Ligand cross-feeding resolves bacterial vitamin B12 auxotrophies.

Cobalamin (vitamin B12, herein referred to as B12) is an essential cofactor for most marine prokaryotes and eukaryotes1,2. Synthesized by a limited number of prokaryotes, its scarcity affects microbial interactions and community dynamics2-4. Here we show that two bacterial B12 auxotrophs can salvage different B12 building blocks and cooperate to synthesize B12. A Colwellia sp. synthesizes and releases the activated lower ligand α-ribazole, which is used by another B12 auxotroph, a Roseovarius sp., to produce the corrin ring and synthesize B12. Release of B12 by Roseovarius sp. happens only in co-culture with Colwellia sp. and only coincidently with the induction of a prophage encoded in Roseovarius sp. Subsequent growth of Colwellia sp. in these conditions may be due to the provision of B12 by lysed cells of Roseovarius sp. Further evidence is required to support a causative role for prophage induction in the release of B12. These complex microbial interactions of ligand cross-feeding and joint B12 biosynthesis seem to be widespread in marine pelagic ecosystems. In the western and northern tropical Atlantic Ocean, bacteria predicted to be capable of salvaging cobinamide and synthesizing only the activated lower ligand outnumber B12 producers. These findings add new players to our understanding of B12 supply to auxotrophic microorganisms in the ocean and possibly in other ecosystems.

Variations of intact phospholipid compositions in the digestive system of Antarctic krill, Euphausia superba, between summer and autumn.

The biochemical composition of Antarctic krill, Euphausia superba, is largely determined by their feeding behaviour. As they supply energy for animals of a higher trophic level and are also commercialized for human consumption, the interest in research on the species is high. Lipids, especially phospholipids, make up a high proportion of dry weight in krill. Seasonal changes are well documented in the fingerprint of free fatty acids analysed after hydrolysis of phospholipids, but the underlying intact polar lipids are rarely considered. In this study, we evaluated the compositions of intact phospholipids (IPLs) in the stomach, digestive gland and hind gut of Antarctic krill caught in summer and autumn at the Antarctic Peninsula region. Using high-resolution mass spectrometry, the fatty acid composition of 179 intact phospholipids could be resolved. Most IPLs were phosphatidylcholines, followed by phosphatidylethanolamines. Several very long chain polyunsaturated fatty acids up to 38:8, which have not been reported in krill before, were identified. The composition shifted to higher molecular weight IPLs with a higher degree of unsaturation for summer samples, especially for samples of the digestive gland. The data supplied in this paper provides new insights into lipid dynamics between summer and autumn usually described by free fatty acid biomarkers.

Vitamin B12 is not shared by all marine prototrophic bacteria with their environment.

Vitamin B12 (cobalamin, herein B12) is an essential cofactor involved in amino acid synthesis and carbon resupply to the TCA cycle for most prokaryotes, eukaryotic microorganisms, and animals. Despite being required by most, B12 is produced by only a minor fraction of prokaryotes and therefore leads to complex interaction between prototrophs and auxotrophs. However, it is unknown how B12 is provided by prototrophs to auxotrophs. In this study, 33 B12 prototrophic alphaproteobacterial strains were grown in co-culture with Thalassiosira pseudonana, a B12 auxotrophic diatom, to determine the bacterial ability to support the growth of the diatom by sharing B12. Among these strains, 18 were identified to share B12 with the diatom, while nine were identified to retain B12 and not support growth of the diatom. The other bacteria either shared B12 with the diatom only with the addition of substrate or inhibited the growth of the diatom. Extracellular B12 measurements of B12-provider and B12-retainer strains confirmed that the cofactor could only be detected in the environment of the tested B12-provider strains. Intracellular B12 was measured by LC-MS and showed that the concentrations of the different B12-provider as well as B12-retainer strains differed substantially. Although B12 is essential for the vast majority of microorganisms, mechanisms that export this essential cofactor are still unknown. Our results suggest that a large proportion of bacteria that can synthesise B12 de novo cannot share the cofactor with their environment.

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.

Simultaneous quantification of all B vitamins and selected biosynthetic precursors in seawater and bacteria by means of different mass spectrometric approaches.

B vitamins have high microbiological relevance in the marine environment, but their very low concentrations and the chemical heterogeneity of the individual vitamins make their analysis challenging. Mass spectrometric analysis of B vitamins in environmental samples at trace levels has mainly been performed using triple quadrupole mass spectrometers operated in targeted analysis mode. The development of such a method can be laborious and error prone. Additionally, high-resolution mass spectrometers can be used to measure a sample in full scan mode and subsequently search the total ion current chromatogram for extracted ion chromatograms of targeted vitamins. Three different analytical approaches for trace analysis of all B vitamins and some of their biosynthetic precursors were optimized and compared on two different mass spectrometers. A triple quadrupole mass spectrometer in selected reaction monitoring mode, and a high-resolution orbitrap mass spectrometer in parallel reaction monitoring, as well as in full scan mode were employed. Detection limits down to 10 ng/L were achieved with all three techniques. The methods were applied to a marine water sample from the North Sea and to the cell extract of a bacterial culture of Phaeobacter inhibens. Most vitamins and precursors were found in the bacterial cell extract and the seawater sample with all three measuring methods. The results of this study emphasize that, in addition to tandem mass spectrometry, high-resolution full scan mass spectrometry is a promising technique for the simultaneous detection of structurally diverse B vitamins in complex natural samples. This enables highly sensitive measurements without loss of detailed mass spectrometric information, which is inevitable when using a triple quadrupole system in MS/MS mode.

The overlooked role of a biotin precursor for marine bacteria - desthiobiotin as an escape route for biotin auxotrophy.

Biotin (vitamin B7) is involved in a wide range of essential biochemical reactions and a crucial micronutrient that is vital for many pro- and eukaryotic organisms. The few biotin measurements in the world's oceans show that availability is subject to strong fluctuations. Numerous marine microorganisms exhibit biotin auxotrophy and therefore rely on supply by other organisms. Desthiobiotin is the primary precursor of biotin and has recently been detected at concentrations similar to biotin in seawater. The last enzymatic reaction in the biotin biosynthetic pathway converts desthiobiotin to biotin via the biotin synthase (BioB). The role of desthiobiotin as a precursor of biotin synthesis in microbial systems, however, is largely unknown. Here we demonstrate experimentally that bacteria can overcome biotin auxotrophy if they retain the bioB gene and desthiobiotin is available. A genomic search of 1068 bacteria predicts that the biotin biosynthetic potential varies greatly among different phylogenetic groups and that 20% encode solely bioB and thus can potentially overcome biotin auxotrophy. Many Actino- and Alphaproteobacteria cannot synthesize biotin de novo, but some possess solely bioB, whereas the vast majority of Gammaproteobacteria and Flavobacteriia exhibit the last four crucial biotin synthesis genes. We detected high intra- and extracellular concentrations of the precursor relative to biotin in the prototrophic bacterium, Vibrio campbellii, with extracellular desthiobiotin reaching up to 1.09 ± 0.15*106 molecules per cell during exponential growth. Our results provide evidence for the ecological role of desthiobiotin as an escape route to overcome biotin auxotrophy for bacteria in the ocean and presumably in other ecosystems.

Availability of vitamin B12 and its lower ligand intermediate α-ribazole impact prokaryotic and protist communities in oceanic systems.

Genome analyses predict that the cofactor cobalamin (vitamin B12, called B12 herein) is produced by only one-third of all prokaryotes but almost all encode at least one B12-dependent enzyme, in most cases methionine synthase. This implies that the majority of prokaryotes relies on exogenous B12 supply and interacts with producers. B12 consists of a corrin ring centred around a cobalt ion and the lower ligand 5'6-dimethylbenzimidazole (DMB). It has never been tested whether availability of this pivotal cofactor, DMB or its intermediate α-ribazole affect growth and composition of prokaryotic microbial communities. Here we show that in the subtropical, equatorial and polar frontal Pacific Ocean supply of B12 and α-ribazole enhances heterotrophic prokaryotic production and alters the composition of prokaryotic and heterotrophic protist communities. In the polar frontal Pacific, the SAR11 clade and Oceanospirillales increased their relative abundances upon B12 supply. In the subtropical Pacific, Oceanospirillales increased their relative abundance upon B12 supply as well but also downregulated the transcription of the btuB gene, encoding the outer membrane permease for B12. Surprisingly, Prochlorococcus, known to produce pseudo-B12 and not B12, exhibited significant upregulation of genes encoding key proteins of photosystem I + II, carbon fixation and nitrate reduction upon B12 supply in the subtropical Pacific. These findings show that availability of B12 and α-ribazole affect growth and composition of prokaryotic and protist communities in oceanic systems thus revealing far-reaching consequences of methionine biosynthesis and other B12-dependent enzymatic reactions on a community level.

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.

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.

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.

Vegetation state changes in the course of shrub encroachment in an African savanna since about 1850 CE and their potential drivers.

Shrub encroachment has far-reaching ecological and economic consequences in many ecosystems worldwide. Yet, compositional changes associated with shrub encroachment are often overlooked despite having important effects on ecosystem functioning.We document the compositional change and potential drivers for a northern Namibian Combretum woodland transitioning into a Terminalia shrubland. We use a multiproxy record (pollen, sedimentary ancient DNA, biomarkers, compound-specific carbon (δ13C) and deuterium (δD) isotopes, bulk carbon isotopes (δ13Corg), grain size, geochemical properties) from Lake Otjikoto at high taxonomical and temporal resolution.We provide evidence that state changes in semiarid environments may occur on a scale of one century and that transitions between stable states can span around 80 years and are characterized by a unique vegetation composition. We demonstrate that the current grass/woody ratio is exceptional for the last 170 years, as supported by n-alkane distributions and the δ13C and δ13Corg records. Comparing vegetation records to environmental proxy data and census data, we infer a complex network of global and local drivers of vegetation change. While our δD record suggests physiological adaptations of woody species to higher atmospheric pCO2 concentration and drought, our vegetation records reflect the impact of broad-scale logging for the mining industry, and the macrocharcoal record suggests a decrease in fire activity associated with the intensification of farming. Impact of selective grazing is reflected by changes in abundance and taxonomical composition of grasses and by an increase of nonpalatable and trampling-resistant taxa. In addition, grain-size and spore records suggest changes in the erodibility of soils because of reduced grass cover. Synthesis. We conclude that transitions to an encroached savanna state are supported by gradual environmental changes induced by management strategies, which affected the resilience of savanna ecosystems. In addition, feedback mechanisms that reflect the interplay between management legacies and climate change maintain the encroached state.

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.

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.

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.

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.

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. Sediment microbiomes at Keri Lake were studied, and their structure and functional potential were compared to other ecosystems with oil exposure histories of various time periods. RESULTS: Replicate sediment cores (up to 3-m depth) were retrieved from one site exposed to oil as well as a non-exposed control site. Samples from three different depths were subjected to chemical analysis and metagenomic shotgun sequencing. At the oil-exposed site, we observed high amounts of asphalt oil compounds and a depletion of sulfate compared to the non-exposed control site. The numbers of reads assigned to genes involved in the anaerobic degradation of hydrocarbons were similar between the two sites. The numbers of denitrifiers and sulfate reducers were clearly lower in the samples from the oil-exposed site, while a higher abundance of methanogens was detected compared to the non-exposed site. Higher abundances of the genes of methanogenesis were also observed in the metagenomes from other ecosystems with a long history of oil exposure, compared to short-term exposed environments. CONCLUSIONS: The analysis of Keri Lake metagenomes revealed that microbiomes in the oil-exposed sediment have a higher potential for methanogenesis over denitrification/sulfate reduction, compared to those in the non-exposed site. Comparison with metagenomes from various oil-impacted environments suggests that syntrophic interactions of hydrocarbon degraders with methanogens are favored in the ecosystems with a long-term presence of oil.

Membrane Lipids as Indicators for Viable Bacterial Communities Inhabiting Petroleum Systems.

Microbial activity in petroleum reservoirs has been implicated in a suite of detrimental effects including deterioration of petroleum quality, increases in oil sulfur content, biofouling of steel pipelines and other infrastructures, and well plugging. Here, we present a biogeochemical approach, using phospholipid fatty acids (PLFAs), for detecting viable bacteria in petroleum systems. Variations within the bacterial community along water flow paths (producing well, topside facilities, and injection well) can be elucidated in the field using the same technique, as shown here within oil production plants in the Molasse Basin of Upper Austria. The abundance of PLFAs is compared to total cellular numbers, as detected by qPCR of the 16S rDNA gene, to give an overall comparison between the resolutions of both methods in a true field setting. Additionally, the influence of biocide applications on lipid- and DNA-based quantification was investigated. The first oil field, Trattnach, showed significant PLFA abundances and cell numbers within the reservoir and topside facilities. In contrast, the second field (Engenfeld) showed very low PLFA levels overall, likely due to continuous treatment of the topside facilities with a glutaraldehyde-based antimicrobial. In comparison, Trattnach is dosed once per week in a batch fashion. Changes within PLFA compositions across the flow path, throughout the petroleum production plants, point to cellular adaptation within the system and may be linked to shifts in the dominance of certain bacterial types in oil reservoirs versus topside facilities. Overall, PLFA-based monitoring provides a useful tool to assess the abundance and high-level taxonomic diversity of viable microbial populations in oil production wells, topside infrastructure, pipelines, and other related facilities.

Metabolism of Hydrocarbons in n-Alkane-Utilizing Anaerobic Bacteria.

The glycyl radical enzyme-catalyzed addition of n-alkanes to fumarate creates a C-C-bond between two concomitantly formed stereogenic carbon centers. The configurations of the two diastereoisomers of the product resulting from n-hexane activation by the n-alkane-utilizing denitrifying bacterium strain HxN1, i.e. (1-methylpentyl)succinate, were assigned as (2S,1'R) and (2R,1'R). Experiments with stereospecifically deuterated n-(2,5-2H2)hexanes revealed that exclusively the pro-S hydrogen atom is abstracted from C2 of the n-alkane by the enzyme and later transferred back to C3 of the alkylsuccinate formed. These results indicate that the alkylsuccinate-forming reaction proceeds with an inversion of configuration at the carbon atom (C2) of the n-alkane forming the new C-C-bond, and thus stereochemically resembles a SN2-type reaction. Therefore, the reaction may occur in a concerted manner, which may avoid the highly energetic hex-2-yl radical as an intermediate. The reaction is associated with a significant primary kinetic isotope effect (kH/kD ≥3) for hydrogen, indicating that the homolytic C-H-bond cleavage is involved in the first irreversible step of the reaction mechanism. The (1-methylalkyl)succinate synthases of n-alkane-utilizing anaerobic bacteria apparently have very broad substrate ranges enabling them to activate not only aliphatic but also alkyl-aromatic hydrocarbons. Thus, two denitrifiers and one sulfate reducer were shown to convert the nongrowth substrate toluene to benzylsuccinate and further to the dead-end product benzoyl-CoA. For this purpose, however, the modified ß-oxidation pathway known from alkylbenzene-utilizing bacteria was not employed, but rather the pathway used for n-alkane degradation involving CoA ligation, carbon skeleton rearrangement and decarboxylation. Furthermore, various n-alkane- and alkylbenzene-utilizing denitrifiers and sulfate reducers were found to be capable of forming benzyl alcohols from diverse alkylbenzenes, putatively via dehydrogenases. The thermophilic sulfate reducer strain TD3 forms n-alkylsuccinates during growth with n-alkanes or crude oil, which, based on the observed patterns of homologs, do not derive from a terminal activation of n-alkanes.