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.

Combining omics and enzymatic approaches, catabolic routes of nine selected amino acids (tryptophan, phenylalanine, methionine, leucine, isoleucine, valine, histidine, lysine and threonine) were elucidated in substrate-adapted cells of Phaeobacter inhibens DSM 17395 (displaying conspicuous morphotypes). The catabolic network [excluding tricarboxylic acid (TCA) cycle] was reconstructed from 71 genes (scattered across the chromosome; one-third newly assigned), with 69 encoded proteins and 20 specific metabolites identified, and activities of 10 different enzymes determined. For example, Ph. inhibens DSM 17395 does not degrade lysine via the widespread saccharopine pathway but might rather employ two parallel pathways via 5-aminopentanoate or 2-aminoadipate. Tryptophan degradation proceeds via kynurenine and 2-aminobenzoate; the latter is metabolized as known from Azoarcus evansii. Histidine degradation is analogous to the Pseudomonas-type Hut pathway via N-formyl-l-glutamate. For threonine, only one of the three genome-predicted degradation pathways (employing threonine 3-dehydrogenase) is used. Proteins of the individual peripheral degradation sequences in Ph. inhibens DSM 17395 were apparently substrate-specifically formed contrasting the non-modulated TCA cycle enzymes. Comparison of genes for the reconstructed amino acid degradation network in Ph. inhibens DSM 17395 across 27 other complete genomes of Roseobacter clade members revealed most of them to be widespread among roseobacters.

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.

Background:The bacterium Dinoroseobacter shibae was exposed to environmental anoxia. Results: Systems biology analyses showed the time-resolved cellular adaptation processes of D. shibae during oxygen depletion. Conclusion:Oxygen depletion led to a metabolic crisis due to the missing regeneration of ATP and reduction equivalents, until denitrification was established. Significance: Here we have elucidated the adaptation processes of marine bacteria to anoxic respiration.

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.