Dynamics of amino acid utilization in Phaeobacter inhibensDSM 17395

Abstract

Time-resolved utilization of multiple amino acids by Phaeobacter inhibens DSM 17395 was studied during growth with casamino acids. The 15 detected amino acids could be grouped according to depletion rate into four different categories, i.e. from rapid (category I) to nondepletion (category IV). Upon entry into stationary growth phase, amino acids of category I (e.g. glutamate) were (almost) completely depleted, while those of categories II (e.g. leucine) and III (e.g. serine) were further consumed at varying rates and to different extents. Thus, cultures entered stationary growth phase despite the ample presence of organic nutrients, i.e. under nonlimiting conditions. Integrated proteomic and metabolomic analysis identified 1747 proteins and 94 intracellular metabolites. Of these, 180 proteins and 86 metabolites displayed altered abundance levels during growth. Most strikingly, abundance and activity profiles of alanine dehydrogenase concomitantly increased with the onset of enhanced alanine utilization during transition into stationary growth phase. Most enzymes of amino acid and central metabolism, however, displayed unaltered abundances across exponential and stationary growth phases. In contrast, metabolites of the Entner-Doudoroff pathway and gluconeogenesis as well as cellular fatty acids increased markedly in abundance in early stationary growth phase.

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

“…Starting from a glycerol stock, 3 passages were conducted before six main cultures (250 ml) were inoculated with 2% [vol/vol] preculture (25). At half-maximal optical density (½ OD max ), 20 ml of each main culture was further incubated in 100-ml Erlenmeyer flasks (see Fig.…”

“…Furthermore, after phase separation, 1 ml of the upper polar phase was transferred to a 2-ml reaction tube and was dried in a vacuum concentrator for gas chromatography (GC)-MS analysis. The two-step derivatization reaction was performed essentially as described previously (25). Intracellular metabolites were analyzed by GC-MS as described previously (26) using a Leco Pegasus 4D GCϫGC-TOF MS (Leco Instrumente, Mönchengladbach, Germany) operated in GC-TOF mode and equipped with an MPS 2 XL autosampler (Gerstel, Mülheim an der Ruhr, Germany).…”

“…After 3.8 min (solvent delay time), full-scan mass spectra were collected from m/z 45 to 600 at 5 scans s Ϫ1 with MassCenter Main software (version 2.3.0.1; JEOL). Data were analyzed as described recently (25,26). The internal standard ribitol and the cell mass were used for normalization, and finally, data were centralized to the reference state succinate-grown cells.…”

“…During growth with complex Marine Broth (MB) as a surrogate for the complex nutritional conditions typically resulting from the collapse of algal blooms, P. inhibens DSM 17395 simultaneously degraded various amino acids and other substrates (24). A subsequent time-resolved study revealed differing degrees of substrate preferences (high to low) during growth with Casamino Acids, containing 15 different detectable amino acids (25). Genomically unclear degradation pathways of nine amino acids could be elucidated by combined proteomics, enzymatics, and metabolomics and could be shown by comparative genomics to be archetypical for roseobacters (26).…”

Microbial Production of Aliphatic ( S )-Epoxyalkanes by Using Rhodococcus sp. Strain ST-10 Styrene Monooxygenase Expressed in Organic-Solvent-Tolerant Kocuria rhizophila DC2201

“…The production level decreased above a glucose concentration of 5% because of the osmotic pressure of high glucose concentrations on the host cells. Moreover, it has been reported that the protein abundance profile and metabolism of microorganisms change dramatically with the nutrient conditions and growth phases (38,39). Therefore, we speculate that the redox balance and overproduction of RhSMO in the host cells were affected by the carbon source and its concentration.…”