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

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.

Native plasmids restrict growth of Phaeobacter inhibens DSM 17395: Energetic costs of plasmids assessed by quantitative physiological analyses

“…The present study investigates the physiological consequences of ECR carriage for P. inhibens DSM 17395, cultivated in process‐controlled bioreactors with casamino acids as complex organic substrate. The composition of casamino acids matches well with the distribution of oceanic dissolved amino acids (Wiegmann, ) and with relative amino acid abundances found in algal hydrolysates (Brown, ). For reasons of simplicity, ECRs are further on designated as “plasmids”, irrespective of their previous classification as “chromids”.…”

“…Its nutritional versatility qualifies Phaeobacter inhibens DSM 17395 as model system to investigate the physiology and catabolic capabilities of heterotrophic roseobacters (Dr€ uppel et al, 2014;Wiegmann et al, 2014;Zech et al, 2013a). In addition to the 3.2 Mb chromosome, P. inhibens DSM 17395 harbors three ECRs of 65, 78, and 262 kb size (Thole et al, 2012) that, amongst others, provide the strain with the ability to form biofilms (Frank et al, 2015), to produce the antibacterial secondary metabolite tropodithietic acid (TDA) (e.g.…”

Recognition cascade and metabolite transfer in a marine bacteria‐phytoplankton model system

“…Diatoms synthesize chitin as part of their cell wall (Durkin et al ., ), and marine bacteria have been shown to target chitinous regions of diatom cells for attachment (Frischkorn et al ., ; Li et al ., ). Chitin monomers such as N ‐acetylglucosamine (GlcNAc) can also regulate bacterial virulence factors (Konopka, ) as well as serve as bacterial substrates (Wiegmann et al ., ). Indeed, we detected enrichment (though not significant) of chitin monomers GlcNAc‐1/6‐P and UDP‐GlcNAc in the R. pomeroyi metabolome (Fig.…”

Anaerobic Activation of p -Cymene in Denitrifying Betaproteobacteria: Methyl Group Hydroxylation versus Addition to Fumarate

“…Each sample lane was cut in four slices, and each slice was cut into ϳ1-mm 3 pieces, which were washed, reduced, alkylated, and tryptically digested (43). Generated peptides were separated with an UltiMate 3000 RSLCnano system (Thermo Fisher Scientific, Germering, Germany) in a trap column setup equipped with an analytical column (C 18 ; pore size, 100 Å; bead size, 2 m; inner diameter, 75 m; length, 25 cm [Thermo Fisher Scientific]) applying a linear gradient as described previously (44). Continuous analysis of the eluent was performed with an online-coupled ion trap mass spectrometer (amaZon ETD; Bruker Daltonik GmbH) operated as described previously (43).…”