parentheses) Chimerys (>2), Mascot 2.8 (>30), Comet (>3), MS Amanda 3.0 (>300), Sequest HT (>3) with and without INFERYS Rescoring. Two missed cleavages were allowed for the tryptic digestion. The precursor mass toler-ance was set to 10 ppm, and the fragment mass tolerance was set to 0.02 Da. Modifications were defined as dynamic Met oxidation, phosphorylation of Ser, Thr, and Tyr, protein N-terminal acetyla-tion with or without Met loss, and static Cys carbamidomethylation. A strict false discovery rate (FDR) < 1% (peptide and protein level) was required for positive protein hits. The Percolator node of PD3.1 and a reverse-decoy database were used to validate q-values for spectral matches. Only rank one proteins and peptides of the top-scoring proteins were counted. Label-free protein quan-tification was based on the Minora algorithm in PD3.1, using precursor abundances determined by intensity and a signal-to-noise ratio greater than 5. Normalization was performed by using the total peptide amount method. Imputation of missing quan values was performed using abundance val-ues corresponding to 75% of the lowest abundance identified per sample. Differential protein abundance was defined as a fold change of >2, pvalue/ABS (log4ratio) <0.05 (pvalue/log4ratio), and at least identified in 2 of 3 replicates of the sample group with the highest abundance. Statistics and data visualization were performed with R 4.3.2 and RStudio 2023.12.0."],"omics_type":["Proteomics"],"labhead":["Axel A. Brakhage"],"instrument_platform":[""],"submission_type":["COMPLETE"],"labhead_affiliation":["Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology / Hans Knöll Institute Jena (Leibniz-HKI), Jena, Germany"],"species":["Clostridium Ljungdahlii"],"publication":["10.1111/1751-7915.70398"],"submitter_mail":["thomas.krueger@leibniz-hki.de"],"submitter_affiliation":["Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute"],"submitter_country":["Germany"],"doi":["10.6019/PXD070356"],"additional_accession":[]},"is_claimable":false,"name":"The stress within: Microbial electrosynthesis challenges Clostridium ljungdahlii physiology","description":"Microbial electrosynthesis (MES) enables certain microorganisms to utilize electrical energy (electrons) to produce valuable compounds using CO2 as a carbon source. It is closely related to gas fermentation in which hydrogen gas (H2) is used as the energy source, rather than in-situ electrochemically produced H2 in MES. Despite its potential for energy and carbon storage and the hype created around it, MES persists to face major limitations, such as low efficiency, unattractive products, and poor microbial growth. In this study, we compare the physiology of the model acetogen Clostridium ljungdahlii cultivated in gas fermenters and H-type electrobioreactors to identify the key stress factors limiting MES. We observed severe cellular stress and distinct physiological changes in MES through transcriptomics, proteomics, and electron microscopy analysis, showing that the electrochemical operation directly affects cellular metabolism. Most impressively, cell integrity was strongly impaired during growth in MES. Our results strongly indicate that this is because of a struggle to maintain the membrane potential and ATP synthesis. MES significantly impacted the central metabolic flux of the Wood-Ljungdahl pathway for CO₂ fixation and a diversion towards the glycine synthase-reductase pathway (GSRP), resulting in a broader spectrum of reduced products, including two amino compounds that appeared exclusively under MES conditions, ethanolamine, and glycine. We show that this struggle for ATP is compensated by the activation of the arginine metabolism to produce ATP. Multiple evidences indicate that this reaction could be fueled by the degradation of internal cyanophycin storage compounds, which have not been reported for C. ljungdahlii before. Additionally, our research highlights the expression of bacterial microcompartments (BMCs), which raises questions about their role during MES. This work demonstrates that MES drives C. ljungdahlii into a distinct physiological state and challenges its fitness, reshaping how we view MES process development. Our findings highlight the need to design MES strategies that mitigate the effects of the electrochemical environment on cellular physiology.","dates":{"publication":"2026-06-08","submission":"2025-11-05"},"accession":"PXD070356","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:1131","NEWT:35795","NEWT:2042546","NEWT:259447","NEWT:295546","NCBITaxon:2719036","NEWT:1334565","NEWT:625351","NEWT:258594","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:247480","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:475174","NEWT:1599","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:12215","NEWT:1590","NEWT:10034","NEWT:33114","NEWT:1596","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:376619","NCBITaxon:79857","NEWT:1096976","NEWT:3765","NEWT:3767","NEWT:1589","NEWT:135622","NEWT:4615","NEWT:672127","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:1582","NEWT:3760","NEWT:1584","NEWT:36630","NEWT:1585","NEWT:383379","NEWT:381198","NEWT:418106","NCBITaxon:12721","NEWT:10029","NEWT:913645","NEWT:2492962","NEWT:641809","NEWT:1392488","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:7719","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:224308","NEWT:3347","NEWT:49928","NEWT:3349","NEWT:160621","NEWT:212790","NEWT:110368","NEWT:1916073","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:410658","NEWT:5516","NEWT:44688","NEWT:44689","NEWT:370038","NEWT:498211","NEWT:345076","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:1310173","NEWT:200795","NEWT:2579791","NEWT:44685","NEWT:161934","NEWT:156587","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:260499","NEWT:410661","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:2649329","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:150808","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:3714","NCBITaxon:37162","NEWT:270643","NEWT:3715","NEWT:2065263","NEWT:177437","NEWT:717966","NEWT:10891","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:28174","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:244366","NEWT:1246791","NEWT:176597","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:926571","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1348799","NEWT:33995","NEWT:55784","NEWT:1303443","NEWT:9483","NEWT:485","NEWT:56636","NEWT:2499525","NEWT:2853422","NEWT:487","NEWT:1538553","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:45972","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:1502","NEWT:128017","NEWT:2836","NEWT:376686","NEWT:237610","NEWT:95486","EFO:0001352","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:11757","NEWT:987059","NEWT:109760","NEWT:29031","NEWT:317047","NEWT:1509","NEWT:491","NEWT:460523","NEWT:45954","NEWT:235443","NEWT:1579","NEWT:1757312","NEWT:85694","NEWT:109786","NEWT:108458","NEWT:5936","NEWT:85692","NEWT:321967","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:415978","NEWT:1255228","NEWT:410289","NEWT:191701","NEWT:373153","NEWT:1640278","NEWT:472","NEWT:940825","NEWT:29459","NEWT:529507","NEWT:1071661","NEWT:28128","NEWT:470","NEWT:41122","NEWT:5911","NEWT:748693","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:11723","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:1547","NEWT:1290454","NEWT:3726","NEWT:906914","NEWT:81231006","NEWT:28141","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:3728","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:548","NEWT:150475","NEWT:267872","NEWT:381666","NEWT:172269","NEWT:9534","NEWT:2126348","NEWT:5180","NEWT:256737","NEWT:9541","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:542","NEWT:2903","NEWT:185579","NEWT:872227","NEWT:13076","NEWT:2587410","NEWT:207340","NEWT:1006581","NEWT:8673","NEWT:33940","NEWT:2126337","NEWT:550","NEWT:10804","NEWT:33941","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:36185","NEWT:1054211","NEWT:1225786","NEWT:771870","NEWT:51338","NEWT:9995","NEWT:575412","NEWT:28112","NEWT:49012","NEWT:28110","NEWT:6493","NEWT:10832","NEWT:6494","NEWT:6491","NEWT:507601","NEWT:520","NEWT:64657","NEWT:186441","NEWT:643680","NEWT:214092","NCBITaxon:6157","NEWT:13095","NEWT:162425"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