Systems Biology of Aromatic Compound Catabolism in Facultative Anaerobic EbN1.

Members of the genus thrive in diverse habitats and use a broad range of recalcitrant organic molecules coupled to denitrification or O respiration. To gain a holistic understanding of the model organism EbN1, we studied its catabolic network dynamics in response to 3-(4-hydroxyphenyl)propanoate, phenylalanine, 3-hydroxybenzoate, benzoate, and acetate utilized under nitrate-reducing versus oxic conditions. Integrated multi-omics (transcriptome, proteome, and metabolome) covered most of the catabolic network (199 genes) and allowed for the refining of knowledge of the degradation modules studied.

Global Response of Phaeobacter inhibens DSM 17395 to Deletion of Its 262-kb Chromid Encoding Antibiotic Synthesis.

The marine alphaproteobacterium Phaeobacter inhibens DSM 17395, a member of the Roseobacter group, was recently shown to markedly enhance growth upon deletion of its 262-kb chromid encoding biosynthesis of tropodithietic acid (TDA). To scrutinize the metabolic/regulatory adaptations that underlie enhanced growth of the Δ262 mutant, its transcriptome and proteome compared to the wild type were investigated in process-controlled bioreactors with Casamino Acids as growth substrate. Genome resequencing revealed only few additional genetic changes (a heterogenic insertion, prophage activation, and several point mutations) between wild type and Δ262 mutant, albeit with no conceivable effect on the studied growth physiology.

Amino Acid and Sugar Catabolism in the Marine Bacterium Phaeobacter inhibens DSM 17395 from an Energetic Viewpoint.

Growth energetics and metabolic efficiency contribute to the lifestyle and habitat imprint of microorganisms. Roseobacters constitute one of the most abundant and successful marine bacterioplankton groups. Here, we reflect on the energetics and metabolic efficiency of DSM 17395, a versatile heterotrophic roseobacter.

Applications of Difference Gel Electrophoresis (DIGE) in the Study of Microorganisms.

OMICs-based investigations of microorganisms are becoming more and more widespread in the upcoming era of systems and synthetic biology. Here, proteomics plays a key role and two-dimensional difference gel electrophoresis (2D DIGE) remains the "gold-standard" for globally determining protein abundance changes on a quantitative and statistically confident level-in particular also for laboratories not having full-cycle proteomic facilities at their disposal. In this contribution we summarize our methodological procedures and experiences with 2D DIGE accumulated over the past 15 years.

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.

More than 2500 years of oil exposure shape sediment microbiomes with the potential for syntrophic degradation of hydrocarbons linked to methanogenesis.

Background: Natural oil seeps offer the opportunity to study the adaptation of ecosystems and the associated microbiota to long-term oil exposure. In the current study, we investigated a land-to-sea transition ecosystem called "Keri Lake" in Zakynthos Island, Greece. This ecosystem is unique due to asphalt oil springs found at several sites, a phenomenon already reported 2500 years ago.

Non-Redfield, nutrient synergy and flexible internal elemental stoichiometry in a marine bacterium.

The stoichiometric constraints of algal growth are well understood, whereas there is less knowledge for heterotrophic bacterioplankton. Growth of the marine bacterium Phaeobacter inhibens DSM 17395, belonging to the globally distributed Roseobacter group, was studied across a wide concentration range of NH4+ and PO43-. The unique dataset covers 415 different concentration pairs, corresponding to 207 different molar N:P ratios (from 10-2 to 105).

Photometric Determination of Ammonium and Phosphate in Seawater Medium Using a Microplate Reader.

To more efficiently process the large sample numbers for quantitative determination of ammonium (NH4+) and phosphate (orthophosphate, PO43-) generated during comprehensive growth experiments with the marine Roseobacter group member Phaeobacter inhibens DSM 17395, specific colorimetric assays employing a microplate reader (MPR) were established. The NH4+ assay is based on the reaction of NH4+ with hypochlorite and salicylate, yielding a limit of detection of 14 µM, a limit of quantitation of 36 µM, and a linear range for quantitative determination up to 200 µM. The PO43-assay is based on the complex formation of PO43- with ammonium molybdate in the presence of ascorbate and zinc acetate, yielding a limit of detection of 13 µM, a limit of quantitation of 50 µM, and a linear range for quantitative determination up to 1 mM.

High performance CCD camera system for digitalisation of 2D DIGE gels.

An essential step in 2D DIGE-based analysis of differential proteome profiles is the accurate and sensitive digitalisation of 2D DIGE gels. The performance progress of commercially available charge-coupled device (CCD) camera-based systems combined with light emitting diodes (LED) opens up a new possibility for this type of digitalisation. Here, we assessed the performance of a CCD camera system (Intas Advanced 2D Imager) as alternative to a traditionally employed, high-end laser scanner system (Typhoon 9400) for digitalisation of differential protein profiles from three different environmental bacteria.

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).

Benzoate mediates the simultaneous repression of anaerobic 4-methylbenzoate and succinate utilization in Magnetospirillum sp. strain pMbN1.

Background: At high concentrations of organic substrates, microbial utilization of preferred substrates (i.e., supporting fast growth) often results in diauxic growth with hierarchical substrate depletion.

Anaerobic activation of p-cymene in denitrifying betaproteobacteria: methyl group hydroxylation versus addition to fumarate.

The betaproteobacteria "Aromatoleum aromaticum" pCyN1 and "Thauera" sp. strain pCyN2 anaerobically degrade the plant-derived aromatic hydrocarbon p-cymene (4-isopropyltoluene) under nitrate-reducing conditions. Metabolite analysis of p-cymene-adapted "A.

Towards habitat-oriented systems biology of "Aromatoleum aromaticum" EbN1: chemical sensing, catabolic network modulation and growth control in anaerobic aromatic compound degradation.

The denitrifying betaproteobacterium "Aromatoleum aromaticum" EbN1 is a well-studied model organism for anaerobic degradation of aromatic compounds. Following publication of its genome in 2005, comprehensive physiological-proteomic studies were conducted to deduce functional understanding from the genomic blueprint. A catabolic network (85 predicted, 65 identified proteins) for anaerobic degradation of 24 aromatic growth substrates (including 11 newly recognized) was established.

Pathways and substrate-specific regulation of amino acid degradation in Phaeobacter inhibens DSM 17395 (archetype of the marine Roseobacter clade).

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.

Proteomic tools for environmental microbiology--a roadmap from sample preparation to protein identification and quantification.

The steadily increasing amount of (meta-)genomic sequence information of diverse organisms and habitats has a strong impact on research in microbial physiology and ecology. In-depth functional understanding of metabolic processes and overall physiological adaptation to environmental changes, however, requires application of proteomics, as the context specific proteome constitutes the true functional output of a cell. Considering the enormous structural and functional diversity of proteins, only rational combinations of various analytical approaches allow a holistic view on the overall state of the cell.

Dynamics of amino acid utilization in Phaeobacter inhibens DSM 17395.

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.

Adaptation of Phaeobacter inhibens DSM 17395 to growth with complex nutrients.

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.

Proteogenomic evidence for β-oxidation of plant-derived 3-phenylpropanoids in "Aromatoleum aromaticum" EbN1.

The betaproteobacterium "Aromatoleum aromaticum" EbN1 utilizes eight different plant-derived nonhydroxylated (e.g. cinnamate) and hydroxylated (e.

Physiological and proteomic adaptation of "Aromatoleum aromaticum" EbN1 to low growth rates in benzoate-limited, anoxic chemostats.

"Aromatoleum aromaticum" EbN1 was cultivated at different growth rates in benzoate-limited chemostats under nitrate-reducing conditions. Physiological characteristics, proteome dynamics, phospholipid-linked fatty acid (PLFA) composition, and poly(3-hydroxybutyrate) (PHB) content were analyzed in steady-state cells at low (μ(low)) (0.036 h(-1)), medium (μ(med)) (0.

Benzoate mediates repression of C(4)-dicarboxylate utilization in "Aromatoleum aromaticum" EbN1.

Diauxic growth was observed in anaerobic C(4)-dicarboxylate-adapted cells of "Aromatoleum aromaticum" EbN1 due to preferred benzoate utilization from a substrate mixture of a C(4)-dicarboxylate (succinate, fumarate, or malate) and benzoate. Differential protein profiles (two-dimensional difference gel electrophoresis [2D DIGE]) revealed dynamic changes in abundance for proteins involved in anaerobic benzoate catabolism and C(4)-dicarboxylate uptake. In the first active growth phase, benzoate utilization was paralleled by maximal abundance of proteins involved in anaerobic benzoate degradation (e.

Solvent stress response of the denitrifying bacterium "Aromatoleum aromaticum" strain EbN1.

The denitrifying betaproteobacterium "Aromatoleum aromaticum" strain EbN1 degrades several aromatic compounds, including ethylbenzene, toluene, p-cresol, and phenol, under anoxic conditions. The hydrophobicity of these aromatic solvents determines their toxic properties. Here, we investigated the response of strain EbN1 to aromatic substrates at semi-inhibitory (about 50% growth inhibition) concentrations under two different conditions: first, during anaerobic growth with ethylbenzene (0.

[Can diagnosis and subsequent trauma management of the multiple trauma patient with blunt thoracic trauma be improved by early computerized tomography of the thorax?].

Objective: The aim of this prospective study was to evaluate, whether early thoracic computed tomography (TCT) is superior to routine chest x-ray (CXR) in the diagnostic work up of blunt thoracic trauma and whether these additional informations influence subsequent therapeutical decisions in the early management of severely injured patients.

Patients And Methods: In a prospective study of 103 consecutive patients with clinical or radiological signs of chest trauma (94 multiple injured patients with chest trauma, 9 patients with isolated chest trauma) with an average ISS of 30 and an average AIS thorax of 3 initial CXR and TCT were compared after first assessment in our emergency department of a level I trauma center.

Results: In 67 patients (65%) TCT detected major complications of chest trauma, that have been missed on CXR [lung contusion (n = 33), pneumothorax (n = 27), residual pneumothorax after chest tube placement (n = 7), hemothorax (n = 21), displaced chest tube (n = 5), diaphragmatic rupture (n = 2), myocardial rupture (n = 1)], in 11 patients only minor additional pathologic findings (dystelectasis, small pleural effusion) were visualized on TCT and in 14 patients CXR and TCT showed the same pathological results.

[Percutaneous transhepatic stent implantation versus hepaticojejunostomy in palliative therapy of distal malignant bile duct stenosis].

The value of radiologically controlled stent implantation compared to hepatojejunostomy in palliative treatment of tumor induced cholestasis was examined in a non randomised retrospective study. 54 patients suffering of adenocarcinoma of the pancreas (n = 34), carcinoma of the distal bile duct (n = 10) and metastatic induced stenosis of the common bile duct (n = 10) were compared in regard to survival time, normalisation of cholestasis and complication rate. The survival time following stenting procedure (n = 31) is 6.

Site-directed mutagenesis of bovine pancreatic ribonuclease: lysine-41 and aspartate-121.

Chemical modification studies suggest that two residues of bovine pancreatic ribonuclease A (RNase A), Lys-41 and Asp-121, are important for catalysis. Three mutants of RNase A have been prepared, two point mutants with Lys-41 altered to Arg-41 and Asp-121 altered to Glu-121, and a double mutant where both residues are altered. The Lys-41 Arg mutant has ca.