Crystal structure of the arginine repressor protein in complex with the DNA operator from Mycobacterium tuberculosis

The arginine repressor (ArgR) from Mycobacterium tuberculosis (Mtb) is a gene product encoded by the open reading frame Rv1657. It regulates the l-arginine concentration in cells by interacting with ARG boxes in the promoter regions of the arginine biosynthesis and catabolism operons. Here we present a 2.5-Å structure of MtbArgR in complex with a 16-bp DNA operator in the absence of arginine. A biological trimer of the protein-DNA complex is formed via the crystallographic 3-fold symmetry axis. The N-terminal domain of MtbArgR has a winged helix-turn-helix motif that binds to the major groove of the DNA. This structure shows that, in the absence of arginine, the ArgR trimer can bind three ARG box half-sites. It also reveals the structure of the whole MtbArgR molecule itself containing both N-terminal and C-terminal domains.     

Fine Structure of Body Wall Cuticle of Females of Eight Genera of Heteroderidae

Body wall cuticle of adult females of eight genera within the Heteroderidae was examined by transmission electron microscopy for comparison with previously studied species within the family. Cuticle structure was used to test some current hypotheses of phylogeny of Heteroderidae and to evaluate intrageneric variability in cuticle layering. Verutus, Rhizonema, and Meloidodera possess striated cuticle surfaces and have the simplest layering, suggesting that striations have not necessarily arisen repeatedly in Heteroderidae through convergent or parallel evolution. Atalodera and Thecavermiculatus possess similar cuticles with derived characteristics, strengthening the hypothesis that the two genera are sister groups. Similarly, the cuticle of Cactodera resembles the specialized cuticle of Globodera and Punctodera in having a basal layer (D) and a surface layer infused with electron-dense substance. Heterodera betulae has a unique cuticle in which the thickest layer (C) is infiltrated with an electron-dense matrix. Little intrageneric difference was found between cuticles of two species of Meloidodera or between two species of Atalodera. However, Atalodera ucri has a basal layer (E) not found in other Heteroderidae. The most striking intrageneric variation in cuticle structure was observed between the thin three-layered cuticle of Sarisodera africana and the much thicker four-layered cuticle of Sarisodera hydrophila; results do not support monophyly of Sarisodera.     

Molecular characterization and expression analysis of Acmago and AcY14 in Antrodia cinnamomea

Mago nashi (Mago) and Y14 proteins, highly conserved among eukaryotes, participate in mRNA localization and splicing, and as such play important roles in oogenesis, embryogenesis and germ-line sex determination during animal development. Here we identified mago (Acmago) and Y14 (AcY14) homologues derived from Antrodia cinnamomea. Acmago encodes 149 amino acids and AcY14 encodes 168 amino acids. Multiple amino acid sequence alignment as well as secondary and tertiary structure prediction showed that AcMago and AcY14 have similar protein structure to the reported crystal structures of other Mago and Y14 proteins. During fungal development both Acmago and AcY14 genes were abundantly expressed in natural basidiomes. This is the first report of the molecular characterization and expression analysis of the mago and Y14 genes from fungi.     

思茅松天然林树冠结构模型

以云南省普洱市思茅区思茅松天然林为研究对象,采用枝解析调查了34株思茅松样木的树冠数据,分析了一级枝枝长、枝径、着枝角度、弦长和树冠半径5个树冠形状变量的变化规律,分别构建其预估模型;分析了树冠结构变化,分别构建了一级枝轮枝高度预估模型、一级枝枝条数量预估模型和一级枝枝条数量累积预估模型,并采用独立样本进行模型统计精度检验。结果表明:8个预估模型的预测效果良好,精度达到91%以上,尤其是一级枝着枝角度模型和一级枝轮枝高度模型预测精度达到97%以上。研究结果合理准确描述思茅松树冠结构的变化,为思茅松天然林的经营管理提供科学依据。     

The effect of a tube-building phoronid on associated infaunal species diversity, composition and community structure

Habitat modifying organisms can alter the distribution of associated species. We surveyed soft-sediment patches in Bodega Harbor, California and found that patches with high densities of the phoronid Phoronopsis harmeri (Pixell, 1912), a chemically-defended tube-building lophophorate, have higher infaunal abundance and richness than similar patches with low densities of P. harmeri. To determine whether this difference was driven by P. harmeri and whether this difference is attributable to the activities of the organism, or simply its physical structure, we conducted a field experiment with four treatments: live phoronids, mimics of phoronid structure, phoronid-free sediments (bare) and unmanipulated sediments. Although the field experiment did not detect differences in the overall abundance or richness of infauna among the manipulated treatments, some of the individual species did show a positive response to the presence of phoronids and phoronid structure (i.e., mimics). In particular, the polychaete Boccardia proboscidea, the amphipod Monocorophium uenoi, and harpacticoid copepods were facilitated by the presence of phoronids and phoronid structure when there was sediment disturbance. The inconsistency between the results of the survey and of the manipulative experiment may be largely driven by the disturbance caused by the manipulation. However, where P. harmeri has an effect, it is generally positively associated with infaunal abundance that may be attributable to the stabilization of sediments.     

A male cone of Pseudofrenelopsis dalatzensis with in situ pollen grains from the Lower Cretaceous of Northeast China

A Classopollis-containing male cone found in close association with Pseudofrenelopsis dalatzensis from the Lower Cretaceous Dalazi Formation of Yanji Basin, eastern Jilin, China was studied using scanning electron microscopy and described as Classostrobus dalatzensis sp. nov. The small oval cone, borne on the top of a stalk (fertile short shoot), consists of helically arranged microsporophylls, each with a rhomboid and expanded distal head. The short shoot is divided into indistinct nodes and internodes with fine longitudinal striations. Cuticles of the microsporophyll and the internode of the short shoot are generally similar to Pseudofrenelopsis dalatzensis internode cuticles, all of them having papillate outer surfaces. Stomata are similar in structure and arrangement, with papillae of subsidiary cells overhanging the stomatal pit. Epidermal cells are rectangular or isodiametrical, each with a robust papilla on the periclinal wall. Hypodermal cells are present between the stomatal files. The internal structure of the in situ Classopollis pollen grains was studied in detail. Pollen grain is small, with thin sexine, poorly developed nexine and may be immature. This is the first record of in situ pollen grains Classopollis in China of which the internal structure is known in detail.     

Structure and development of the style base in Abildgaardia, Bulbostylis, and Fimbristylis (Cyperaceae,Cyperoideae, Abildgaardieae)

The Abildgaardieae tribe within the family Cyperaceae comprises six or seven genera, among which Abildgaardia, Bulbostylis and Fimbristylis pose a challenge regarding their morphological delimitation. Molecular phylogenetic analyses including species of Abildgaardieae are rare, but in most of those studies, Abildgaardia and Fimbristylis appear as more closely related to each other than to the Bulbostylis genus. Duration of the style base has been one of the most widely used characters for delimiting these three genera. The style base is a persistent structure in most species of Bulbostylis and deciduous in Abildgaardia and Fimbristylis. The reasons why the style base may persist or fall off have been scarcely discussed. The assumption that abscission layers are present in the style base of all three genera and the fact that tracheids have been observed in the style base of Bulbostylis suggest that this structure might have histological complexity. In view of this, a complete ontogenetic and anatomical study of the gynoecium has been carried out for all these three genera. It turned out that the style base is histologically simple in Abildgaardia, Bulbostylis and Fimbristylis and shows similar structure and development in all three genera. The fact that the style base has a shorter duration in Abildgaardia and Fimbristylis than in Bulbostylis might be related to the lower number of sclerotised cells that make up such structures in the mature fruit of the former two genera. Abscission of the style and style base may be the result of much simpler reasons than the differentiation of an abscission layer, resulting merely from mechanical shear force effects. Differences among genera have been observed in the shape of the style base and the development of the style. The histological simplicity of the style base is consistent with the homoplastic appearance of this structure in genera that are not closely related (e.g. Rhynchospora). Because of this, while the presence of the thickened style base seems to be a synapomorphy in species of Abildgaardieae, its persistence on or detachment from the fruit might have emerged repeatedly during this clade evolution and might not be a suitable character for genera delimitation.     

Structures of the O-antigens of Escherichia coli O13, O129, and O135 related to the O-antigens of Shigella flexneri

O-Polysaccharides (O-antigens) were isolated from Escherichia coli O13, O129, and O135 and studied by chemical analyses along with 2D ¹H and ¹³C NMR spectroscopy. They were found to possess a common →2)-l-Rha-(α1→2)-l-Rha-(α1→3)-l-Rha-(α1→3)-d-GlcNAc-(β1→ backbone, which is a characteristic structural motif of the O-polysaccharides of Shigella flexneri types 1-5. In both the bacterial species, the backbone is decorated with lateral glucose residues or/and O-acetyl groups. In E. coli O13, a new site of glycosylation on 3-substituted Rha was revealed and the following O-polysaccharide structure was established:The structure of the E. coli O129 antigen was found to be identical to the O-antigen structure of S. flexneri type 5a specified in this work and that of E. coli O135 to S. flexneri type 4b reported earlier.     

Divergence of cofactor recognition across evolution: coenzyme A binding in a prokaryotic arylamine N-acetyltransferase

Arylamine N-acetyltransferase (NAT) enzymes are widespread in nature. They serve to acetylate xenobiotics and/or endogenous substrates using acetyl coenzyme A (CoA) as a cofactor. Conservation of the architecture of the NAT enzyme family from mammals to bacteria has been demonstrated by a series of prokaryotic NAT structures, together with the recently reported structure of human NAT1. We report here the cloning, purification, kinetic characterisation and crystallographic structure determination of NAT from Mycobacterium marinum, a close relative of the pathogenic Mycobacterium tuberculosis. We have also determined the structure of M. marinum NAT in complex with CoA, shedding the first light on cofactor recognition in prokaryotic NATs. Surprisingly, the principal CoA recognition site in M. marinum NAT is located some 30 Å from the site of CoA recognition in the recently deposited structure of human NAT2 bound to CoA. The structure explains the Ping-Pong Bi-Bi reaction mechanism of NAT enzymes and suggests mechanisms by which the acetylated enzyme intermediate may be protected. Recognition of CoA in a much wider groove in prokaryotic NATs suggests that this subfamily may accommodate larger substrates than is the case for human NATs and may assist in the identification of potential endogenous substrates. It also suggests the cofactor-binding site as a unique subsite to target in drug design directed against NAT in mycobacteria.     

Phylogenetic utility of the nuclear genes AGAMOUS 1 and PHYTOCHROME B in palms (Arecaceae): an example within Bactridinae

Background and Aims

Molecular phylogenetic studies of palms (Arecaceae) have not yet provided a fully resolved phylogeny of the family. There is a need to increase the current set of markers to resolve difficult groups such as the Neotropical subtribe Bactridinae (Arecoideae: Cocoseae). We propose the use of two single-copy nuclear genes as valuable tools for palm phylogenetics.

Methods

New primers were developed for the amplification of the AGAMOUS 1 (AG1) and PHYTOCHROME B (PHYB) genes. For the AGAMOUS gene, the paralogue 1 of Elaeis guineensis (EgAG1) was targeted. The region amplified contained coding sequences between the MIKC K and C MADS-box domains. For the PHYB gene, exon 1 (partial sequence) was first amplified in palm species using published degenerate primers for Poaceae, and then specific palm primers were designed. The two gene portions were sequenced in 22 species of palms representing all genera of Bactridinae, with emphasis on Astrocaryum and Hexopetion, the status of the latter genus still being debated.

Key Results

The new primers designed allow consistent amplification and high-quality sequencing within the palm family. The two loci studied produced more variability than chloroplast loci and equally or less variability than PRK, RPBII and ITS nuclear markers. The phylogenetic structure obtained with AG1 and PHYB genes provides new insights into intergeneric relationships within the Bactridinae and the intrageneric structure of Astrocaryum. The Hexopetion clade was recovered as monophyletic with both markers and was weakly supported as sister to Astrocaryum sensu stricto in the combined analysis. The rare Astrocaryum minus formed a species complex with Astrocaryum gynacanthum. Moreover, both AG1 and PHYB contain a microsatellite that could have further uses in species delimitation and population genetics.

Conclusions

AG1 and PHYB provide additional phylogenetic information within the palm family, and should prove useful in combination with other genes to improve the resolution of palm phylogenies.     

Effects of native predators and eelgrass habitat structure on the introduced Asian mussel Musculista senhousia (Benson in Cantor) in southern California

The ability of predators to control the abundance of non-native species has been little explored in marine systems. Native predators may be used to control non-native species or may confer invasion resistance to communities if predation rates on invaders are density-dependent. We studied the response of southern California native predators to the density of Musculista senhousia (Benson in Cantor, 1842), a small, fast growing mussel that has been introduced from Japan to several coastlines worldwide. We performed field experiments to determine if M. senhousia proportional mortality is density-dependent and if eelgrass Zostera marina L. habitat structure influenced mussel density-dependent mortality. We also evaluated the effect of seagrass habitat structure on the aggregative and functional responses of the predatory gastropod Pteropurpura festiva (Hinds, 1844) to Asian mussel density. In the summer of 2002, P. festiva aggregated in plots with high mussel density and was responsible for nearly all predation on M. senhousia. However, M. senhousia proportional mortality was inversely density-dependent at all levels of eelgrass above-ground and below-ground habitat structure. Asian mussel proportional mortality also was inversely density-dependent and was not influenced by eelgrass habitat structure in the spring of 2004 when wading birds were the chief predator of mussels. In contrast to results for mussel proportional mortality, the aggregative and functional responses of P. festiva varied with seagrass habitat structure. P. festiva density increased with Asian mussel density in plots with low simulated habitat structure, but the relationship between P. festiva density and Asian mussel density was parabolic at zero, intermediate and high levels of habitat structure. In field enclosures, P. festiva exhibited a Type I (linear) functional response to Asian mussel density at low levels of eelgrass structure, and a Type II (hyperbolic) functional response to mussel density at high levels of eelgrass structure. Our results and those of others suggest that the degree to which local benthic communities in southern California are resistant to Asian mussel invasion depends on habitat structure, mussel settlement rates, and the density and diversity of predators.     

Contrasting population makeup of two intertidal gastropod species that differ in dispersal opportunities

This study of population structure of two intertidal monodontine species: Diloma nigerrima and Diloma subrostrata, revealed the level of genetic connectedness among populations. Despite their markedly different geographic ranges (D. nigerrima is a geographically widespread species, inhabiting both Chile and New Zealand, including its subantarctic islands, whereas D. subrostrata is endemic to New Zealand), both species are believed to possess short-lived lecithotrophic larval stages. Polymorphic DNA microsatellite sequences were used to reveal the level of genetic connectedness among populations, thus inferring the two species' relative effective dispersal abilities. For each species F statistics, AMOVA values and the strength of the relationship between geographic and genetic distance were calculated. We observed a higher within-species level of genetic variation (Φ_ST = 0.099 vs. Φ_ST = 0.016) and a higher proportion of variance (11.15% vs. 0%) among populations of D. nigerrima than of D. subrostrata. A larger fraction of significant F_ST values was observed among D. nigerrima population pairs (65%) than among D. subrostrata population pairs (33%). Significant correlation between genetic and geographic distance was observed for D. nigerrima but not for D. subrostrata, but this relationship was not consistent among pairwise D. nigerrima population comparisons and PCA analysis confirms that, for each species, population structure does not follow a consistent pattern of increasing with geographic distance. The lack of population structure among D. subrostrata populations is probably due to its ubiquitous distribution, meaning little opportunity exists for genetic structure. D. nigerrima, by contrast has a patchier distribution, which allows for greater opportunities for genetic differentiation to occur. We argue that, despite the probable short larval stage in this species, the lack of geographical pattern in the genetic structure found in D. nigerrima is best explained by occasional dispersal over relatively short distances around the coast of New Zealand, over longer distances from New Zealand to the subantarctic islands and even across the Pacific Ocean from New Zealand to Chile.     

Crystal Structure of the First Eubacterial Mre11 Nuclease Reveals Novel Features that May Discriminate Substrates During DNA Repair

Mre11 nuclease plays a central role in the repair of cytotoxic and mutagenic DNA double-strand breaks. As X-ray structural information has been available only for the Pyrococcus furiosus enzyme (PfMre11), the conserved and variable features of this nuclease across the domains of life have not been experimentally defined. Our crystal structure and biochemical studies demonstrate that TM1635 from Thermotoga maritima, originally annotated as a putative nuclease, is an Mre11 endo/exonuclease (TmMre11) and the first such structure from eubacteria. TmMre11 and PfMre11 display similar overall structures, despite sequence identity in the twilight zone of only ∼20%. However, they differ substantially in their DNA-specificity domains and in their dimeric organization. Residues in the nuclease domain are highly conserved, but those in the DNA-specificity domain are not. The structural differences likely affect how Mre11 from different organisms recognize and interact with single-stranded DNA, double-stranded DNA and DNA hairpin structures during DNA repair. The TmMre11 nuclease active site has no bound metal ions, but is conserved in sequence and structure with the exception of a histidine that is important in PfMre11 nuclease activity. Nevertheless, biochemical characterization confirms that TmMre11 possesses both endonuclease and exonuclease activities on single-stranded and double-stranded DNA substrates, respectively.     

Initiation of rubber biosynthesis: In vitro comparisons of benzophenone-modified diphosphate analogues in three rubber-producing species

Natural rubber, cis-1,4-polyisoprene, is a vital industrial material synthesized by plants via a side branch of the isoprenoid pathway by the enzyme rubber transferase. While the specific structure of this enzyme is not yet defined, based on activity it is probably a cis-prenyl transferase. Photoactive functionalized substrate analogues have been successfully used to identify isoprenoid-utilizing enzymes such as cis- and trans-prenyltransferases, and initiator binding of an allylic pyrophosphate molecule in rubber transferase has similar features to these systems. In this paper, a series of benzophenone-modified initiator analogues were shown to successfully initiate rubber biosynthesis in vitro in enzymatically-active washed rubber particles from Ficus elastica, Heveabrasiliensis and Parthenium argentatum.Rubber transferases from all three species initiated rubber biosynthesis most efficiently with farnesyl pyrophosphate. However, rubber transferase had a higher affinity for benzophenone geranyl pyrophosphate (Bz-GPP) and dimethylallyl pyrophosphate (Bz-DMAPP) analogues with ether-linkages than the corresponding GPP or DMAPP. In contrast, ester-linked Bz-DMAPP analogues were less efficient initiators than DMAPP. Thus, rubber biosynthesis depends on both the size and the structure of Bz-initiator molecules. Kinetic studies thereby inform selection of specific probes for covalent photolabeling of the initiator binding site of rubber transferase.     

The Molecular Structure of Ornithine Acetyltransferase from Mycobacterium tuberculosis Bound to Ornithine, a Competitive Inhibitor

Mycobacterium tuberculosis ornithine acetyltransferase (Mtb OAT; E.C. 2.3.1.35) is a key enzyme of the acetyl recycling pathway during arginine biosynthesis. It reversibly catalyzes the transfer of the acetyl group from N-acetylornithine (NAORN) to l-glutamate. Mtb OAT is a member of the N-terminal nucleophile fold family of enzymes. The crystal structures of Mtb OAT in native form and in its complex with ornithine (ORN) have been determined at 1.7 and 2.4 Å resolutions, respectively. ORN is a competitive inhibitor of this enzyme against l-glutamate as substrate. Although the acyl-enzyme complex of Streptomyces clavuligerus ornithine acetyltransferase has been determined, ours is the first crystal structure to be reported of an ornithine acetyltransferase in complex with an inhibitor. ORN binding does not alter the structure of Mtb OAT globally. However, its presence stabilizes the three C-terminal residues that are disordered and not observed in the native structure. Also, stabilization of the C-terminal residues by ORN reduces the size of the active-site pocket volume in the structure of the ORN complex. The interactions of ORN and the protein residues of Mtb OAT unambiguously delineate the active-site residues of this enzyme in Mtb. Moreover, modeling studies carried out with NAORN based on the structure of the ORN-Mtb OAT complex reveal important interactions of the carbonyl oxygen of the acetyl group of NAORN with the main-chain nitrogen atom of Gly128 and with the side-chain oxygen of Thr127. These interactions likely help in the stabilization of oxyanion formation during enzymatic reaction and also will polarize the carbonyl carbon-oxygen bond, thereby enabling the side-chain atom O^γ1 of Thr200 to launch a nucleophilic attack on the carbonyl-carbon atom of the acetyl group of NAORN.