Cloning and Characterization of a Gene Cluster for Hatomarubigin Biosynthesis in Streptomyces sp. Strain 2238-SVT4

Streptomyces sp. strain 2238-SVT4 produces hatomarubigins A, B, C, and D, which belong to the angucycline family. Among them, hatomarubigin D has a unique dimeric structure with a methylene linkage. PCR using aromatase and cyclase gene-specific primers identified the hrb gene cluster for angucycline biosynthesis in Streptomyces sp. 2238-SVT4. The cluster consisted of 30 open reading frames, including those for the minimal polyketide synthase, ketoreductase, aromatase, cyclase, O-methyltransferase, oxidoreductase, and oxygenase genes. Expression of a part of the gene cluster containing hrbR1 to hrbX in Streptomyces lividans TK23 resulted in the production of hatomarubigins A, B, and C. Hatomarubigin D was obtained from the conversion of hatomarubigin C by a purified enzyme encoded by hrbY, among the remaining genes.The angucycline antibiotics are a large group of naturally occurring aromatic polyketides of microbial origin (11, 15). They exhibit a wide range of biological activities, which include antibacterial, antiviral, antitumor, enzyme inhibitory, and platelet aggregation inhibitory effects. Although all the members contain a benz[a]anthraquinone skeleton of decaketide origin, their structural diversity is very broad and they have a wide variety of oxidation states. Hatomarubigins A, B, C, and D (Fig. ​(Fig.1)1) belong to the angucycline family and reverse colchicine resistance in multidrug-resistant tumor cells (8). Among them, hatomarubigin D is a unique hatomarubigin C dimer with a methylene linkage. Such a dimer has not been reported previously, and little is known about the mechanism of the methylene bridge formation between two aromatic rings. In this study, a gene cluster for hatomarubigin biosynthesis was identified in Streptomyces sp. strain 2238-SVT4, and a part of the gene cluster was expressed in Streptomyces lividans to produce the hatomarubigins.Open in a separate windowFIG. 1.Structures of angucycline antibiotics.     

Open and Closed Conformations of the Isolated Transmembrane Domain of Death Receptor 5 Support a New Model of Activation

It has long been presumed that activation of the apoptosis-initiating Death Receptor 5, as well as other structurally homologous members of the TNF-receptor superfamily, relies on ligand-stabilized trimerization of noninteracting receptor monomers. We and others have proposed an alternate model in which the TNF-receptor dimer—sitting at the vertices of a large supramolecular receptor network of ligand-bound receptor trimers—undergoes a closed-to-open transition, propagated through a scissorslike conformational change in a tightly bundled transmembrane (TM) domain dimer. Here we have combined electron paramagnetic resonance spectroscopy and potential-of-mean force calculations on the isolated TM domain of the long isoform of DR5. The experiments and calculations both independently validate that the opening transition is intrinsic to the physical character of the TM domain dimer, with a significant energy barrier separating the open and closed states.Death receptor 5 (DR5) is a member of the tumor necrosis factor receptor (TNFR) superfamily that mediates apoptosis when bound by its cognate ligand, TNF-related apoptosis-inducing ligand (1). Upregulated in cancer cells, DR5 is among the most actively pursued anticancer targets (2). TNF-related apoptosis-inducing ligand binds to preassembled DR5 trimers at their extracellular domains, causing the formation of oligomeric ligand-receptor networks that are held together by receptor dimers (3). In the long-isoform of DR5, this dimer is crosslinked via ligand-induced disulfide bond formation between two transmembrane (TM) domain α-helices at Cys-209, and is further stabilized by a GxxxG motif one helix-turn downstream (3).Our recent study of the structurally homologous TNFR1 showed that receptor activation involves a conformational change that propagates from the extracellular domain to the cytosolic domain through a separation (or opening) of the TM domains of the dimer (4). We have therefore hypothesized that the activation of DR5, and indeed all structurally homologous TNF-receptors, involves a scissorslike opening of the TM domain dimer (Fig. 1).Open in a separate windowFigure 1Activation model of the DR5-L TM dimer. The sequence and positions of the disulfide bond and TOAC spin label (top), along with our previously published model (bottom, left) are shown. We propose an activation model (bottom, right) in which the transmembrane dimer pivots at its disulfide bond to reach an active open conformation.Using electron paramagnetic resonance (EPR) spectroscopy, a technique that has been used previously to study TM helix architecture and dynamics (5,6), and potential-of-mean force (PMF) calculations (7,8), this study addresses the question of whether the isolated disulfide-linked DR5-L TM domain dimer occupies distinct open and closed states (Fig. 1), and how its dynamic behavior contributes to the free-energy landscape of the opening transition of the full-length receptor.The DR5-L TM domain was synthesized with TOAC, an amino acid with a nitroxide spin label rigidly fixed to the α-carbon (9), incorporated at position 32 (Fig. 1), with some minor modification to facilitate EPR measurements. Previous work confirmed that this peptide forms disulfide-linked dimers (e.g., via comparison to 2-ME treated sample) and a negligible population of higher-order oligomers (further supported by model fitting of the EPR data below). For peptide work, residues were renumbered such that Thr-204 corresponds to Thr-1, and so on. The cytosolic Cys-29 (which we previously showed does not participate in a disulfide bond in cells) was replaced with serine to prevent the formation of antiparallel disulfide-linked dimers, and Trp-34 was replaced with tyrosine to prevent intrinsic fluorescence in fluorescence studies (not published). Continuous-wave (CW) dipolar EPR (sensitive only to spin-spin distances <25 Å) was used to measure TOAC-TOAC distances within the TM dimers and revealed an ordered Gaussian distribution centered at 16 Å (full width half-maximum (FWHM) = 4 Å), corresponding to a closed state (Fig. 2 A). Double electron-electron resonance (DEER) (sensitive to spin-spin distances from 15 to 60 Å) also detected a short distance consistent with the dipolar EPR data, along with a longer, disordered component (32.9 Å, FWHM = 28 Å) (Fig. 2 B). Together, these measurements indicate the presence of a compact, ordered closed state and a broader, disordered open state. EPR on oriented membranes also indicated two structural states. Global fitting revealed two populations of spin-label tilt angles (orientation of the nitroxide principal axis relative to the membrane normal): a narrow conformation (24°, FWHM = 20°), and a disordered conformation (50°, FWHM = 48°) (Fig. 2 C). This bimodal orientational distribution (Fig. 2 C) is remarkably consistent with the bimodal distance distribution (Fig. 2 B).Open in a separate windowFigure 2EPR spectra (left) of 32-TOAC-DR5 in lipid, and resulting structural distributions (right). (A) CW dipolar EPR spectra (left) of dimer (1 mM diamide) and monomer (1 mM 2-mercaptoethanol). Best-fit spin-spin distance distribution was a single Gaussian centered at 16 ± 2 Å (right). (B) The DEER waveform (left) of 32-TOAC-DR5 dimer was best fit (right) to a two-Gaussian distribution. The short distance was constrained to agree with the CW data, because DEER has poor sensitivity for distances <20 Å. The long-distance distribution is centered at 32.9 Å and is much broader. (C) CW EPR spectra (left) of 32-TOAC-DR5, with the membrane-normal oriented parallel (red) and perpendicular (blue) to the field. Simultaneous (global) fitting of these spectra reveals narrow and broad components (right). (In panels B and C, the overall distribution is plotted as black, while the closed and open components are plotted as green and magenta, respectively.)We subsequently conducted a PMF calculation (10) using the DR5-L TM dimer starting configuration developed by our group previously (3), embedded in a DMPC bilayer, with the Leu-32/Leu-32 C_α distance as the reaction coordinate. Three calculations were run from independent starting configurations, each using 50 windows spaced in 0.5° increments, and run for 20 ns at each window (totaling 3 μs). Each of the calculations yielded a similar result, and the averaged free energy curve (Fig. 3 A) agrees remarkably well with our EPR measurements: a narrow distribution at the closed conformation (∼16 Å, Fig. 3 B) separated by an ∼3 kcal/mol energy barrier from a broad distribution of accessible open conformations at ∼27 Å, (Fig. 3 C). Each of the three individual PMF plots can be found in Fig. S1 in the Supporting Material.Open in a separate windowFigure 3(A) PMF calculation of the DR5 TM domain dimer along the Leu-32/Leu-32 distance reaction coordinate. The PMF calculation reveals a narrow closed state and a broader open state separated by a free energy barrier. Representative snapshots of the (B) closed state and (C) open state.In the closed state, the helices are tightly packed at the GxxxG interfacial motif and all the way down the juxtaposed helix faces at residues Ala-18, Leu-22, Ala-25, and Val-26. The tight packing is aided by kinking and twisting of the two helices around their common axis, increasing the interacting surface area. In the open conformations, the Ala-18, Leu-22, Ala-25, and Val-26 pairs are dissociated and, interestingly, the GxxxG motif at Gly-10 and Gly-14 remains tightly packed. The open state energy well is only slightly less favorable than the closed state (by ∼2 kcal/mol), and its free energy profile is relatively broad and flat. The increased crossing angle in the open state is facilitated by straightening of the helix kink and is not accommodated by a change in bilayer thickness (see Fig. S3, A and B).The observed change in helix-helix distance (11 Å between the two minima in the PMF) is extremely close to that observed previously in live-cell FRET studies of a constitutively active form of TNFR1 (∼8 Å change between states using large fluorescence probes at the cytosolic domains) (4). The change observed in the EPR data (17 Å) may be an overestimate because the measurement is made between TOAC spin labels that likely protrude from the two helices, depending on rotational orientation. These results collectively show that activation of these receptors requires a small, but clearly significant conformational opening of the TM domains. One important note is that our EPR experiments recapitulate the equilibrium distribution of the two states despite there being no driving force to traverse the barrier between them (∼3 kcal/mol in the closed-to-open transition and ∼1 kcal/mol in the open-to-closed transition, Fig. 3). We do not interpret the results to mean that the dimer necessarily traverses these barriers at 4°C. Rather, there likely exist multiple reaction paths for dimerization of the abstracted TM domains. Finally, in the context of the full-length receptor, how the ligand induces a conformational change capable of overcoming the closed-to-open barrier remains an important question.Whether the observed structural transition in the TM domain dimer of the long-isoform of DR5 is a ubiquitous conformational switch that acts over the entire TNFR superfamily remains unknown. Vilar et al. (11) first proposed a similar scissors-model for activation of p75 neurotrophin receptor, which has a cysteine at the center of its TM helix. The short isoform of DR5 lacks a TM domain cysteine, but does form noncovalent dimers in cells, with likely TM domain dimer contacts (3). Among the other closely related and structurally homologous members of the TNFR superfamily, TNFR1 contains a cysteine at the center of the TM domain, but lacks any discernible small residue motifs (e.g., GxxxG). TNFR2 lacks a TM cysteine on the extracellular side, but does have a GxxxG motif positioned similarly to that of DR5. On the other hand, Death Receptor 4, whose functional distinction from DR5 has remained somewhat elusive, lacks both a cysteine and any recognizable small-residue hydrophobic motif.In summary, we have extended recent findings that point to the TM domain of DR5 as an essential structural component in the conformational change associated with activation. Our findings that the DR5-L TM domain occupies distinct open and closed states, separated by a substantial energy barrier, points the way to further studies across the TNF-receptor superfamily.     

Streptomyces Development in Colonies and Soils

Streptomyces development was analyzed under conditions resembling those in soil. The mycelial growth rate was much lower than that in standard laboratory cultures, and the life span of the previously named first compartmentalized mycelium was remarkably increased.Streptomycetes are gram-positive, mycelium-forming, soil bacteria that play an important role in mineralization processes in nature and are abundant producers of secondary metabolites. Since the discovery of the ability of these microorganisms to produce clinically useful antibiotics (2, 15), they have received tremendous scientific attention (12). Furthermore, its remarkably complex developmental features make Streptomyces an interesting subject to study. Our research group has extended our knowledge about the developmental cycle of streptomycetes, describing new aspects, such as the existence of young, fully compartmentalized mycelia (5-7). Laboratory culture conditions (dense inocula, rich culture media, and relatively elevated temperatures [28 to 30°C]) result in high growth rates and an orderly-death process affecting these mycelia (first death round), which is observed at early time points (5, 7).In this work, we analyzed Streptomyces development under conditions resembling those found in nature. Single colonies and soil cultures of Streptomyces antibioticus ATCC 11891 and Streptomyces coelicolor M145 were used for this analysis. For single-colony studies, suitable dilutions of spores of these species were prepared before inoculation of plates containing GYM medium (glucose, yeast extract, malt extract) (11) or GAE medium (glucose, asparagine, yeast extract) (10). Approximately 20 colonies per plate were obtained. Soil cultures were grown in petri dishes with autoclaved oak forest soil (11.5 g per plate). Plates were inoculated directly with 5 ml of a spore suspension (1.5 × 10⁷ viable spores ml⁻¹; two independent cultures for each species). Coverslips were inserted into the soil at an angle, and the plates were incubated at 30°C. To maintain a humid environment and facilitate spore germination, the cultures were irrigated with 3 ml of sterile liquid GAE medium each week.The development of S. coelicolor M145 single colonies growing on GYM medium is shown in Fig. ​Fig.1.1. Samples were collected and examined by confocal microscopy after different incubation times, as previously described (5, 6). After spore germination, a viable mycelium develops, forming clumps which progressively extend along the horizontal (Fig. 1a and b) and vertical (Fig. 1c and d) axes of a plate. This mycelium is fully compartmentalized and corresponds to the first compartmentalized hyphae previously described for confluent surface cultures (Fig. 1e, f, and j) (see below) (5); 36 h later, death occurs, affecting the compartmentalized hyphae (Fig. 1e and f) in the center of the colony (Fig. ​(Fig.1g)1g) and in the mycelial layers below the mycelial surface (Fig. 1d and k). This death causes the characteristic appearance of the variegated first mycelium, in which alternating live and dead segments are observed (Fig. 1f and j) (5). The live segments show a decrease in fluorescence, like the decrease in fluorescence that occurs in solid confluent cultures (Fig. ​(Fig.11 h and i) (5, 9). As the cycle proceeds, the intensity of the fluorescence in these segments returns, and the segments begin to enlarge asynchronously to form a new, multinucleated mycelium, consisting of islands or sectors on the colony surfaces (Fig. 1m to o). Finally, death of the deeper layers of the colony (Fig. ​(Fig.1q)1q) and sporulation (Fig. ​(Fig.1r)1r) take place. Interestingly, some of the spores formed germinate (Fig. ​(Fig.1s),1s), giving rise to a new round of mycelial growth, cell death, and sporulation. This process is repeated several times, and typical, morphologically heterogeneous Streptomyces colonies grow (not shown). The same process was observed for S. antibioticus ATCC 11891, with minor differences mainly in the developmental time (not shown).Open in a separate windowFIG. 1.Confocal laser scanning fluorescence microscopy analysis of the development-related cell death of S. coelicolor M145 in surface cultures containing single colonies. Developmental culture times (in hours) are indicated. The images in panels l and n were obtained in differential interference contrast mode and correspond to the same fields as in panels k and m, respectively. The others are culture sections stained with SYTO 9 and propidium iodide. Panels c, d, k, l, p, and q are cross sections; the other images are longitudinal sections (see the methods). Panels h and i are images of the same field taken with different laser intensities, showing low-fluorescence viable hyphae in the center of the colonies that develop into a multinucleated mycelium. The arrows in panels e and s indicate septa (e) and germinated spores (s). See the text for details.Figure ​Figure22 shows the different types of mycelia present in S. coelicolor cultures under the conditions described above, depending on the compartmentalization status. Hyphae were treated with different fluorescent stains (SYTO 9 plus propidium iodide for nucleic acids, CellMask plus FM4-64 for cell membranes, and wheat germ agglutinin [WGA] for cell walls). Samples were processed as previously described (5). The young initial mycelia are fully compartmentalized and have membranous septa (Fig. 2b to c) with little associated cell wall material that is barely visible with WGA (Fig. ​(Fig.2d).2d). In contrast, the second mycelium is a multinucleated structure with fewer membrane-cell wall septa (Fig. 2e to h). At the end of the developmental cycle, multinucleated hyphae begin to undergo the segmentation which precedes the formation of spore chains (Fig. 2i to m). Similar results were obtained for S. antibioticus (not shown), but there were some differences in the numbers of spores formed. Samples of young and late mycelia were freeze-substituted using the methodology described by Porta and Lopez-Iglesias (13) and were examined with a transmission electron microscope (Fig. 2n and o). The septal structure of the first mycelium (Fig. ​(Fig.2n)2n) lacks the complexity of the septal structure in the second mycelium, in which a membrane with a thick cell wall is clearly visible (Fig. ​(Fig.2o).2o). These data coincide with those previously described for solid confluent cultures (4).Open in a separate windowFIG. 2.Analysis of S. coelicolor hyphal compartmentalization with several fluorescent indicators (single colonies). Developmental culture times (in hours) are indicated. (a, e, and i) Mycelium stained with SYTO 9 and propidium iodide (viability). (b, f, and j) Hyphae stained with Cell Mask (a membrane stain). (c, g, and l) Hyphae stained with FM 4-64 (a membrane stain). (d, h, and m) Hyphae stained with WGA (cell wall stain). Septa in all the images in panels a to j, l, and m are indicated by arrows. (k) Image of the same field as panel j obtained in differential interference contrast mode. (n and o) Transmission electron micrographs of S. coelicolor hyphae at different developmental phases. The first-mycelium septa (n) are comprised of two membranes separated by a thin cell wall; in contrast, second-mycelium septa have thick cell walls (o). See the text for details. IP, propidium iodide.The main features of S. coelicolor growing in soils are shown in Fig. ​Fig.3.3. Under these conditions, spore germination is a very slow, nonsynchronous process that commences at about 7 days (Fig. 3c and d) and lasts for at least 21 days (Fig. 3i to l), peaking at around 14 days (Fig. 3e to h). Mycelium does not clump to form dense pellets, as it does in colonies; instead, it remains in the first-compartmentalized-mycelium phase during the time analyzed. Like the membrane septa in single colonies, the membrane septa of the hyphae are stained with FM4-64 (Fig. 3j and k), although only some of them are associated with thick cell walls (WGA staining) (Fig. ​(Fig.3l).3l). Similar results were obtained for S. antibioticus cultures (not shown).Open in a separate windowFIG. 3.Confocal laser scanning fluorescence microscopy analysis of the development-related cell death and hyphal compartmentalization of S. coelicolor M145 growing in soil. Developmental culture times (in days) are indicated. The images in panels b, f, and h were obtained in differential interference contrast mode and correspond to the same fields as the images in panels a, e, and g, respectively. The dark zone in panel h corresponds to a particle of soil containing hyphae. (a, c, d, e, g, i, j, and k) Hyphae stained with SYTO 9, propidium iodide (viability stain), and FM4-64 (membrane stain) simultaneously. (i) SYTO 9 and propidium iodide staining. (j) FM4-64 staining. The image in panel k is an overlay of the images in panels i and j and illustrates that first-mycelium membranous septa are not always apparent when they are stained with nucleic acid stains (SYTO 9 and propidium iodide). (l) Hyphae stained with WGA (cell wall stain), showing the few septa with thick cell walls present in the cells. Septa are indicated by arrows. IP, propidium iodide.In previous work (8), we have shown that the mycelium currently called the substrate mycelium corresponds to the early second multinucleated mycelium, according to our nomenclature, which still lacks the hydrophobic layers characteristic of the aerial mycelium. The aerial mycelium therefore corresponds to the late second mycelium which has acquired hydrophobic covers. This multinucleated mycelium as a whole should be considered the reproductive structure, since it is destined to sporulate (Fig. ​(Fig.4)4) (8). The time course of lysine 6-aminotransferase activity during cephamycin C biosynthesis has been analyzed by other workers using isolated colonies of Streptomyces clavuligerus and confocal microscopy with green fluorescent protein as a reporter (4). A complex medium and a temperature of 29°C were used, conditions which can be considered similar to the conditions used in our work. Interestingly, expression did not occur during the development of the early mycelium and was observed in the mycelium only after 80 h of growth. This suggests that the second mycelium is the antibiotic-producing mycelium, a hypothesis previously confirmed using submerged-growth cultures of S. coelicolor (9).Open in a separate windowFIG. 4.Cell cycle features of Streptomyces growing under natural conditions. Mycelial structures (MI, first mycelium; MII, second mycelium) and cell death are indicated. The postulated vegetative and reproductive phases are also indicated (see text).The significance of the first compartmentalized mycelium has been obscured by its short life span under typical laboratory culture conditions (5, 6, 8). In previous work (3, 7), we postulated that this structure is the vegetative phase of the bacterium, an hypothesis that has been recently corroborated by proteomic analysis (data not shown). Death in confluent cultures begins shortly after germination (4 h) and continues asynchronously for 15 h. The second multinucleated mycelium emerges after this early programmed cell death and is the predominant structure under these conditions. In contrast, as our results here show, the first mycelium lives for a long time in isolated colonies and soil cultures. As suggested in our previous work (5, 6, 8), if we assume that the compartmentalized mycelium is the Streptomyces vegetative growth phase, then this phase is the predominant phase in individual colonies (where it remains for at least 36 h), soils (21 days), and submerged cultures (around 20 h) (9). The differences in the life span of the vegetative phase could be attributable to the extremely high cell densities attained under ordinary laboratory culture conditions, which provoke massive differentiation and sporulation (5-7, 8).But just exactly what are “natural conditions”? Some authors have developed soil cultures of Streptomyces to study survival (16, 17), genetic transfer (14, 17-19), phage-bacterium interactions (3), and antibiotic production (1). Most of these studies were carried out using amended soils (supplemented with chitin and starch), conditions under which growth and sporulation were observed during the first few days (1, 17). These conditions, in fact, might resemble environments that are particularly rich in organic matter where Streptomyces could conceivably develop. However, natural growth conditions imply discontinuous growth and limited colony development (20, 21). To mimic such conditions, we chose relatively poor but more balanced carbon-nitrogen soil cultures (GAE medium-amended soil) and less dense spore inocula, conditions that allow longer mycelium growth times. Other conditions assayed, such as those obtained by irrigating the soil with water alone, did not result in spore germination and mycelial growth (not shown). We were unable to detect death, the second multinucleated mycelium described above, or sporulation, even after 1 month of incubation at 30°C. It is clear that in nature, cell death and sporulation must take place at the end of the long vegetative phase (1, 17) when the imbalance of nutrients results in bacterial differentiation.In summary, the developmental kinetics of Streptomyces under conditions resembling conditions in nature differs substantially from the developmental kinetics observed in ordinary laboratory cultures, a fact that should be born in mind when the significance of development-associated phenomena is analyzed.     

Poring over exosome structure

The authors analyse the eukaryotic exosome structure, published in EMBO reports, in light of the known archaeal and prokaryotic exosomes, and discuss its striking flexibility and the conservation of the RNA channelling mechanism.EMBO Rep (2010) advance online publication. doi: 10.1038/embor.2010.164Almost all RNA molecules are processed by RNases to form mature RNAs. In addition, many RNAs are degraded, either because they are no longer needed or because they are aberrant. All of these functions—RNA processing, normal RNA degradation and RNA quality control—are carried out by the eukaryotic RNA exosome complex. In this issue of EMBO reports, the Lorentzen group provide structural insight into the eukaryotic exosome and the mechanism by which it degrades RNA from 3′ to 5′ (Malet et al, 2010).The crystal structures of overlapping parts of the eukaryotic exosome (Liu et al, 2006; Bonneau et al, 2009) and the related bacterial PNPase (Symmons et al, 2000) and archaeal exosome (Lorentzen et al, 2007) have been solved, and show that these RNA-degrading machines from the three domains of life have a similar structure (Fig 1). They are all composed of a ring of six RNase PH domains, one side of which has a cap that contains putative RNA-binding domains. Although this overall structure is conserved, the way that it is formed is not. Bacterial PNPase is a homotrimer of which each monomer contains two RNase PH domains, an S1 domain and a KH domain. The archaeal PH ring consists of three copies of two proteins and the cap is made of three copies of either one of two proteins. Finally, the eukaryotic exosome core is composed of nine proteins: six with one RNase PH domain each and three cap proteins.Open in a separate windowFigure 1Exosome structures. The bacterial PNPase (left), the archaeal exosome (middle) and eukaryotic core exosome (right) have a common overall structure. The top panels are schematic views from above, showing the cap proteins. The bottom panels show a view from the side, with one-third of the exosome cut away to reveal the RNA in the central channel.In PNPase and the archaeal exosome, substrates enter the PH ring from the cap-side. The putative RNA-binding domains of the cap are therefore probably important for controlling entry to the PH ring. In both archaea and bacteria, the active sites are on the inner side of the PH ring and thus the ribonucleic catalysis occurs inside the central channel. However, in humans and yeast each of the RNase PH domains have point mutations that make the exosome ring catalytically inactive (Dziembowski et al, 2007). Instead, catalysis is carried out by a tenth subunit—Rrp44/Dis3—which binds to the PH ring on the opposite side to the cap proteins (Bonneau et al, 2009; Wang et al, 2007). This organization made it unclear whether RNA also enters the central channel of the exosome in eukaryotes (Fig 1), or whether substrate RNAs directly access the catalytic subunit.Malet and colleagues now provide structural information that resolves this by reconstituting the ten-subunit yeast exosome and analysing its structure with electron microscopy, in the presence and absence of RNA. This analysis suggests that the RNase PH ring of the exosome is stable, but that the cap and catalytic subunits are more flexible than previously appreciated. It is the first structural evidence that in eukaryotes RNA is threaded through the central channel before being degraded by Rrp44.     

Overexpression of the groESL Operon Enhances the Heat and Salinity Stress Tolerance of the Nitrogen-Fixing Cyanobacterium Anabaena sp. Strain PCC7120

The bicistronic groESL operon, encoding the Hsp60 and Hsp10 chaperonins, was cloned into an integrative expression vector, pFPN, and incorporated at an innocuous site in the Anabaena sp. strain PCC7120 genome. In the recombinant Anabaena strain, the additional groESL operon was expressed from a strong cyanobacterial P_psbA1 promoter without hampering the stress-responsive expression of the native groESL operon. The net expression of the two groESL operons promoted better growth, supported the vital activities of nitrogen fixation and photosynthesis at ambient conditions, and enhanced the tolerance of the recombinant Anabaena strain to heat and salinity stresses.Nitrogen-fixing cyanobacteria, especially strains of Nostoc and Anabaena, are native to tropical agroclimatic conditions, such as those of Indian paddy fields, and contribute to the carbon (C) and nitrogen (N) economy of these soils (22, 30). However, their biofertilizer potential decreases during exposure to high temperature, salinity, and other such stressful environments (1). A common target for these stresses is cellular proteins, which are denatured and inactivated during stress, resulting in metabolic arrest, cessation of growth, and eventually loss of viability. Molecular chaperones play a major role in the conformational homeostasis of cellular proteins (13, 16, 24, 26) by (i) proper folding of nascent polypeptide chains; (ii) facilitating protein translocation and maturation to functional conformation, including multiprotein complex assembly; (iii) refolding of misfolded proteins; (iv) sequestering damaged proteins to aggregates; and (v) solubilizing protein aggregates for refolding or degradation. Present at basal levels under optimum growth conditions in bacteria, the expression of chaperonins is significantly enhanced during heat shock and other stresses (2, 25, 32).The most common and abundant cyanobacterial chaperones are Hsp60 proteins, and nitrogen-fixing cyanobacteria possess two or more copies of the hsp60 or groEL gene (http://genome.kazusa.or.jp/cyanobase). One occurs as a solitary gene, cpn60 (17, 21), while the other is juxtaposed to its cochaperonin encoding genes groES and constitutes a bicistronic operon groESL (7, 19, 31). The two hsp60 genes encode a 59-kDa GroEL and a 61-kDa Cpn60 protein in Anabaena (2, 20). Both the Hsp60 chaperonins are strongly expressed during heat stress, resulting in the superior thermotolerance of Anabaena, compared to the transient expression of the Hsp60 chaperonins in Escherichia coli (20). GroEL and Cpn60 stably associate with thylakoid membranes in Anabaena strain PCC7120 (14) and in Synechocystis sp. strain PCC6803 (15). In Synechocystis sp. strain PCC6803, photosynthetic inhibitors downregulate, while light and redox perturbation induce cpn60 expression (10, 25, 31), and a cpn60 mutant exhibits a light-sensitive phenotype (http://genome.kazusa.or.jp/cyanobase), indicating a possible role for Cpn60 in photosynthesis. GroEL, a lipochaperonin (12, 28), requires a cochaperonin, GroES, for its folding activity and has wider substrate selectivity. In heterotrophic nitrogen-fixing bacteria, such as Klebsiella pneumoniae and Bradyrhizobium japonicum, the GroEL protein has been implicated in nif gene expression and the assembly, stability, and activity of the nitrogenase proteins (8, 9, 11).Earlier work from our laboratory demonstrated that the Hsp60 family chaperonins are commonly induced general-stress proteins in response to heat, salinity, and osmotic stresses in Anabaena strains (2, 4). Our recent work elucidated a major role of the cpn60 gene in the protection from photosynthesis and the nitrate reductase activity of N-supplemented Anabaena cultures (21). In this study, we integrated and constitutively overexpressed an extra copy of the groESL operon in Anabaena to evaluate the importance and contribution of GroEL chaperonin to the physiology of Anabaena during optimal and stressful conditions.Anabaena sp. strain PCC7120 was photoautotrophically grown in combined nitrogen-free (BG11⁻) or 17 mM NaNO₃-supplemented (BG11⁺) BG11 medium (5) at pH 7.2 under continuous illumination (30 μE m⁻² s⁻¹) and aeration (2 liters min⁻¹) at 25°C ± 2°C. Escherichia coli DH5α cultures were grown in Luria-Bertani medium at 37°C at 150 rpm. For E. coli DH5α, kanamycin and carbenicillin were used at final concentrations of 50 μg ml⁻¹ and 100 μg ml⁻¹, respectively. Recombinant Anabaena clones were selected on BG11⁺ agar plates supplemented with 25 μg ml⁻¹ neomycin or in BG11⁻ liquid medium containing 12.5 μg ml⁻¹ neomycin. The growth of cyanobacterial cultures was estimated either by measuring the chlorophyll a content as described previously (18) or the turbidity (optical density at 750 nm). Photosynthesis was measured as light-dependent oxygen evolution at 25 ± 2°C by a Clark electrode (Oxy-lab 2/2; Hansatech Instruments, England) as described previously (21). Nitrogenase activity was estimated by acetylene reduction assays, as described previously (3). Protein denaturation and aggregation were measured in clarified cell extracts containing ∼500 μg cytosolic proteins treated with 100 μM 8-anilino-1-naphthalene sulfonate (ANS). The pellet (protein aggregate) was solubilized in 20 mM Tris-6 M urea-2% sodium dodecyl sulfate (SDS)-40 mM dithiothreitol for 10 min at 50°C. The noncovalently trapped ANS was estimated using a fluorescence spectrometer (model FP-6500; Jasco, Japan) at a λ_excitation of 380 nm and a λ_emission of 485 nm, as described previously (29).The complete bicistronic groESL operon (2.040 kb) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ608815","term_id":"222354886","term_text":"FJ608815"}}FJ608815) was PCR amplified from PCC7120 genomic DNA using specific primers (Table ​(Table1)1) and the amplicon cloned into the NdeI-BamHI restriction sites of plasmid vector pFPN, which allows integration at a defined innocuous site in the PCC7120 genome and expression from a strong cyanobacterial P_psbA1 promoter (6). The resulting construct, designated pFPNgro (Table ​(Table1),1), was electroporated into PCC7120 using an exponential-decay wave form electroporator (200 J capacitive energy at a full charging voltage of 2 kV; Pune Polytronics, Pune, India), as described previously (6). The electroporation was carried out at 6 kV cm⁻¹ for 5 ms, employing an external autoclavable electrode with a 2-mm gap. The electroporation buffer contained high concentrations of salt (10 mM HEPES, 100 mM LiCl, 50 mM CaCl₂), as have been recommended for plant cells (23) and other cell types (27). The electrotransformants, selected on BG11⁺ agar plates supplemented with 25 μg ml⁻¹ neomycin by repeated subculturing for at least 25 weeks to achieve complete segregation, were designated AnFPNgro.

TABLE 1.

Plasmids, strains, and primers used in this study

Structural Study of the Serratia entomophila Antifeeding Prophage: Three-Dimensional Structure of the Helical Sheath

The sheath of the Serratia entomophila antifeeding prophage, which is pathogenic to the New Zealand grass grub Costelytra zealandica, is a 3-fold helix formed by a 4-fold symmetric repeating motif disposed around a helical inner tube. This structure, determined by electron microscopy and image processing, is distinct from that of the other known morphologically similar bacteriophage sheaths.The antifeeding prophage (Afp) of Serratia entomophila and Serratia proteamaculans is a naturally occurring virus tail-like structure which delivers a putative toxin molecule that leads to starvation of the New Zealand grass grub Costelytra zealandica (5). Afp is composed of 18 different gene products (molecular masses of 6.5 to 263 kDa). The first 16 open reading frames have orthologues (Photorhabdus virulence cassettes [PVC]) in the insecticidal bacterium Photorhabdus luminescens TTO1 genome (5). Afp and PVCs morphologically resemble a typical R-type bacteriocin (6, 12, 16) However, Afp is the only known phage tail-like protein complex that is not a bacteriocin-protein complex of considerable medical relevance that targets the same or closely related bacterial strains (1, 3, 8, 12). The major component of Afp is a contractile cylindrical outer sheath encasing an inner tube speculated to house the toxin molecule (6). A dome-shaped “head” defines one extremity of the tube, while the other end is attached to a “bell-shaped” structure with a base morphologically similar to the base plate of the T4 bacteriophage tail (9).Transmission electron micrographs of two-dimensional (2D) projections of negatively stained (Fig. ​(Fig.11 A) or frozen-hydrated and vitrified (Fig. ​(Fig.1B)1B) recombinant Afp particles (see Fig. S1 in the supplemental material) were used for computational image analysis. A globally averaged image of the Afp particle in the major configuration (called E here) (Fig. ​(Fig.1C),1C), generated using negatively stained specimens, clearly distinguished the morphologies of the various constituent structural parts. Thus, the cylindrical sheath appears to be formed by a periodic structure harboring a distinctive, inverted-V-shaped feature. A minor population of Afp particles displays an alternate configuration (called C here) where, concomitant with contraction of the sheath (averaged axial compression of ∼52% [see Table S1 in the supplemental material]), the inner tube, shorn off the bell-shaped structure, is revealed (Fig. ​(Fig.1A)1A) (6). Several other bacteriocins undergo such a high degree of compression, which has been characterized in detail for the tail sheath of bacteriophage T4 (9). We also generated individual global averages for the periodic sheath structure, for the bell-shaped structure, and for the inner tube (Fig. ​(Fig.1C)1C) which provide more accurate dimensions of these different sections (see Table S1 in the supplemental material) than those reported earlier (6).Open in a separate windowFIG. 1.(A) Electron micrograph of a negatively stained preparation of partially purified recombinant Afp particles. The gray, white, and black arrows point to an Afp particle in the extended (E) configuration, to an Afp particle with the sheath contracted, exposing the inner tube in the contracted (C) configuration, and to an inner tube with the bell-shaped structure attached at one end, respectively. (B) Cryoelectron micrograph recorded from a preparation similar to that shown in panel A. (C) Globally averaged images of Afp particles (3,026 images) in the E state and the three distinguishable parts visualized by negative staining. Bars, 200 Å. (D) Averaged power spectrum of the sheath of vitrified Afp particles. The black and white arrows indicate reflections delineating the axial rise (1/78.5 Å) and the helical pitch (1/118 Å), respectively. In panels A and C, lighter regions represent protein and the contrast is reversed in panel B. Global averages were created using classalign2 of the EMAN suite (10) and visualized in bshow (4).For a better insight, we determined the 3D structure of the central periodic section of the Afp particle in the E state. A global power spectrum derived from the cryoimages established the structure to be helical with a clear first meridional reflection at 1/78.5 Å and the first, strongest nonmeridional reflection at 1/118 Å (Fig. ​(Fig.1D).1D). These reflections correspond to the axial rise (Δx) and the pitch of the helix, respectively, and reflect a turn angle (Δψ) of about ±240° (3₂ helix) for the repeating motif. The correct sign, i.e., the hand, of the helix remains to be determined. Computationally excised overlapping segments of this helical section from images of vitrified and negatively stained Afp particles were subjected to 3D reconstruction using the iterative helical real-space reconstruction (IHRSR) algorithm (2) using the determined helical parameters (see Fig. S2 in the supplemental material). After a few iterations, the presence of an in-plane 4-fold symmetry (C4) was apparent in the density map (see Fig. S2 in the supplemental material), which was then imposed in the subsequent reconstruction exercises. However, no stable solution was forthcoming, even after many (e.g., 30) iterative cycles. This is generally indicative of the presence of heterogeneity in the form of variations in helix translation and/or twist angle (15) in the structure. As a first step, we focused our attention on the pitch value, and following classification (see Fig. S3 in the supplemental material), we found that the majority of the image segments correspond to a pitch of 120 Å. These segments were then selected out of the full data set and led to a stable and refined 3D reconstruction. We also obtained very comparable results for the helical section when images of negatively stained Afp sheath sections were used, thus supporting our computational approach (see Fig. S4 in the supplemental material) and general conclusions about the E state described below.Figure ​Figure22 A is an isosurface representation of the density map of the helical Afp sheath in the E state calculated at ∼21.5-Å resolution (see Fig. S5 in the supplemental material). To the best of our knowledge, a 4-fold rotational symmetry has not been seen for any other contractile T4 bacteriophage taillike structure, which points to the unique architecture of the Afp sheath. A power spectrum generated using the 2D projection from the final density map, compared to the experimental global power spectrum (Fig. ​(Fig.2D),2D), showed strong agreement, confirming the fidelity of the computational image analysis. The density map displays protein layers, ∼80 Å thick, that are stacked on each other in a periodic fashion. The uneven outer surface of the sheath is perforated and decorated with ∼35-Å protrusions. When rendered with a raised threshold, a characteristic feature of the map is a contiguous, high-electron-density region having an inverted-Y-shaped structure (Fig. 2C and E; see Fig. S4 in the supplemental material). At the modest ∼21.5-Å resolution, the boundary of the repeating subunit cannot be defined. A 25 ± 3-Å-wide central lumen is seen clearly when viewed along the helix axis (Fig. ​(Fig.2B)2B) and likely represents the pore of the inner tube (see also below). Using scanning transmission electron microscopy (STEM) (see Fig. S6 in the supplemental material), we estimated the averaged molecular mass of the central helical section of an Afp particle to be 9.8 ± 0.4 kDa/Å (Fig. ​(Fig.3)3) (14) and that of only the inner tube to be ∼2.5 kDa/Å, based on a relatively small pool of such images. These values translate to a mass contribution of approximately 145 kDa of the subunit whose periodic arrangement forms the outer component of the sheath (i.e., excluding the inner tube) (see Fig. S6 in the supplemental material). This value is not very different from the cumulative mass of the different proteins, i.e., homologous afp2, afp3, and afp4, thought to be involved in Afp sheath formation (5) (see Fig. S7 in the supplemental material).Open in a separate windowFIG. 2.Orthogonal isosurface rendering, at 1 σ (standard deviation) of the computed ∼21.5-Å density map of the helical sheath of the vitrified Afp particle viewed normal (A) and parallel (B) to the helix. The images were generated using the software package CHIMERA (13). The arrow indicates a surface perforation. (C) The Afp density map rendered at 3.5 σ to highlight the largest contiguous high-electron-density regions; one circumscribed by a black ellipse is computationally extracted and shown in panel E. (D) Comparison of the experimental, averaged power spectrum of the helical sheath of Afp (left) with that computed (right) from the 2D projection of the calculated density map. (F) Global average of the inner tube of the Afp particle and a plot of the surface density variation (scaled from 0 to 1) along the helical (y) axis. The dimension along the tube is plotted on the x axis.Open in a separate windowFIG. 3.(A) Dark-field micrograph of a freeze-dried, unstained preparation of Afp particles used in STEM measurements. An Afp particle in the E state, an Afp inner tube with the attached bell-shaped structure, and a tobacco mosaic virus particle, used as a calibration standard, are marked by the arrowhead and the gray and white arrows, respectively. (B) Histogram plot of the measured distributions of mass per unit length corresponding to the uniform periodic section of the Afp particles overlaid with a fitted Gaussian curve produced by using the ORIGIN6 software package.A paucity of images of the C state (∼5% of the complete data set) precluded a full, refined 3D reconstruction, but based on the available 2,774 overlapping image segments of the isolated inner tube, a global average was calculated. A plot of contrast variation (Fig. ​(Fig.2F)2F) indicates that the surface is characterized by ∼40-Å spaced elevated crests and invaginated grooves, in agreement with the calculated axial rise of ∼39 Å for the subunit (see Fig. S8 in the supplemental material) comprising the tube. Based on these preliminary results, it appears that the helical symmetry of the inner tube is markedly different from that of the sheath.Our observation that the pitch of the helix in the E state can vary by as much as ∼50 Å attests to the flexible nature of the sheath, which is required for compressibility and may be facilitated by the somewhat porous nature of the sheath (Fig. ​(Fig.2).2). Preliminary deductions (data not shown) based on a small pool of images of the C state suggest profound rearrangement of the elements of the sheath. How that translates to extrusion of the toxin remains to be revealed.      

Discovery and Characterization of a Novel Lachrymatory Factor Synthase in Petiveria alliacea and Its Influence on Alliinase-Mediated Formation of Biologically Active Organosulfur Compounds

A novel lachrymatory factor synthase (LFS) was isolated and purified from the roots of the Amazonian medicinal plant Petiveria alliacea. The enzyme is a heterotetrameric glycoprotein comprised of two α-subunits (68.8 kD each), one γ-subunit (22.5 kD), and one δ-subunit (11.9 kD). The two α-subunits are glycosylated and connected by a disulfide bridge. The LFS has an isoelectric point of 5.2. It catalyzes the formation of a sulfine lachrymator, (Z)-phenylmethanethial S-oxide, only in the presence of P. alliacea alliinase and its natural substrate, S-benzyl-l-cysteine sulfoxide (petiveriin). Depending on its concentration relative to that of P. alliacea alliinase, the LFS sequesters, to varying degrees, the sulfenic acid intermediate formed by alliinase-mediated breakdown of petiveriin. At LFS:alliinase of 5:1, LFS sequesters all of the sulfenic acid formed by alliinase action on petiveriin, and converts it entirely to (Z)-phenylmethanethial S-oxide. However, starting at LFS:alliinase of 5:2, the LFS is unable to sequester all of the sulfenic acid produced by the alliinase, with the result that sulfenic acid that escapes the action of the LFS condenses with loss of water to form S-benzyl phenylmethanethiosulfinate (petivericin). The results show that the LFS and alliinase function in tandem, with the alliinase furnishing the sulfenic acid substrate on which the LFS acts. The results also show that the LFS modulates the formation of biologically active thiosulfinates that are downstream of the alliinase in a manner dependent upon the relative concentrations of the LFS and the alliinase. These observations suggest that manipulation of LFS-to-alliinase ratios in plants displaying this system may provide a means by which to rationally modify organosulfur small molecule profiles to obtain desired flavor and/or odor signatures, or increase the presence of desirable biologically active small molecules.Lachrymatory factor synthase (LFS) is the term coined to refer to the recently discovered enzyme shown to catalyze the formation of the sulfine responsible for the lachrymatory effect of onion (Allium cepa), (Z)-propanethial S-oxide (PTSO; Imai et al., 2002). Until the discovery of the onion LFS, the formation of the onion lachrymatory factor (LF) was thought to be mediated by only a single enzyme, onion alliinase. Alliinases, which are pyridoxal 5′-P (PLP)-dependent Cys sulfoxide lyases most often found in members of the Allium genus, catalyze the breakdown of Cys sulfoxide derivatives to yield fleeting sulfenic acid intermediates and α-aminoacrylic acid (Scheme 1; Block, 1992; Shimon et al., 2007). Once formed, the sulfenic acids are most often observed to spontaneously condense with loss of water to form thiosulfinates, whereas the α-aminoacrylic acid is further hydrolyzed with loss of ammonia to form pyruvate. The S-substituted Cys sulfoxides that are acted upon by alliinases differ from one another by the identity of the sulfur-bound R group. In Allium plants, the R groups are alk(en)yl, with R = methyl and 2-propenyl appearing in large quantities in garlic (Allium sativum) and R = methyl and (E)-1-propenyl preponderating in onion (Scheme 1). The Cys sulfoxide that serves as the precursor of the onion lachrymator is (E)-S-(1-propenyl)-l-Cys sulfoxide (isoalliin). It is structurally distinct from other naturally occurring S-substituted Cys sulfoxides so far reported in that it is α,β-unsaturated. This structural feature affords its corresponding 1-propenylsulfenic acid (PSA) the possibility of undergoing a [1,4]-sigmatropic rearrangement that, in principle, would furnish the onion lachrymator, PTSO. Indeed, the formation of the onion lachrymator was proposed to occur by such a mechanism (Scheme 2; Block, 1992). Thus, it was surmised that were the α,β-unsaturation to be absent in the precursor S-substituted Cys sulfoxide, the [1,4]-sigmatropic rearrangement that would lead to sulfine formation could not occur. Consequently, it was not surprising that other S-substituted Cys sulfoxides constitutively present in garlic, onion, and other alliinase-containing plants, but devoid of this α,β-unsaturation in the sulfur-bound R group, did not themselves yield lachrymators on plant tissue wounding. It has since been discovered, however, that formation of the onion lachrymator is not catalyzed by onion alliinase, but instead by a novel class of enzyme—LFS. Imai et al. (2002) observed that although a crude preparation of onion alliinase yielded both the LF and the corresponding thiosulfinate, the protein fraction with lachrymator-forming ability could be completely separated from that with alliinase activity by passing the crude onion protein preparation through a hydroxyapatite column. The LFS was subsequently purified and shown to be highly substrate specific, producing the LF from only (E)-S-(1-propenyl)-l-Cys sulfoxide (isoalliin), which occurs constitutively in onion. Interestingly, the LF was detected only when three components, namely, the purified onion alliinase, isoalliin, and the onion LFS, were present in the reaction mixture simultaneously (Imai et al., 2002). Omission of the LFS from the reaction mixture resulted in an increased yield of thiosulfinates, but no LF. Although the complete cDNA sequence of the onion LFS has been determined (Imai et al., 2002), to our knowledge, full biochemical characterization of the enzyme has yet to be reported.Open in a separate windowScheme 1.Alliinase-mediated formation of thiosulfinates from Cys sulfoxide precursors (Block, 1992; Shimon et al., 2007). Alliin is S-allyl-l-Cys sulfoxide, isoalliin is (E)-S-(1-propenyl)-l-Cys sulfoxide, methiin is S-methyl-l-Cys sulfoxide, and propiin is S-propyl-l-Cys sulfoxide.Open in a separate windowScheme 2.Mechanism advanced by Block (1992) to account for formation of the onion lachrymator, PTSO. Alliinase-bound PLP forms a Schiff base with bound isoalliin. General base catalysis at the active site yields an α,β-unsaturated sulfenic acid that can undergo a [1,4]-sigmatropic rearrangement to furnish the sulfine.In the course of our studies on the organosulfur chemistry of non-Allium plants, we isolated and characterized the S-benzyl-l-Cys sulfoxides (petiveriins) and S-(2-hydroxyethyl)-l-Cys sulfoxides (2-hydroxyethiins) from the Amazonian medicinal plant Petiveria alliacea (Fig. 1; Kubec and Musah, 2001; Kubec et al., 2002). These compounds are S-substituted Cys sulfoxide derivatives with R = benzyl and 2-hydroxyethyl, respectively, that, to our knowledge, had never before been isolated from plants. We showed that, as has been observed in garlic and onion, symmetrical and mixed thiosulfinate derivatives of the corresponding petiveriin and 2-hydroxyethiin precursors could be extracted with ether solvent (Fig. 1; Kubec et al., 2002) upon root tissue disruption. We have also shown that an alliinase that mediates the transformation of the petiveriins and 2-hydroxyethiins to their corresponding thiosulfinates is present in P. alliacea (Musah et al., 2009). Interestingly, while working with P. alliacea root extracts, we noted the presence of a potent lachrymator that we subsequently determined to be a sulfine—(Z)-phenylmethanethial S-oxide (PMTSO; Fig. 2; Kubec et al., 2003). However, the biochemical precursor of PMTSO and the pathway(s) leading to its formation upon disruption of P. alliacea tissue remain to be determined. Given that the onion LF (PTSO), whose formation is mediated by an LFS, is also a sulfine, we were prompted to investigate the possibility of the presence of a LFS in P. alliacea. In this report, we describe our confirmation of the existence of a LFS in P. alliacea, and detail biochemical characterization of this novel class of enzymes.Open in a separate windowFigure 1.Cys sulfoxides and their corresponding thiosulfinate derivatives isolated from the Amazonian medicinal plant P. alliacea. The breakdown of the Cys sulfoxides is mediated by P. alliacea alliinase.Open in a separate windowFigure 2.Lachrymatory sulfine isolated from P. alliacea.     

Efficient Production of 2-Oxobutyrate from 2-Hydroxybutyrate by Using Whole Cells of Pseudomonas stutzeri Strain SDM

2-Oxobutyrate is an important intermediate in the chemical, drug, and food industries. Whole cells of Pseudomonas stutzeri SDM, containing NAD-independent lactate dehydrogenases, effectively converted 2-hydroxybutyrate into 2-oxobutyrate. Under optimal conditions, the biocatalytic process produced 2-oxobutyrate at a high concentration (44.4 g liter⁻¹) and a high yield (91.5%).2-Oxobutyrate (2-OBA) is used as a raw material in the synthesis of chiral 2-aminobutyric acid, isoleucine, and some kinds of medicines (1, 8). There is no suitable starting material for 2-OBA production by chemical synthesis; therefore, the development of innovative biotechnology-based techniques for 2-OBA production is desirable (12).2-Hydroxybutyrate (2-HBA) is cheaper than 2-OBA and can be substituted for 2-OBA in the production of isoleucine, as reported previously (9, 10). The results of those studies also indicated that it might be possible to produce 2-OBA from 2-HBA by a suitable biocatalytic process. In the presence of NAD, NAD-dependent 2-hydroxybutyrate dehydrogenase can catalyze the oxidation of 2-HBA to 2-OBA (4). However, due to the high cost of pyridine cofactors (11), it is preferable to use a biocatalyst that directly catalyzes the formation of 2-OBA from 2-HBA without any requirement for NAD as a cofactor.In our previous report, we confirmed that NAD-independent lactate dehydrogenases (iLDHs) in the pyruvate-producing strain Pseudomonas stutzeri SDM (China Center for Type Culture Collection no. M206010) could oxidize lactate and 2-HBA (6). Therefore, in addition to pyruvate production from lactate, P. stutzeri SDM might also have a potential application in 2-OBA production.To determine the 2-OBA production capability of P. stutzeri SDM, the strain was first cultured at 30°C in a minimal salt medium (MSM) supplemented with 5.0 g liter⁻¹ dl-lactate as the sole carbon source (5). The whole-cell catalyst was prepared by centrifuging the medium and resuspending the cell pellet, and biotransformation was then carried out under the following conditions using 2-HBA as the substrate and whole cells of P. stutzeri SDM as the biocatalyst: 2-HBA, 10 g liter⁻¹; dry cell concentration, 6 g liter⁻¹; buffer, 100 mM potassium phosphate (pH 7.0); temperature, 30°C; shaking speed, 300 rpm. After 4 h of reaction, the mixture was analyzed by high-performance liquid chromatography (HPLC; Agilent 1100 series; Hewlett-Packard) using a refractive index detector (3). The HPLC system was fitted with a Bio-Rad Aminex HPX-87 H column. The mobile phase consisted of 10 mM H₂SO₄ pumped at 0.4 ml min⁻¹ (55°C). Biotransformation resulted in the production of a compound that had a retention time of 19.57 min, which corresponded to the peak of authentic 2-OBA (see Fig. S1 in the supplemental material).After acidification and vacuum distillation, the new compound was analyzed by negative-ion mass spectroscopy. The molecular ion ([M − H]⁻, m/z 101.1) signal of the compound was consistent with the molecular weight of 2-OBA, i.e., 102.1 (see Fig. S2 in the supplemental material). These results confirmed that 2-HBA was oxidized to 2-OBA by whole cells of P. stutzeri SDM.To investigate whether iLDHs are responsible for 2-OBA production in the above-described biocatalytic process, 2-HBA oxidation activity in P. stutzeri SDM was probed by native polyacrylamide gel electrophoresis. After electrophoresis, the gels were soaked in a substrate solution [50 mM Tris-HCl buffer (pH 8.0) containing 0.1 mM phenazine methosulfate, 0.1 mM 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, and 1 mM l-lactate, dl-lactate, or dl-2-HBA] and gently shaken. As shown in Fig. ​Fig.1,1, d- and l-iLDH migrated as two bands with distinct mobilities. The activities responsible for d- and l-2-HBA oxidation were located at the same positions as the d- and l-iLDH activities, respectively. No other bands responsible for d- and l-2-HBA oxidation were detected. Moreover, the dialysis of the crude cell extract did not lead to loss of 2-HBA oxidation activity and the addition of 10 mM NAD⁺ could not stimulate the reaction (see Table S1 in the supplemental material). These results implied that in the biocatalytic system, 2-HBA was oxidized to 2-OBA by iLDHs present in P. stutzeri SDM.Open in a separate windowFIG. 1.Activity staining of iLDHs after native polyacrylamide gel electrophoresis with lactate or 2-HBA as the substrate.Although the SDM strain could not use 2-HBA or 2-OBA for growth (see Fig. S3 in the supplemental material), 2-HBA might induce some of the enzymes responsible for 2-OBA production in the biocatalytic process. To exclude this possibility, the SDM strain was cultured in MSM containing dl-lactate or pyruvate as the sole carbon source. As shown in Fig. ​Fig.2,2, the enzyme activities that catalyzed lactate and 2-HBA oxidation were simultaneously present in the cells cultured on lactate and were absent in those cultured on pyruvate. After the lactate or pyruvate was exhausted, 5.05 g liter⁻¹ dl-2-HBA was added to the medium. It was observed that dl-2-HBA was efficiently converted to 2-OBA in the medium containing dl-lactate (Fig. ​(Fig.2a).2a). No 2-OBA production was detected in the medium containing pyruvate. Because 2-HBA addition did not induce the enzymes involved in 2-HBA oxidation (Fig. 2a and b), we concluded that the iLDHs induced by dl-lactate catalyzed 2-HBA oxidation in this biocatalytic process.Open in a separate windowFIG. 2.Time course of P. stutzeri SDM growth on media containing dl-lactate (a) and pyruvate (b). 2-HBA was added to the medium after the exhaustion of lactate or pyruvate. Symbols: ▴, lactate; ▵, pyruvate; •, 2-HBA; ○, 2-OBA; ▪, cell density; ▧, iLDHs activity with dl-lactate as the substrate; ▒, iLDHs activity with dl-2-HBA as the substrate.iLDHs could catalyze the oxidation of the substrate in a flavin-dependent manner and might use membrane quinone as the electron acceptor. Unlike the oxidases, which directly use the oxygen as the electron acceptor, this substrate oxidation mechanism could prevent the formation of H₂O₂ (see Fig. S4 in the supplemental material). The P. stutzeri SDM strain efficiently converted dl-2-HBA to 2-OBA with high yields (4.97 g liter⁻¹ 2-OBA was produced from 5.05 g liter⁻¹ dl-2-HBA); therefore, 2-OBA production by this strain can be a valuable and technically feasible process. To increase the efficiency of P. stutzeri SDM in the biotechnological production of 2-OBA, the conditions for biotransformation using whole cells of P. stutzeri SDM were first optimized. The influence of the reaction pH and 2-HBA concentration on 2-OBA production was determined in 100 mM phosphate buffer containing whole cells harvested from the medium containing dl-lactate as the sole carbon source. The reaction was initiated by adding the whole cells and 2-HBA at 37°C, followed by incubation for 10 min. After stopping the reaction by adding 1 M HCl, the 2-OBA concentration was determined by HPLC.As shown in Fig. ​Fig.3a,3a, ​,2-OBA2-OBA production was highest at pH 7.0. Under acidic or alkaline conditions, the transformation of 2-HBA to 2-OBA decreased. The optimal 2-HBA concentration was found to be 0.4 M, as shown in Fig. ​Fig.3b.3b. 2-OBA production increased as the 2-HBA concentration increased up to about 0.4 M and decreased thereafter. The concentration of the whole-cell catalyst was then optimized using 0.4 M 2-HBA as the substrate at pH 7.0. As shown in Fig. ​Fig.3c,3c, the highest 2-OBA concentration was obtained with 20 g (dry cell weight [DCW]) liter⁻¹ of P. stutzeri SDM. The 2-OBA concentration decreased with any increase beyond this cell concentration.Open in a separate windowFIG. 3.Optimization of the biocatalysis conditions. (a) Effect of pH on 2-OBA production activity. (b) Effect of 2-HBA concentrations on 2-OBA production activity. (c) Effect of the concentration of P. stutzeri SDM on biotransformation. OD, optical density.After optimizing the biocatalytic conditions, we studied the biotechnological production of 2-OBA from 2-HBA by using the whole-cell catalyst P. stutzeri SDM. As shown in Fig. ​Fig.4,4, when 20 g (DCW) liter⁻¹ P. stutzeri SDM was used as the biocatalyst, 48.5 g liter⁻¹ 2-HBA was biotransformed into 44.4 g liter⁻¹ 2-OBA in 24 h.Open in a separate windowFIG. 4.Time course of production of 2-OBA from 2-HBA under the optimum conditions. Symbols: ▪, 2-OBA; •, 2-HBA.Biocatalytic production of 2-OBA was carried out using crotonic acid, propionaldehyde, 1,2-butanediol, or threonine as the substrate (2, 7, 8, 12). Resting cells of the strain Rhodococcus erpi IF0 3730 produced 15.7 g liter⁻¹ 2-OBA from 20 g liter⁻¹ 1,2-butanediol, which is the highest reported yield of 2-OBA to date (8). By using the whole-cell catalyst P. stutzeri SDM, it was possible to produce 2-OBA at a high concentration (44.4 g liter⁻¹) and a high yield (91.5%). Due to the simple composition of the biocatalytic system (see Fig. S5 in the supplemental material), 2-HBA and 2-OBA could be easily separated on a column using a suitable resin. Separation of 2-OBA from the biocatalytic system was relatively inexpensive. The biocatalytic process presented in this report could be a promising alternative for the biotechnological production of 2-OBA.      

The Structure and Function of the Eukaryotic Ribosome

Structures of the bacterial ribosome have provided a framework for understanding universal mechanisms of protein synthesis. However, the eukaryotic ribosome is much larger than it is in bacteria, and its activity is fundamentally different in many key ways. Recent cryo-electron microscopy reconstructions and X-ray crystal structures of eukaryotic ribosomes and ribosomal subunits now provide an unprecedented opportunity to explore mechanisms of eukaryotic translation and its regulation in atomic detail. This review describes the X-ray crystal structures of the Tetrahymena thermophila 40S and 60S subunits and the Saccharomyces cerevisiae 80S ribosome, as well as cryo-electron microscopy reconstructions of translating yeast and plant 80S ribosomes. Mechanistic questions about translation in eukaryotes that will require additional structural insights to be resolved are also presented.All ribosomes are composed of two subunits, both of which are built from RNA and protein (Figs. ​(Figs.11 and ​and2).2). Bacterial ribosomes, for example of Escherichia coli, contain a small subunit (SSU) composed of one 16S ribosomal RNA (rRNA) and 21 ribosomal proteins (r-proteins) (Figs. ​(Figs.1A1A and ​and1B)1B) and a large subunit (LSU) containing 5S and 23S rRNAs and 33 r-proteins (Fig. 2A). Crystal structures of prokaryotic ribosomal particles, namely, the Thermus thermophilus SSU (Schluenzen et al. 2000; Wimberly et al. 2000), Haloarcula marismortui and Deinococcus radiodurans LSU (Ban et al. 2000; Harms et al. 2001), and E. coli and T. thermophilus 70S ribosomes (Yusupov et al. 2001; Schuwirth et al. 2005; Selmer et al. 2006), reveal the complex architecture that derives from the network of interactions connecting the individual r-proteins with each other and with the rRNAs (Brodersen et al. 2002; Klein et al. 2004). The 16S rRNA can be divided into four domains, which together with the r-proteins constitute the structural landmarks of the SSU (Wimberly et al. 2000) (Fig. 1A): The 5′ and 3′ minor (h44) domains with proteins S4, S5, S12, S16, S17, and S20 constitute the body (and spur or foot) of the SSU; the 3′ major domain forms the head, which is protein rich, containing S2, S3, S7, S9, S10, S13, S14, and S19; whereas the central domain makes up the platform by interacting with proteins S1, S6, S8, S11, S15, and S18 (Fig. 1B). The rRNA of the LSU can be divided into seven domains (including the 5S rRNA as domain VII), which—in contrast to the SSU—are intricately interwoven with the r-proteins as well as each other (Ban et al. 2000; Brodersen et al. 2002) (Fig. 2A). Structural landmarks on the LSU include the central protuberance (CP) and the flexible L1 and L7/L12 stalks (Fig. 2A).Open in a separate windowFigure 1.The bacterial and eukaryotic small ribosomal subunit. (A,B) Interface (upper) and solvent (lower) views of the bacterial 30S subunit (Jenner et al. 2010a). (A) 16S rRNA domains and associated r-proteins colored distinctly: b, body (blue); h, head (red); pt, platform (green); and h44, helix 44 (yellow). (B) 16S rRNA colored gray and r-proteins colored distinctly and labeled. (C–E) Interface and solvent views of the eukaryotic 40S subunit (Rabl et al. 2011), with (C) eukaryotic-specific r-proteins (red) and rRNA (pink) shown relative to conserved rRNA (gray) and r-proteins (blue), and with (D,E) 18S rRNA colored gray and r-proteins colored distinctly and labeled.Open in a separate windowFigure 2.The bacterial and eukaryotic large ribosomal subunit. (A) Interface (upper) and solvent (lower) views of the bacterial 50S subunit (Jenner et al. 2010b), with 23S rRNA domains and bacterial-specific (light blue) and conserved (blue) r-proteins colored distinctly: cp, central protuberance; L1, L1 stalk; and St, L7/L12 stalk (or P-stalk in archeaa/eukaryotes). (B–E) Interface and solvent views of the eukaryotic 60S subunit (Klinge et al. 2011), with (B) eukaryotic-specific r-proteins (red) and rRNA (pink) shown relative to conserved rRNA (gray) and r-proteins (blue), (C) eukaryotic-specific expansion segments (ES) colored distinctly, and (D,E) 28S rRNA colored gray and r-proteins colored distinctly and labeled.In contrast to their bacterial counterparts, eukaryotic ribosomes are much larger and more complex, containing additional rRNA in the form of so-called expansion segments (ES) as well as many additional r-proteins and r-protein extensions (Figs. 1C–E and ​and2C–E).2C–E). Compared with the ∼4500 nucleotides of rRNA and 54 r-proteins of the bacterial 70S ribosome, eukaryotic 80S ribosomes contain >5500 nucleotides of rRNA (SSU, 18S rRNA; LSU, 5S, 5.8S, and 25S rRNA) and 80 (79 in yeast) r-proteins. The first structural models for the eukaryotic (yeast) ribosome were built using 15-Å cryo–electon microscopy (cryo-EM) maps fitted with structures of the bacterial SSU (Wimberly et al. 2000) and archaeal LSU (Ban et al. 2000), thus identifying the location of a total of 46 eukaryotic r-proteins with bacterial and/or archaeal homologs as well as many ES (Spahn et al. 2001a). Subsequent cryo-EM reconstructions led to the localization of additional eukaryotic r-proteins, RACK1 (Sengupta et al. 2004) and S19e (Taylor et al. 2009) on the SSU and L30e (Halic et al. 2005) on the LSU, as well as more complete models of the rRNA derived from cryo-EM maps of canine and fungal 80S ribosomes at ∼9 Å (Chandramouli et al. 2008; Taylor et al. 2009). Recent cryo-EM reconstructions of plant and yeast 80S translating ribosomes at 5.5–6.1 Å enabled the correct placement of an additional six and 10 r-proteins on the SSU and LSU, respectively, as well as the tracing of many eukaryotic-specific r-protein extensions (Armache et al. 2010a,b). The full assignment of the r-proteins in the yeast and fungal 80S ribosomes, however, only became possible with the improved resolution (3.0–3.9 Å) resulting from the crystal structures of the SSU and LSU from Tetrahymena thermophila (Klinge et al. 2011; Rabl et al. 2011) and the Saccharomyces cerevisiae 80S ribosome (Figs. ​(Figs.1D,E1D,E and ​and2D,E)2D,E) (Ben-Shem et al. 2011).     

High-Resolution Cryo-Electron Microscopy Structures of Murine Norovirus 1 and Rabbit Hemorrhagic Disease Virus Reveal Marked Flexibility in the Receptor Binding Domains

Our previous structural studies on intact, infectious murine norovirus 1 (MNV-1) virions demonstrated that the receptor binding protruding (P) domains are lifted off the inner shell of the virus. Here, the three-dimensional (3D) reconstructions of recombinant rabbit hemorrhagic disease virus (rRHDV) virus-like particles (VLPs) and intact MNV-1 were determined to ∼8-Å resolution. rRHDV also has a raised P domain, and therefore, this conformation is independent of infectivity and genus. The atomic structure of the MNV-1 P domain was used to interpret the MNV-1 reconstruction. Connections between the P and shell domains and between the floating P domains were modeled. This observed P-domain flexibility likely facilitates virus-host receptor interactions.Murine norovirus 1 (MNV-1) (3, 14, 15) and rabbit hemorrhagic disease virus (RHDV) are members of the genera Norovirus and Lagovirus of the family Caliciviridae that offer a comparison to recombinant human norovirus (rNV) virus-like particles (VLPs) for assessing the structures and roles of domains within the capsid proteins of this family of viruses. Calicivirus particles contain 180 copies of the 56- to 76-kDa major capsid protein (Orf2), which is comprised of the internal/buried N terminus (N), shell (S), and protruding (P) domains (9, 10). The S domain, an eight-stranded β-barrel, forms an ∼300-Å contiguous shell around the RNA genome. A flexible hinge connects the shell to a “protruding” (P) domain at the C-terminal half of the capsid protein, which can be further divided into a globular head region (P2) and a stem region (P1) that connects the shell domain to P2. The accompanying article (13) describes the determination of the structure of the P domain of MNV-1 to a resolution of 2.0 Å.We recently determined the cryo-transmission electron microscopy (TEM) structure of MNV-1 to ∼12-Å resolution (4) and found that, compared to rNV VLPs (10) and San Miguel sea lion virus (SMSV) (1, 2), the protruding domains are rotated by ∼40° in a clockwise fashion and lifted up by ∼16 Å. To better understand the unusual conformation of MNV-1 and whether it is unique to this particular member of the calicivirus family, the ∼8-Å cryo-TEM structures of infectious MNV-1 and the VLPs of RHDV were determined.MNV-1 was produced as previously described (4). Three liters of cell culture yielded 0.5 to 1.0 mg of purified virus with a particle/PFU ratio of less than 100. Baculovirus expression and purification of recombinant RHDV (rRHDV) VLPs were performed as previously described (8). Cryo-electron microscopy (EM) data were collected at the National Resource for Automated Molecular Microscopy (NRAMM) facility in San Diego, CA (4). Images were collected at a nominal magnification of ×50,000 at a pixel size of 0.1547 nm at the specimen level using Leginon software (12) and processed with Appion software (5). The contrast transfer function for each set of particles from each image was estimated and corrected using ACE2 (a variation of ACE [7]). Particle images were automatically selected (11). The final stacks of particle images contained 20,425 MNV virions and 7,856 rRHDV VLPs, and EMAN 3D (6) was used for the reconstructions. Resolutions were estimated by Fourier shell correlations (FSC) of the three-dimensional (3D) reconstructions and application of a cutoff of 0.5. An amplitude correction of the final electron density was performed using GroEL small-angle X-ray scattering (SAXS) data.3D reconstructions of MNV-1 and rRHDV were calculated to resolutions of 8 Å and 8.1 Å, respectively (Fig. ​(Fig.1).1). The P domains of rNV VLPs rest directly on top of the shell domain (10) (Fig. ​(Fig.1A).1A). In contrast, the P domains of MNV-1 are lifted and rotated above the shell of the capsid (4) (Fig. ​(Fig.1B).1B). At this higher resolution, there was a clear connection between the P1 domain and the shell domain in all three capsid subunits (Fig. ​(Fig.1B,1B, arrow A). Unlike the smooth protruding domains of rNV, MNV-1 has two clear “horns” (arrow B), not dissimilar to those observed for the sapoviruses (1, 2). There also are islands of density in the interior of the shell, directly beneath the 5-fold axes, that may represent ordered regions of RNA.Open in a separate windowFIG. 1.Stereo diagrams (left) and thin sections (right), with radius coloring, of rNV (A), MNV-1 (B), and an rRHDV VLP (C). For rNV, the atomic coordinates (10) were used. In MNV, arrow A indicates the thin connector between the P1 and S domains. Arrow B denotes the horns found at the tips of the P2 domains. Arrow C denotes the large gap between the P1 and S domains in the rRHDV VLP. Arrow D denotes the false connectivity in rRHDV VLPs between the P1 domain and the S domain near the 5-fold axes.As with MNV-1, there is a marked gap between the P and S domains in the rRHDV VLP (Fig. ​(Fig.1C,1C, arrow C). This gap is not as pronounced as in MNV-1 because the P domains are not rotated as in MNV-1. In this electron density map, the A/B dimers appear to be touching the shell domain near the 5-fold axes. This contact difference between the A/B dimers and the C/C dimers could be the reason why the tops of the C/C dimers appear to be markedly disordered compared to the A/B dimers in rRHDV and the C/C dimers in MNV-1.Shown in Fig. ​Fig.22 is the fitting of the atomic structures of the MNV-1 P domain (13) and the rNV S domains into the MNV-1 3D reconstruction electron density. The horns (arrow A, loops A′-B′ and E′-F′) observed at the tips of the P domain match exceedingly well with the electron density. As discussed in the accompanying publication (13), the A′-B′ and E′-F′ loops displayed two discrete conformations, a closed structure, where the two loops were tightly associated, and an open structure, where the loops were splayed apart. The horns of the closed conformation fit better into the reconstruction, as the E′-F′ loop in the open form jutted out of the density at the base of the horns. The unmodified density in the lower panel of Fig. ​Fig.22 shows fine features in the shell domain and a very clear connection between the shell and P1 domains. The connections between the P1 and S domains were of sufficient quality to build a basic backbone model by uncoiling the linker region (arrow B). The P domain in the unfiltered 3D reconstruction was far less ordered than the S domain (Fig. ​(Fig.2).2). This was likely due to movement of the entire P domain with respect to the shell.Open in a separate windowFIG. 2.Fitting of the MNV-1 P domain and the rNV shell domain into the MNV-1 electron density. A, B, and C subunits are represented by blue, green, and red, respectively. The electron density is shown in transparent gray. The top panel is the 8.0-Å-resolution 3D reconstruction modified using a low-pass filter. The bottom panel is the reconstruction without modification. The horns on the tops of the P domains are denoted by arrow A. Arrow B denotes the connection between the S and P domains.Using the structure of rNV VLP P domains for modeling, the rRHDV P domains are lifted off the surface of the shell, but not rotated as with MNV-1. This places the bottom edge of the A subunit P1 domain near the S domain at the 5-fold axes. The P-domain dimers of rNV and rRHDV have a more “arch-like” shape than MNV-1. Unlike in MNV-1, the electron densities of the C/C dimers in rRHDV are far more diffuse than those of the A/B dimers (Fig. ​(Fig.3B)3B) and the connector between the S and P1 domains is not clear. During fitting, the connector region was not as extended as with MNV-1. This may afford greater flexibility, leading to more diffuse electron density.Open in a separate windowFIG. 3.Fitting of the rNV atomic structure into the rRHDV VLP electron density. The upper stereo image shows the 8.1-Å-resolution 3D reconstruction after modification by a low-pass filter. Below is the same reconstruction prior to density modification.When the atomic models for the MNV-1 P domains (13) were placed into the cryo-TEM electron density (Fig. ​(Fig.4),4), the C termini extended deep into the cores of adjacent P domains. Possible connections not accounted for by the P-domain structures were also observed in the electron density between the P domains. A bulge between the P1 and P2 domains in the 3D reconstruction indicated a possible interaction between the C termini and the adjacent P domains. These same interactions were observed in the crystal lattice. This highly mobile C terminus may be a flexible tether between the P domains in the intact virion.Open in a separate windowFIG. 4.Possible carboxyl-terminus interactions between the P domains of MNV-1. (A) Stereo image of MNV-1 calculated to 12-Å resolution with (red) and without (yellow) the last 10 residues of the P domain. (B) The calculated MNV-1 density with the carboxyl terminus removed (yellow) overlaid onto the 3D reconstruction of MNV-1 (blue). Note the strands of difference density that roughly correspond to the C terminus in panel A. (C) The C-terminus interactions observed in the structure of the MNV-1 P domains. Shown in blue and green are ribbon diagrams of an A/B P-domain dimer. In mauve is a surface rendering of the C terminus from a crystallographically related dimer. (D) Surface rendering of the final MNV-1 model with possible interactions between the P domains in MNV-1. The carboxyl termini of the A subunits (blue) interact with the counterclockwise-related B subunits around the 5-fold axes (white arrows). Around the 3-fold (quasi-6-fold) axes, the C subunits interact with the A subunits and the B subunits interact with the C subunits (orange arrows).It is absolutely clear that the hinge region between the S and P domains affords a remarkable degree of flexibility in the P domains that is not genus specific or related to differences between rVLPs and authentic virions. The simplest explanation for the role of this transition is that it gives the P domains flexibility that may be used to optimize interactions with cell receptors during attachment and entry. In this way, the P domains can increase their avidity for the cell surface by being more facile in adapting to the presentation of cellular recognition motifs.     

Spotlight on Zebrafish: Translational Impact

In recent years, the zebrafish has emerged as an increasingly prominent model in biomedical research. To showcase the translational impact of the model across multiple disease areas, Disease Models & Mechanisms has compiled a Special Issue that includes thought-provoking reviews, original research reporting new and important insights into disease mechanisms, and novel resources that expand the zebrafish toolkit. This Editorial provides a summary of the issue’s contents, highlighting the diversity of zebrafish disease models and their clinical applications.Open in a separate windowE. Elizabeth PattonOpen in a separate windowJames F. AmatrudaOpen in a separate windowLalita Ramakrishnan     

CLM1 of Fusarium graminearum Encodes a Longiborneol Synthase Required for Culmorin Production

Fusarium graminearum is a fungal pathogen of cereal crops (e.g., wheat, barley, maize) and produces a number of mycotoxins, including 15-acetyldeoxynivalenol, butenolide, zearalenone, and culmorin. To identify a biosynthetic gene for the culmorin pathway, an expressed-sequence-tag database was examined for terpene cyclase genes. A gene designated CLM1 was expressed under trichothecene-inducing conditions. Expression of CLM1 in yeast (Saccharomyces cerevisiae) resulted in the production of a sesquiterpene alcohol, longiborneol, which has the same ring structure as culmorin. Gene disruption and add-back experiments in F. graminearum showed that CLM1 was required for culmorin biosynthesis. CLM1 gene disruptants were able to convert exogenously added longiborneol to culmorin. Longiborneol accumulated transiently in culmorin-producing strains. The results indicate that CLM1 encodes a longiborneol synthase and is required for culmorin biosynthesis in F. graminearum.Fusarium graminearum Schwabe produces a number of mycotoxins, including the sesquiterpenoid trichothecene, 15-acetyldeoxynivalenol (15ADON) (Fig. ​(Fig.1).1). The TRI5 gene encodes trichodiene synthase, a terpene cyclase which converts farnesyl pyrophosphate to trichodiene, the hydrocarbon precursor of trichothecenes (12). Fusarium cultures often produce additional nontrichothecene sesquiterpenes. Some of these, including sambucinol and apotrichodiol (Fig. ​(Fig.1),1), lack the epoxide moiety characteristic of trichothecenes but are likely derived from trichodiene. Two Fusarium sesquiterpenes that are not products of trichodiene are cyclonerodiol and culmorin (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Chemical structures of 15ADON, sambucinol, cyclonerodiol, apotrichodiol, culmorin, longiborneol, and longifolene.Culmorins are tricyclic sesquiterpene diols that have been reported from Fusarium culmorum, F. graminearum, Fusarium venenatum, and the marine ascomycete Leptosphaeria oraemaris (17, 32). Although culmorin was not toxic in some animal and cell systems, nor mutagenic in the Ames test (25), it has antifungal activity (32). Dowd (6) reported that culmorin enhanced the toxicity of deoxynivalenol to insects, impacting both growth and mortality. Culmorin has some phytotoxicity to wheat coleoptile tissue (37), but its role in wheat head scab is not known. Contaminated grain samples are not commonly screened for culmorins. However, Ghebremeskel and Langseth (8) found that naturally contaminated Norwegian wheat, barley, and oat samples with high deoxynivalenol concentrations also contained culmorin and hydroxyculmorins at relatively high levels.In order to determine if culmorin is a virulence factor, we first looked for a culmorin biosynthetic terpene cyclase gene to disrupt. No culmorin biosynthetic genes have been previously identified. A candidate terpene cyclase gene, fg10397, was found in an F. graminearum expressed sequence tag (EST) database that had been previously used to identify butenolide and trichothecene biosynthetic genes (11, 20). Here, we report the characterization of the terpene cyclase gene CLM1. Time course, gene disruption, and add-back experiments, as well as expression in Saccharomyces cerevisiae, indicated that CLM1 encodes longiborneol synthase that is required for the synthesis of culmorin (Fig. ​(Fig.11).