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The denitrifying "Aromatoleum aromaticum" strain EbN1 utilizes a wide range of aromatic and nonaromatic compounds under anoxic and oxic conditions. The recently determined genome revealed corresponding degradation pathways and predicted a fine-tuned regulatory network. In this study, differential proteomics (2-D DIGE and MS) was used to define degradation pathway-specific subproteomes and to determine their growth condition dependent regulation. Differential protein profiles were determined for cultures adapted to growth under 22 different substrate and redox conditions. In total, 354 different proteins were identified, 199 of which displayed significantly changed abundances. These regulated proteins mainly represented enzymes of the different degradation pathways, and revealed different degrees of growth condition specific regulation. In case of three substrate conditions (e.g. phenylalanine, anoxic), proteins previously predicted to be involved in their degradation were apparently not involved (e.g. Pdh, phenylacetaldehyde dehydrogenase). Instead, previously not considered proteins were specifically increased in abundance (e.g. EbA5005, predicted aldehyde:ferredoxin oxidoreductase), shedding new light on the respective pathways. Moreover, strong evidence was obtained for thus far unpredicted degradation pathways of three hitherto unknown substrates (e.g. o-aminobenzoate, anoxic). Comparing all identified regulated and nonregulated proteins provided first insights into regulatory hierarchies of special degradation pathways versus general metabolism in strain EbN1.

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.

Among the dominant deltaproteobacterial sulfate-reducing bacteria (SRB), members of the genus Desulfobacula are not only present in (hydrocarbon-rich) marine sediments, but occur also frequently in the anoxic water bodies encountered in marine upwelling areas. Here, we present the 5.2 Mbp genome of Desulfobacula toluolica Tol2, which is the first of an aromatic compound-degrading, marine SRB. The genome has apparently been shaped by viral attacks (e.g. CRISPRs) and its high plasticity is reflected by 163 detected genes related to transposases and integrases, a total of 494 paralogous genes and 24 group II introns. Prediction of the catabolic network of strain Tol2 was refined by differential proteome and metabolite analysis of substrate-adapted cells. Toluene and p-cresol are degraded by separate suites of specific enzymes for initial arylsuccinate formation via addition to fumarate (p-cresol-specific enzyme HbsA represents a new phylogenetic branch) as well as for subsequent modified β-oxidation of arylsuccinates to the central intermediate benzoyl-CoA. Proteogenomic evidence suggests specific electron transfer (EtfAB) and membrane proteins to channel electrons from dehydrogenation of both arylsuccinates directly to the membrane redox pool. In contrast to the known anaerobic degradation pathways in other bacteria, strain Tol2 deaminates phenylalanine non-oxidatively to cinnamate by phenylalanine ammonia-lyase and subsequently forms phenylacetate (both metabolites identified in (13) C-labelling experiments). Benzoate degradation involves CoA activation, reductive dearomatization by a class II benzoyl-CoA reductase and hydrolytic ring cleavage as found in the obligate anaerobe Geobacter metallireducens GS-15. The catabolic sub-proteomes displayed high substrate specificity, reflecting the genomically predicted complex and fine-tuned regulatory network of strain Tol2. Despite the genetic equipment for a TCA cycle, proteomic evidence supports complete oxidation of acetyl-CoA to CO2 via the Wood-Ljungdahl pathway. Strain Tol2 possesses transmembrane redox complexes similar to that of other Desulfobacteraceae members. The multiple heterodisulfide reductase-like proteins (more than described for Desulfobacterium autotrophicum HRM2) may constitute a multifaceted cytoplasmic electron transfer network.

Anaerobic biodegradation of toluene and ethylbenzene is of environmental concern and biochemical interest due to toxicity and novel reactions, respectively. The denitrifying strain EbN1 is unique in anaerobically degrading both alkylbenzenes via different pathways which converge at benzoyl coenzyme A. The organization of genes involved in both pathways was only recently determined for strain EbN1. In the present study, global expression analysis (DNA microarray and proteomics) indicated involvement of several thus-far-unknown proteins in the degradation of both alkylbenzenes. For example, orf68 and orf57, framing the ebd operon, are implicated in ethylbenzene degradation, and the ebA1932 and ebA1936 genes, located 7.2 kb upstream of the bbs operon, are implicated in toluene degradation. In addition, expression studies were now possible on the level of the complete pathways. Growth experiments demonstrated that degradative capacities for toluene and ethylbenzene could be simultaneously induced, regardless of the substrate used for adaptation. Regulation was studied at the RNA (real-time reverse transcription-PCR and DNA microarray) and protein (two-dimensionaldifference gel electrophoresis) level by using cells adapted to anaerobic growth with benzoate, toluene, ethylbenzene, or a mixture of toluene and ethylbenzene. Expression of the two toluene-related operons (bss and bbs) was specifically induced in toluene-adapted cells. In contrast, genes involved in anaerobic ethylbenzene degradation were induced in ethylbenzene-and toluene-adapted cells, suggesting that toluene may act as a gratuitous inducer. In agreement with the predicted sequential regulation of the ethylbenzene pathway, Ebd proteins (encoding subunits of ethylbenzene dehydrogenase) were formed in ethylbenzene-but not in acetophenone-adapted cells, while Apc proteins (subunits of predicted acetophenone carboxylase) were formed under both conditions.

The denitrifying "Aromatoleum aromaticum" strain EbN1 was demonstrated to utilize p-ethylphenol under anoxic conditions and was suggested to employ a degradation pathway which is reminiscent of known anaerobic ethylbenzene degradation in the same bacterium: initial hydroxylation of p-ethylphenol to 1-(4-hydroxyphenyl)-ethanol followed by dehydrogenation to p-hydroxyacetophenone. Possibly, subsequent carboxylation and thiolytic cleavage yield p-hydroxybenzoyl-coenzyme A (CoA), which is channeled into the central benzoyl-CoA pathway. Substrate-specific formation of three of the four proposed intermediates was confirmed by gas chromatographic-mass spectrometric analysis and also by applying deuterated p-ethylphenol. Proteins suggested to be involved in this degradation pathway are encoded in a single large operon-like structure (ϳ15 kb). Among them are a p-cresol methylhydroxylase-like protein (PchCF), two predicted alcohol dehydrogenases (ChnA and EbA309), a biotin-dependent carboxylase (XccABC), and a thiolase (TioL). Proteomic analysis (two-dimensional difference gel electrophoresis) revealed their specific and coordinated upregulation in cells adapted to anaerobic growth with p-ethylphenol and p-hydroxyacetophenone (e.g., PchF up to 29-fold). Coregulated proteins of currently unknown function (e.g., EbA329) are possibly involved in p-ethylphenol-and p-hydroxyacetophenone-specific solvent stress responses and related to other aromatic solvent-induced proteins of strain EbN1.Alkylphenols, such as p-ethylphenol, are present in coal tars and crude oils (2, 45). Besides the accidental release of fuelderived alkylphenols to the environment, phenolic compounds are also prominent constituents of petrochemical wastewaters arising from spent caustic and coal gasification. Alkylphenol concentrations in these effluents (mainly phenol, cresols, and ethylphenols) range from 100 to 68,000 mg liter Ϫ1 , depending on the source (2). Moreover, p-ethylphenol can also be plant derived; e.g., native olive oils contain up to 52 mg p-ethylphenol per kg, and the content can reach 470 mg kg Ϫ1 during storage (5). p-Ethylphenol can also be formed from p-coumaric acid, a major component of cereal cell walls, by several yeast and Lactobacillus species (11,50). Due to their cytotoxicity and relatively high water solubility (www.epa.gov/safewater/mcl .html), alkylphenols are of environmental concern.Anaerobic degradation of aromatic compounds requires reactions independent of molecular oxygen. They are fundamentally different from the oxygenase-catalyzed reactions employed under oxic conditions (for an overview, see references 14, 17, and 18). A variety of anaerobic aromatic compounddegrading bacteria were newly isolated during the last decades (for an overview, see references 46 and 51). Most of them are denitrifiers belonging to the "Aromatoleum"/Azoarcus/Thauera cluster within Betaproteobacteria (R. Rabus et al., unpublished data). "Aromatoleum aromaticum" strain EbN1, a metabolically versatile representative of this group, was originall...

Cryptochromes are ubiquitously expressed in various animal tissues including the retina. Some cryptochromes are involved in regulating circadian activity. Cryptochrome proteins have also been suggested to mediate the primary mechanism in light-dependent magnetic compass orientation in birds. Cryptochrome 1b (Cry1b) exhibits a unique carboxy terminus exclusively found in birds so far, which might be indicative for a specialised function. Cryptochrome 1a (Cry1a) is so far the only cryptochrome protein that has been localised to specific cell types within the retina of migratory birds. Here we show that Cry1b, an alternative splice variant of Cry1a, is also expressed in the retina of migratory birds, but it is primarily located in other cell types than Cry1a. This could suggest different functions for the two splice products. Using diagnostic bird-specific antibodies (that allow for a precise discrimination between both proteins), we show that Cry1b protein is found in the retinae of migratory European robins (Erithacus rubecula), migratory Northern Wheatears (Oenanthe oenanthe) and pigeons (Columba livia). In all three species, retinal Cry1b is localised in cell types which have been discussed as potentially well suited locations for magnetoreception: Cry1b is observed in the cytosol of ganglion cells, displaced ganglion cells, and in photoreceptor inner segments. The cytosolic rather than nucleic location of Cry1b in the retina reported here speaks against a circadian clock regulatory function of Cry1b and it allows for the possible involvement of Cry1b in a radical-pair-based magnetoreception mechanism.

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 (0.32 mM) or toluene (0.74 mM); and second, when anaerobic succinate-utilizing cultures were shocked with ethylbenzene (0.5 mM), toluene (1.2 mM), p-cresol (3.0 mM), and phenol (6.5 mM) as single stressors or as a mixture (total solvent concentration, 2.7 mM). Under all tested conditions impaired growth was paralleled by decelerated nitrate-nitrite consumption. Additionally, alkylbenzene-utilizing cultures accumulated poly(3-hydroxybutyrate) (PHB) up to 10% of the cell dry weight. These physiological responses were also reflected on the proteomic level (as determined by two-dimensional difference gel electrophoresis), e.g., up-regulation of PHB granule-associated phasins, cytochrome cd 1 nitrite reductase of denitrification, and several proteins involved in oxidative (e.g., SodB) and general (e.g., ClpB) stress responses.