Drosophila katanin-60 depolymerizes and severs at microtubule defects

Microtubule (MT) length and location is tightly controlled in cells. One novel family of MT-associated proteins that regulates MT dynamics is the MT-severing enzymes. In this work, we investigate how katanin (p60), believed to be the first discovered severing enzyme, binds and severs MTs via single molecule total internal reflection fluorescence microscopy. We find that severing activity depends on katanin concentration. We also find that katanin can remove tubulin dimers from the ends of MTs, appearing to depolymerize MTs. Strikingly, katanin localizes and severs at the interface of GMPCPP-tubulin and GDP-tubulin suggesting that it targets to protofilament-shift defects. Finally, we observe that binding duration, mobility, and oligomerization are ATP dependent.     

Effects of combining <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Nosema pyrausta</Emphasis> on the mortality of <Emphasis Type="Italic">Ostrinia nubilalis</Emphasis>

We tested the combined effect of the fungus Beauveria bassiana and the microsporidium Nosema pyrausta on the European corn borer larvae, Ostrinia nubilalis, in the laboratory. The first instar of O. nubilalis larvae was the most sensitive to the B. bassiana infection followed by the fifth, second, third, and fourth instar (LC₅₀s were 4.91, 6.67, 7.13, 9.15, and 6.51 × 10⁵ conidia/ml for the first to fifth instars, respectively). Mortality of each instar increases positively with concentration of conidia. When B. bassiana and N. pyrausta were used in combination, mortality increased significantly in all instars. Relative to the B. bassiana treatment alone, the B. bassiana + N. pyrausta treatment decreased the LC₅₀s by 42.16%, 37.63%, 21.60%, 27.11%, and 33.95% for the first to fifth instars, respectively. The combined effects of the two pathogens were mostly additive. However, at the two highest concentrations the pathogens interacted synergistically in the first and second instar. Individuals that survived the B. bassiana and B. bassiana + N. pyrausta treatments and developed into adults had significantly shorter lifespans and females oviposited fewer eggs than non-exposed insects. The effects on the longevity and the egg production were most pronounced at high concentration of B. bassiana conidia.     

XylS domain interactions can be deduced from intraallelic dominance in double mutants of Pseudomonas putida.

Summary The XylS protein is the positive regulator of the TOL plasmid-encoded meta-cleavage pathway for the metabolism of alkylbenzoates in Pseudomonas putida. This protein is activated by a variety of benzoate analogues. To elucidate the functional domains of the regulator and their interactions, several fusions of the XylS C-terminus to MS2 polymerase and of the N-terminus to -galactosidase were constructed but all are inactive. In addition, 15 double mutant xylS genes were constructed in vitro by fusing parts of various mutant genes to produce mutant regulators exhibiting C-terminal and N-terminal amino acid substitutions. The phenotypic properties of the parental single mutant genes, and those of the double mutant genes, suggest that the C-terminal region is involved in binding to DNA sequences at the promoter of the meta-cleavage pathway operon, and that the benzoate effector binding pocket includes critical residues present at both the N-terminal and C-terminal ends of the protein. The intraallelic dominance of the Ile229 (Ser229 Ile) and Val274 (Asp274 Val) substitutions over the N-terminal His4l (Arg4l His) substitution, and the intraallelic dominance of Thr45 (Arg45 Thr) over Ile229 and Val274, support the proposal that these two regions of the regulator interact functionally. Combination of the Leu88 (Trp88 Leu) and Arg256 (Pro256 Arg) substitutions did not suppress the semiconstitutive phenotype conferred by Leu88, but resulted in a protein with altered ability to recognize benzoates. In contrast, the Leu88 semiconstitutive phenotype was suppressed by Va1288 (Asp288 Val), and the double mutant was susceptible to activation by benzoates. The results suggest that intramolecular interactions between the C- and N-terminal regions of XylS are critical for activation of the regulator by the effector.     

Redesign of LAOBP to bind novel l‐amino acid ligands

Computational protein design is still a challenge for advancing structure‐function relationships. While recent advances in this field are promising, more information for genuine predictions is needed. Here, we discuss different approaches applied to install novel glutamine (Gln) binding into the Lysine/Arginine/Ornithine binding protein (LAOBP) from Salmonella typhimurium. We studied the ligand binding behavior of two mutants: a binding pocket grafting design based on a structural superposition of LAOBP to the Gln binding protein QBP from Escherichia coli and a design based on statistical coupled positions. The latter showed the ability to bind Gln even though the protein was not very stable. Comparison of both approaches highlighted a nonconservative shared point mutation between LAOBP_graft and LAOBP_sca. This context dependent L117K mutation in LAOBP turned out to be sufficient for introducing Gln binding, as confirmed by different experimental techniques. Moreover, the crystal structure of LAOBP_L117K in complex with its ligand is reported.     

Isolation of the facA (acetyl-CoA synthetase) gene of Phycomyces blakesleeanus

A 5.6 kb DNA fragment from the fungus Phycomyces blakesleeanus has been cloned and sequenced. The fragment contains a gene that probably codes for the enzyme acetyl-coenzyme A synthetase (facA). The amino acid sequence deduced for the P. blakesleeanns protein is highly homologous to those of acetyl-coA-synthetases from other organisms. When placed under the control of a constitutive promoter from Aspergillus nidulans, the cloned gene complemented a facA mutation of this organism. In P. blakesleeanns, the expression of facA is induced by acetate.     

From lignocellulosic residues to market: Production and commercial potential of xylooligosaccharides

The updated definition of prebiotic expands the range of potential applications in which emerging xylooligosaccharides (XOS) can be used. It has been demonstrated that XOS exhibit prebiotic effects at lower amounts compared to others, making them competitively priced prebiotics. As a result, the industry is focused on developing alternative approaches to improve processes efficiency that can meet the increasing demand while reducing costs. Recent advances have been made towards greener and more efficient processes, by applying process integration strategies to produce XOS from costless lignocellulosic residues and using genetic engineering to create microorganisms that convert these residues to XOS. In addition, collecting more in vivo data on their performance will be key to achieve regulatory claims, greatly increasing XOS commercial value.     

The potential role of polyploidy and hybridisation in the further evolution of the highly invasive <Emphasis Type="Italic">Fallopia</Emphasis> taxa in Europe

Japanese knotweed s.l. comprises Fallopia japonica, F. sachalinensis, F. × bohemica and any F2s or backcrosses. The parental taxa were introduced from the East to the West as garden ornamentals in the nineteenth century, and soon spread beyond the confines of the garden to become widespread and persistent weeds. Since only female F. japonica var. japonica was introduced, its impressive spread has occurred solely by vegetative means. However, the initial lack of genetic variability has been complemented by an extensive series of hybridisations in the adventive range. We examine the history, spread, reproductive biology and ecological impact of these species in the West. The role and importance of polyploidy and hybridisation in their invasion of the West is discussed, as are the implications of these factors for the potential further evolution of the group.     

Phylogenetic position of Salinibacter ruber based on concatenated protein alignments

A total of 22 genes from the genome of Salinibacter ruber strain M31 were selected in order to study the phylogenetic position of this species based on protein alignments. The selection of the genes was based on their essential function for the organism, dispersion within the genome, and sufficient informative length of the final alignment. For each gene, an individual phylogenetic analysis was performed and compared with the resulting tree based on the concatenation of the 22 genes, which rendered a single alignment of 10,757 homologous positions. In addition to the manually chosen genes, an automatically selected data set of 74 orthologous genes was used to reconstruct a tree based on 17,149 homologous positions. Although single genes supported different topologies, the tree topology of both concatenated data sets was shown to be identical to that previously observed based on small subunit (SSU) rRNA gene analysis, in which S. ruber was placed together with Bacteroidetes. In both concatenated data sets the bootstrap was very high, but an analysis with a gradually lower number of genes indicated that the bootstrap was greatly reduced with less than 12 genes. The results indicate that tree reconstructions based on concatenating large numbers of protein coding genes seem to produce tree topologies with similar resolution to that of the single 16S rRNA gene trees. For classification purposes, 16S rRNA gene analysis may remain as the most pragmatic approach to infer genealogic relationships.