Conspicuous chloroplast with light harvesting-photosystem I/II megacomplex in marine Prorocentrum cordatum.

Marine photosynthetic (micro)organisms drive multiple biogeochemical cycles and display a large diversity. Among them, the bloom-forming, free-living dinoflagellate Prorocentrum cordatum CCMP 1329 (formerly P. minimum) stands out with its distinct cell biological features. Here, we obtained insights into the structural properties of the chloroplast and the photosynthetic machinery of P. cordatum using microscopic and proteogenomic approaches. High-resolution FIB/SEM analysis revealed a single large chloroplast (∼40% of total cell volume) with a continuous barrel-like structure, completely lining the inner face of the cell envelope and enclosing a single reticular mitochondrium, the Golgi apparatus, as well as diverse storage inclusions. Enriched thylakoid membrane fractions of P. cordatum were comparatively analyzed with those of the well-studied model-species Arabidopsis (Arabidopsis thaliana) using 2D BN DIGE. Strikingly, P. cordatum possessed a large photosystem-light harvesting megacomplex (>1.5 MDa), which is dominated by photosystems I and II (PSI, PSII), chloroplast complex I, and chlorophyll a-b binding light harvesting complex proteins. This finding parallels the absence of grana in its chloroplast and distinguishes from the predominant separation of PSI and PSII complexes in A. thaliana, indicating a different mode of flux balancing. Except for the core elements of the ATP synthase and the cytb6f-complex, the composition of the other complexes (PSI, PSII, and pigment-binding proteins, PBPs) of P. cordatum differed markedly from those of A. thaliana. Furthermore, a high number of PBPs was detected, accounting for a large share of the total proteomic data (∼65%) and potentially providing P. cordatum with flexible adaptation to changing light regimes.

A Novel Coenzyme A Analogue in the Anaerobic, Sulfate-Reducing, Marine Bacterium Desulfobacula toluolica Tol2T.

Coenzyme A (CoA) thioesters are formed during anabolic and catabolic reactions in every organism. Degradation pathways of growth-supporting substrates in bacteria can be predicted by differential proteogenomic studies. Direct detection of proposed metabolites such as CoA thioesters by high-performance liquid chromatography coupled with high-resolution mass spectrometry can confirm the reaction sequence and demonstrate the activity of these degradation pathways. In the metabolomes of the anaerobic sulfate-reducing bacterium Desulfobacula toluolica Tol2T grown with different substrates various CoA thioesters, derived from amino acid, fatty acid or alcohol metabolism, have been detected. Additionally, the cell extracts of this bacterium revealed a number of CoA analogues with molecular masses increased by 1 dalton. By comparing the chromatographic and mass spectrometric properties of synthetic reference standards with those of compounds detected in cell extracts of D. toluolica Tol2T and by performing co-injection experiments, these analogues were identified as inosino-CoAs. These CoA thioesters contain inosine instead of adenosine as the nucleoside. To the best of our knowledge, this finding represents the first detection of naturally occurring inosino-CoA analogues.

Transcriptome-proteome compendium of the Antarctic krill (Euphausia superba): Metabolic potential and repertoire of hydrolytic enzymes.

The Antarctic krill (Euphausia superba Dana) is a keystone species in the Southern Ocean that uses an arsenal of hydrolases for biomacromolecule decomposition to effectively digest its omnivorous diet. The present study builds on a hybrid-assembled transcriptome (13,671 ORFs) combined with comprehensive proteome profiling. The analysis of individual krill compartments allowed detection of significantly more different proteins compared to that of the entire animal (1464 vs. 294 proteins). The nearby krill sampling stations in the Bransfield Strait (Antarctic Peninsula) yielded rather uniform proteome datasets. Proteins related to energy production and lipid degradation were particularly abundant in the abdomen, agreeing with the high energy demand of muscle tissue. A total of 378 different biomacromolecule hydrolysing enzymes were detected, including 250 proteases, 99 CAZymes, 14 nucleases and 15 lipases. The large repertoire in proteases is in accord with the protein-rich diet affiliated with E. superba's omnivorous lifestyle and complex biology. The richness in chitin-degrading enzymes allows not only digestion of zooplankton diet, but also the utilisation of the discharged exoskeleton after moulting.

Complex and flexible catabolism in Aromatoleum aromaticum pCyN1.

Large quantities of organic matter are continuously deposited, and (a)biotic gradients intersect in the soil-rhizosphere, where biodegradation contributes to the global cycles of elements. The betaproteobacterial genus Aromatoleum comprises cosmopolitan, facultative denitrifying degradation specialists. Aromatoleum aromaticum. pCyN1 stands out for anaerobically decomposing plant-derived monoterpenes in addition to monoaromatic hydrocarbons, polar aromatics and aliphatics. The catabolic network's structure and flexibility in A. aromaticum pCyN1 were studied across 34 growth conditions by superimposing proteome profiles onto the manually annotated 4.37 Mbp genome. Strain pCyN1 employs three fundamentally different enzymes for C-H-bond cleavage at the methyl groups of p-cymene/4-ethyltoluene, toluene and p-cresol respectively. Regulation of degradation modules displayed substrate specificities ranging from narrow (toluene and cyclohexane carboxylate) via medium-wide (one module shared by p-cymene, 4-ethyltoluene, α-phellandrene, α-terpinene, γ-terpinene and limonene) to broad (central benzoyl-CoA pathway serving 16 aromatic substrates). Remarkably, three variants of ATP-dependent (class I) benzoyl-CoA reductase and four different ß-oxidation routes establish a degradation hub that accommodates the substrate diversity. The respiratory system displayed several conspicuous profiles, e.g. the presence of nitrous oxide reductase under oxic and of low-affinity oxidase under anoxic conditions. Overall, nutritional versatility in conjunction with network regulation endow A. aromaticum pCyN1 with broad adaptability.

Proteome, Bioinformatic, and Functional Analyses Reveal a Distinct and Conserved Metabolic Pathway for Bile Salt Degradation in the Sphingomonadaceae.

Bile salts are amphiphilic steroids with digestive functions in vertebrates. Upon excretion, bile salts are degraded by environmental bacteria. Degradation of the bile salt steroid skeleton resembles the well-studied pathway for other steroids, like testosterone, while specific differences occur during side chain degradation and the initiating transformations of the steroid skeleton. Of the latter, two variants via either Δ1,4- or Δ4,6-3-ketostructures of the steroid skeleton exist for 7-hydroxy bile salts. While the Δ1,4 variant is well known from many model organisms, the Δ4,6 variant involving a 7-hydroxysteroid dehydratase as a key enzyme has not been systematically studied. Here, combined proteomic, bioinformatic, and functional analyses of the Δ4,6 variant in Sphingobium sp. strain Chol11 were performed. They revealed a degradation of the steroid rings similar to that of the Δ1,4 variant except for the elimination of the 7-OH as a key difference. In contrast, differential production of the respective proteins revealed a putative gene cluster for the degradation of the C5 carboxylic side chain encoding a CoA ligase, an acyl-CoA dehydrogenase, a Rieske monooxygenase, and an amidase but lacking most canonical genes known from other steroid-degrading bacteria. Bioinformatic analyses predicted the Δ4,6 variant to be widespread among the Sphingomonadaceae, which was verified for three type strains which also have the predicted side chain degradation cluster. A second amidase in the side chain degradation gene cluster of strain Chol11 was shown to cleave conjugated bile salts while having low similarity to known bile salt hydrolases. This study identifies members of the Sphingomonadaceae that are remarkably well adapted to the utilization of bile salts via a partially distinct metabolic pathway. IMPORTANCE This study highlights the biochemical diversity of bacterial degradation of steroid compounds, in particular bile salts. Furthermore, it substantiates and advances knowledge of a variant pathway for degradation of steroids by sphingomonads, a group of environmental bacteria that are well known for their broad metabolic capabilities. Biodegradation of bile salts is a critical process due to the high input of these compounds from manure into agricultural soils and wastewater treatment plants. In addition, these results may also be relevant for the biotechnological production of bile salts or other steroid compounds with pharmaceutical functions.

Responsiveness of Aromatoleum aromaticum EbN1T to Lignin-Derived Phenylpropanoids.

The betaproteobacterial degradation specialist Aromatoleum aromaticum EbN1T utilizes several plant-derived 3-phenylpropanoids coupled to denitrification. In vivo responsiveness of A. aromaticum EbN1T was studied by exposing nonadapted cells to distinct pulses (spanning 100 µM to 0.1 nM) of 3-phenylpropanoate, cinnamate, 3-(4-hydroxyphenyl)propanoate, or p-coumarate. Time-resolved, targeted transcript analyses via quantitative reverse transcription-PCR of four selected 3-phenylpropanoid genes revealed a response threshold of 30 to 50 nM for p-coumarate and 1 to 10 nM for the other three tested 3-phenylpropanoids. At these concentrations, transmembrane effector equilibration is attained by passive diffusion rather than active uptake via the ABC transporter, presumably serving the studied 3-phenylpropanoids as well as benzoate. Highly substrate-specific enzyme formation (EbA5316 to EbA5321 [EbA5316-21]) for the shared peripheral degradation pathway putatively involves the predicted TetR-type transcriptional repressor PprR. Accordingly, relative transcript abundances of ebA5316-21 are lower in succinate- and benzoate-grown wild-type cells than in an unmarked in-frame ΔpprR mutant. In trans-complementation of pprR into the ΔpprR background restored wild-type-like transcript levels. When adapted to p-coumarate, the three genotypes had relative transcript abundances similar to those of ebA5316-21 despite a significantly longer lag phase of the pprR-complemented mutant (∼100-fold higher pprR transcript level than the wild type). Notably, transcript levels of ebA5316-21 were ∼10- to 100-fold higher in p-coumarate- than succinate- or benzoate-adapted cells across all three genotypes. This indicates the additional involvement of an unknown transcriptional regulator. Furthermore, physiological, transcriptional, and (aromatic) acyl-coenzyme A ester intermediate analyses of the wild type and ΔpprR mutant grown with binary substrate mixtures suggest a mode of catabolite repression of superior order to PprR.IMPORTANCE Lignin is a ubiquitous heterobiopolymer built from a suite of 3-phenylpropanoid subunits. It accounts for more than 30% of the global plant dry material, and lignin-related compounds are increasingly released into the environment from anthropogenic sources, i.e., by wastewater effluents from the paper and pulp industry. Hence, following biological or industrial decomplexation of lignin, vast amounts of structurally diverse 3-phenylpropanoids enter terrestrial and aquatic habitats, where they serve as substrates for microbial degradation. This raises the question of what signaling systems environmental bacteria employ to detect these nutritionally attractive compounds and to adjust their catabolism accordingly. Moreover, determining in vivo response thresholds of an anaerobic degradation specialist such as A. aromaticum EbN1T for these aromatic compounds provides insights into the environmental fate of the latter, i.e., when they could escape biodegradation due to too low ambient concentrations.

Suspect screening and targeted analysis of acyl coenzyme A thioesters in bacterial cultures using a high-resolution tribrid mass spectrometer.

Analysis of acyl coenzyme A thioesters (acyl-CoAs) is crucial in the investigation of a wide range of biochemical reactions and paves the way to fully understand the concerned metabolic pathways and their superimposed networks. We developed two methods for suspect screening of acyl-CoAs in bacterial cultures using a high-resolution Orbitrap Fusion tribrid mass spectrometer. The methods rely on specific fragmentation patterns of the target compounds, which originate from the coenzyme A moiety. They make use of the formation of the adenosine 3',5'-diphosphate key fragment (m/z 428.0365) and the neutral loss of the adenosine 3'-phosphate-5'-diphosphate moiety (506.9952) as preselection criteria for the detection of acyl-CoAs. These characteristic ions are generated either by an optimised in-source fragmentation in a full scan Orbitrap measurement or by optimised HCD fragmentation. Additionally, five different filters are included in the design of method. Finally, data-dependent MS/MS experiments on specifically preselected precursor ions are performed. The utility of the methods is demonstrated by analysing cultures of the denitrifying betaproteobacterium "Aromatoleum" sp. strain HxN1 anaerobically grown with hexanoate. We detected 35 acyl-CoAs in total and identified 24 of them by comparison with reference standards, including all 9 acyl-CoA intermediates expected to occur in the degradation pathway of hexanoate. The identification of additional acyl-CoAs provides insight into further metabolic processes occurring in this bacterium. The sensitivity of the method described allows detecting acyl-CoAs present in biological samples in highly variable abundances. Graphical abstract.

Nanomolar Responsiveness of an Anaerobic Degradation Specialist to Alkylphenol Pollutants.

Anaerobic degradation of p-cresol (4-methylphenol) by the denitrifying betaproteobacterium Aromatoleum aromaticum EbN1 is regulated with high substrate specificity, presumed to be mediated by the predicted σ54-dependent two-component system PcrSR. An unmarked, in-frame ΔpcrSR deletion mutant showed reduced expression of the genes cmh (21-fold) and hbd (8-fold) that encode the two enzymes for initial oxidation of p-cresol to p-hydroxybenzoate compared to their expression in the wild type. The expression of cmh and hbd was restored by in trans complementation with pcrSR in the ΔpcrSR background to even higher levels than in the wild type. This is likely due to ∼200-/∼30-fold more transcripts of pcrSR in the complemented mutant. The in vivo responsiveness of A. aromaticum EbN1 to p-cresol was studied in benzoate-limited anaerobic cultures by the addition of p-cresol at various concentrations (from 100 µM down to 0.1 nM). Time-resolved transcript profiling by quantitative reverse transcription-PCR (qRT-PCR) revealed that the lowest p-cresol concentrations just affording cmh and hbd expression (response threshold) ranged between 1 and 10 nM, which is even more sensitive than the respective odor receptors of insects. A similar response threshold was determined for another alkylphenol, p-ethylphenol, which strain EbN1 anaerobically degrades via a different route and senses by the σ54-dependent one-component system EtpR. Based on these data and theoretical considerations, p-cresol or p-ethylphenol added as a single pulse (10 nM) requires less than a fraction of a second to reach equilibrium between intra- and extracellular space (∼20 molecules per cell), with an estimated Kd (dissociation constant) of <100 nM alkylphenol (p-cresol or p-ethylphenol) for its respective sensory protein (PcrS or EtpR).IMPORTANCE Alkylphenols (like p-cresol and p-ethylphenol) represent bulk chemicals for industrial syntheses. Besides massive local damage events, large-scale micropollution is likewise of environmental and health concern. Next to understanding how such pollutants can be degraded by microorganisms, it is also relevant to determine the microorganisms' lower threshold of responsiveness. Aromatoleum aromaticum EbN1 is a specialist in anaerobic degradation of aromatic compounds, employing a complex and substrate-specifically regulated catabolic network. The present study aims at verifying the predicted role of the PcrSR system in sensing p-cresol and at determining the threshold of responsiveness for alkylphenols. The findings have implications for the enigmatic persistence of dissolved organic matter (escape from biodegradation) and for the lower limits of aromatic compounds required for bacterial growth.

Metabolites of the Anaerobic Degradation of n-Hexane by Denitrifying Betaproteobacterium Strain HxN1.

The constitutions of seven metabolites formed during anaerobic degradation of n-hexane by the denitrifying betaproteobacterium strain HxN1 were elucidated by comparison of their GC and MS data with those of synthetic reference standards. The synthesis of 4-methyloctanoic acid derivatives was accomplished by the conversion of 2-methylhexanoyl chloride with Meldrum's acid. The ß-oxoester was reduced with NaBH4 , the hydroxy group was eliminated, and the double bond was displaced to yield the methyl esters of 4-methyl-3-oxooctanoate, 3-hydroxy-4-methyloctanoate, (E)-4-methyl-2-octenoate, and (E)- and (Z)-4-methyl-3-octenoate. The methyl esters of 2-methyl-3-oxohexanoate and 3-hydroxy-2-methylhexanoate were similarly prepared from butanoyl chloride and Meldrum's acid. However, methyl (E)-2-methyl-2-hexenoate was prepared by Horner-Wadsworth-Emmons reaction, followed by isomerization to methyl (E)-2-methyl-3-hexenoate. This investigation, with the exception of 4-methyl-3-oxooctanoate, which was not detectable in the cultures, completes the unambiguous identification of all intermediates of the anaerobic biodegradation of n-hexane to 2-methyl-3-oxohexanoyl coenzyme A (CoA), which is then thiolytically cleaved to butanoyl-CoA and propionyl-CoA; these two metabolites are further transformed according to established pathways.

Metabolites of the anaerobic degradation of diethyl ether by denitrifying betaproteobacterium strain HxN1.

The constitutions of five metabolites formed during co-metabolic, anaerobic degradation of diethyl ether by the denitrifying betaproteobacterium Aromatoleum sp. strain HxN1 were elucidated by comparison of mass spectrometric and gas chromatographic data with those of synthetic reference standards. Furthermore, the absolute configurations of two stereogenic centers in the metabolites were established. Based on these results a degradation pathway for diethyl ether by Aromatoleum sp. HxN1 analogous to that of n-hexane is proposed. Synthesis of both enantiomers of methyl (E)-4-ethoxy-2-pentenoate was accomplished by etherification of ethyl (R)- or (S)-lactate, followed by hydrolysis of the ester group and reduction to furnish 2-ethoxy-1-propanol. The primary alcohol was converted by a Swern oxidation followed by a Horner-Wadsworth-Emmons reaction to methyl (E)-4-ethoxy-2-pentenoate that was finally hydrogenated to methyl 4-ethoxypentanoate. Methyl (S)-4-ethoxy-3-oxopentanoate was prepared by conversion of (S)-2-ethoxypropanoyl chloride with Meldrum's acid. Reduction of the resulting ß-oxoester with NaBH4 or baker's yeast gave both diastereoisomers of methyl 4-ethoxy-3-hydroxypentanoate. The stereocenter at C-3 of the main diastereoisomer produced with baker's yeast was determined by Mosher ester analysis to be (R)-configurated. Dimethyl 2-(1-ethoxyethyl)succinate was prepared by Michael addition of nitroethane to diethyl maleate, followed by conjugate addition of sodium ethanolate, hydrolysis and esterification with diazomethane.

Method development and validation for the quantification of organic acids in microbial samples using anionic exchange solid-phase extraction and gas chromatography-mass spectrometry.

Organic acids play a key role in central metabolic functions of organisms, are crucial for understanding regulatory processes and are ubiquitous inside the cell. Therefore, quantification of these compounds provides a valuable approach for studying dynamics of metabolic processes, in particular when the organism faces changing environmental conditions. However, the extraction and analysis of organic acids can be challenging and validated methods available in this field are limited. In this study, we developed a method for the extraction and quantification of organic acids from microbial samples based on solid-phase extraction on a strong anionic exchange cartridge and gas chromatographic-mass spectrometric analysis. Full method validation was conducted to determine quality parameters of the new method. Recoveries for 12 of the 15 aromatic and aliphatic acids were between 100 and 111% and detection limits between 3 and 272 ng/mL. The ranges for the regression coefficients and process standard deviations for these compound classes were 0.9874-0.9994 and 0.04-0.69 µg/mL, respectively. Limitations were encountered when targeting aliphatic acids with hydroxy, oxo or enol ester functions. Finally, we demonstrated the applicability of the method on cell extracts of the bacterium Escherichia coli and the dinoflagellate Prorocentrum minimum. Graphical abstract.

A simple protocol for the isolation of proteorhodopsins of the dinoflagellate Oxyrrhis marina.

For the first time, native proteorhodopsins of the marine dinoflagellate Oxyrrhis marina were isolated. Total cell membrane fractions were minced in a bead beater and solubilized with the detergent Triton X-100. Subsequent sucrose density gradient centrifugation resulted in three or four red-colored bands. Nonsolubilized, but still red colored, membranes sedimented at the bottom. For each of these bands, absorbance maxima were registered at approximately 514-516 nm with shoulders toward shorter wavelengths (470-490 nm). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the uppermost band represented free retinal chromophore, as it contained no protein. The other bands were almost pure proteorhodopsin fractions as the banding patterns showed one major protein of 25 kDa. Tryptic, in-gel digestion of the 25 kDa proteins and of faint protein bands above and below 25 kDa was followed by mass spectrometry, confirming these protein bands to consist, nearly exclusively, proteorhodopsins. Only single peptides of few other proteins were detected. In total, at least seven predicted proteorhodopsin protein sequences were experimentally verified.

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 Phaeobacter inhibens DSM 17395, a versatile heterotrophic roseobacter. Fourteen different substrates (five sugars and nine amino acids) and their degradation pathways were assessed for energetic efficiencies based on catabolic ATP yields, calculated from net formed ATP and reducing equivalents. The latter were converted into ATP by employing the most divergent coupling ratios (i.e., ions per ATP) currently known for F1Fo ATP synthases in heterotrophic bacteria. The catabolic ATP yields of the pathways studied in P. inhibens differed ∼3-fold. The actual free energy costs for ATP synthesis were estimated at 81.6 kJ per mol ATP (3.3 ions per ATP) or 104.2 kJ per mol ATP (4.3 ions per ATP), yielding an average thermodynamic efficiency of ∼37.7% or ∼29.5%, respectively. Growth performance (rates, yields) and carbon assimilation efficiency were determined for P. inhibens growing in process-controlled bioreactors with 10 different single substrates (Glc, Man, N-acetylglucosamine [Nag], Phe, Trp, His, Lys, Thr, Val, or Leu) and with 2 defined substrate mixtures. The efficiencies of carbon assimilation into biomass ranged from ∼28% to 61%, with His/Trp and Thr/Leu yielding the lowest and highest levels. These efficiencies correlated with catabolic and ATP yields only to some extent. Substrate-specific metabolic demands and/or functions, as well as the compositions of the substrate mixtures, apparently affected the energetic costs of growth. These include energetic burdens associated with, e.g., slow growth, stress, and/or the production of tropodithietic acid.IMPORTANCE Heterotrophic members of the bacterioplankton serve the marine ecosystem by transforming organic matter, an activity that is governed by the bacterial growth efficiencies (BGEs) obtained under given environmental conditions. In marine ecology, the concept of BGE refers to the carbon assimilation efficiency within natural communities. The marine bacterium studied here, Phaeobacter inhibens DSM 17395, is a copiotrophic representative of the globally abundant Roseobacter group, and the 15 catabolic pathways investigated are widespread among these marine heterotrophs. Combining pathway-specific catabolic ATP yields with in-depth quantitative physiological data could (i) provide a new baseline for the study of growth energetics and efficiency in further Roseobacter group members and other copiotrophic marine bacteria in productive coastal ecosystems and (ii) contribute to a better understanding of the factors controlling BGE (including the additional energetic burden arising from widespread secondary-metabolite formation) based on laboratory studies with pure cultures.

Stereochemical Insights into the Anaerobic Degradation of 4-Isopropylbenzoyl-CoA in the Denitrifying Bacterium Strain pCyN1.

The constitutions and absolute configurations of two previously unknown intermediates, (1S,2S,4S)-2-hydroxy-4-isopropylcyclohexane-1-carboxylate and (S)-3-isopropylpimelate, of anaerobic degradation of p-cymene in the bacterium Aromatoleum aromaticum pCyN1 are reported. These intermediates (as CoA esters) are involved in the further degradation of 4-isopropylbenzoyl-CoA formed by methyl group hydroxylation and subsequent oxidation of p-cymene. Proteogenomics indicated 4-isopropylbenzoyl-CoA degradation involves (i) a novel member of class I benzoyl-CoA reductase (BCR) as known from Thauera aromatica K172 and (ii) a modified ß-oxidation pathway yielding 3-isopropylpimeloyl-CoA analogously to benzoyl-CoA degradation in Rhodopseudomonas palustris. Reference standards of all four diastereoisomers of 2-hydroxy-4-isopropylcyclohexane-1-carboxylate as well as both enantiomers of 3-isopropylpimelate were obtained by stereoselective syntheses via methyl 4-isopropyl-2-oxocyclohexane-1-carboxylate. The stereogenic center carrying the isopropyl group was established using a rhodium-catalyzed asymmetric conjugate addition. X-ray crystallography revealed that the thermodynamically most stable stereoisomer of 2-hydroxy-4-isopropylcyclohexane-1-carboxylate is formed during p-cymene degradation. Our findings imply that the reductive dearomatization of 4-isopropylbenzoyl-CoA by the BCR of A. aromaticum pCyN1 stereospecifically forms (S)-4-isopropyl-1,5-cyclohexadiene-1-carbonyl-CoA.

Aromatoleum gen. nov., a novel genus accommodating the phylogenetic lineage including Azoarcus evansii and related species, and proposal of Aromatoleum aromaticum sp. nov., Aromatoleum petrolei sp. nov., Aromatoleum bremense sp. nov., Aromatoleum toluolicum sp. nov. and Aromatoleum diolicum sp. nov.

Comparative 16S rRNA gene sequence analysis and major physiological differences indicate two distinct sublineages within the genus Azoarcus: the Azoarcus evansii lineage, comprising Azoarcusevansii (type strain KB740T=DSM 6898T=CIP 109473T=NBRC 107771T), Azoarcusbuckelii (type strain U120T=DSM 14744T=LMG 26916T), Azoarcusanaerobius (type strain LuFRes1T=DSM 12081T=LMG 30943T), Azoarcustolulyticus (type strain Tol-4T=ATCC 51758T=CIP 109470T), Azoarcustoluvorans (type strain Td21T=ATCC 700604T=DSM 15124T) and Azoarcustoluclasticus (type strain MF63T=ATCC 700605T), and the Azoarcus indigens lineage, comprising Azoarcusindigens (type strain VB32T=ATCC 51398T=LMG 9092T), Azoarcus communis (type strain SWub3T=ATCC 51397T=LMG 9095T) and Azoarcusolearius (type strain DQS-4T=BCRC 80407T=KCTC 23918T=LMG 26893T). Az. evansii lineage members have remarkable anaerobic degradation capacities encompassing a multitude of alkylbenzenes, aromatic compounds and monoterpenes, often involving novel biochemical reactions. In contrast, Az. indigens lineage members are diazotrophic endophytes lacking these catabolic capacities. It is proposed that species of the Az. evansii lineage should be classified in a novel genus, Aromatoleum gen. nov. Finally, based on the literature and new growth, DNA-DNA hybridization and proteomic data, the following five new species are proposed: Aromatoleum aromaticum sp. nov. (type strain EbN1T=DSM 19018T=LMG 30748T and strain pCyN1=DSM 19016=LMG 31004), Aromatoleum petrolei sp. nov. (type strain ToN1T=DSM 19019T=LMG 30746T), Aromatoleumbremense sp. nov. (type strain PbN1T=DSM 19017T=LMG 31005T), Aromatoleum toluolicum sp. nov. (type strain TT=DSM 19020T=LMG 30751T) and Aromatoleum diolicum sp. nov. (type strain 22LinT=DSM 15408T=LMG 30750T).

Towards the Response Threshold for p-Hydroxyacetophenone in the Denitrifying Bacterium "Aromatoleum aromaticum" EbN1.

The denitrifying betaproteobacterium "Aromatoleum aromaticum" EbN1 regulates the capacity to anaerobically degrade p-ethylphenol (via p-hydroxyacetophenone) with high substrate specificity. This process is mediated by the σ54-dependent transcriptional regulator EtpR, which apparently recognizes both aromatic compounds, yielding congruent expression profiles. The responsiveness of this regulatory system was studied with p-hydroxyacetophenone, which is more easily administered to cultures and traced analytically. Cultures of A. aromaticum EbN1 were initially cultivated under nitrate-reducing conditions with a growth-limiting supply of benzoate, upon the complete depletion of which p-hydroxyacetophenone was added at various concentrations (from 500 µM down to 0.1 nM). Depletion profiles of this aromatic substrate and presumptive effector were determined by highly sensitive micro-high-performance liquid chromatography (microHPLC). Irrespective of the added concentration of p-hydroxyacetophenone, depletion commenced after less than 5 min and suggested a response threshold of below 10 nM. This approximation was corroborated by time-resolved transcript profiles (quantitative reverse transcription-PCR) of selected degradation and efflux relevant genes (e.g., pchF, encoding a subunit of predicted p-ethylphenol methylenehydroxylase) and narrowed down to a range of 10 to 1 nM. The most pronounced transcriptional response was observed, as expected, for genes located at the beginning of the two operon-like structures, related to catabolism (i.e., acsA) and potential efflux (i.e., ebA335).IMPORTANCE Aromatic compounds are widespread microbial growth substrates with natural as well as anthropogenic sources, albeit with their in situ concentrations and their bioavailabilities varying over several orders of magnitude. Even though degradation pathways and underlying regulatory systems have long been studied with aerobic and, to a lesser extent, with anaerobic bacteria, comparatively little is known about the effector concentration-dependent responsiveness. A. aromaticum EbN1 is a model organism for the anaerobic degradation of aromatic compounds with the architecture of the catabolic network and its substrate-specific regulation having been intensively studied by means of differential proteogenomics. The present study aims at unraveling the minimal concentration of an aromatic growth substrate (p-hydroxyacetophenone here) required to initiate gene expression for its degradation pathway and to learn in principle about the lower limit of catabolic responsiveness of an anaerobic degradation specialist.

Impact of Extraction Methods on the Detectable Protein Complement of Metaproteomic Analyses of Marine Sediments.

Metaproteomic analysis targets proteins, the catalytic entities in the habitat, thereby providing direct insights into the metabolic activity of the community studied. A major challenge still remaining for metaproteomics is the effective and comprehensive extraction of proteins from environmental samples, due to their high complexity with respect to organismic diversity and abundance range. Moreover, in certain habitats, the inherent matrix may interfere with protein extraction. In recent years, several studies reported different protein extraction methods for soils known for their complex geochemistry, but only three analyzed marine sediments that generally comprise different though similarly complex geochemistry. In this study, the impact of four different extraction methods was investigated for coastal North Sea and deep sea Pacific Ocean sediments. The extraction methods comprised (i) phenol, (ii) SDS, (iii) a mixture of SDS and phenol, and (iv) urea and thiourea. Prior to extraction, a cell and protein standard (CPS) was added to the sediment samples to trace recovery of proteins from different subcellular locations as well as dissolved BSA. While each extraction method detected distinct peptide complements, SDS-phenol extraction generally achieved highest protein yield and most comprehensive CPS protein identification. Application of two different methods was shown to further improve proteome coverage.

Differential proteomic analysis of the metabolic network of the marine sulfate-reducer Desulfobacterium autotrophicum HRM2.

The marine sulfate-reducing bacterium Desulfobacterium autotrophicum HRM2 belongs to the deltaproteobacterial family Desulfobacteraceae and stands out for its capacity of facultative chemolithoautotrophic growth (next to heterotrophy). Here, proteomics-driven metabolic reconstruction was based on a combination of 2D-DIGE, shotgun proteomics, and analysis of the membrane protein enriched fraction applied to eight different substrate adaptation conditions (seven aliphatic compounds plus H2 /CO2 ). In total, 1344 different proteins were identified (∼27% of the 4947 genome-predicted), from which a complex metabolic network was reconstructed consisting of 136 proteins (124 detected; ∼91%). Peripheral degradation routes for organic substrates feed directly or via the methylmalonyl-CoA pathway into the Wood-Ljungdahl pathway (WLP) for terminal oxidation to CO2 . Chemolithoautotrophic growth apparently involves the periplasmic [Ni/Fe/Se]-containing hydrogenase HysAB (H2 oxidation), the reductively operating WLP (CO2 fixation), and classical gluconeogenesis. Diverse soluble proteins (e.g., Hdr, Etf) probably establish a fine balanced cytoplasmic electron transfer network connecting individual catabolic reactions with the membrane menaquinone pool. In addition, multiple membrane protein complexes (Nqr, Qmo, Qrc, Rnf1, Rnf2, and Tmc) provide ample routes for interacting with the reducing equivalent pool and delivering electrons to dissimilatory sulfate reduction (both localized in the cytoplasm). Overall, this study contributes to the molecular understanding of the habitat-relevant Desulfobacteraceae.

Analysis of membrane-protein complexes of the marine sulfate reducer Desulfobacula toluolica Tol2 by 1D blue native-PAGE complexome profiling and 2D blue native-/SDS-PAGE.

Sulfate-reducing bacteria (SRB) obtain energy from cytoplasmic reduction of sulfate to sulfide involving APS-reductase (AprAB) and dissimilatory sulfite reductase (DsrAB). These enzymes are predicted to obtain electrons from membrane redox complexes, i.e. the quinone-interacting membrane-bound oxidoreductase (QmoABC) and DsrMKJOP complexes. In addition to these conserved complexes, the genomes of SRB encode a large number of other (predicted) membrane redox complexes, the function and actual formation of which is unknown. This study reports the establishment of 1D Blue Native-PAGE complexome profiling and 2D BN-/SDS-PAGE for analysis of the membrane protein complexome of the marine sulfate reducer Desulfobacula toluolica Tol2. Analysis of normalized score profiles of >800 proteins in combination with hierarchical clustering and identification of 2D BN-/SDS-PAGE separated spots demonstrated separation of membrane complexes in their native form, e.g. ATP synthase. In addition to the QmoABC and DsrMKJOP complexes, other complexes were detected that constitute the basic membrane complexome of D. toluolica Tol2, e.g. transport proteins (e.g. sodium/sulfate symporters) or redox complexes involved in Na(+) -based bioenergetics (RnfABCDEG). Notably, size estimation indicates dimer and quadruple formation of the DsrMKJOP complex in vivo. Furthermore, cluster analysis suggests interaction of this complex with a rhodanese-like protein (Tol2_C05230) possibly representing a periplasmic electron transfer partner for DsrMKJOP.

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. Overall, the performance of the CCD camera system was comparable to the laser scanner, as evident from very similar protein abundance changes (irrespective of spot position and volume), as well as from linear range and limit of detection.