The GATC-binding protein SeqA is required for bile resistance and virulence in Salmonella enterica serovar typhimurium

Disruption of the seqA gene of Salmonella enterica serovar Typhimurium causes defects similar to those described in E. coli: filament formation, aberrant nucleoid segregation, induction of the SOS response, envelope instability, and increased sensitivity to membrane-damaging agents. Differences between SeqA⁻ mutants of E. coli and S. enterica, however, are found. SeqA⁻ mutants of S. enterica form normal colonies and do not exhibit alterations in phage plaquing morphology. Lack of SeqA causes attenuation of S. enterica virulence by the oral route but not by the intraperitoneal route, suggesting a virulence defect in the intestinal stage of infection. However, SeqA⁻ mutants are fully proficient in the invasion of epithelial cells. We hypothesize that attenuation of SeqA⁻ mutants by the oral route may be caused by bile sensitivity, which in turn may be a consequence of envelope instability.     

The minimum information required for reporting a molecular interaction experiment (MIMIx)

A wealth of molecular interaction data is available in the literature, ranging from large-scale datasets to a single interaction confirmed by several different techniques. These data are all too often reported either as free text or in tables of variable format, and are often missing key pieces of information essential for a full understanding of the experiment. Here we propose MIMIx, the minimum information required for reporting a molecular interaction experiment. Adherence to these reporting guidelines will result in publications of increased clarity and usefulness to the scientific community and will support the rapid, systematic capture of molecular interaction data in public databases, thereby improving access to valuable interaction data.     

Novel pH-Stable Glycoside Hydrolase Family 3 β-Xylosidase from Talaromyces amestolkiae: an Enzyme Displaying Regioselective Transxylosylation

This paper reports on a novel β-xylosidase from the hemicellulolytic fungus Talaromyces amestolkiae. The expression of this enzyme, called BxTW1, could be induced by beechwood xylan and was purified as a glycoprotein from culture supernatants. We characterized the gene encoding this enzyme as an intronless gene belonging to the glycoside hydrolase gene family 3 (GH3). BxTW1 exhibited transxylosylation activity in a regioselective way. This feature would allow the synthesis of oligosaccharides or other compounds not available from natural sources, such as alkyl glycosides displaying antimicrobial or surfactant properties. Regioselective transxylosylation, an uncommon combination, makes the synthesis reproducible, which is desirable for its potential industrial application. BxTW1 showed high pH stability and Cu²⁺ tolerance. The enzyme displayed a pI of 7.6, a molecular mass around 200 kDa in its active dimeric form, and K_m and V_max values of 0.17 mM and 52.0 U/mg, respectively, using commercial p-nitrophenyl-β-d-xylopyranoside as the substrate. The catalytic efficiencies for the hydrolysis of xylooligosaccharides were remarkably high, making it suitable for different applications in food and bioenergy industries.     

Molecules, wing pattern and distribution: an approach to species delimitation in the "loxurina group" (Lepidoptera: Lycaenidae: Penaincisalia)

The wide range of morphological variations in the "loxurina group" makes taxa identification difficult, and despite several reviews, serious taxonomical confusion remains. We make use of DNA data in conjunction with morphological appearance and available information on species distribution to delimit the boundaries of the "loxurina" group species previously established based on morphology. A fragment of 635 base pairs within the mtDNA gene cytochrome oxidase I (COI) was analysed for seven species of the "loxurina group". Phylogenetic relationships among the included taxa were inferred using maximum parsimony and maximum likelihood methods. Penaincisalia sigsiga (Bálint et al), P. cillutincarae (Draudt), P. atymna (Hewitson) and P. loxurina (C. Felder & R. Felder) were easily delimited as the morphological, geographic and molecular data were congruent. Penaincisalia ludovica (Bálint & Wojtusiak) and P. loxurina astillero (Johnson) represent the same entity and constitute a sub-species of P. loxurina. However, incongruence among morphological, genetic, and geographic data is shown in P. chachapoya (Bálint & Wojtusiak) and P. tegulina (Bálint et al). Our results highlight that an integrative approach is needed to clarify the taxonomy of these neotropical taxa, but more genetic and geographical studies are still required.     

Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex

Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca²⁺-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of ³²[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca²⁺-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component.     

Natural Regeneration in Plantations of Native Trees in Lowland Brazilian Atlantic Forest: Community Structure,Diversity, and Dispersal Syndromes

Plantations of native‐tree species are often recommended for ecological restoration, but the understanding of how these techniques catalyze natural ecological processes is limited. We investigated natural regeneration in five plantations of native trees in the Poço das Antas Biological Reserve (PABR) in the state of Rio de Janeiro, Brazil. The plantations were 9–11 years old, and contained 8–14 native‐tree species with different compositions and relative density of species. We analyzed floristic composition, structure (density and basal area) of overstory and understory strata, as well as other ecological attributes (dispersal syndromes, fruit or seed size, and the availability of fruit for frugivores). Zoochorous species comprised 77% of the community, with a prevalence of the two smallest size classes of propagules (< 0.6 and 0.6–1.6 cm) in natural regeneration. The density of zoochorous plants in the understory was positively correlated with their density in the overstory, indicating their influence on natural regeneration (r² = 0.36; p < 0.0002). Fruit availability for frugivores (density and richness of plants fruiting during the year) was also positively correlated with the density of stems in the understory. Therefore, attributes such as dispersal syndrome and fruiting season should be considered in selecting species to be planted. The differences in natural regeneration observed in each of the native‐tree plantations indicated that the performance of plantations as a restoration strategy may differ, depending on initial species composition, planting density, and site conditions.     

Characterization of a novel immobilized biocatalyst obtained by matrix-assisted refolding of recombinant polyhydroxyoctanoate depolymerase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2442 isolated from inclusion bodies

Purification and matrix-assisted refolding of recombinant His-tagged polyhydroxyalkanoate (PhaZ) depolymerase from Pseudomonas putida KT2442 was carried out. His-tagged enzyme was overproduced as inclusion bodies in recombinant E. coli M15 (pREP4, pPAZ3), which were denatured by 8 M urea, immobilized on Ni²⁺-nitrilotriacetate-agarose matrix, and refolded by gradual removal of the chaotropic agent. The refolded enzyme could not be eluted with 1 M imidazole buffer, leading to an immobilized biocatalyst where PhaZ depolymerase was homogeneously distributed in the agarose support as shown by confocal scanning microscopy. Polyhydroxyoctanoate could not be hydrolyzed by this novel immobilized biocatalyst, whereas the attached enzyme was active in the hydrolysis of p-nitrophenyl alkanoate esters, which differed in their alkyl chain length. Taking advantage of the observed esterase activity on p-nitrophenylacetate, functional characterization of immobilized PhaZ depolymerase was carried out. The immobilized enzyme was more stable than its soluble counterpart and showed optimal hydrolytic activity at 37°C and 50 mM phosphate buffer pH 8.0. Kinetic parameters were obtained with both p-nitrophenylacetate and p-nitrophenyloctanoate, which had not been described so far for the soluble enzyme, representing an attractive and alternative chromogenic assay for the study of this paradigmatic enzyme.     

Structures of wall heterogalactomannans isolated from three genera of entomopathogenic fungi

O-linked heterogalactomannans with similar structural features have been purified from the fungal walls of the entomopathogenic fungi Lecanicillium muscarium, Beauveria bassiana, Beauveriabrongniartii, and Cordyceps sphingum. Their composition and structure have been determined using acid hydrolysis, methylation analysis, gas-liquid chromatography-mass spectrometry (GC-MS) and Nuclear magnetic resonance spectroscopy (NMR). All structures have an α-(1→6)-mannose backbone, but one of the two strains of L. muscarium included in this study contained an acidic heterogalactomannan instead of the neutral polysaccharide isolated in the rest of the species analyzed. Sequence analysis of the internal transcribed spacer (ITS) region of this strain indicated that it belongs to the related genus Simplicillium, displaying low identity (83%) with the closest Lecanicillium species. This is a new demonstration of the structural diversity of fungal wall heteromannans and validates their interest as chemotaxonomic markers. The production of a pullulan-like extracellular polysaccharide in strain CBS 413.70C of L. muscarium is also reported.     

Ursodeoxycholic acid is conjugated with taurine to promote secretin-stimulated biliary hydrocholeresis in the normal rat

Background & Aims

Secretin induces bicarbonate-rich hydrocholeresis in healthy individuals, but not in untreated patients with primary biliary cirrhosis (PBC). Ursodeoxycholic acid (UDCA) – the first choice treatment for PBC – restores the secretin response. Compared with humans, secretin has poor effect in experimental normal-rat models with biliary drainage, although it may elicit hydrocholeresis when the bile-acid pool is maintained. In view of the benefits of UDCA in PBC, we used normal-rat models to unravel the acute contribution of UDCA (and/or taurine-conjugated TUDCA) for eliciting the biliary secretin response.

Methods

Intravascular and/or intrabiliary administration of agonists and inhibitors was performed in normal rats with biliary monitoring. Secretin/bile-acid interplay was analyzed in 3D cultured rat cholangiocytes that formed expansive cystic structures with intralumenal hydroionic secretion.

Results

In vivo, secretin stimulates hydrocholeresis upon UDCA/TUDCA infusion, but does not modify the intrinsic hypercholeretic effect of dehydrocholic acid (DHCA). The former effect is dependent on microtubule polymerization, and involves PKCα, PI3K and MEK pathways, as shown by colchicine (i.p.) and retrograde biliary inhibitors. In vitro, while secretin alone accelerates the spontaneous expansion of 3D-cystic structures, this effect is enhanced in the presence of TUDCA, but not UDCA or DHCA. Experiments with inhibitors and Ca²⁺-chelator confirmed that the synergistic effect of secretin plus TUDCA involves microtubules, intracellular Ca²⁺, PKCα, PI3K, PKA and MEK pathways. Gene silencing also demonstrated the involvement of the bicarbonate extruder Ae2.

Conclusions

UDCA is conjugated in order to promote secretin-stimulated hydrocholeresis in rats through Ae2, microtubules, intracellular Ca²⁺, PKCα, PI3K, PKA, and MEK.