The novel NhaE-type Na+/H+ antiporter of the pathogenic bacterium Neisseria meningitidis

Neisseria meningitidis is a pathogenic bacterium responsible for meningitis. The mechanisms underlying the control of Na⁺ transmembrane movement, presumably important to pathogenicity, have been barely addressed. To elucidate the function of the components of the Na⁺ transport system in N. meningitidis, an open reading frame from the genome of this bacterium displaying similarity with the NhaE type of Na⁺/H⁺ antiporters was expressed in Escherichia coli and characterized for sodium transport ability. The N. meningitidis antiporter (NmNhaE) was able to complement an E. coli strain devoid of Na⁺/H⁺ antiporters (KNabc) respecting the ability to grow in the presence of NaCl and LiCl. Ion transport assays in everted vesicles prepared from KNabc expressing NmNhaE from a plasmid confirmed its ability to translocate Na⁺ and Li⁺. Here is presented the characterization of the first NhaE from a pathogen, an important contribution to the comprehension of sodium ion metabolism in this kind of microorganisms.     

Efficient in vitro regeneration systems for Vaccinium species

Efficient protocols for shoot regeneration from leaf explants suitable for micropropagation as well as for the development of transgenic plants were developed for blueberry (Vaccinium corymbosum) and lingonberry (Vaccinium vitis-idaea) cultivars. Nodal segments were used to initiate in vitro shoot cultures of lingonberry cultivar ‘Red Pearl’ and southern highbush blueberry cultivar ‘Ozarkblue’. In order to develop an optimized regeneration procedure, different types and concentrations of plant growth regulators were tested to induce adventitious shoot regeneration on excised leaves from micropropagated shoots of both cultivars. The effect on percentage regeneration and number of shoots per explant was investigated. Results indicated that zeatin was superior to TDZ and meta-topolin in promoting adventitious shoot formation. A concentration of 20 μM zeatin was most effective in promoting shoot regeneration in both cultivars, in case of ‘Red Pearl’ along with 1 μM NAA. Shoots were either allowed to root in vitro on medium containing IBA or NAA or ex vitro in a fog tunnel. IBA was superior to NAA for induction of root development in vitro in both Vaccinium cultivars. Ex vitro rooting under high humidity was tested with cuttings from mature field-grown plants, from acclimatized tissue culture derived plants and with unrooted in vitro proliferated shoots planted directly. It was found that in vitro shoots rooted better under fog than cuttings from the other plant sources and rooting was equivalent to that achieved in vitro.     

Genetic Barriers to Historical Gene Flow between Cryptic Species of Alpine Bumblebees Revealed by Comparative Population Genomics

Evidence is accumulating that gene flow commonly occurs between recently diverged species, despite the existence of barriers to gene flow in their genomes. However, we still know little about what regions of the genome become barriers to gene flow and how such barriers form. Here, we compare genetic differentiation across the genomes of bumblebee species living in sympatry and allopatry to reveal the potential impact of gene flow during species divergence and uncover genetic barrier loci. We first compared the genomes of the alpine bumblebee Bombus sylvicola and a previously unidentified sister species living in sympatry in the Rocky Mountains, revealing prominent islands of elevated genetic divergence in the genome that colocalize with centromeres and regions of low recombination. This same pattern is observed between the genomes of another pair of closely related species living in allopatry (B. bifarius and B. vancouverensis). Strikingly however, the genomic islands exhibit significantly elevated absolute divergence (d_XY) in the sympatric, but not the allopatric, comparison indicating that they contain loci that have acted as barriers to historical gene flow in sympatry. Our results suggest that intrinsic barriers to gene flow between species may often accumulate in regions of low recombination and near centromeres through processes such as genetic hitchhiking, and that divergence in these regions is accentuated in the presence of gene flow.     

Development of isoform selective PI3-kinase inhibitors as pharmacological tools for elucidating the PI3K pathway

Using a parallel synthesis approach to target a non-conserved region of the PI3K catalytic domain a pan-PI3K inhibitor 1 was elaborated to provide alpha, delta and gamma isoform selective Class I PI3K inhibitors 21, 24, 26 and 27. The compounds had good cellular activity and were selective against protein kinases and other members of the PI3K superfamily including mTOR and DNA-PK.     

New Insights into the Fructosyltransferase Activity of Schwanniomyces occidentalis β-Fructofuranosidase,Emerging from Nonconventional Codon Usage and Directed Mutation

Schwanniomyces occidentalis β-fructofuranosidase (Ffase) releases β-fructose from the nonreducing ends of β-fructans and synthesizes 6-kestose and 1-kestose, both considered prebiotic fructooligosaccharides. Analyzing the amino acid sequence of this protein revealed that it includes a serine instead of a leucine at position 196, caused by a nonuniversal decoding of the unique mRNA leucine codon CUG. Substitution of leucine for Ser196 dramatically lowers the apparent catalytic efficiency (k_cat/K_m) of the enzyme (approximately 1,000-fold), but surprisingly, its transferase activity is enhanced by almost 3-fold, as is the enzymes'' specificity for 6-kestose synthesis. The influence of 6 Ffase residues on enzyme activity was analyzed on both the Leu196/Ser196 backgrounds (Trp47, Asn49, Asn52, Ser111, Lys181, and Pro232). Only N52S and P232V mutations improved the transferase activity of the wild-type enzyme (about 1.6-fold). Modeling the transfructosylation products into the active site, in combination with an analysis of the kinetics and transfructosylation reactions, defined a new region responsible for the transferase specificity of the enzyme.β-Fructofuranosidases (EC 3.2.1.26) are enzymes of biotechnological interest that catalyze the release of β-fructose from the nonreducing termini of various β-d-fructofuranoside substrates. In general, they exhibit a high degree of sequence homology, and based on their amino acid sequences, they fall into family 32 of the glycosyl-hydrolases (GH), along with invertases, inulinases, and fructosyltransferases (http://www.cazy.org). The GH32 family has been studied intensely, and some three-dimensional structures are now available, such as that of inulinase from Aspergillus awamorii (26), fructan-exohydrolase from Cichorium intybus (CiFEH) (34, 36), or invertase from Thermotoga maritima (2, 3) and Arabidopsis thaliana (35). These proteins contain a five-blade β-propeller N-terminal catalytic module and a C-terminal β-sandwich domain (19). Multiple-sequence alignment of GH32 proteins, which are included in the GH-J clan together with the GH68 proteins of the inulosucrase family, reveals the presence of three conserved motifs, each containing a key acidic residue (in boldface) implicated in substrate binding and hydrolysis: Asn-Asp-Pro-Asn-Gly (NDPNG), Arg-Asp-Pro (RDP), and Glu-Cys (EC) (28). These conserved residues are implicated in a double-displacement reaction in which a covalent glycosyl-enzyme intermediate is formed. Thus, the catalytic mechanism proposed for the Saccharomyces cerevisiae invertase implies that Asp23 (NDPNG) acts as a nucleophile and Glu204 (EC) acts as the acid/base catalyst (29), whereas Asp309 (RDP) of Acetobacter diazotropicus levansucrase influences the efficiency of sucrose hydrolysis (7) and Arg188 and Asp189 of the latter motif define the substrate binding and specificity of exoinulinase from A. awamorii toward fructopyranosyl residues (26).As well as hydrolyzing sucrose, β-fructofuranosidases may also catalyze the synthesis of short-chain fructooligosaccharides (FOS), in which one to three fructosyl moieties are linked to the sucrose skeleton by different glycosidic bonds, depending on the source of the enzyme (12, 21, 31). FOS act as prebiotics, and they exert a beneficial effect on human health, participating in the prevention of cardiovascular diseases, colon cancer, and osteoporosis (16). Currently, FOS are mainly produced by Aspergillus fructosyltransferase in industry (10, 31), providing a mixture of FOS with an inulin-type structure that contains β-(2→1)-linked fructose oligomers (¹F-FOS: 1-kestose or nystose). Curiously, when the link between two fructose units (⁶F-FOS: 6-kestose) or between fructose and the glucosyl moiety (⁶G-FOS: neokestose) involves a β-(2→6) link, the prebiotic properties of the FOS may be enhanced beyond that of commercial FOS (23).The yeast Schwanniomyces occidentalis (also called Debaryomyces occidentalis) produces a number of extracellular enzymes that make it of interest in biotechnology. Several of its amylolytic enzymes have been characterized, including amylases and glucoamylase (1, 9), as well as an invertase (17). In addition, we also characterized an extracellular β-fructofuranosidase (Ffase) from this yeast that hydrolyzes sucrose, 1-kestose, and nystose (5). This enzyme exhibited a transfructosylating activity that efficiently produces the trisaccharides 6-kestose and 1-kestose in the ratio 3:1, generating the highest 6-kestose yield yet reported, as far as we know. The Ffase three-dimensional structure has recently been solved (6) and represented as a homodimer, each modular subunit arranged like other GH32 enzymes. The Asp50 (NDPNG) and Glu230 (EC) located at the center of the propeller are the catalytic residues implicated in substrate binding and hydrolysis, whereas Arg178 and Asp179 form the RDP motif (6).The genetic codes of some yeasts incorporate certain variations. For example, while CUG was believed to be a universal codon for leucine, in the cytoplasm of certain species of the genus Candida (15) it encodes a serine, as in Pichia farinosa (33). The reassignment of this codon is mediated by a novel serine-tRNA that acquired a leucine 5′-CAG-3′ anticodon (25).Here, we show that deviation from the standard use of the CUG leucine codon to encode serine was correlated with the transferase capacity and specificity of the Ffase enzyme. Indeed, the S196L substitution enhanced the transferase activity of the enzyme 3-fold. Several site-directed mutants were generated and characterized to study their transferase capacities. These results are considered on the basis of the enzymes'' three-dimensional structure, which enables a novel putative binding site of sucrose that serves as a water substitute donor in the hydrolytic reaction yielding the tranglycosylation product 6-kestose to be identified.     

Sensing and signalling in response to oxygen deprivation in plants and other organisms

AIMS AND SCOPE: All aerobic organisms require molecular di-oxygen (O2) for efficient production of ATP though oxidative phosphorylation. Cellular depletion of oxygen results in rapid molecular and physiological acclimation. The purpose of this review is to consider the processes of low oxygen sensing and response in diverse organisms, with special consideration of plant cells. CONCLUSIONS: The sensing of oxygen deprivation in bacteria, fungi, metazoa and plants involves multiple sensors and signal transduction pathways. Cellular responses result in a reprogramming of gene expression and metabolic processes that enhance transient survival and can enable long-term tolerance to sub-optimal oxygen levels. The mechanism of sensing can involve molecules that directly bind or react with oxygen (direct sensing), or recognition of altered cellular homeostasis (indirect sensing). The growing knowledge of the activation of genes in response to oxygen deprivation has provided additional information on the response and acclimation processes. Conservation of calcium fluxes and reactive oxygen species as second messengers in signal transduction pathways in metazoa and plants may reflect the elemental importance of rapid sensing of cellular restriction in oxygen by aerobic organisms.     

Comparative linkage mapping of the Grey coat colour gene in horses

Grey horses are born coloured, turn progressively grey and often develop melanomas late in life. Grey shows an autosomal dominant inheritance and the locus has previously been mapped to horse chromosome 25 (ECA25), around the TXN gene. We have now developed eight new single nucleotide polymorphisms (SNPs) associated with genes on ECA25 using information on the linear order of genes on human chromosome 9q, as well as the human and mouse coding sequences. These SNPs were mapped in relation to the Grey locus using more than 300 progeny from matings between two Swedish Warmblood grey stallions and non-grey mares. Grey was firmly assigned to an interval with flanking markers NANS and ABCA1. This corresponds to a region of approximately 6.9 Mb on human chromosome 9q. Furthermore, no recombination was observed between Grey, TGFBR1 and TMEFF1, the last two being 1.4 Mb apart in human. There are no obvious candidate genes in this region and none of the genes has been associated with pigmentation disorders or melanoma development, suggesting that the grey phenotype is caused by a mutation in a novel gene.