Subcellular protein localization (cell envelope) in Phaeobacter inhibens DSM 17395

Abstract

Phaeobacter inhibens DSM 17395 is a metabolically versatile, secondary metabolite producing and surface colonizing member of the alphaproteobacterial Roseobacter clade. Proteins compartmentalized across the Gram-negative cell envelope are expected to be relevant for the habitat success of P. inhibens DSM 17395. Subcellular fractionation was followed by gel- or nano-LC-based separation of proteins and peptides, respectively. Subsequent MS-based identification of in total 1187 proteins allowed allocation to cytoplasm (303 proteins), cytoplasmic membrane (346), periplasm (325), outer membrane (76), and extracellular milieu (22). Multidimensional scaling was used to visualize the spreading of heuristically allocated proteins across the five different compartments. Experimentally inferred subcellular protein localization was compared with PSORTb prediction of protein secretion and membrane localization. Determined subcellular localizations of identified proteins were interpreted to reconstruct the functional traits of the different cell envelope compartments, in particular protein secretion and sorting, direct effector molecule transit, and cell envelope biogenesis. From a proteogenomic perspective, functional prediction of 74 genes (including 17 coding for proteins of hitherto unknown function) could be refined.

The marine bacterium Phaeobacter inhibens secures external ammonium by rapid buildup of intracellular nitrogen stocks

“…2012), including P. inhibens DSM 17395 (Koßmehl et al. 2013), are dominated by potential RTX ( R epeats-in- T o X in) proteins exported via type I secretion systems, also at hand in P. inhibens DSM 17395 (Koßmehl et al. 2013).…”

“…Subsequent washing steps, in-gel tryptic digestion, spotting onto Anchorchip steel targets (Bruker Daltonik GmbH, Bremen, Germany) and mass spectrometric analysis with an UltrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker Daltonik GmbH) was performed as described recently (Koßmehl et al. 2013). Proteins were identified by PMF and PFF mapping, respectively, using an in-house Mascot server (version 2.3; Matrix Science, London, UK) via the ProteinScape platform (version 3.1; Bruker Daltonik GmbH).…”

“…2012; Koßmehl et al. 2013). Electrophoretic separation of similar amounts of membrane proteins was conducted with 7 cm long, 1 mm thick 12.5% (v/v) SDS-PAGE gels using the Mini-Protean Tetra System (Bio-Rad) (Zech et al.…”

Carbohydrate Catabolism in Phaeobacter inhibens DSM 17395, a Member of the Marine Roseobacter Clade

“…(ii) Analysis of proteins from the cytoplasmic and outer membrane protein-enriched fractions by nano-liquid chromatography (nano-LC)-ESI-MS-MS. Cytoplasmic and outer membrane proteins from two biological replicates per substrate condition were prepared via sequential solubilization with sarcosyl and SDS essentially as described recently (32). Outer membrane-enriched (2.5 g) and cytoplasmic membrane-enriched (4.0 g) protein fractions were separated according to molecular mass on 7-cm-long, 1-mm-thick 12.5% SDS-PAGE gels.…”

“…Outer membrane-enriched (2.5 g) and cytoplasmic membrane-enriched (4.0 g) protein fractions were separated according to molecular mass on 7-cm-long, 1-mm-thick 12.5% SDS-PAGE gels. Four slices per gel lane were cut into smaller pieces and were subjected to in-gel-digestion as described previously (32). The tryptic peptides generated were separated with an UltiMate 3000 RSLCnano system (Thermo Fisher Scientific, Germering, Germany) equipped with a trap column (PepMap C 18 Nano-Trap column; pore size, 100 Å; bead size, 3 m; inner diameter, 75 m; length, 2 cm; Thermo Fisher Scientific) and a 15-cm analytical column (C 18 ; pore size, 100 Å; bead size, 2 m; inner diameter, 75 m; length, 15 cm; Thermo Fisher Scientific).…”