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

Plasmid carriage is associated with energetic costs, and thus only those plasmids providing fitness benefits are stably maintained in the host lineage. Marine bacteria of the Roseobacter clade harbor up to 11 extrachromosomal replicons, adding lifestyle-relevant and possibly habitat success-promoting functions to their genomic repertoire. Phaeobacter inhibens DSM 17395 is a nutritionally versatile representative, carrying three stable and functionally distinct plasmids (65, 78, and 262 kb). The present study investigates the physiological and energetic consequences of plasmid carriage in P. inhibens DSM 17395, employing mutants cured from all native plasmids in every possible combination (seven different). Cultivation in process-controlled bioreactors with casamino acids as organic substrate revealed a complex physiological response, suggesting existence of functional interconnections between the replicons. Deletion of the 262 kb plasmid boosted growth rate (>3-fold) and growth efficiency (yields for carbon, O2 and CO2 ), which was not observed for the 65 or 78 kb plasmid. Carriage of the 262 kb plasmid was most costly for the wild type, i.e. contributing ∼50% to its energetic (dissimilatory) expenditures. Cost-benefit analysis of plasmid carriage reflects the high value of plasmids for niche specialization of P. inhibens DSM 17395 and most likely also for related Phaeobacter species.

Carbohydrate catabolism in Phaeobacter inhibens DSM 17395, a member of the marine roseobacter clade.

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.

The marine bacterium Phaeobacter inhibens secures external ammonium by rapid buildup of intracellular nitrogen stocks.

Reduced nitrogen species are key nutrients for biological productivity in the oceans. Ammonium is often present in low and growth-limiting concentrations, albeit peaks occur during collapse of algal blooms or via input from deep sea upwelling and riverine inflow. Autotrophic phytoplankton exploit ammonium peaks by storing nitrogen intracellularly. In contrast, the strategy of heterotrophic bacterioplankton to acquire ammonium is less well understood. This study revealed the marine bacterium Phaeobacter inhibens DSM 17395, a Roseobacter group member, to have already depleted the external ammonium when only ∼⅓ of the ultimately attained biomass is formed. This was paralleled by a three-fold increase in cellular nitrogen levels and rapid buildup of various nitrogen-containing intracellular metabolites (and enzymes for their biosynthesis) and biopolymers (DNA, RNA and proteins). Moreover, nitrogen-rich cells secreted potential RTX proteins and the antibiotic tropodithietic acid, perhaps to competitively secure pulses of external ammonium and to protect themselves from predation. This complex response may ensure growing cells and their descendants exclusive provision with internal nitrogen stocks. This nutritional strategy appears prevalent also in other roseobacters from distant geographical provenances and could provide a new perspective on the distribution of reduced nitrogen in marine environments, i.e. temporary accumulation in bacterioplankton cells.