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Abstract

Since genome analysis did not allow unambiguous reconstruction of transport, catabolism, and substrate-specific regulation for several important carbohydrates in Phaeobacter inhibens DSM 17395, proteomic and metabolomic analyses of N-acetylglucosamine-, mannitol-, sucrose-, glucose-, and xylose-grown cells were carried out to close this knowledge gap. These carbohydrates can pass through the outer membrane via porins identified in the outer membrane fraction. For transport across the cytoplasmic membrane, carbohydrate-specific ABC transport systems were identified. Their coding genes mostly colocalize with the respective "catabolic" and "regulatory" genes. The degradation of N-acetylglucosamine proceeds via N-acetylglucosamine-6-phosphate and glucosamine-6-phosphate directly to fructose-6-phosphate; two of the three enzymes involved were newly predicted and identified. Mannitol is catabolized via fructose, sucrose via fructose and glucose, glucose via glucose-6-phosphate, and xylose via xylulose-5-phosphate. Of the 30 proteins predicted to be involved in uptake, regulation, and degradation, 28 were identified by proteomics and 19 were assigned to their respective functions for the first time. The peripheral degradation pathways feed into the Entner-Doudoroff (ED) pathway, which is connected to the lower branch of the Embden-Meyerhof-Parnas (EMP) pathway. The enzyme constituents of these pathways displayed higher abundances in P. inhibens DSM 17395 cells grown with any of the five carbohydrates tested than in succinate-grown cells. Conversely, gluconeogenesis is turned on during succinate utilization. While tricarboxylic acid (TCA) cycle proteins remained mainly unchanged, the abundance profiles of their metabolites reflected the differing growth rates achieved with the different substrates tested. Homologs of the 74 genes involved in the reconstructed catabolic pathways and central metabolism are present in various Roseobacter clade members.