Cloning, characterization, and engineering of fungal L-arabinitol dehydrogenases

L-Arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of L-arabinitol to L-xylulose with concomitant NAD⁺ reduction in fungal L-arabinose catabolism. It is an important enzyme in the development of recombinant organisms that convert L-arabinose to fuels and chemicals. Here, we report the cloning, characterization, and engineering of four fungal LADs from Penicillium chrysogenum, Pichia guilliermondii, Aspergillus niger, and Trichoderma longibrachiatum, respectively. The LAD from P. guilliermondii was inactive, while the other three LADs were NAD⁺-dependent and showed high catalytic activities, with P. chrysogenum LAD being the most active. T. longibrachiatum LAD was the most thermally stable and showed the maximum activity in the temperature range of 55–65°C with the other LADs showed the maximum activity in the temperature range of 40–50°C. These LADs were active from pH 7 to 11 with an optimal pH of 9.4. Site-directed mutagenesis was used to alter the cofactor specificity of these LADs. In a T. longibrachiatum LAD mutant, the cofactor preference toward NADP⁺ was increased by 2.5 × 10⁴-fold, whereas the cofactor preference toward NADP⁺ of the P. chrysogenum and A. niger LAD mutants was also drastically improved, albeit at the expense of significantly reduced catalytic efficiencies. The wild-type LADs and their mutants with altered cofactor specificity could be used to investigate the functionality of the fungal L-arabinose pathways in the development of recombinant organisms for efficient microbial L-arabinose utilization.     

Speciation and phylogeography of giant petrels Macronectes

We examine global phylogeography of the two forms of giant petrel Macronectes spp. Although previously considered to be a single taxon, and despite debate over the status of some populations and the existence of minimal genetic data (one mitochondrial cytochrome b sequence per form), the current consensus based on morphology is that there are two species, Northern Giant Petrel M. halli and Southern Giant Petrel M. giganteus. This study examined genetic variation at cytochrome b as well as six microsatellite loci in giant petrels from 22 islands, representing most island groups at which the two species breed. Both markers support separate species status, although sequence divergence in cytochrome b was only 0.42% (corrected). Divergence was estimated to have occurred approximately 0.2 mya, but with some colonies apparently separated for longer (up to 0.5 my). Three clades were found within giant petrels, which separated approximately 0.7 mya, with the Southern Giant Petrel paraphyletic to a monophyletic Northern Giant Petrel. There was evidence of past fragmentation during the Pleistocene, with subsequent secondary contact within Southern Giant Petrels. The analysis also suggested a period of past population expansion that corresponded roughly to the timing of speciation and the separation of an ancestral giant petrel population from the fulmar Fulmarus clade.     

The subtleties of error management

Error management theory is a theory of considerable scope and emerging influence. The theory claims that cognitive biases do not necessarily reflect flaws in evolutionary design, but that they may be best conceived as design features. Unfortunately, existing accounts are vague with respect to the key concept of bias. The result is that it is unclear that the cognitive biases that the theory seeks to defend are not simply a form of behavioral bias, in which case the theory reduces to a version of expected utility theory. We propose some clarifications and refinements of error management theory by emphasizing important distinctions between different forms of behavioral and cognitive bias. We also highlight a key assumption, that the capacity for Bayesian beliefs is subject to constraints. This assumption is necessary for what we see as error management theory's genuinely novel claim: that behavioral tendencies to avoid costly errors can rest on systematic departures from Bayesian beliefs and that the latter can be adaptive insofar as they generate the former.     

Genome-wide association analyses of common wheat (Triticum aestivum L.) germplasm identifies multiple loci for aluminium resistance

Aluminium (Al3+) toxicity restricts productivity and profitability of wheat (Triticum aestivum L.) crops grown on acid soils worldwide. Continued gains will be obtained by identifying superior alleles and novel Al3+ resistance loci that can be incorporated into breeding programs. We used association mapping to identify genomic regions associated with Al3+ resistance using 1055 accessions of common wheat from different geographic regions of the world and 178 polymorphic diversity arrays technology (DArT) markers. Bayesian analyses based on genetic distance matrices classified these accessions into 12 subgroups. Genome-wide association analyses detected markers that were significantly associated with Al3+ resistance on chromosomes 1A, 1B, 2A, 2B, 2D, 3A, 3B, 4A, 4B, 4D, 5B, 6A, 6B, 7A, and 7B. Some of these genomic regions correspond to previously identified loci for Al3+ resistance, whereas others appear to be novel. Among the markers targeting TaALMT1 (the major Al3+-resistance gene located on chromosome 4D), those that detected alleles in the promoter explained most of the phenotypic variance for Al3+ resistance, which is consistent with this region controlling the level of TaALMT1 expression. These results demonstrate that genome-wide association mapping cannot only confirm known Al3+-resistance loci, such as those on chromosomes 4D and 4B, but they also highlight the utility of this technique in identifying novel resistance loci.     

Compared effects of missense mutations in Very-Long-Chain Acyl-CoA Dehydrogenase deficiency: Combined analysis by structural,functional and pharmacological approaches

Very-Long-Chain Acyl-CoA Dehydrogenase deficiency (VLCADD) is an autosomal recessive disorder considered as one of the more common ß-oxidation defects, possibly associated with neonatal cardiomyopathy, infantile hepatic coma, or adult-onset myopathy. Numerous gene missense mutations have been described in these VLCADD phenotypes, but only few of them have been structurally and functionally analyzed, and the molecular basis of disease variability is still poorly understood. To address this question, we first analyzed fourteen disease-causing amino acid changes using the recently described crystal structure of VLCAD. The predicted effects varied from the replacement of amino acid residues lining the substrate binding cavity, involved in holoenzyme–FAD interactions or in enzyme dimerisation, predicted to have severe functional consequences, up to amino acid substitutions outside key enzyme domains or lying on near enzyme surface, with predicted milder consequences. These data were combined with functional analysis of residual fatty acid oxidation (FAO) and VLCAD protein levels in patient cells harboring these mutations, before and after pharmacological stimulation by bezafibrate. Mutations identified as detrimental to the protein structure in the 3-D model were generally associated to profound FAO and VLCAD protein deficiencies in the patient cells, however, some mutations affecting FAD binding or monomer–monomer interactions allowed a partial response to bezafibrate. On the other hand, bezafibrate restored near-normal FAO rates in some mutations predicted to have milder consequences on enzyme structure. Overall, combination of structural, biochemical, and pharmacological analysis allowed assessment of the relative severity of individual mutations, with possible applications for disease management and therapeutic approach.     

Broad-Host-Range Plasmids for Red Fluorescent Protein Labeling of Gram-Negative Bacteria for Use in the Zebrafish Model System

To observe real-time interactions between green fluorescent protein-labeled immune cells and invading bacteria in the zebrafish (Danio rerio), a series of plasmids was constructed for the red fluorescent protein (RFP) labeling of a variety of fish and human pathogens. The aim of this study was to create a collection of plasmids that would express RFP pigments both constitutively and under tac promoter regulation and that would be nontoxic and broadly transmissible to a variety of Gram-negative bacteria. DNA fragments encoding the RFP dimeric (d), monomeric (m), and tandem dimeric (td) derivatives d-Tomato, td-Tomato, m-Orange, and m-Cherry were cloned into the IncQ-based vector pMMB66EH in Escherichia coli. Plasmids were mobilized into recipient strains by conjugal mating. Pigment production was inducible in Escherichia coli, Pseudomonas aeruginosa, Edwardsiella tarda, and Vibrio (Listonella) anguillarum strains by isopropyl-β-d-thiogalactopyranoside (IPTG) treatment. A spontaneous mutant exconjugant of P. aeruginosa PA14 was isolated that expressed td-Tomato constitutively. Complementation analysis revealed that the constitutive phenotype likely was due to a mutation in lacI^q carried on pMMB66EH. DNA sequence analysis confirmed the presence of five transitions, four transversions, and a 2-bp addition within a 14-bp region of lacI. Vector DNA was purified from this constitutive mutant, and structural DNA sequences for RFP pigments were cloned into the constitutive vector. Exconjugants of P. aeruginosa, E. tarda, and V. anguillarum expressed all pigments in an IPTG-independent fashion. Results from zebrafish infectivity studies indicate that RFP-labeled pathogens will be useful for the study of real-time interactions between host cells of the innate immune system and the infecting pathogen.The use of fluorescent proteins as probes has proven to be a vital tool in elucidating spatial and temporal patterns of gene expression, protein localization, and protein-protein interactions in vivo. In addition to well-known contributions to cell biology and biochemistry, the use of fluorescent proteins to label bacteria has provided insight into complex host-pathogen interactions and routes of entry (14, 23, 29).The zebrafish has become an attractive model for in vivo infection studies. As an optically clear organism at the embryo and larval developmental stages, zebrafish are amenable to visualization during challenge with fluorescently labeled pathogens. In addition, fertilization in the zebrafish occurs ex utero, thereby simplifying infection by either static immersion or microinjection. The visualization of infection in situ can be conducted on the whole embryo using confocal or wide-field epifluorescence microscopy, an approach not possible in other model systems. To date, several zebrafish infection models have been established employing both fish and mammalian pathogens, including Edwardsiella tarda (21), E. ictaluri (14, 23), Staphylococcus aureus (16), Streptococcus pyogenes and S. iniae (18), and Pseudomonas aeruginosa (4). Illustrating the value of fluorescently labeled bacteria in studies of microbial pathogenesis, Van der Sar and colleagues established the zebrafish as a model for Salmonella enterica serovar Typhimurium infections using DsRed-labeled bacteria (30). Similarly, O''Toole and colleagues made use of a green fluorescent protein (GFP)-labeled strain of Vibrio (Listonella) anguillarum to elucidate the natural route of invasion of this fish pathogen (20). However, obstacles to the construction of fluorescently labeled microbes have prevented many from taking advantage of this technique to permit the real-time in situ analysis of infection. These obstacles include the nontransformable nature of many pathogens and the toxicity of RFP expression from high-copy-number plasmids.Recently, two transgenic lines of zebrafish that express GFP in cells of the myeloid lineage have been developed. Lawson et al. described a transgenic (Tg) zebrafish that utilizes the protooncogene fli1 promoter to drive the expression of enhanced GFP (EGFP) (15). The fli1 gene is preferentially expressed in endothelial and hematopoietic cells. Intended for the study of vascular development, Tg(fli1::EGFP) zebrafish not only have fluorescent endothelial cells but also macrophages (15), making this zebrafish line attractive to those who study innate immunity. It has been shown that zebrafish macrophages possess strong phagocytic properties and migrate to sites of bacterial infection (13). In 2006, Renshaw and his colleagues engineered a novel Tg(mpo::GFP) zebrafish that expresses GFP under the control of the neutrophil-specific myeloperoxidase promoter (22). In this study, they characterized the GFP-labeled neutrophils, noting their capability of chemotaxis at wound sites (22). Given the recent progress in zebrafish genetics with regard to these GFP-expressing transgenic lines, there was a clear need for a simple method for the RFP labeling of Gram-negative bacteria. Such a method would facilitate further studies of microbial pathogenesis in the zebrafish and could further elucidate phagocyte (GFP labeled)-pathogen (RFP labeled) interactions, kinetics of bacterial clearance, and mechanisms of phagocyte migration to sites of bacterial infection.For the purposes of this study, we employed dimeric (d) and monomeric (m) red fluorescent protein variants d-Tomato, td-Tomato, m-Orange, and m-Cherry, which were constructed by Shaner et al. (25). These variants were chosen based on combinations of their high relative brightness and decreased RFP maturation times compared to those of the original RFPs (25). We describe here a simple method for the RFP labeling of Gram-negative bacteria.     

The I4895T mutation in the type 1 ryanodine receptor induces fiber-type specific alterations in skeletal muscle that mimic premature aging

The I4898T (IT) mutation in type 1 ryanodine receptor (RyR1), the Ca(2+) release channel of the sarcoplasmic reticulum (SR) is linked to a form of central core disease (CCD) in humans and results in a nonleaky channel and excitation-contraction uncoupling. We characterized age-dependent and fiber-type-dependent alterations in muscle ultrastructure, as well as the magnitude and spatiotemporal properties of evoked Ca(2+) release in heterozygous Ryr1(I4895T/WT) (IT/+) knock-in mice on a mixed genetic background. The results indicate a classical but mild CCD phenotype that includes muscle weakness and the presence of mitochondrial-deficient areas in type I fibers. Electrically evoked Ca(2+) release is significantly reduced in single flexor digitorum brevis (FDB) fibers from young and old IT/+ mice. Structural changes are strongly fiber-type specific, affecting type I and IIB/IIX fibers in very distinct ways, and sparing type IIA fibers. Ultrastructural alterations in our IT/+ mice are also present in wild type, but at a lower frequency and older ages, suggesting that the disease mutation on the mixed background promotes an acceleration of normal age-dependent changes. The observed functional and structural alterations and their similarity to age-associated changes are entirely consistent with the known properties of the mutated channel, which result in reduced calcium release as is also observed in normal aging muscle. In strong contrast to these observations, a subset of patients with the analogous human heterozygous mutation and IT/+ mice on an inbred 129S2/SvPasCrl background exhibit a more severe disease phenotype, which is not directly consistent with the mutated channel properties.     

Guidance receptor degradation is required for neuronal connectivity in the Drosophila nervous system

Axon pathfinding and synapse formation rely on precise spatiotemporal localization of guidance receptors. However, little is known about the neuron-specific intracellular trafficking mechanisms that underlie the sorting and activity of these receptors. Here we show that loss of the neuron-specific v-ATPase subunit a1 leads to progressive endosomal guidance receptor accumulations after neuronal differentiation. In the embryo and in adult photoreceptors, these accumulations occur after axon pathfinding and synapse formation is complete. In contrast, receptor missorting occurs sufficiently early in neurons of the adult central nervous system to cause connectivity defects. An increase of guidance receptors, but not of membrane proteins without signaling function, causes specific gain-of-function phenotypes. A point mutant that promotes sorting but prevents degradation reveals spatiotemporally specific guidance receptor turnover and accelerates developmental defects in photoreceptors and embryonic motor neurons. Our findings indicate that a neuron-specific endolysosomal degradation mechanism is part of the cell biological machinery that regulates guidance receptor turnover and signaling.