Metabolic capability and in situ activity of microorganisms in an oil reservoir.

BACKGROUND: Microorganisms have long been associated with oxic and anoxic degradation of hydrocarbons in oil reservoirs and oil production facilities. While we can readily determine the abundance of microorganisms in the reservoir and study their activity in the laboratory, it has been challenging to resolve what microbes are actively participating in crude oil degradation in situ and to gain insight into what metabolic pathways they deploy. RESULTS: Here, we describe the metabolic potential and in situ activity of microbial communities obtained from the Jiangsu Oil Reservoir (China) by an integrated metagenomics and metatranscriptomics approach. Almost complete genome sequences obtained by differential binning highlight the distinct capability of different community members to degrade hydrocarbons under oxic or anoxic condition. Transcriptomic data delineate active members of the community and give insights that Acinetobacter species completely oxidize alkanes into carbon dioxide with the involvement of oxygen, and Archaeoglobus species mainly ferment alkanes to generate acetate which could be consumed by Methanosaeta species. Furthermore, nutritional requirements based on amino acid and vitamin auxotrophies suggest a complex network of interactions and dependencies among active community members that go beyond classical syntrophic exchanges; this network defines community composition and microbial ecology in oil reservoirs undergoing secondary recovery. CONCLUSION: Our data expand current knowledge of the metabolic potential and role in hydrocarbon metabolism of individual members of thermophilic microbial communities from an oil reservoir. The study also reveals potential metabolic exchanges based on vitamin and amino acid auxotrophies indicating the presence of complex network of interactions between microbial taxa within the community.

Glycerol Phosphate Cytidylyltransferase Stereospecificity Is Key to Understanding the Distinct Stereochemical Compositions of Glycerophosphoinositol in Bacteria and Archaea.

Glycerophosphoinositol (GPI) is a compatible solute present in a few hyperthermophiles. Interestingly, different GPI stereoisomers accumulate in Bacteria and Archaea, and the basis for this domain-dependent specificity was investigated herein. The archaeon Archaeoglobus fulgidus and the bacterium Aquifex aeolicus were used as model organisms. The synthesis of GPI involves glycerol phosphate cytidylyltransferase (GCT), which catalyzes the production of CDP-glycerol from CTP and glycerol phosphate, and di-myo-inositol phosphate-phosphate synthase (DIPPS), catalyzing the formation of phosphorylated GPI from CDP-glycerol and l-myo-inositol 1-phosphate. DIPPS of A. fulgidus recognized the two CDP-glycerol stereoisomers similarly. This feature and the ability of 31P nuclear magnetic resonance (NMR) to distinguish the GPI diastereomers provided a means to study the stereospecificity of GCTs. The AF1418 gene and genes aq_185 and aq_1368 are annotated as putative GCT genes in the genomes of A. fulgidus and Aq. aeolicus, respectively. The functions of these genes were determined by assaying the activity of the respective recombinant proteins: AQ1368 and AQ185 are GCTs, while AF1418 has flavin adenine dinucleotide (FAD) synthetase activity. AQ185 is absolutely specific for sn-glycerol 3-phosphate, while AQ1368 recognizes the two enantiomers but has a 2:1 preference for sn-glycerol 3-phosphate. In contrast, the partially purified A. fulgidus GCT uses sn-glycerol 1-phosphate preferentially (4:1). Significantly, the predominant GPI stereoforms found in the bacterium and the archaeon reflect the distinct stereospecificities of the respective GCTs: i.e., A. fulgidus accumulates predominantly sn-glycero-1-phospho-3-l-myo-inositol, while Aq. aeolicus accumulates sn-glycero-3-phospho-3-l-myo-inositol. IMPORTANCE: Compatible solutes of hyperthermophiles show high efficacy in thermal protection of proteins in comparison with solutes typical of mesophiles; therefore, they are potentially useful in several biotechnological applications. Glycerophosphoinositol (GPI) is synthesized from CDP-glycerol and l-myo-inositol 1-phosphate in a few hyperthermophiles. In this study, the molecular configuration of the GPI stereoisomers accumulated by members of the Bacteria and Archaea was established. The stereospecificity of glycerol phosphate cytidylyltransferase (GCT), the enzyme catalyzing the synthesis of CDP-glycerol, is crucial to the stereochemistry of GPI. However, the stereospecific properties of GCTs have not been investigated thus far. We devised a method to characterize GCT stereospecificity which does not require sn-glycerol 1-phosphate, a commercially unavailable substrate. This led us to understand the biochemical basis for the distinct GPI stereoisomer composition observed in archaea and bacteria.

Optimization of Expression and Purification of Recombinant Archeoglobus fulgidus F420H2:NADP+ Oxidoreductase, an F420 Cofactor Dependent Enzyme.

Methanogens play a critical role in carbon cycling and contain a number of intriguing biosynthetic pathways. One unusual cofactor found in methanogenic and sulfate reducing archaea is Factor 420 (F420), which can be interconverted between its reduced and oxidized forms by the F420H2:NADP(+) oxidoreductase (Fno) through hydride transfer mechanisms. Here, we report an optimized expression and purification method for recombinant Fno derived from the extreme thermophile Archeoglobus fulgidus. An expression vector that is codon-optimized for heterologous expression in Escherichia coli, modified growth conditions, and a modified purification protocol involving a key polyethyleneimine precipitation step results in a highly purified, homogeneous preparation of Fno that displays high catalytic activity with a truncated F420 analog. This method should accelerate studies on how Fno uses the unusual F420 cofactor during catalysis.

Measurement of word frequencies in genomic DNA sequences based on partial alignment and fuzzy set.

Accompanied with the rapid increase of the amount of data registered in the databases of biological sequences, the need for a fast method of sequence comparison applicable to sequences of large size is also increasing. In general, alignment is used for sequence comparison. However, the alignment may not be appropriate for comparison of sequences of large size such as whole genome sequences due to its large time complexity. In this article, we propose a semi alignment-free method of sequence comparison based on word frequency distributions, in which we partially use the alignment to measure word frequencies along with the idea of fuzzy set theory. Experiments with ten bacterial genome sequences demonstrated that the fuzzy measurements has the effect that facilitates discrimination between close relatives and distant relatives.

Carbon dioxide fixation in 'Archaeoglobus lithotrophicus': are there multiple autotrophic pathways?

Several representatives of the euryarchaeal class Archaeoglobi are able to grow facultative autotrophically using the reductive acetyl-CoA pathway, with 'Archaeoglobus lithotrophicus' being an obligate autotroph. However, genome sequencing revealed that some species harbor genes for key enzymes of other autotrophic pathways, i.e. 4-hydroxybutyryl-CoA dehydratase of the dicarboxylate/hydroxybutyrate cycle and the hydroxypropionate/hydroxybutyrate cycle and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the Calvin-Benson cycle. This raised the question of whether only one or multiple autotrophic pathways are operating in these species. We searched for the presence of enzyme activities specific for the dicarboxylate/hydroxybutyrate or the hydroxypropionate/hydroxybutyrate cycles in 'A. lithotrophicus', but such enzymes could not be detected. Low Rubisco activity was detected that could not account for the carbon dioxide (CO(2)) fixation rate; in addition, phosphoribulokinase activity was not found. The generation of ribulose 1,5-bisphosphate from 5-phospho-D-ribose 1-pyrophosphate was observed, but not from AMP; these sources for ribulose 1,5-bisphosphate have been proposed before. Our data indicate that the reductive acetyl-CoA pathway is the only functioning CO(2) fixation pathway in 'A. lithotrophicus'.

Archaeoglobus sulfaticallidus sp. nov., a thermophilic and facultatively lithoautotrophic sulfate-reducer isolated from black rust exposed to hot ridge flank crustal fluids.

A novel thermophilic and lithoautotrophic sulfate-reducing archaeon was isolated from black rust formed on the steel surface of a borehole observatory (CORK 1026B) retrieved during IODP Expedition 301 on the eastern flank of Juan de Fuca Ridge, eastern Pacific Ocean. Cells of the strain were lobe-shaped or triangular. The optimum temperature, pH and NaCl concentration for growth were 75°C, pH 7 and 2 % (w/v), respectively. The isolate was strictly anaerobic, growing lithoautotrophically on H(2) and CO(2) using sulfate, sulfite or thiosulfate as electron acceptors. Lactate and pyruvate could serve as alternative energy and carbon sources. The G+C content of the genomic DNA was 42 mol%. Phylogenetic analyses of the 16S rRNA gene indicated that the isolate was closely related to members of the family Archaeoglobaceae, with sequence similarities of 90.3-94.4 %. Physiological and molecular properties showed that the isolate represents a novel species of the genus Archaeoglobus. The name Archaeoglobus sulfaticallidus sp. nov. is proposed; the type strain is PM70-1(T) (=DSM 19444(T)=JCM 14716(T)).

Archaeoglobus infectus sp. nov., a novel thermophilic, chemolithoheterotrophic archaeon isolated from a deep-sea rock collected at Suiyo Seamount, Izu-Bonin Arc, western Pacific Ocean.

A novel thermophilic, strictly anaerobic archaeon, designated strain Arc51T, was isolated from a rock sample collected from a deep-sea hydrothermal field in Suiyo Seamount, Izu-Bonin Arc, western Pacific Ocean. Cells of the isolate were irregular cocci with single flagella and exhibited blue-green fluorescence at 436 nm. The optimum temperature, pH and NaCl concentration for growth were 70 degrees C, pH 6.5 and 3 % (w/v), respectively. Strain Arc51T could grow on thiosulfate or sulfite as an electron acceptor in the presence of hydrogen. This strain required acetate as a carbon source for its growth, suggesting that the reductive acetyl CoA pathway for CO2 fixation was incomplete. In addition, coenzyme M (2-mercaptoethanesulfonic acid), which is a known methyl carrier in methanogenesis, was also a requirement for growth of the strain. Analysis of the 16S rRNA gene sequence revealed that the isolate was similar to members of the genus Archaeoglobus, with sequence similarities of 93.6-97.2 %; the closest relative was Archaeoglobus veneficus. Phylogenetic analyses of the dsrAB and apsA genes, encoding the alpha and beta subunits of dissimilatory sulfite reductase and the alpha subunit of adenosine-5'-phosphosulfate reductase, respectively, produced results similar to those inferred from comparisons based on the 16S rRNA gene sequence. On the basis of phenotypic and phylogenetic data, strain Arc51T represents a novel species of the genus Archaeoglobus, for which the name Archaeoglobus infectus sp. nov. is proposed. The type strain is Arc51T (=NBRC 100649T=DSM 18877T).

Effect of nitrate and nitrite on sulfide production by two thermophilic, sulfate-reducing enrichments from an oil field in the North Sea.

Thermophilic sulfate-reducing bacteria (tSRB) can be major contributors to the production of H(2)S (souring) in oil reservoirs. Two tSRB enrichments from a North Sea oil field, NS-tSRB1 and NS-tSRB2, were obtained at 58 degrees C with acetate-propionate-butyrate and with lactate as the electron donor, respectively. Analysis by rDNA sequencing indicated the presence of Thermodesulforhabdus norvegicus in NS-tSRB1 and of Archaeoglobus fulgidus in NS-tSRB2. Nitrate (10 mM) had no effect on H(2)S production by mid-log phase cultures of NS-tSRB1 and NS-tSRB2, whereas nitrite (0.25 mM or higher) inhibited sulfate reduction. NS-tSRB1 did not recover from inhibition, whereas sulfate reduction activity of NS-tSRB2 recovered after 500 h. Nitrite was also effective in souring inhibition and H(2)S removal in upflow bioreactors, whereas nitrate was similarly ineffective. Hence, nitrite may be preferable for souring prevention in some high-temperature oil fields because it reacts directly with sulfide and provides long-lasting inhibition of sulfate reduction.

Enzyme-driven speciation: crystallizing Archaea via lipid capture.

As the origin(s) of life on Earth remains an open question, detailed characteristics about the "last universal ancestor" (LUA) continue to be obscured. Here we provide arguments that strengthen the bacterial-like nature of the LUA. Our view attempts to recreate the evolution of archaeal lipids, the major components of the distinctive membrane that encapsulates these ancient prokaryotes. We show that (S)- 3-O-geranylgeranylglyceryl phosphate synthase (GGGPS), a TIM-barrel protein that performs the committed step in archaeal lipid synthesis, likely evolved from the duplication and fusion of a (betaalpha)4 half-barrel ancestor. By comparison to the well-characterized HisA and HisF TIM-barrel proteins, we propose a time line for the invention of this diagnostic archaeal biosynthetic pathway. After excluding the possibility of horizontal gene transfer, we conclude that the evolutionary history of GGGPS mirrors the emergence of Archaea from the LUA. We illustrate aspects of this "lipid capture" model that support its likelihood in recreating key evolutionary events and, as our hypothesis is built on a single initiating event, we suggest that the appearance of GGGPS represents an example of enzyme-driven speciation.

Elucidation of an archaeal replication protein network to generate enhanced PCR enzymes.

Thermostable DNA polymerases are an important tool in molecular biology. To exploit the archaeal repertoire of proteins involved in DNA replication for use in PCR, we elucidated the network of proteins implicated in this process in Archaeoglobus fulgidus. To this end, we performed extensive yeast two-hybrid screens using putative archaeal replication factors as starting points. This approach yielded a protein network involving 30 proteins potentially implicated in archaeal DNA replication including several novel factors. Based on these results, we were able to improve PCR reactions catalyzed by archaeal DNA polymerases by supplementing the reaction with predicted polymerase co-factors. In this approach we concentrated on the archaeal proliferating cell nuclear antigen (PCNA) homologue. This protein is known to encircle DNA as a ring in eukaryotes, tethering other proteins to DNA. Indeed, addition of A. fulgidus PCNA resulted in marked stimulation of PCR product generation. The PCNA-binding domain was determined, and a hybrid DNA polymerase was constructed by grafting this domain onto the classical PCR enzyme from Thermus aquaticus, Taq DNA polymerase. Addition of PCNA to PCR reactions catalyzed by the fusion protein greatly stimulated product generation, most likely by tethering the enzyme to DNA. This sliding clamp-induced increase of PCR performance implies a promising novel micromechanical principle for the development of PCR enzymes with enhanced processivity.

Plasmid pGS5 from the hyperthermophilic archaeon Archaeoglobus profundus is negatively supercoiled.

We present evidence that, in contrast to plasmids from other hyperthermophilic archaea, which are in the relaxed to positively supercoiled state, plasmid pGS5 (2.8 kb) from Archaeoglobus profundus is negatively supercoiled. This might be due to the presence of a gyrase introducing negative supercoils, since gyrase genes are present in the genome of its close relative A. fulgidus, and suggests that gyrase activity predominates over reverse gyrase whenever the two topoisomerases coexist in cells.

Transcription in archaea.

Using the sequences of all the known transcription-associated proteins from Bacteria and Eucarya (a total of 4,147), we have identified their homologous counterparts in the four complete archaeal genomes. Through extensive sequence comparisons, we establish the presence of 280 predicted transcription factors or transcription-associated proteins in the four archaeal genomes, of which 168 have homologs only in Bacteria, 51 have homologs only in Eucarya, and the remaining 61 have homologs in both phylogenetic domains. Although bacterial and eukaryotic transcription have very few factors in common, each exclusively shares a significantly greater number with the Archaea, especially the Bacteria. This last fact contrasts with the obvious close relationship between the archaeal and eukaryotic transcription mechanisms per se, and in particular, basic transcription initiation. We interpret these results to mean that the archaeal transcription system has retained more ancestral characteristics than have the transcription mechanisms in either of the other two domains.

Newly discovered archaebacterial flap endonucleases show a structure-specific mechanism for DNA substrate binding and catalysis resembling human flap endonuclease-1.

Mammalian flap endonuclease-1 (FEN-1) is a structure-specific metalloenzyme that acts in processing of both the Okazaki fragments during lagging strand DNA synthesis and flap intermediates during DNA damage repair. We identified and cloned three open reading frames encoding a flap endonuclease from Archaeglobus fulgidus, Methanococcus jannaschii, and Pyrococcus furiosus, respectively. The deduced FEN-1 protein sequences share approximately 75% similarity with the human FEN-1 nuclease in the conserved nuclease domains, and extensive biochemical experiments indicate that the substrate specificities and catalytic activities of these enzymes have overall similarities with those of the human enzyme. Thus, FEN-1 enzymes and likely reaction mechanisms are conserved across the eukaryotic and archaeal kingdoms. Detailed comparative analysis, however, reveals subtle differences among these four enzymes including distinctive substrate specificity, tolerance of the archaebacterial enzymes for acidic pHs and elevated temperatures, and variations in the metal-ion dependence of substrate cleavage. Although the archaebacterial enzymes were inactive at temperatures below 30 degreesC, DNA binding occurred at temperatures as low as 4 degreesC and with or without metal ions. Thus, these archaeal enzymes may provide a means to dissect the specific binding and catalytic mechanisms of the entire FEN-1 family of structure-specific nucleases.

The ribosomal gene spacer region in archaebacteria.

Sequences for the spacer regions that separate the 16S and 23S ribosomal RNA genes have been determined for four more (strategically placed) archaebacteria. These confirm the general rule that methanogens and extreme halophiles have spacers that contain a single tRNAala gene, while tRNA genes are not found in the spacer region of the true extreme thermophiles. The present study also shows that the spacer regions from the sulfate reducing Archaeglobus and the extreme thermophile Thermococcus (both of which cluster phylogenetically with the methanogens and extreme halophiles) contain each a tRNAala gene. Thus, not only all methanogens and extreme halophiles show this characteristic, but all organisms on the "methanogen branch" of the archaebacterial tree appear to do so. The finding of a tRNA gene in the spacer region of the extreme thermophile Thermococcus celer is the first known phenotypic property that links this organism with its phylogenetic counterparts, the methanogens, rather than with its phenotypic counterparts, the sulfur-dependent extreme thermophiles.

A possible biochemical missing link among archaebacteria.

Until recently all archaebacteria isolated conformed to one of three basic phenotypes: they were either methanogens, extreme halophiles, or ('sulphur-dependent') extreme thermophiles. However, a novel phenotype, that fits none of these categories, has recently been described. The organism, strain VC-16 (tentatively called "Archaeoglobus fulgidus") reduces sulphate--the only archaebacterium so far known to do so--and makes very small quantities of methane, although it lacks some of the cofactors normally associated with methanogenesis. These characteristics suggest that strain VC-16 might represent a transition form between an anaerobic thermophilic sulfur-based type of metabolism (which seems to be the ancestral metabolism for archaebacteria and methanogenesis (which somehow then derives from it). We here show that the lineage represented by strain VC-16 arises from the archaebacterial tree precisely where such an interpretation would predict that it would, between the Methanococcus lineage (which is the deepest of the methanogen branchings) and that of Thermococcus (the deepest of all branchings on the methanogen side of the tree).