Functional screening of a soil metagenome for DNA endonucleases by acquired resistance to bacteriophage infection

Endonucleases play a crucial role as reagents in laboratory research and diagnostics. Here, metagenomics was used to functionally screen a fosmid library for endonucleases. A fosmid library was constructed using metagenomic DNA isolated from soil sampled from the unique environment of the Kogelberg Nature Reserve in the Western Cape of South Africa. The principle of acquired immunity against phage infection was used to develop a plate-based screening technique for the isolation of restriction endonucleases from the library. Using next-generation sequencing and bioinformatics tools, sequence data were generated and analysed, revealing 113 novel open reading frames (ORFs) encoding putative endonuclease genes and ORFs of unknown identity and function. One endonuclease designated Endo52 was selected from the putative endonuclease ORFs and was recombinantly produced in Escherichia coli Rosetta? (DE3) pLysS. Endo52 was purified by immobilised metal affinity chromatography and yielded 0.437 g per litre of cultivation volume. Its enzyme activity was monitored by cleaving lambda DNA and pUC19 plasmid as substrates, and it demonstrated non-specific endonuclease activity. In addition to endonuclease-like genes, the screen identified several unknown genes. These could present new phage resistance mechanisms and are an opportunity for future investigations.

     

Iron(III) protoporphyrin IX complexes of the antimalarial Cinchona alkaloids quinine and quinidine

The antimalarial properties of the Cinchona alkaloids quinine and quinidine have been known for decades. Surprisingly, 9-epiquinine and 9-epiquinidine are almost inactive. A lack of definitive structural information has precluded a clear understanding of the relationship between molecular structure and biological activity. In the current study, we have determined by single crystal X-ray diffraction the structures of the complexes formed between quinine and quinidine and iron(III) protoporphyrin IX (Fe(III)PPIX). Coordination of the alkaloid to the Fe(III) center is a key feature of both complexes, and further stability is provided by an intramolecular hydrogen bond formed between a propionate side chain of Fe(III)PPIX and the protonated quinuclidine nitrogen atom of either alkaloid. These interactions are believed to be responsible for inhibiting the incorporation of Fe(III)PPIX into crystalline hemozoin during its in vivo detoxification. It is also possible to rationalize the greater activity of quinidine compared to that of quinine.     

A conserved interdomain interaction is a determinant of folding cooperativity in the GST fold

The canonical glutathione transferase (GST) fold found in many monomeric and dimeric proteins consists of two domains that differ in structure and conformational dynamics. However, no evidence exists that the two domains unfold/fold independently at equilibrium, indicating the significance of interdomain interactions in governing cooperativity between domains. Bioinformatics analyses indicate the interdomain interface of the GST fold is large, predominantly hydrophobic with a high packing density explaining cooperative interdomain behavior. Structural alignments reveal a topologically conserved lock-and-key interaction across the domain interface in which a bulky hydrophobic residue ("key") protrudes from the surface of the N-domain and inserts into a pocket ("lock") in the C-domain. To better understand the molecular basis for the contribution of interdomain interactions toward cooperativity within the GST fold in the absence of any influence from quaternary interactions, studies were done with two monomeric GST proteins: Escherichia coli Grx2 (EcGrx2) and human CLIC1 (hCLIC1). Replacing the methionine "key" residue with alanine is structurally nondisruptive, whereas it significantly diminishes the folding cooperativity of both proteins. The loss in cooperativity between domains in the mutants is reflected by a change in the equilibrium folding mechanism from a wild-type two-state process to a three-state process, populating a stable folding intermediate.     

Stability of the domain interface contributes towards the catalytic function at the H-site of class alpha glutathione transferase A1-1

Cytosolic glutathione transferases (GSTs) are major detoxification enzymes in aerobes. Each subunit has two distinct domains and an active site consisting of a G-site for binding GSH and an H-site for an electrophilic substrate. While the active site is located at the domain interface, the role of the stability of this interface in the catalytic function of GSTs is poorly understood. Domain 1 of class alpha GSTs has a conserved tryptophan (Trp21) in helix 1 that forms a major interdomain contact with helices 6 and 8 in domain 2. Replacing Trp21 with an alanine is structurally non-disruptive but creates a cavity between helices 1, 6 and 8 thus reducing the packing density and van der Waals contacts at the domain interface. This results in destabilization of the protein and a marked reduction in catalytic activity. While functionality at the G-site is not adversely affected by the W21A mutation, the H-site becomes more accessible to solvent and less favorable for the electrophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB). Not only does the mutation result in a reduction in the energy for stabilizing the transition state formed in the S_NAr reaction between the substrates GSH and CDNB, it also compromises the ability of the enzyme to form and stabilize a transition state analogue (Meisenheimer complex) formed between GSH and 1,3,5-trinitrobenzene (TNB). The study demonstrates that the stability of the domain–domain interface plays a role in mediating the catalytic functionality of the active site, particularly the H-site, of class alpha GSTs.     

From the Neotropics to the Namib: evidence for rapid ecological divergence following extreme long‐distance dispersal

Extreme long‐distance dispersal is an important process in plant biogeography. Such events can lead to rapid diversification due to founder effects, genetic drift and novel selection in recipient environments. Balloon vines (Cardiospermum spp.) are mainly Neotropical, but include two native southern African species, the endemic desert‐adapted C. pechuelii and the moist subtropical C. corindum (which also occurs in the Neotropics). We used phylogenetic approaches (internal transcribed spacer (ITS), rpl32 and trnL‐trnF DNA sequencing data) and population genetics (amplified fragment length polymorphism (AFLP) analyses) to confirm the long‐distance dispersal of C. corindum to southern Africa and to reveal the subsequent divergence of the morphologically and ecologically extreme but genetically close C. pechuelii. We could not judge whether incongruences between ecological requirements and morphology and gene trees for the African species resulted from ongoing gene flow or incomplete lineage sorting, but our findings do support recent divergence of C. pechuelii from C. corindum in Africa following transoceanic dispersal of the lineage. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 477–486.     

Major and minor groups in evolution

Kerr and Godfrey-Smith argue that two mathematically equivalent, alternative formal representations drawn from population genetics, the contextualist and collectivist formalisms, may be equally good for quantifying the dynamics of some natural systems, despite important differences between the formalisms. I draw on constraints on causal representation from Woodward (Making things happen, Oxford University Press, New York, 2003) and Eberhardt and Scheines (Philos Sci 74(5):981–995, 2006) to argue that one or the other formalism will be superior for arbitrary natural systems in which individuals form different types of groups.     

Darwin, Herschel, and the role of analogy in Darwin’s origin

In what follows, I consider the role of analogy in the first edition of Darwin’s Origin. I argue that Darwin follows Herschel’s methodology and hence exploits an analogy between artificial and natural selection that allows him generalize selection as a cause of evolutionary change. This argument strategy is not equivalent to an argument from analogy. Reading Darwin’s argument as conforming to Herschel’s two-step methodology of causal analysis followed by generalization allows us to understand the role and placement of Darwin’s discussion of artificial selection in the Origin, without making the mistake of portraying Darwin’s argument for the existence and character of natural selection as an analogical argument.     

Righteous modeling: the competence of classical population genetics

In a recent article, ??Wayward Modeling: Population Genetics and Natural Selection,?? Bruce Glymour claims that population genetics is burdened by serious predictive and explanatory inadequacies and that the theory itself is to blame. Because Glymour overlooks a variety of formal modeling techniques in population genetics, his arguments do not quite undermine a major scientific theory. However, his arguments are extremely valuable as they provide definitive proof that those who would deploy classical population genetics over natural systems must do so with careful attention to interactions between individual population members and environmental causes. Glymour??s arguments have deep implications for causation in classical population genetics.     

How Well Do Computer-Generated Faces Tap Face Expertise?

The use of computer-generated (CG) stimuli in face processing research is proliferating due to the ease with which faces can be generated, standardised and manipulated. However there has been surprisingly little research into whether CG faces are processed in the same way as photographs of real faces. The present study assessed how well CG faces tap face identity expertise by investigating whether two indicators of face expertise are reduced for CG faces when compared to face photographs. These indicators were accuracy for identification of own-race faces and the other-race effect (ORE)–the well-established finding that own-race faces are recognised more accurately than other-race faces. In Experiment 1 Caucasian and Asian participants completed a recognition memory task for own- and other-race real and CG faces. Overall accuracy for own-race faces was dramatically reduced for CG compared to real faces and the ORE was significantly and substantially attenuated for CG faces. Experiment 2 investigated perceptual discrimination for own- and other-race real and CG faces with Caucasian and Asian participants. Here again, accuracy for own-race faces was significantly reduced for CG compared to real faces. However the ORE was not affected by format. Together these results signal that CG faces of the type tested here do not fully tap face expertise. Technological advancement may, in the future, produce CG faces that are equivalent to real photographs. Until then caution is advised when interpreting results obtained using CG faces.