Functional proteomic view of metabolic regulation in "Aromatoleum aromaticum" strain EbN1

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.     

Anaerobic degradation of p-ethylphenol by "Aromatoleum aromaticum" strain EbN1: pathway, regulation, and involved proteins

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 (~15 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.     

Functional genomics of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1

Nitrate-reducing bacteria of the recently recognized Azoarcus/Thauera group within the Betaproteobacteria contribute significantly to the biodegradation of aromatic and other refractory compounds in anoxic waters and soils. Strain EbN1 belongs to a distinct cluster (new genus) and is the first member of this phylogenetic group, the genome of which has been determined (4.7 Mb; one chromosome, two plasmids) by [Rabus R, Kube M, Heider J, Beck A, Heitmann K, Widdel F, Reinhardt R (2005) The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1. Arch Microbiol 183:27–36]. Ten anaerobic and four aerobic aromatic-degradation pathways were recognized on the chromosome, with the coding genes mostly forming clusters. Presence of paralogous gene clusters (e.g. for anaerobic ethylbenzene degradation) suggests an even broader degradation spectrum than previously known. Metabolic versatility is also reflected by the presence of multiple respiratory complexes and is apparently controlled by an extensive regulatory network. Strain EbN1 is unique for its capacity to degrade toluene and ethylbenzene anaerobically via completely different pathways. Bioinformatical analysis of their genetic blueprints and global expression analysis (DNA-microarray and proteomics) of substrate-adapted cells [Kühner S, Wöhlbrand L, Fritz I, Wruck W, Hultschig C, Hufnagel P, Kube M, Reinhardt R, Rabus R (2005) Substrate-dependent regulation of anaerobic degradation pathways for toluene and ethylbenzene in a denitrifying bacterium, strain EbN1. J Bacteriol 187:1493–1503] indicated coordinated vs sequential modes of regulation for the toluene and ethylbenzene pathways, respectively.     

The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium,strain EbN1

Recent research on microbial degradation of aromatic and other refractory compounds in anoxic waters and soils has revealed that nitrate-reducing bacteria belonging to the Betaproteobacteria contribute substantially to this process. Here we present the first complete genome of a metabolically versatile representative, strain EbN1, which metabolizes various aromatic compounds, including hydrocarbons. A circular chromosome (4.3 Mb) and two plasmids (0.21 and 0.22 Mb) encode 4603 predicted proteins. Ten anaerobic and four aerobic aromatic degradation pathways were recognized, with the encoding genes mostly forming clusters. The presence of paralogous gene clusters (e.g., for anaerobic phenylacetate oxidation), high sequence similarities to orthologs from other strains (e.g., for anaerobic phenol metabolism) and frequent mobile genetic elements (e.g., more than 200 genes for transposases) suggest high genome plasticity and extensive lateral gene transfer during metabolic evolution of strain EbN1. Metabolic versatility is also reflected by the presence of multiple respiratory complexes. A large number of regulators, including more than 30 two-component and several FNR-type regulators, indicate a finely tuned regulatory network able to respond to the fluctuating availability of organic substrates and electron acceptors in the environment. The absence of genes required for nitrogen fixation and specific interaction with plants separates strain EbN1 ecophysiologically from the closely related nitrogen-fixing plant symbionts of the Azoarcus cluster. Supplementary material on sequence and annotation are provided at the Web page .Electronic Supplementary Material Supplementary material is available for this article at Dedicated to Prof. Dr. h.c. Gerhard Gottschalk on the occasion of his 70th birthday.     

Development of a genetic system for the denitrifying bacterium 'Aromatoleum aromaticum' strain EbN1

Members of the betaproteobacterial 'Aromatoleum'/Azoarcus/Thauera cluster contribute to the biodegradation of aromatic and other recalcitrant compounds in anoxic soils and sediments. The metabolically versatile 'Aromatoleumaromaticum' strain EbN1 represents a model organism for this cluster, having already been studied on the physiological, proteogenomic and biochemical level. Here we report the development of a genetic system for 'A. aromaticum' strain EbN1 enabling unmarked deletion mutagenesis by heterologous recombination and subsequent complementation. The antibiotic sensitivity of strain EbN1 was characterized and optimal conditions for efficient cultivation on solid medium were established. A procedure for introducing foreign DNA into strain EbN1 by conjugation was developed. The effectiveness of the genetic system was demonstrated by unmarked in-frame deletion of ebdC2, encoding the gamma-subunit of a paralogous ethylbenzene dehydrogenase.     

Acetone and butanone metabolism of the denitrifying bacterium "Aromatoleum aromaticum" demonstrates novel biochemical properties of an ATP-dependent aliphatic ketone carboxylase

The anaerobic and aerobic metabolism of acetone and butanone in the betaproteobacterium "Aromatoleum aromaticum" is initiated by their ATP-dependent carboxylation to acetoacetate and 3-oxopentanoic acid, respectively. Both reactions are catalyzed by the same enzyme, acetone carboxylase, which was purified and characterized. Acetone carboxylase is highly induced under growth on acetone or butanone and accounts for at least 5.5% of total cell protein. The enzyme consists of three subunits of 85, 75, and 20 kDa, respectively, in a (αβγ)(2) composition and contains 1 Zn and 2 Fe per heterohexamer but no organic cofactors. Chromatographic analysis of the ATP hydrolysis products indicated that ATP was exclusively cleaved to AMP and 2 P(i). The stoichiometry was determined to be 2 ATP consumed per acetone carboxylated. Purified acetone carboxylase from A. aromaticum catalyzes the carboxylation of acetone and butanone as the only substrates. However, the enzyme shows induced (uncoupled) ATPase activity with many other substrates that were not carboxylated. Acetone carboxylase is a member of a protein family that also contains acetone carboxylases of various other organisms, acetophenone carboxylase of A. aromaticum, and ATP-dependent hydantoinases/oxoprolinases. While the members of this family share several characteristic features, they differ with respect to the products of ATP hydrolysis, subunit composition, and metal content.     

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)) (0.108 h(-1)), and high (μ(high)) (0.180 h(-1)) growth rates. A positive correlation to growth rate was observed for cellular parameters (cell size, and DNA and protein contents). The free energy consumed for biomass formation steadily increased with growth rate. In contrast, the energy demand for maintenance increased only from μ(low) to μ(med) and then remained constant until μ(high). The most comprehensive proteomic changes were observed at μ(low) compared to μ(high). Uniformly decreased abundances of protein components of the anaerobic benzoyl coenzyme A (benzoyl-CoA) pathway, central carbon metabolism, and information processing agree with a general deceleration of benzoate metabolism and cellular processes in response to slow growth. In contrast, increased abundances were observed at μ(low) for diverse catabolic proteins and components of uptake systems in the absence of the respective substrate (aromatic or aliphatic compounds) and for proteins involved in stress responses. This potential catabolic versatility and stress defense during slow growth may be interpreted as preparation for future needs.     

Genes involved in the anaerobic degradation of ethylbenzene in a denitrifying bacterium,strain EbN1

Genes involved in anaerobic degradation of the petroleum hydrocarbon ethylbenzene in the denitrifying Azoarcus-like strain EbN1 were identified on a 56-kb DNA contig obtained from shotgun sequencing. Ethylbenzene is first oxidized via ethylbenzene dehydrogenase to (S)-1-phenylethanol; this is converted by (S)-1-phenylethanol dehydrogenase to acetophenone. Further degradation probably involves acetophenone carboxylase forming benzoylacetate, a ligase forming benzoylacetyl-CoA, and a thiolase forming acetyl-CoA and benzoyl-CoA. Genes of this pathway were identified via N-terminal sequences of proteins isolated from strain EbN1 and by sequence similarities to proteins from other bacteria. Ethylbenzene dehydrogenase is encoded by three genes (ebdABC), in accordance with the heterotrimeric enzyme structure. Binding domains for a molybdenum cofactor (in subunit EbdA) and iron/sulfur-clusters (in subunits EbdA and EbdB) were identified. The previously observed periplasmic location of the enzyme was corroborated by the presence of a twin-arginine leader peptide characteristic of the Tat system for protein export. A fourth gene (ebdD) was identified, the product of which may act as an enzyme-specific chaperone in the maturation of the molybdenum-containing subunit. A distinct gene (ped) coding for (S)-1-phenylethanol dehydrogenase apparently forms an operon with the ebdABCD genes. The ped gene product with its characteristic NAD(P)-binding motif in the N-terminal domain belongs to the short-chain dehydrogenase/reductase (SDR) superfamily. A further operon apparently contains five genes (apc1-5) suggested to code for subunits of acetophenone carboxylase. Four of the five gene products are similar to subunits of acetone carboxylase from Xanthobacter autotrophicus. Upstream of the apc genes, a single gene (bal) was identified which possibly codes for a benzoylacetate CoA-ligase and which is co-transcribed with the apc genes. In addition, an apparent operon containing almost all genes required for beta-oxidation of fatty acids was detected; one of the gene products may be involved in thiolytic cleavage of benzoylacetyl-CoA. The DNA fragment also included genes for regulatory systems; these were two sets of two-component systems, two LysR homologs, and a TetR homolog. Some of these proteins may be involved in ethylbenzene-dependent gene expression.     

Crystal structure and enzyme kinetics of the (S)-specific 1-phenylethanol dehydrogenase of the denitrifying bacterium strain EbN1

(S)-1-Phenylethanol dehydrogenase (PED) from the denitrifying bacterium strain EbN1 catalyzes the NAD+-dependent, stereospecific oxidation of (S)-1-phenylethanol to acetophenone and the biotechnologically interesting reverse reaction. This novel enzyme belongs to the short-chain alcohol dehydrogenase/aldehyde reductase family. The coding gene (ped) was heterologously expressed in Escherichia coli and the purified protein was crystallized. The X-ray structures of the apo-form and the NAD+-bound form were solved at a resolution of 2.1 and 2.4 A, respectively, revealing that the enzyme is a tetramer with two types of hydrophobic dimerization interfaces, similar to beta-oxoacyl-[acyl carrier protein] reductase (FabG) from E. coli. NAD+-binding is associated with a conformational shift of the substrate binding loop of PED from a crystallographically unordered "open" to a more ordered "closed" form. Modeling the substrate acetophenone into the active site revealed the structural prerequisites for the strong enantioselectivity of the enzyme and for the catalytic mechanism. Studies on the steady-state kinetics of PED indicated a highly positive cooperativity of both catalytic directions with respect to the substrates. This is contrasted by the behavior of FabG. Moreover, PED exhibits extensive regulation on the enzyme level, being inhibited by elevated concentrations of substrates and products, as well as the wrong enantiomer of 1-phenylethanol. These regulatory properties of PED are consistent with the presence of a putative "transmission module" between the subunits. This module consists of the C-terminal loops of all four subunits, which form a special interconnected structural domain and mediate close contact of the subunits, and of a phenylalanine residue in each subunit that reaches out between substrate-binding loop and C-terminal domain of an adjacent subunit. These elements may transmit the substrate-induced conformational change of the substrate binding loop from one subunit to the others in the tetrameric complex and thus mediate the cooperative behavior of PED.     

Crystal structure of ethylbenzene dehydrogenase from Aromatoleum aromaticum

Anaerobic degradation of hydrocarbons was discovered a decade ago, and ethylbenzene dehydrogenase was one of the first characterized enzymes involved. The structure of the soluble periplasmic 165 kDa enzyme was established at 1.88 A resolution. It is a heterotrimer. The alpha subunit contains the catalytic center with a molybdenum held by two molybdopterin-guanine dinucleotides, one with an open pyran ring, and an iron-sulfur cluster with a histidine ligand. During catalysis, electrons produced by substrate oxidation are transferred to a heme in the gamma subunit and then presumably to a separate cytochrome involved in nitrate respiration. The beta subunit contains four iron-sulfur clusters and is structurally related to ferredoxins. The gamma subunit is the first known protein with a methionine and a lysine as axial heme ligands. The catalytic product was modeled into the active center, showing the reaction geometry. A mechanism consistent with activity and inhibition data of ethylbenzene-related compounds is proposed.     

Anaerobic degradation of ethylbenzene and toluene in denitrifying strain EbN1 proceeds via independent substrate-induced pathways

Denitrifying strain EbN1 utilizes either ethylbenzene or toluene as the sole source of organic carbon under strictly anoxic conditions. When cells were grown on ethylbenzene, 1-phenylethanol and acetophenone were detected in the culture supernatant. However, these two compounds were not observed when cells were grown on benzoate. Growth on ethylbenzene, 1-phenylethanol, or acetophenone strictly depended on the presence of CO2, whereas growth on benzoate did not. These results suggest that strain EbN1 degrades ethylbenzene via 1-phenylethanol and acetophenone as intermediates, and that acetophenone is subsequently carboxylated. In suspensions of benzoate-grown cells, induction was required for degradation of ethylbenzene, 1-phenylethanol, and acetophenone. Induction was also required for toluene-grown cells to gain activity to degrade ethylbenzene, and, conversely, for ethylbenzene-grown cells to degrade toluene. In accordance with our findings from these studies, two-dimensional gel electrophoretic analysis of extracts of cells grown on benzoate, acetophenone, ethylbenzene, or toluene showed that a number of substrate-specific proteins were induced in strain EbN1. Growth on toluene or ethylbenzene induced a distinct set of proteins. However, some of the induced proteins in ethylbenzene or acetophenone grown cells were identical. This agrees with the finding that acetophenone is an intermediate in the degradation of ethylbenzene.     

Anaerobic degradation of 4-methylbenzoate by a newly isolated denitrifying bacterium, strain pMbN1

A novel alphaproteobacterium isolated from freshwater sediments, strain pMbN1, degrades 4-methylbenzoate to CO(2) under nitrate-reducing conditions. While strain pMbN1 utilizes several benzoate derivatives and other polar aromatic compounds, it cannot degrade p-xylene or other hydrocarbons. Based on 16S rRNA gene sequence analysis, strain pMbN1 is affiliated with the genus Magnetospirillum.     

Initial reactions in anaerobic ethylbenzene oxidation by a denitrifying bacterium, strain EB1.

Initial reactions in anaerobic oxidation of ethylbenzene were investigated in a denitrifying bacterium, strain EB1. Cells of strain EB1 mineralized ethylbenzene to CO2 under denitrifying conditions, as demonstrated by conversion of 69% of [14C]ethylbenzene to 14CO2. In anaerobic suspensions of strain EB1 cells metabolizing ethylbenzene, the transient formation and consumption of 1-phenylethanol, acetophenone, and an as yet unidentified compound were observed. On the basis of growth experiments and spectroscopic data, the unknown compound is proposed to be benzoyl acetate. Cell suspension experiments using H2(18)O demonstrated that the hydroxyl group of the first product of anoxic ethylbenzene oxidation, 1-phenylethanol, is derived from water. A tentative pathway for anaerobic ethylbenzene mineralization by strain EB1 is proposed.     

Mass spectrometric identification of proteins in complex post-genomic projects. Soluble proteins of the metabolically versatile, denitrifying 'Aromatoleum' sp. strain EbN1

The rapidly developing proteomics technologies help to advance the global understanding of physiological and cellular processes. The lifestyle of a study organism determines the type and complexity of a given proteomic project. The complexity of this study is characterized by a broad collection of pathway-specific subproteomes, reflecting the metabolic versatility as well as the regulatory potential of the aromatic-degrading, denitrifying bacterium 'Aromatoleum' sp. strain EbN1. Differences in protein profiles were determined using a gel-based approach. Protein identification was based on a progressive application of MALDI-TOF-MS, MALDI-TOF-MS/MS and LC-ESI-MS/MS. This progression was result-driven and automated by software control. The identification rate was increased by the assembly of a project-specific list of background signals that was used for internal calibration of the MS spectra, and by the combination of two search engines using a dedicated MetaScoring algorithm. In total, intelligent bioinformatics could increase the identification yield from 53 to 70% of the analyzed 5,050 gel spots; a total of 556 different proteins were identified. MS identification was highly reproducible: most proteins were identified more than twice from parallel 2DE gels with an average sequence coverage of >50% and rather restrictive score thresholds (Mascot >or=95, ProFound >or=2.2, MetaScore >or=97). The MS technologies and bioinformatics tools that were implemented and integrated to handle this complex proteomic project are presented. In addition, we describe the basic principles and current developments of the applied technologies and provide an overview over the current state of microbial proteome research.     

Two distinct pathways for anaerobic degradation of aromatic compounds in the denitrifying bacterium Thauera aromatica strain AR-1

Denitrifying bacteria degrade many different aromatic compounds anaerobically via the well-described benzoyl-CoA pathway. We have shown recently that the denitrifiers Azoarcus anaerobius and Thauera aromatica strain AR-1 use a different pathway for anaerobic degradation of resorcinol (1,3-dihydroxybenzene) and 3,5-dihydroxybenzoate, respectively. Both substrates are converted to hydroxyhydroquinone (1,2,4-trihydroxybenzene). In the membrane fraction of T. aromatica strain AR-1 cells grown with 3,5-dihydroxybenzoate, a hydroxyhydroquinone-dehydrogenating activity of 74 nmol min(-1)(mg protein)-1 was found. This activity was significantly lower in benzoate-grown cells. Benzoate-grown cells were not induced for degradation of 3,5-dihydroxybenzoate, and cells grown with 3,5-dihydroxybenzoate degraded benzoate only at a very low rate. With a substrate mixture of benzoate plus 3,5-dihydroxybenzoate, the cells showed diauxic growth. Benzoate was degraded first, while complete degradation of 3,5-dihydroxybenzoate occurred only after a long lag phase. The 3,5-dihydroxybenzoate-oxidizing and the hydroxyhydroquinone-dehydrogenating activities were fully induced only during 3,5-dihydroxybenzoate degradation. Synthesis of benzoyl-CoA reductase appeared to be significantly lower in 3,5-dihydroxybenzoate-grown cells as shown by immunoblotting. These results confirm that T. aromatica strain AR-1 harbors, in addition to the benzoyl-CoA pathway, a second, mechanistically distinct pathway for anaerobic degradation of aromatic compounds. This pathway is inducible and subject to catabolite repression by benzoate.