RESUMEN

Graphene has attracted much attention as an impermeable membrane and a protective coating against oxidation. While many theoretical studies have shown that defect-free graphene is impermeable, in reality graphene inevitably has defects in the form of grain boundaries and vacancies. Here, we study the effects of N-dopants on the impermeability of few-layered graphene (FLG) grown on copper using chemical vapor deposition. The grain boundaries in FLG have minimal impact on their permeability to oxygen as they do not provide a continuous channel for gas transport due to high tortuosity. However, we experimentally show that the N-dopants in FLG display multiple configurations that create structural imperfections to selectively allow gas molecules to permeate. We used a comprehensive array of tools including Raman spectroscopy, X-ray photoelectron spectroscopy, optically stimulated electron emission measurements, and density functional theory of N-doped graphene on copper to elucidate the effects of dopant configuration on the impermeability of graphene. Our results clearly show that oxygen can permeate through graphene with non-graphitic nitrogen dopants that create pores in graphene and oxidize the underlying Cu substrate while graphitic nitrogen dopants do not show any changes compared to the pristine form. Furthermore, we observed that the work function of graphene can be tuned effectively by changing the dopant configuration.

RESUMEN

BACKGROUND: Bifidobacteria are frequently proposed to be associated with good intestinal health primarily because of their overriding dominance in the feces of breast fed infants. However, clinical feeding studies with exogenous bifidobacteria show they don't remain in the intestine, suggesting they may lose competitive fitness when grown outside the gut. RESULTS: To further the understanding of genetic attenuation that may be occurring in bifidobacteria cultures, we obtained the complete genome sequence of an intestinal isolate, Bifidobacterium longum DJO10A that was minimally cultured in the laboratory, and compared it to that of a culture collection strain, B. longum NCC2705. This comparison revealed colinear genomes that exhibited high sequence identity, except for the presence of 17 unique DNA regions in strain DJO10A and six in strain NCC2705. While the majority of these unique regions encoded proteins of diverse function, eight from the DJO10A genome and one from NCC2705, encoded gene clusters predicted to be involved in diverse traits pertinent to the human intestinal environment, specifically oligosaccharide and polyol utilization, arsenic resistance and lantibiotic production. Seven of these unique regions were suggested by a base deviation index analysis to have been precisely deleted from strain NCC2705 and this is substantiated by a DNA remnant from within one of the regions still remaining in the genome of NCC2705 at the same locus. This targeted loss of genomic regions was experimentally validated when growth of the intestinal B. longum in the laboratory for 1,000 generations resulted in two large deletions, one in a lantibiotic encoding region, analogous to a predicted deletion event for NCC2705. A simulated fecal growth study showed a significant reduced competitive ability of this deletion strain against Clostridium difficile and E. coli. The deleted region was between two IS30 elements which were experimentally demonstrated to be hyperactive within the genome. The other deleted region bordered a novel class of mobile elements, termed mobile integrase cassettes (MIC) substantiating the likely role of these elements in genome deletion events. CONCLUSION: Deletion of genomic regions, often facilitated by mobile elements, allows bifidobacteria to adapt to fermentation environments in a very rapid manner (2 genome deletions per 1,000 generations) and the concomitant loss of possible competitive abilities in the gut.

Asunto(s)

RESUMEN

Lactic acid-producing bacteria are associated with various plant and animal niches and play a key role in the production of fermented foods and beverages. We report nine genome sequences representing the phylogenetic and functional diversity of these bacteria. The small genomes of lactic acid bacteria encode a broad repertoire of transporters for efficient carbon and nitrogen acquisition from the nutritionally rich environments they inhabit and reflect a limited range of biosynthetic capabilities that indicate both prototrophic and auxotrophic strains. Phylogenetic analyses, comparison of gene content across the group, and reconstruction of ancestral gene sets indicate a combination of extensive gene loss and key gene acquisitions via horizontal gene transfer during the coevolution of lactic acid bacteria with their habitats.

Asunto(s)

RESUMEN

Eukaryotic histone proteins condense DNA into compact structures called nucleosomes. Nucleosomes were viewed as a distinguishing feature of eukaryotes prior to identification of histone orthologs in methanogens. Although evolutionarily distinct from methanogens, the methane-producing hyperthermophile Methanopyrus kandleri produces a novel, 154-residue histone (HMk). Amino acid sequence comparisons show that HMk differs from both methanogenic and eukaryotic histones, in that it contains two histone-fold ms within a single chain. The two HMk histone-fold ms, N and C terminal, are 28% identical in amino acid sequence to each other and approximately 21% identical in amino acid sequence to other histone proteins. Here we present the 1.37-A-resolution crystal structure of HMk and report that the HMk monomer structure is homologous to the eukaryotic histone heterodimers. In the crystal, HMk forms a dimer homologous to [H3-H4](2) in the eukaryotic nucleosome. Based on the spatial similarities to structural ms found in the eukaryotic nucleosome that are important for DNA-binding, we infer that the Methanopyrus histone binds DNA in a manner similar to the eukaryotic histone tetramer [H3-H4](2).

Asunto(s)

Asunto(s)

RESUMEN

Previously we have characterized type IB DNA topoisomerase V (topo V) in the hyperthermophile Methanopyrus kandleri. The enzyme has a powerful topoisomerase activity and is abundant in M. kandleri. Here we report two characterizations of topo V. First, we found that its N-terminal domain has sequence homology with both eukaryotic type IB topoisomerases and the integrase family of tyrosine recombinases. The C-terminal part of the sequence includes 12 repeats, each repeat consisting of two similar but distinct helix-hairpin-helix motifs; the same arrangement is seen in recombination protein RuvA and mammalian DNA polymerase beta. Second, on the basis of sequence homology between topo V and polymerase beta, we predict and demonstrate that topo V possesses apurinic/apyrimidinic (AP) site-processing activities that are important in base excision DNA repair: (i) it incises the phosphodiester backbone at the AP site, and (ii) at the AP endonuclease cleaved AP site, it removes the 5' 2-deoxyribose 5-phosphate moiety so that a single-nucleotide gap with a 3'-hydroxyl and 5'-phosphate can be filled by a DNA polymerase. Topo V is thus the prototype for a new subfamily of type IB topoisomerases and is the first example of a topoisomerase with associated DNA repair activities.

Asunto(s)

RESUMEN

Archaeal histones have significant sequence and structural similarity to their eukaryal counterparts. However, whereas DNA is wrapped in negatively constrained supercoils in eukaryal nucleosomes, it has been reported that DNA is positively supercoiled by archaeal nucleosomes. This was inferred from experiments performed at low temperature and low salt concentrations, conditions markedly different from those expected for many archaea in vivo. Here, we report that the archaeal histones HMf and HTz wrap DNA in negatively constrained supercoils in buffers containing potassium glutamate (K-Glu) above 300 mM, either at 37 degrees C or at 70 degrees C. This suggests that high salt concentrations allow an alternate archaeal nucleosome topology: a left-handed tetramer rather than the right-handed tetramer seen in low salt conditions. In contrast, the archaeal histone MkaH produces DNA negative supercoiling at all salt concentrations, suggesting that this duality of structure is not possible for this atypical protein, which is formed by the association of two histone folds in a single polypeptide. These results extend the already remarkable similarity between archaeal and eukaryal nucleosomes, as it has been recently shown that DNA can be wrapped into either positive or negative supercoils around the H3/H4 tetramer. Negative supercoiling could correspond to the predominant physiological mode of DNA supercoiling in archaeal nucleosomes.

Asunto(s)

RESUMEN

Histones have been identified recently in many prokaryotes. These histones, unlike their eukaryotic homologs, are of a single uniform type that is thought to resemble the archetypal ancestor of the eukaryotic histone family. In this paper we report the finding, the cloning and the phylogenetic analysis of the sequence of a prokaryotic histone from the hyperthermophile Methanopyrus kandleri . Unlike previously described prokaryotic histones, the Methanopyrus sequence has a novel structure consisting of two tandemly repeated histone fold motifs in a single polypeptide. Sequence analyses indicate that the N-terminal repeat is most closely related to eukaryotic H2A and H4 histones, whereas the C-terminal repeat resembles that found in prokaryotic histones. These results imply an early divergence within the histone gene family prior to the emergence of eukaryotes and may represent an evolutionary step leading to eukaryotic histones.

Asunto(s)

RESUMEN

A recently described reverse gyrase from the hyperthermophilic methanogen Methanopyrus kandleri is the only known example of a heterodimeric type I topoisomerase. The enzyme is made up of a 42-kDa subunit which covalently interacts with DNA (RgyA) and a 138-kDa subunit which binds ATP (RgyB). We have now cloned and sequenced the genes for both subunits of this enzyme. Surprisingly, the universally conserved type I topoisomerase domain [Lima, C. D., Wang, J. C. & Mondragon, A. (1994) Nature (London) 367, 138-146] which has been found as a contiguous polypeptide in the prokaryotes and eukaryotes is shared between the protomers. The subdomain with the active-site tyrosine is entirely within RgyA, whereas the subdomain implicated in noncovalent binding of the cleaved DNA strand is contained entirely in RgyB. The appearance of this unique structure in a highly conserved enzyme family supports the hypothesis that the methanogens branched from other prokaryotes and eukaryotes very early in evolution.

Asunto(s)

RESUMEN

The widespread application of polymerase chain reaction and related techniques in biology and medicine has led to a heightened interest in thermophilic enzymes of DNA metabolism. Some of these enzymes are stable for hours at 100 degrees C, but no enzymatic activity on duplex DNA at temperatures above 100 degrees C has so far been demonstrated. Recently, we isolated topoisomerase V from the hyperthermophile Methanopyrus kandleri, which grows up to 110 degrees C. This novel enzyme is similar to eukaryotic topoisomerase I and acts on duplex DNA regions. We now show that topoisomerase V catalyzes the unlinking of double-stranded circular DNA at temperatures up to 122 degrees C. In this in vitro system, maximal DNA unlinking occurs at 108 degrees C and corresponds to complementary strands being linked at most once. These results further imply that in the presence of sufficient positive supercoiling DNA can exist as a double helix even at 122 degrees C.

Asunto(s)

RESUMEN

Reverse gyrase, an ATP-dependent topoisomerase that positively supercoils DNA, has been purified to near-homogeneity from the hyperthermophile Methanopyrus kandleri. It migrates on SDS-polyacrylamide gel electrophoresis as two principal bands with apparent molecular masses of 150 and 50 kDa. Both proteins remain associated throughout all chromatographic steps. Transfer of a radioactive phosphate from DNA to the 50-kDa protein and gel retardation experiments indicate that this protein forms the covalent complex with DNA. A blot overlay assay identifies the 150-kDa protein as the potential ATPase. This is the first evidence that a reverse gyrase can be a topoisomerase consisting of two protomers. In analogy with the DNA gyrase A subunit (DNA breakage and reunion activity) and the B subunit (ATPase), the 50- and 150-kDa components of Mka reverse gyrase have been designated the A and B subunits, respectively. Methanopyrus reverse gyrase changes DNA linking number in steps of one and its A subunit covalently binds to the 5'-DNA phosphoryl group. It nicks DNA at sites that predominantly have a cytosine at the -4-position. The same rule was derived previously for monomeric reverse gyrase from sulfur-metabolizing hyperthermophiles and for topoisomerase I from mesophilic bacteria. Based on these results, Mka reverse gyrase is classified as belonging to group A of type I topoisomerases. The structural diversity of type I group A topoisomerases parallels the diversity of type II enzymes and suggests the evolution of an essential function by gene fusion.

Asunto(s)

RESUMEN

DNA topoisomerase V is a novel prokaryotic enzyme related to eukaryotic topoisomerase I. The enzyme is a type I DNA topoisomerase and is recognized by polyclonal antibody against human topoisomerase I. We describe its purification from the hyperthermophilic methanogen Methanopyrus kandleri. The enzyme has high activity in crude extracts and is present in at least 1,500 copies/cell. Topoisomerase V migrates as a 110-kDa polypeptide in SDS-polyacrylamide gel electrophoresis and as a 142-kDa globular protein in gel filtration. It is active up to at least 100 degrees C on both positively and negatively supercoiled DNA and is not inhibited by single-stranded DNA. The enzyme works from 1 to 650 mM NaCl and up to 3.1 M potassium glutamate. It acts processively at low ionic strength and distributively at high NaCl or KCl concentration. Magnesium is not required and does not stimulate the enzymatic activity. Under DNA denaturing conditions, topoisomerase V catalyzes an unlinking reaction which results in substantial reduction in the linking number of closed circular DNA. The driving force for this process is DNA melting. Camptothecin is not nearly as good an inhibitor for topoisomerase V as it is for eukaryotic topoisomerase I. The unique occurrence of two major type I topoisomerases (reverse gyrase and topoisomerase V) in M. kandleri may shed new light on the evolution of this family of enzymes and supports the concept of a distant but significant relationship between some hyperthermophilic organisms and eukaryotes.

Asunto(s)

RESUMEN

The DNA topoisomerases are ubiquitous enzymes that fulfil vital roles in the replication, transcription and recombination of DNA by carrying out DNA-strand passage reactions. Here we characterize a prokaryotic counterpart to the eukaryotic topoisomerase I in the hyperthermophilic methanogen Methanopyrus kandleri. The new enzyme, called topoisomerase V, has the following properties in common with eukaryotic topoisomerase I, which distinguish it from all other known prokaryotic topoisomerases: (1) its activity is Mg(2+)-independent; (2) it relaxes both negatively and positively supercoiled DNA; (3) it makes a covalent complex with the 3' end of the broken DNA strand; and (4) it is recognized by antibody raised against human topoisomerase I. Eukaryotic-like enzymes have been discovered in some hyperthermophilic prokaryotes, namely the eocytes and the extremely thermophilic archaebacteria, and hyperthermophilic homologues of eukaryotic DNA polymerase-alpha, transcription factor IIB and DNA ligase have all been reported. Thus our findings support the idea that some essential parts of the eukaryotic transcription-translation and replication machineries were in place before the emergence of eukaryotes, and that the closest living relatives of eukaryotes may be hyperthermophiles.

Asunto(s)

RESUMEN

A second type I topoisomerase was purified from the extremely thermophilic archaebacterium Desulfurococcus amylolyticus. In contrast to the previously described reverse gyrase from this organism, the novel enzyme designated as Dam topoisomerase III is an ATP-independent relaxing topoisomerase. It is a monomer with Mr 108,000, as determined by electrophoresis under denaturing conditions and by size exclusion chromatography. Dam topoisomerase III, like other bacterial type I topoisomerases, absolutely requires Mg2+ for activity and is specific for single-stranded DNA. At 60-80 degrees C, it relaxes negatively but not positively supercoiled DNA and is inhibited by single-stranded M13 DNA. At 95 degrees C, the enzyme unwinds both positively and negatively supercoiled substrates and produces extensively unwound form I* and I** DNA. The peculiarities of DNA topoisomerization at high temperatures are discussed.

Asunto(s)

RESUMEN

Whether or not DNA gyrase can supercoil DNA so that alternative structures will arise in it is the major question of this work. We have shown gyrase to produce in pAO3 DNA a superhelix density sufficient for cruciform formation. However, the transition does not take place because of too slow kinetics. A change of ionic conditions in favour of more intense DNA supercoiling by gyrase shifts the midpoint of the equilibrium transition to the cruciform structure toward more supercoiled topoisomers. The width of the equilibrium transition to the cruciform as a function of linking number has been revealed to be an order of magnitude larger in buffers containing magnesium and spermidine than in buffers with monovalent cations only. We ascribe this effect to the influence that the counter ions surrounding the DNA molecule have on its elasticity, the coefficient of elasticity being dependent on superhelix density sigma. Thus, the free energy of supercoiling (a) depends on the ionic conditions and (b) is not a quadratic function of sigma in the physiological range of parameters. We propose a description of DNA as a system of links that can be either closed or open; we also introduce a new concept of the DNA linking potential akin to the chemical and electric potentials. The linking potential is a suitable parameter for describing the equilibrium distribution of links in heterogeneous DNA, the coexistence of various DNA structures, the equilibrium input and output of DNA links by enzymes, and the nonequilibrium movement of links along DNA chains. Within the framework of this approach DNA gyrase is considered as the source of the DNA linking potential.

Asunto(s)

RESUMEN

ATP-dependent type I topoisomerases from extremely thermophilic archaebacteria--reverse gyrases--drive positive supercoiling of DNA. We showed that reverse gyrase from Desulfurococcus amylolyticus breaks the DNA at specific sites and covalently binds to the 5' end. In 30 out of 31 sites located in pBR322 DNA fragments, cleavage occurs at the sequence 5'---CNNN/---(N is any base). The same rule was previously shown to hold for single-stranded DNA breakage by eubacterial topoisomerases I. The relative cleavage frequencies at different sites depend on Mg2+ and temperature. We discuss the possible physiological and mechanistic role of the above specificity of the bacterial topoisomerases I.

Asunto(s)

RESUMEN

It has been shown earlier that eukaryotic type I DNA topoisomerases act on duplex DNA regions, while eubacterial type I topoisomerases require single-stranded regions. The present paper demonstrates that the type I topoisomerase from extremely thermophilic archaebacteria, reverse gyrase, winds DNA by binding to single-stranded DNA regions. Thus, type I topoisomerases, both relaxing one in eubacteria and reverse gyrase in extremely thermophilic archaebacteria share a substrate specificity to melted DNA regions. The important consequence of this specificity is that the cellular DNA superhelical stress actively controlled by bacterial topoisomerases is confined to a narrow range characterized by a low stability of the double helix. Hence we suppose that bacterial topoisomerase systems control duplex stability near its minimum, for which purpose they create an appropriate negative superhelicity at moderate temperatures or a positive one at extremely high temperatures, the feedback being ensured by the aforesaid specificity of type I bacterial topoisomerases.

Asunto(s)

RESUMEN

A topoisomerase capable of introducing positive supercoils into closed-circular DNA has been isolated from the extremely thermophilic anaerobic archaebacterium Desulfurococcus amylolyticus. This polypeptide has an Mr of 135,000, as determined by electrophoresis under denaturing conditions. The enzyme is active in the temperature range from 65 degrees C to 100 degrees C and catalyzes positive supercoiling both in negatively supercoiled DNA and in relaxed DNA. These reactions require the presence of ATP. The enzyme's action on a single topoisomer has shown the linking number to increase by an integral number upon the relaxation of negative supercoils and the introduction of positive ones. This means that the reverse gyrase from D. amylolyticus is a type I topoisomerase. The presence of an extended AT sequence within the closed-circular DNA enhances the activity of the Desulfurococcus topoisomerase. Even though the enzyme is isolated from a strictly anaerobic bacterium, it is fully active in the presence of oxygen.

Asunto(s)

Asunto(s)