Adaptation of Phaeobacter inhibensDSM 17395 to growth with complex nutrients

Abstract

Phaeobacter inhibens DSM 17395, a member of the Roseobacter clade, was studied for its adaptive strategies to complex and excess nutrient supply, here mimicked by cultivation with Marine Broth (MB). During growth in process-controlled fermenters, P. inhibens DSM 17395 grew faster (3.6-fold higher μmax ) and reached higher optical densities (2.2-fold) with MB medium, as compared to the reference condition of glucose-containing mineral medium. Apparently, in the presence of MB medium, metabolism was tuned to maximize growth rate at the expense of efficiency. Comprehensive proteomic analysis of cells harvested at ½ ODmax identified 1783 (2D DIGE, membrane and extracellular protein-enriched fractions, shotgun) different proteins (50.5% coverage), 315 (based on 2D DIGE) of which displayed differential abundance profiles. Moreover, 145 different metabolites (intra- and extracellular combined) were identified, almost all of which (140) showed abundance changes. During growth with MB medium, P. inhibens DSM 17395 specifically formed the various proteins required for utilization of phospholipids and several amino acids, as well as for gluconeogenesis. Metabolic tuning on amino acid utilization is also reflected by massive discharge of urea to dispose the cell of excess ammonia. Apparently, P. inhibens DSM 17395 modulated its metabolism to simultaneously utilize diverse substrates from the complex nutrient supply.

Transposon Mutagenesis Identified Chromosomal and Plasmid Genes Essential for Adaptation of the Marine Bacterium Dinoroseobacter shibae to Anaerobic Conditions

“…Following the reduction and alkylation of 50 g total cellular protein, proteolytic digestion was performed overnight with 0.5 g trypsin GOLD (Promega, Mannheim, Germany). Finally, 1 g of digested protein was separated with an UltiMate 3000 nanoLC system (Thermo Scientific, Bremen, Germany) by applying a linear gradient of increasing acetonitrile concentrations over 215 min coupled online to an ESI ion trap mass spectrometer (amaZon ETD; Bruker Daltonik GmbH, Bremen, Germany) as described before (14). Three biological replicates were analyzed.…”

“…Preparation and SDS-PAGE separation of the membrane protein-enriched fraction were performed as described recently (14). For each sample, one gel lane was cut into 11 slices that were further cut into smaller pieces for washing, reduction, alkylation, and tryptic digestion as described before (14). Separation of the peptides generated was performed by UltiMate 3000 nanoLC (Thermo Scientific, Bremen, Germany) with a 95-min linear gradient of increasing acetonitrile concentrations (14).…”

“…Cell pellets of approximately 50 mg (wet weight) from bioreactor growth were resuspended in 200 l lysis buffer, and cells were disrupted with the PlusOne grinding kit (GE Healthcare, Munich, Germany) as described before (14). Protein concentrations were determined as described before (15).…”

“…Preparation and SDS-PAGE separation of the membrane protein-enriched fraction were performed as described recently (14). For each sample, one gel lane was cut into 11 slices that were further cut into smaller pieces for washing, reduction, alkylation, and tryptic digestion as described before (14).…”

“…For each sample, one gel lane was cut into 11 slices that were further cut into smaller pieces for washing, reduction, alkylation, and tryptic digestion as described before (14). Separation of the peptides generated was performed by UltiMate 3000 nanoLC (Thermo Scientific, Bremen, Germany) with a 95-min linear gradient of increasing acetonitrile concentrations (14). Mass spectrometric analysis of the LC eluent was performed with an online-coupled ion trap mass spectrometer (amazon ETD; Bruker Daltonik GmbH) as described before (14).…”