Probing the ubiquinol-binding site of recombinant Sauromatum guttatum alternative oxidase expressed in E. coli membranes through site-directed mutagenesis

In the present paper we have investigated the effect of mutagenesis of a number of highly conserved residues (R159, D163, L177 and L267) which we have recently shown to line the hydrophobic inhibitor/substrate cavity in the alternative oxidases (AOXs). Measurements of respiratory activity in rSgAOX expressed in Escherichia coli FN102 membranes indicate that all mutants result in a decrease in maximum activity of AOX and in some cases (D163 and L177) a decrease in the apparent K_m (O₂). Of particular importance was the finding that when the L177 and L267 residues, which appear to cause a bottleneck in the hydrophobic cavity, are mutated to alanine the sensitivity to AOX antagonists is reduced. When non-AOX anti-malarial inhibitors were also tested against these mutants widening the bottleneck through removal of isobutyl side chain allowed access of these bulkier inhibitors to the active-site and resulted in inhibition. Results are discussed in terms of how these mutations have altered the way in which the AOX's catalytic cycle is controlled and since maximum activity is decreased we predict that such mutations result in an increase in the steady state level of at least one O₂-derived AOX intermediate. Such mutations should therefore prove to be useful in future stopped-flow and electron paramagnetic resonance experiments in attempts to understand the catalytic cycle of the alternative oxidase which may prove to be important in future rational drug design to treat diseases such as trypanosomiasis. Furthermore since single amino acid mutations in inhibitor/substrate pockets have been found to be the cause of multi-drug resistant strains of malaria, the decrease in sensitivity to main AOX antagonists observed in the L-mutants studied in this report suggests that an emergence of drug resistance to trypanosomiasis may also be possible. Therefore we suggest that the design of future AOX inhibitors should have structures that are less reliant on the orientation by the two-leucine residues. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

The origin of annelids

Annelids are a phylum of segmented bilaterian animals that have become important components of ecosystems spanning terrestrial realms to the deep sea. Annelids are remarkably diverse, possessing high taxonomic diversity and exceptional morphological disparity, and have evolved numerous feeding strategies and ecologies. Their interrelationships and evolution have been the source of much controversy over the past century with the composition of the annelid crown group, the relationship of major groups and the body plan of the ancestral annelid having undergone major recent revisions. There is a convincing body of molecular evidence that polychaetes form a paraphyletic grade and that clitellates are derived polychaetes. The earliest stem group annelids from Cambrian Lagerstätten are errant, epibenthic polychaetes, confirming that biramous parapodia, head appendages and diverse, simple chaetae are primitive for annelids. Current evidence from molecular clocks and the fossil record suggest that crown group annelids are a Late Cambrian – Ordovician radiation, with clitellates radiating in the Late Palaeozoic. Their body fossil record is largely confined to deposits showing exceptional preservation and is punctuated by the acquisition of hard parts in major groups. The discovery of an Ordovician fossil with soft tissues has shown that machaeridians are in fact a clade of crown polychaetes. They were in existence for more than 200 million years and possess unique calcitic dorsal armour, allowing their mode of life and phylogeny to be interpreted in the context of the annelid body plan. We identify a novel clade of machaeridians, the Cuniculepadida, which exhibit a series of adaptations for burrowing.     

Benzothiazinones Mediate Killing of Corynebacterineae by Blocking Decaprenyl Phosphate Recycling Involved in Cell Wall Biosynthesis

Benzothiazinones (BTZs) are a new class of sulfur containing heterocyclic compounds that target DprE1, an oxidoreductase involved in the epimerization of decaprenyl-phosphoribose (DPR) to decaprenyl-phosphoarabinose (DPA) in the Corynebacterineae, such as Corynebacterium glutamicum and Mycobacterium tuberculosis. As a result, BTZ inhibition leads to inhibition of cell wall arabinan biosynthesis. Previous studies have demonstrated the essentiality of dprE1. In contrast, Cg-UbiA a ribosyltransferase, which catalyzes the first step of DPR biosynthesis prior to DprE1, when genetically disrupted, produced a viable mutant, suggesting that although BTZ biochemically targets DprE1, killing also occurs through chemical synthetic lethality, presumably through the lack of decaprenyl phosphate recycling. To test this hypothesis, a derivative of BTZ, BTZ043, was examined in detail against C. glutamicum and C. glutamicum::ubiA. The wild type strain was sensitive to BTZ043; however, C. glutamicum::ubiA was found to be resistant, despite possessing a functional DprE1. When the gene encoding C. glutamicum Z-decaprenyl-diphosphate synthase (NCgl2203) was overexpressed in wild type C. glutamicum, resistance to BTZ043 was further increased. This data demonstrates that in the presence of BTZ, the bacilli accumulate DPR and fail to recycle decaprenyl phosphate, which results in the depletion of decaprenyl phosphate and ultimately leads to cell death.     

Establishing the A. E. Watkins landrace cultivar collection as a resource for systematic gene discovery in bread wheat

Key message

A high level of genetic diversity was found in the A. E. Watkins bread wheat landrace collection. Genotypic information was used to determine the population structure and to develop germplasm resources.

Abstract

In the 1930s A. E. Watkins acquired landrace cultivars of bread wheat (Triticum aestivum L.) from official channels of the board of Trade in London, many of which originated from local markets in 32 countries. The geographic distribution of the 826 landrace cultivars of the current collection, here called the Watkins collection, covers many Asian and European countries and some from Africa. The cultivars were genotyped with 41 microsatellite markers in order to investigate the genetic diversity and population structure of the collection. A high level of genetic diversity was found, higher than in a collection of modern European winter bread wheat varieties from 1945 to 2000. Furthermore, although weak, the population structure of the Watkins collection reveals nine ancestral geographical groupings. An exchange of genetic material between ancestral groups before commercial wheat-breeding started would be a possible explanation for this. The increased knowledge regarding the diversity of the Watkins collection was used to develop resources for wheat research and breeding, one of them a core set, which captures the majority of the genetic diversity detected. The understanding of genetic diversity and population structure together with the availability of breeding resources should help to accelerate the detection of new alleles in the Watkins collection.