Mechanism of foreign DNA recognition by a CRISPR RNA-guided surveillance complex from Pseudomonas aeruginosa

The Type I-F CRISPR-mediated (clustered regularly interspaced short palindromic repeats) adaptive immune system in Pseudomonas aeruginosa consists of two CRISPR loci and six CRISPR-associated (cas) genes. Foreign DNA surveillance is performed by a complex of Cas proteins (Csy1–4) that assemble with a CRISPR RNA (crRNA) into a 350-kDa ribonucleoprotein called the Csy complex. Here, we show that foreign nucleic acid recognition by the Csy complex proceeds through sequential steps, initiated by detection of two consecutive guanine–cytosine base pairs (G–C/G–C) located adjacent to the complementary DNA target. We show that this motif, called the PAM (protospacer adjacent motif), must be double-stranded and that single-stranded PAMs do not provide significant discriminating power. Binding assays performed with G–C/G–C-rich competitor sequences indicate that the Csy complex interacts directly with this dinucleotide motif, and kinetic analyses reveal that recognition of a G–C/G–C motif is a prerequisite for crRNA-guided binding to a target sequence. Together, these data indicate that the Csy complex first interacts with G–C/G–C base pairs and then samples adjacent target sequences for complementarity to the crRNA guide.     

Structures and mechanisms of CRISPR RNA-guided effector nucleases

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Multiple adaptations underly co-option of a CRISPR surveillance complex for RNA-guided DNA transposition

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Cells depleted of mitochondrial DNA (rho0) yield insight into physiological mechanisms.

A resurgence of interest in mitochondrial physiology has recently developed as a result of new experimental data demonstrating that mitochondria function as important participants in a diverse collection of novel intracellular signaling pathways. Cells depleted of mitochondrial DNA, or rho0 cells, lack critical respiratory chain catalytic subunits that are encoded in the mitochondrial genome. Although rho0 cells contain petit mitochondria, they cannot support normal oxidative phosphorylation and must survive and replicate using ATP derived solely from glycolysis. Without a functional electron transport chain, rho0 cells cannot normally regulate redox potential and their mitochondria appear to be incapable of generating reactive oxygen species. Emerging evidence suggests that these signals are important components in a number of mitochondria-initiated signaling pathways. The present article focuses on how rho0 cells have contributed to an understanding of the role that mitochondria play in distinct physiological pathways involved with apoptosis, glucose-induced insulin secretion, and oxygen sensing.     

New insight into the molecular mechanisms of two-partner secretion

Two-partner secretion (TPS) systems, which export large proteins to the surface and/or extracellular milieu of Gram-negative bacteria, are members of a large superfamily of protein translocation systems that are widely distributed in animals, plants and fungi, in addition to nearly all groups of Gram-negative bacteria. Recent intense research on TPS systems has provided new insight into the structure and topology of the outer membrane translocator proteins and the large exoproteins that they secrete, the interactions between them, and mechanisms for retention of some of the secreted proteins on the bacterial surface. Evidence for secretion-dependent folding of mature exoproteins has also been obtained. Together, these findings provide a deeper understanding of the molecular mechanisms underlying these simple but elegant secretion systems.     

Structural insight into mechanisms for dynamic regulation of PKM2

Pyruvate kinase isoform M2 (PKM2) converts phosphoenolpyruvate (PEP) to pyruvate and plays an important role in cancer metabolism. Here, we show that posttranslational modifications and a patient-derived mutation regulate pyruvate kinase activity of PKM2 through modulating the conformation of the PKM2 tetramer. We determined crystal structures of human PKM2 mutants and proposed a “seesaw” model to illustrate conformational changes between an inactive T-state and an active R-state tetramers of PKM2. Biochemical and structural analyses demonstrate that PKM2^Y105E (phosphorylation mimic of Y105) decreases pyruvate kinase activity by inhibiting FBP (fructose 1,6-bisphosphate)-induced R-state formation, and PKM2^K305Q (acetylation mimic of K305) abolishes the activity by hindering tetramer formation. K422R, a patient-derived mutation of PKM2, favors a stable, inactive T-state tetramer because of strong intermolecular interactions. Our study reveals the mechanism for dynamic regulation of PKM2 by posttranslational modifications and a patient-derived mutation and provides a structural basis for further investigation of other modifications and mutations of PKM2 yet to be discovered.     

Transgenic plants: An insight into oxidative stress tolerance mechanisms

Environmental stresses considerably limit plant productivity. At the molecular level the negative effect of stress is often mediated by reactive oxygen species-initiated oxidative damage. Hence, it was hypothesised that increased tolerance to several environmental constraints could be achieved through enhanced tolerance to oxidative stress. In recent years much effort has been undertaken to improve oxidative stress tolerance by transforming plants with native or bacterial genes coding either for reactive oxygen species-scavenging enzymes or for enzymes modulating the cellular antioxidant capacity. This review deals with data on transgenic plants with altered antioxidant capacity and focuses on the new insight into the antioxidant defence mechanism given by this type of experimental model.     

Structural insight into the mechanisms of Wnt signaling antagonism by Dkk

Dickkopf (Dkk) proteins are antagonists of the canonical Wnt signaling pathway and are crucial for embryonic cell fate and bone formation. Wnt antagonism of Dkk requires the binding of the C-terminal cysteine-rich domain of Dkk to the Wnt coreceptor, LRP5/6. However, the structural basis of the interaction between Dkk and low density lipoprotein receptor-related protein (LRP) 5/6 is unknown. In this study, we examined the structure of the Dkk functional domain and elucidated its interactions with LRP5/6. Using NMR spectroscopy, we determined the solution structure of the C-terminal cysteine-rich domain of mouse Dkk2 (Dkk2C). Then, guided by mutagenesis studies, we docked Dkk2C to the YWTD beta-propeller domains of LRP5/6 and showed that the ligand binding site of the third LRP5/6 beta-propeller domain matches Dkk2C best, suggesting that this domain binds to Dkk2C with higher affinity. Such differential binding affinity is likely to play an essential role in Dkk function in the canonical Wnt pathway.     

Structural insight into the substrate specificity of DNA Polymerase mu

DNA polymerase mu (Pol mu) is a family X enzyme with unique substrate specificity that contributes to its specialized role in nonhomologous DNA end joining (NHEJ). To investigate Pol mu's unusual substrate specificity, we describe the 2.4 A crystal structure of the polymerase domain of murine Pol mu bound to gapped DNA with a correct dNTP at the active site. This structure reveals substrate interactions with side chains in Pol mu that differ from other family X members. For example, a single amino acid substitution, H329A, has little effect on template-dependent synthesis by Pol mu from a paired primer terminus, but it reduces both template-independent and template-dependent synthesis during NHEJ of intermediates whose 3' ends lack complementary template strand nucleotides. These results provide insight into the substrate specificity and differing functions of four closely related mammalian family X DNA polymerases.     

An insight into the structure of DNA through melting studies

Helix-coil transition of calf thymus (CT) DNA in 0.1 M NaCl was observed under different environmental conditions for studying structural and conformational changes, if any. Pre-exposure of the DNA to different types of radiations - UV and gamma-brought different degrees of change (lowering) in the melting temperature (Tm). Interaction of drugs (daunomycin and actinomycin-D) increased the Tm inducing structural stability. There was 50% more destabilisation in case of polynucleotide-drug complex as compared to DNA-drug complex exposed to UV radiation. Thermodynamic studies on DNA in D2O (0.1 M NaCl prepared in D2O) were carried out and compared with the corresponding results in H2O. Presence of D2O increased the stability of the DNA structure. Structural and conformational aspects have been discussed in the light of the results obtained.     

New insight into the biochemical mechanisms regulating auxin transport in plants

The transport of the plant hormone auxin has been under intense investigation since its identification 80 years ago. Studies have gradually refined our understanding of the importance of auxin transport in many aspects of plant signalling and development, and the focus has intensified in recent years towards the identification of the proteins involved in auxin transport and their functional mechanism. Within the past 18 months, the field has progressed rapidly, with confirmation that several distinct classes of proteins, previously dubbed as 'putative auxin permeases' or 'auxin transport facilitators', are bona fide transporters of IAA (indol-3-ylacetic acid). In this review we will appraise the recent transport data and highlight likely future research directions, including the characterization of auxiliary proteins necessary for the regulation of auxin transporters.     

Increasing insight into induced plant defense mechanisms using elicitors and inhibitors

One of the strategies that plants employ to defend themselves against herbivore attack is the induced production of carnivore-attracting volatiles. Using elicitors and inhibitors of different steps of the signal-transduction pathways can improve our understanding of the mechanisms underlying induced plant defenses. For instance, we recently showed that application of jasmonic acid, a key hormone in the octadecanoid pathway involved in herbivore-induced defense, to Brassica oleracea affects gene expression, hormone levels, and volatile emission, as well as oviposition by herbivores and host location behavior by parasitoids. Such defense responses vary with the dose of the elicitor and with time since application. This addendum describes how the use of inhibitors, in addition to the use of elicitors like jasmonic acid, can be applied in bio-assays to investigate the role of signal-transduction pathways involved in induced plant defense. We show how inhibition of different steps of the octadecanoid pathway affects host location behavior by parasitoids.Key words: Brassica oleracea, Cotesia glomerata, elicitor, herbivore-induced plant defense, inhibitor, jasmonic acid, octadecanoid pathway, phenidone, propyl gallate, diethyldithiocarbamic acid (DIECA)Chemical information plays an important role in the interactions between plants and insects. When a plant is damaged, it can respond with the production of specific volatiles and toxins.¹ Insects associated with these plants can use the resulting chemical information to find their host plants and to determine the suitability of a plant for feeding or oviposition. Application of chemicals acting as elicitors can be used to mimic such plant responses, while knowledge of the signal transduction pathways involved can be used to select potential inhibitors of induced plant response. Compared to exposing plants to herbivores, the application of elicitors and inhibitors allows for manipulation of defined steps in signal-transduction pathways, as well as to induce plants in a dose-controlled manner.² However, also with elicitors and inhibitors it is often difficult to link the applied dose to the strength of the induction of the plant, as the plant may use alternatives routes to express certain traits and the manipulation can result in unwanted effects on other processes in the plant, such as flowering or senescence.^3,4 Hence, experiments using elicitors or inhibitors should preferably use rather short incubation times (hours to days), to avoid developmental differences due to treatment.⁵JA is a key hormone in the octadecanoid pathway, involved in direct as well as indirect plant defenses against herbivores. Application of this phytohormone is known to mediate induction of volatile emission, increase toxin levels and to upregulate defense gene expression. In turn, the changes in these plant traits affect members of the insect community associated with the plant and may result in higher parasitism rates of herbivores, attraction of predators, and reduced oviposition and development of herbivores.^6–12JA is often used to mimic herbivory in studies on induced plant responses. However, recent studies on JA-application to e.g., Brassica oleracea var. gemmifera L. (Brussels sprouts) also indicate that the JA-induced volatile emission differs from volatile emission induced by herbivores.¹² More nectar was secreted by flowers of herbivore-infested Brassica nigra L. (black mustard) than by flowers from JA-induced plants.⁶ The intensity of the behavioral responses of herbivores and parasitoids differs between JA- and herbivore-induced plants, but compared to non-induced plants, both treatments are favored by parasitoids on both Brussels sprouts and black mustard plants,^6,12 while Pieris butterflies avoid oviposition on induced Brussels sprouts plants.¹¹ The results indicate that JA-mediated responses do play an important role in plant defense against herbivorous insects, and can be used to induce defense responses in many plant species. However, cross-talk with other phytohormones, as well as visual cues will also affect plant defense responses.While JA application induces the octadecanoid pathway, inhibitors of steps in this pathway are also available (Fig. 1). This approach allows including visual cues of feeding damage while eliminating or reducing chemical cues. Three inhibitors of different steps of the octadecanoid pathway are phenidone (1-phenyl-pyrazolidinone), DIECA (diethyldithiocarbamic acid) and n-propyl gallate (3,4,5-trihydroxybenzoic acid propyl ester; all obtained from Sigma-Aldrich, St. Louis, MO; Fig. 1). The redox-active compound phenidone is known to inhibit the activity of lipoxygenases (LOXs),^13–15 by reducing the active form of LOX to an inactive form. Therefore, phenidone is an effective inhibitor of an early step in the octadecanoid pathway, and thus of the plant’s induced defense system.^16,17 DIECA reduces 13-hydroperoxylinolenic acid to its corresponding alcohol 13-hydroxylinolenic acid, which is not a signaling intermediate and cannot be converted into JA.^18–20 Propyl gallate is a less specific inhibitor inhibiting both LOX and allene oxide cyclase (AOC), an enzyme catalyzing the step to 12-oxo-phytodienoic acid (OPDA) in the octadecanoid pathway.^14,21,22 We investigated the effects of these three inhibitors on herbivore-induced parasitoid attraction. For all three inhibitors 2 mM aqueous solutions with 0.1% Tween were applied to the plants.Open in a separate windowFigure 1Representation of the octadecanoid pathway with indication of which step of the signal-transduction pathway is affected by the different elicitors (+) and inhibitors (−).The response of the parasitoid Cotesia glomerata was tested to Pieris brassicae-infested plants (15 2^nd instar larvae) treated with inhibitor solution, Pieris brassicae-infested plants treated with a solution without inhibitor or intact plants sprayed with inhibitor solution. Recently, Bruinsma et al.¹⁷ showed that Pieris brassicae- infested plants treated with phenidone were less attractive to C. glomerata than infested plants treated with control solution (binomial test, N = 42, p = 0.008, Fig. 2). However, infested plants treated with phenidone were still more attractive than intact plants sprayed with phenidone (binomial test, N = 39, p < 0.001, Fig. 2). Thus, phenidone did reduce the induction of parasitoid attractants, but did not eliminate the induction completely. Here, we present additional experiments with the inhibitors DIECA and propyl gallate. DIECA application shows similar results as phenidone application; infested plants treated with DIECA are less attractive to C. glomerata than infested plants treated with control solution, but are more attractive than uninfested plants treated with DIECA (binomial test, N = 46, p = 0.026 and N = 26, p < 0.001, respectively, Fig. 2). Treatment with propyl gallate resulted in lower attractiveness of infested inhibitor-treated plants compared to infested control plants, but not significantly so (binomial test, N = 45, p = 0.072, Fig. 2), and propyl gallate-treated infested plants were more attractive than propyl gallate-treated intact plants (binomial test, N = 28, p < 0.001; Fig. 2). Summarizing, phenidone and DIECA treatment of Brussels sprouts plants resulted in a reduced attractiveness of caterpillar-infested B. oleracea plants to C. glomerata. Although propyl gallate-treated plants also attracted fewer parasitoids, this difference was marginally insignificant. Of the three inhibitors, the LOX inhibitor phenidone had the largest effect on the attraction of the parasitoid C. glomerata.Open in a separate windowFigure 2Attraction of Cotesia glomerata to plants sprayed with the inhibitors phenidone, DIECA, or propyl gallate, or sprayed with a control solution, with or without infestation with Pieris brassicae. Numbers to the left of the bars indicate the total number of parasitoids tested, numbers between brackets the number of parasitoids that landed on a plant (binomial test, ***p < 0.001, **p < 0.01, *p < 0.05).Our data show that both elicitors and inhibitors can be used in bio-assays to demonstrate the importance of certain steps in defense pathways.^5,23 Although application of the inhibitors to herbivore-infested plants did not abolish the response of the plants and the parasitoids still preferred them over non-induced plants, the inhibition of the octadecanoid pathway did reduce the attractiveness of the plants to the parasitoids. This implies that the octadecanoid pathway is involved in attracting parasitoids, but it is not the only factor determining parasitoid host location. This shows that use of inhibitors can provide interesting opportunities to comparatively investigate ecological interactions of genetically identical plants that differ in the degree of defense expression. Integrating knowledge on mechanisms with studies on ecological interactions and applying this to studies of increasingly complex interactions will further promote the understanding of induced defense in a community ecology context.^24,25     

Structural insight into substrate specificity and regulatory mechanisms of phosphoinositide 3-kinases

Phosphoinositide 3-kinases (PI3Ks) are implicated in a variety of fundamental cellular processes. These enzymes catalyse phosphorylation of the 3'-OH position of myo-inositol lipids that serve as secondary messengers. The catalytic subunit for one of the family members, PI3K gamma, has been structurally characterized, independently, in complexes with kinase inhibitors and with the p21(Ras) GTPase. These atomic structures provide a basis for the rationalization of some PI3K substrate specificities and regulatory mechanisms, establishing links to functional and cellular data. Ongoing comprehensive structural and functional studies are essential to realize the promise of PI3K isozyme-specific therapeutic agents.     

Recent insight into the pathogenicity mechanisms of the emergent pathogen Photorhabdus asymbiotica

Photorhabdus asymbiotica is unique among the entomopathogenic bacteria of this genus in also being able to infect humans, leading to its isolation from some clinical samples. Recent comparative genomics data and the results of studies of interactions between bacteria and cells provide insight into the adaptation of this bacterium to its new niche, the human body.     

Optimization of delivery of foreign DNA into higher-plant chloroplasts

We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of -glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells. Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity. No GUS activity was detected in cells bombarded with pUC118 or pBI101.3. Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants. pHD203-GUS was expressed exclusively in chloroplasts. We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation. Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device. Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate. Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium. This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before.     

Structural basis of target DNA recognition by CRISPR-Cas12k for RNA-guided DNA transposition

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