Quaternary organization of the Staphylothermus marinus phosphoenolpyruvate synthase: angular reconstitution from cryoelectron micrographs with molecular modeling

Digital electron images of frozen-hydrated preparations of the 2.25-MDa Staphylothermus marinus phosphoenolpyruvate synthase (EC 2.7.9.2) have been analyzed by single-particle classification and averaging and iterative quaternion-based angular reconstitution. Contrast transfer function correction of micrographs obtained at different defocus values was used to improve the informational quality of the projection averages. Three-dimensional reconstructions were obtained to roughly 3-nm spatial resolution, in which the 24 identical subunits were arranged to form an octahedral complex, although the amino-terminal nucleotide-binding domain was not resolved. An atomic model of the subunit was generated by homology modeling using as the reference the known X-ray crystallographic structure of the related enzyme pyruvate orthophosphate dikinase (EC 2.7.9.1) from Clostridium symbiosum (Protein Data Bank entry 1DIK). The S. marinus protein could be arranged into an assembly of 12 homodimers to match the three-dimensional reconstruction in terms of shape and size of the homodimers, as well as overall shape and size of the complex. The quaternary model indicated that active sites of three monomers were localized around cavities (or putative channels) centered at the threefold axes of rotational symmetry and that carboxyl-terminal alpha-helical segments of four monomers were localized at the fourfold axes of rotational symmetry where they could facilitate interdimer interaction. The quaternary arrangement also indicated numerous potential hydrophobic and electrostatic interactions at the interdimer interfaces that could contribute further to structural stability.     

Evidence for genetic drift in the diversification of a geographically isolated population of the hyperthermophilic archaeon Pyrococcus

Genetic drift is a mechanism of population divergence that is important in the evolution of plants and animals but is thought to be rare in free-living microorganisms because of their typically large population sizes and unrestricted means of dispersal. We used both phylogenetic and insertion sequence (IS) element analyses in hyperthermophilic archaea of the genus Pyrococcus to test the hypothesis that genetic drift played an important role in the diversification of these microorganisms. Multilocus sequence typing of a collection of 36 isolates of Pyrococcus, from different hydrothermal systems in the Pacific Ocean and the Mediterranean Sea, revealed that Pyrococcus populations from different geographic locations are genetically differentiated. Analysis of IS elements in these isolates exposed their presence in all individuals of only one geographically isolated lineage, that of Vulcano Island in the Mediterranean Sea. Detailed sequence analysis of six selected IS elements in the Vulcano population showed that these elements cause deleterious genomic alterations, including inactivation of gene function. The high frequency of IS elements in the sampled population together with their observed harmful effects in the genome of Pyrococcus provide molecular evidence that the Vulcano Island population of Pyrococcus is geographically isolated and that those genetic mobile elements have been brought up to high frequency by genetic drift. Thus, genetic drift resulting from physical isolation should be considered as a factor influencing differentiation in prokaryotes.     

Recombination shapes the natural population structure of the hyperthermophilic archaeon Sulfolobus islandicus

Although microorganisms make up the preponderance of the biodiversity on Earth, the ecological and evolutionary factors that structure microbial populations are not well understood. We investigated the genetic structure of a thermoacidophilic crenarchaeal species, Sulfolobus islandicus, using multilocus sequence analysis of six variable protein-coding loci on a set of 60 isolates from the Mutnovsky region of Kamchatka, Russia. We demonstrate significant incongruence among gene genealogies and a lack of association between alleles consistent with recombination rates greater than the rate of mutation. The observation of high relative rates of recombination suggests that the structure of this natural population does not fit the periodic selection model often used to describe populations of asexual microorganisms. We propose instead that frequent recombination among closely related individuals prevents periodic selection from purging diversity and provides a fundamental cohesive mechanism within this and perhaps other archaeal species.     

Role of the precorrin 6-X reductase gene in cobamide biosynthesis in Methanococcus maripaludis

In Methanococcus maripaludis strain JJ, deletion of the homolog to cbiJ, which encodes the corrin biosynthetic enzyme precorrin 6-X reductase, yielded an auxotroph that required either cobamide or acetate for good growth. This phenotype closely resembled that of JJ117, a mutant in which tandem repeats were introduced into the region immediately downstream of the homolog of cbiJ. Mutant JJ117 also produced low quantities of cobamides, about 15 nmol g(-1) protein or 1-2% of the amount found in wild-type cells. These results confirm the role of the cbiJ homolog in cobamide biosynthesis in the Archaea and suggest the presence of low amounts of a bypass activity in these organisms.     

Crystal structure of archaeosine tRNA-guanine transglycosylase

Archaeosine tRNA-guanine transglycosylase (ArcTGT) catalyzes the exchange of guanine at position 15 in the D-loop of archaeal tRNAs with a free 7-cyano-7-deazaguanine (preQ(0)) base, as the first step in the biosynthesis of an archaea-specific modified base, archaeosine (7-formamidino-7-deazaguanosine). We determined the crystal structures of ArcTGT from Pyrococcus horikoshii at 2.2 A resolution and its complexes with guanine and preQ(0), at 2.3 and 2.5 A resolutions, respectively. The N-terminal catalytic domain folds into an (alpha/beta)(8) barrel with a characteristic zinc-binding site, showing structural similarity with that of the bacterial queuosine TGT (QueTGT), which is involved in queuosine (7-[[(4,5-cis-dihydroxy-2-cyclopenten-1-yl)-amino]methyl]-7-deazaguanosine) biosynthesis and targets the tRNA anticodon. ArcTGT forms a dimer, involving the zinc-binding site and the ArcTGT-specific C-terminal domain. The C-terminal domains have novel folds, including an OB fold-like "PUA domain", whose sequence is widely conserved in eukaryotic and archaeal RNA modification enzymes. Therefore, the C-terminal domains may be involved in tRNA recognition. In the free-form structure of ArcTGT, an alpha-helix located at the rim of the (alpha/beta)(8) barrel structure is completely disordered, while it is ordered in the guanine-bound and preQ(0)-bound forms. Structural comparison of the ArcTGT.preQ(0), ArcTGT.guanine, and QueTGT.preQ(1) complexes provides novel insights into the substrate recognition mechanisms of ArcTGT.     

A New Type-II NADH Dehydrogenase from the Archaeon Acidianus ambivalens: Characterization and in vitro Reconstitution of the Respiratory Chain

A new type-II NADH dehydrogenase (NDH-II) was isolated from the hyperthermoacidophilic archaeon Acidianus ambivalens. This enzyme is a monomer with an apparent molecular mass of 47 kDa, containing a covalently bound flavin, and no iron–sulfur clusters. Upon isolation, NDH-II loses activity, which can, nevertheless, be restored by incubation with phospholipids. Catalytically, it is a proficient NADH:caldariella quinone oxidoreductase (130 mmol NADH oxidized/mg protein^-1/min^-1) but it can also donate electrons to synthetic quinones, strongly suggesting its involvement in the respiratory chain. The apparent K_m for NADH was found to be 6 M, both for the purified and membrane-integrated enzyme, thus showing that detergent solubilization and purification did not affect the substrate binding site. Further, it is the first example of a type-II NADH dehydrogenase that contains the flavin covalently attached, which may be related to the need to stabilize the otherwise labile cofactor in a thermophilic environment. A fully operative minimal version of Acidianus ambivalens respiratory system was successfully reconstituted into artificial liposomes, using three basic components isolated from the organism: the type-II NADH dehydrogenase, caldariella quinone, the organism-specific quinone, and the aa₃ type quinol oxidase. This system, which mimics the in vivo chain, is efficiently energized by NADH, driving oxygen consumption by means of the terminal oxidase.     

Genomic perspective on the photobiology of Halobacterium species NRC-1, a phototrophic,phototactic, and UV-tolerant haloarchaeon

Halobacterium species display a variety of responses to light, including phototrophic growth, phototactic behavior, and photoprotective mechanisms. The complete genome sequence of Halobacterium species NRC-1 (Proc Natl Acad Sci USA 97: 12176–12181, 2000), coupled with the availability of a battery of methods for its analysis makes this an ideal model system for studying photobiology among the archaea. Here, we review: (1) the structure of the 2.57 Mbp Halobacterium NRC-1 genome, including a large chromosome, two minichromosomes, and 91 transposable IS elements; (2) the purple membrane regulon, which programs the accumulation of large quantities of the light-driven proton pump, bacteriorhodopsin, and allows for a period of phototrophic growth; (3) components of the sophisticated pathways for color-sensitive phototaxis; (4) the gas vesicle gene cluster, which codes for cell buoyancy organelles; (5) pathways for the production of carotenoid pigments and retinal, (6) processes for the repair of DNA damage; and (7) putative homologs of circadian rhythm regulators. We conclude with a discussion of the power of systems biology for comprehensive understanding of Halobacterium NRC-1 photobiology. This revised version was published online in June 2006 with corrections to the Cover Date.     

Agl22 and Agl23 are involved in the synthesis and utilization of the lipid-linked intermediates in the glycosylation pathways of the halophilic archaeaon Haloarcula hispanica

Like both eukaryotes and bacteria, archaea can decorate proteins with N- and O-linked glycans. Whereas pathways and roles of N-glycosylation have been studied in several model archaeal organisms, little is known of O-glycosylation. To explore commonalities and variations of these two versions of glycosylation, we used Haloarcula hispanica as a model. Our previous work showed that H. hispanica S-layer glycoproteins are modified by an N-linked glucose-α-(1, 2)-[sulfoquinovosamine-β-(1, 6)-]galactose trisaccharide and an O-linked glucose-α-(1, 4)-galactose disaccharide. Here, we found that H. hispanica membrane contains C60 dolichol phosphate (DolP) as a lipid carrier for glycosylation. As revealed by bioinformatics, gene deletion and phenotype analysis, gene HAH_1571, renamed agl22, encodes a predicted glucosyltransferase that transfers glucose from glucose-DolP onto galactose-DolP to form the glucose-α-(1, 4)-galactose-DolP precursor of the N-glycosylation. Gene HAH_2016, renamed agl23, encodes a putative flippase-associated protein responsible for flipping of hexose-DolPs across the membrane to face the exterior. Our results also suggested that the synthesis of the N- and O-linked glycans onto target protein occurs on the outer surface of the cell using hexose-DolPs as sugar donors. Deletion mutant showed that N- and O-glycosylation are required for growth in the defined medium mimicking the natural habitat of H. hispanica.     

Stoichiometric model and flux balance analysis for a mixed culture of Leptospirillum ferriphilum and Ferroplasma acidiphilum

The oxidation process of sulfide minerals in natural environments is achieved by microbial communities from the Archaea and Bacteria domains. A metabolic reconstruction of two dominant species, Leptospirillum ferriphilum and Ferroplasma acidiphilum, which are always found together as a mixed culture in this natural environments, was made. The metabolic model, composed of 152 internal reactions and 29 transport reactions, describes the main interactions between these species, assuming that both use ferrous iron as energy source, and F. acidiphilum takes advantage of the organic compounds secreted by L. ferriphilum for chemomixotrophic growth. A first metabolic model for a mixed culture used in bacterial leaching is proposed in this article, which pretends to represent the characteristics of the mixed culture in a simplified manner. It was evaluated with experimental data through flux balance analysis (FBA) using as objective function the maximization of biomass. The growth yields on ferrous iron obtained for each microorganism are consistent with experimental data, and the flux distribution obtained allows understanding of the metabolic capabilities of both microorganisms growing together in a bioleaching process. The model was used to simulate the growth of F. acidiphilum on different substrates, to determine in silico which compounds maximize cell growth, and which are essential. Knockout simulations were carried out for L. ferriphilum and F. acidiphilum metabolic models, predicting key enzymes of central metabolism. The results of this analysis are consistent with experimental data from literature, showing a robust behavior of the metabolic model. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:307–315, 2015