A green fluorescent protein solubility screen in E. coli reveals domain boundaries of the GTP-binding domain in the P element transposase

Guanosine triphosphate (GTP) binding and hydrolysis events often act as molecular switches in proteins, modulating conformational changes between active and inactive states in many signaling molecules and transport systems. The P element transposase of Drosophila melanogaster requires GTP binding to proceed along its reaction pathway, following initial site‐specific DNA binding. GTP binding is unique to P elements and may represent a novel form of transpositional regulation, allowing the bound transposase to find a second site, looping the transposon DNA for strand cleavage and excision. The GTP‐binding activity has been previously mapped to the central portion of the transposase protein; however, the P element transposase contains little sequence identity with known GTP‐binding folds. To identify soluble, active transposase domains, a GFP solubility screen was used testing the solubility of random P element gene fragments in E. coli. The screen produced a single clone spanning known GTP‐binding residues in the central portion of the transposase coding region. This clone, amino acids 275–409 in the P element transposase, was soluble, highly expressed in E.coli and active for GTP‐binding activity, therefore is a candidate for future biochemical and structural studies. In addition, the chimeric screen revealed a minimal N‐terminal THAP DNA‐binding domain attached to an extended leucine zipper coiled‐coil dimerization domain in the P element transposase, precisely delineating the DNA‐binding and dimerization activities on the primary sequence. This study highlights the use of a GFP‐based solubility screen on a large multidomain protein to identify highly expressed, soluble truncated domain subregions.     

Complete genome sequence of Sphaerobacter thermophilus type strain (S 6022)

Sphaerobacter thermophilus Demharter et al. 1989 is the sole and type species of the genus Sphaerobacter, which is the type genus of the family Sphaerobacteraceae, the order Sphaerobacterales and the subclass Sphaerobacteridae. Phylogenetically, it belongs to the genomically little studied class of the Thermomicrobia in the bacterial phylum Chloroflexi. Here, the genome of strain S 6022(T) is described which is an obligate aerobe that was originally isolated from an aerated laboratory-scale fermentor that was pulse fed with municipal sewage sludge. We describe the features of this organism, together with the complete genome and annotation. This is the first complete genome sequence of the thermomicrobial subclass Sphaerobacteridae, and the second sequence from the chloroflexal class Thermomicrobia. The 3,993,764 bp genome with its 3,525 protein-coding and 57 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Vulcanisaeta distributa type strain (IC-017)

Vulcanisaeta distributa Itoh et al. 2002 belongs to the family Thermoproteaceae in the phylum Crenarchaeota. The genus Vulcanisaeta is characterized by a global distribution in hot and acidic springs. This is the first genome sequence from a member of the genus Vulcanisaeta and seventh genome sequence in the family Thermoproteaceae. The 2,374,137 bp long genome with its 2,544 protein-coding and 49 RNA genes is a part of the Genomic Encyclopedia of Bacteriaand Archaea project.     

A small group of sulfated benzofurans induces steady-state submaximal inhibition of thrombin

Despite the development of promising direct oral anticoagulants, which are all orthosteric inhibitors, a sizable number of patients suffer from bleeding complications. We have hypothesized that allosterism based on the heparin-binding exosites presents a major opportunity to induce sub-maximal inhibition of coagulation proteases, thereby avoiding/reducing bleeding risk. We present the design of a group of sulfated benzofuran dimers that display heparin-binding site-dependent partial allosteric inhibition of thrombin against fibrinogen (ΔY?=?55–75%), the first time that a small molecule (MW??<?800) has been found to thwart macromolecular cleavage by a monomeric protease in a controlled manner. The work leads to the promising concept that it should be possible to develop allosteric inhibitors that reduce clotting, but do not completely eliminate it, thereby avoiding major bleeding complications that beset anticoagulants today.     

Implications of host genetic variation on the risk and prevalence of infectious diseases transmitted through the environment

Previous studies have shown that host genetic heterogeneity in the response to infectious challenge can affect the emergence risk and the severity of diseases transmitted through direct contact between individuals. However, there is substantial uncertainty about the degree and direction of influence owing to different definitions of genetic variation, most of which are not in line with the current understanding of the genetic architecture of disease traits. Also, the relevance of previous results for diseases transmitted through environmental sources is unclear. In this article a compartmental genetic-epidemiological model was developed to quantify the impact of host genetic diversity on epidemiological characteristics of diseases transmitted through a contaminated environment. The model was parameterized for footrot in sheep. Genetic variation was defined through continuous distributions with varying shape and degree of dispersion for different disease traits. The model predicts a strong impact of genetic heterogeneity on the disease risk and its progression and severity, as well as on observable host phenotypes, when dispersion in key epidemiological parameters is high. The impact of host variation depends on the disease trait for which variation occurs and on environmental conditions affecting pathogen survival. In particular, compared to homogeneous populations with the same average susceptibility, disease risk and severity are substantially higher in populations containing a large proportion of highly susceptible individuals, and the differences are strongest when environmental contamination is low. The implications of our results for the recording and analysis of disease data and for predicting response to selection are discussed.     

Complete genome sequence of Hirschia baltica type strain (IFAM 1418(T))

The family Hyphomonadaceae within the Alphaproteobacteria is largely comprised of bacteria isolated from marine environments with striking morphologies and an unusual mode of cell growth. Here, we report the complete genome sequence Hirschia baltica, which is only the second a member of the Hyphomonadaceae with a published genome sequence. H. baltica is of special interest because it has a dimorphic life cycle and is a stalked, budding bacterium. The 3,455,622 bp long chromosome and 84,492 bp plasmid with a total of 3,222 protein-coding and 44 RNA genes were sequenced as part of the DOE Joint Genome Institute Program CSP 2008.     

Phylogeny of Sabellidae (Annelida) and relationships with other taxa inferred from morphology and multiple genes

The monophyly of Sabellidae, the phylogenetic relationships of its lineages, and the composition of Sabellida have been debated for many decades. Most studies on sabellid phylogeny have focused on morphological features but little DNA work has been published to date. We performed analyses using maximum‐parsimony methods that included 36 sabellids and members of previously related taxa. We integrated morphological and DNA sequence data to resolve relationships at different hierarchical levels (135 morphological features, fragments of the nuclear ribosomal RNA genes 18S and 28S, and the mitochondrial gene 16S). The results indicate the monophyly of Sabellida, including Sabellidae and Serpulidae. Monophyly of Fabriciinae and Serpulidae is assessed and the two groups are recovered as sister taxa, but with weak support. There is no significant support for the monophyly of Sabellinae. Relationships between members of the Sabellidae are still partially unresolved due to incongruence between partitions and low support for most clades. The evolution and transformation of certain characters within Sabellidae is explored.
© The Willi Hennig Society 2010.     

Development of Nutritionally Enhanced Tortillas

Large interest has recently risen in the development of “functional” foods, products that may provide a health benefit beyond the traditional nutrients. Foods rich in antioxidants and, simultaneously, characterized by a low glycemic index (GI), can reduce, through a double mechanism, the risk of increased postprandial oxidative stress, which is one of the constituent of the onset of several chronic diseases. Nutritionally enhanced tortillas were therefore developed by incorporating ingredients with well-documented nutritional functionality (carrots, soy, and wholemeal kamut) in a standard wheat tortillas formulation, in an attempt to create low GI and antioxidant-rich products while preserving sensory acceptability and physico-chemical properties. Five tortilla prototypes were developed and characterized for sensory acceptability, textural attributes, color, total antioxidant capacity, and in vivo GI. The simultaneous combination of carrot juice, soy, and wholemeal kamut resulted in a very interesting product that was not only the most acceptable by the consumers (although slightly harder than the standard control) but also showed the lowest GI and was relatively high in total antioxidant capacity. This work was presented at the 2nd International Symposium on: Delivery of Functionality in Complex Food Systems, Amherst, MA, USA, October 8–10, 2007.