Factors Defining the Functional Oligomeric State of Escherichia coli DegP Protease

Escherichia coli DegP protein is a periplasmic protein that functions both as a protease and as a chaperone. In the absence of substrate, DegP oligomerizes as a hexameric cage but in its presence DegP reorganizes into 12 and 24-mer cages with large chambers that house the substrate for degradation or refolding. Here, we studied the factors that determine the oligomeric state adopted by DegP in the presence of substrate. Using size exclusion chromatography and electron microscopy, we found that the size of the substrate molecule is the main factor conditioning the oligomeric state adopted by the enzyme. Other factors such as temperature, a major regulatory factor of the activity of this enzyme, did not influence the oligomeric state adopted by DegP. In addition, we observed that substrate concentration exerted an effect only when large substrates (full-length proteins) were used. However, small substrate molecules (peptides) always triggered the same oligomeric state regardless of their concentration. These results clarify important aspects of the regulation of the oligomeric state of DegP

Formation of two-dimensional weak localization in conducting Langmuir-Blodgett films

We report the magnetotransport properties up to 7 T in the organic highly conducting Langmuir-Blodgett(LB) films formed by a molecular association of the electroactive donor molecule bis(ethylendioxy)tetrathiafulvalene (BEDO-TTF) and stearic acid CH$_3$(CH$_2$)$_{16}$COOH. We show the logarithmic decrease of dc conductivity and the negative transverse magnetoresistance at low temperature. They are interpreted in the weak localization of two-dimensional (2D) electronic system based on the homogeneous conducting layer with the molecular size thickness of BEDO-TTF. The electronic length with phase memory is given at the mesoscopic scale, which provides for the first time evidence of the 2D coherent charge transport in the conducting LB films.Comment: 5 pages, 1 Table and 5 figure

Associations between fears related to safety during sleep and self-reported sleep in men and women living in a low-socioeconomic status setting

South Africans living in low socioeconomic areas have self-reported unusually long sleep durations (approximately 9–10 h). One hypothesis is that these long durations may be a compensatory response to poor sleep quality as a result of stressful environments. This study aimed to investigate whether fear of not being safe during sleep is associated with markers of sleep quality or duration in men and women. South Africans (n = 411, 25–50 y, 57% women) of African-origin living in an urban township, characterised by high crime and poverty rates, participated in this study. Participants are part of a larger longitudinal cohort study: Modelling the Epidemiologic Transition Study (METS)–Microbiome. Customised questions were used to assess the presence or absence of fears related to feeling safe during sleep, and the Epworth Sleepiness Scale, Pittsburgh Sleep Quality Index (PSQI) and Insomnia Severity Index were used to assess daytime sleepiness, sleep quality and insomnia symptom severity respectively. Adjusted logistic regression models indicated that participants who reported fears related to safety during sleep were more likely to report poor sleep quality (PSQI > 5) compared to participants not reporting such fears and that this relationship was stronger among men than women. This is one of the first studies outside American or European populations to suggest that poor quality sleep is associated with fear of personal safety in low-SES South African adults

Molecular characterization of the Corynebacterium pseudotuberculosis hsp60-hsp10 operon, and evaluation of the immune response and protective efficacy induced by hsp60 DNA vaccination in mice

<p>Abstract</p> <p>Background</p> <p>Heat shock proteins (HSPs) are important candidates for the development of vaccines because they are usually able to promote both humoral and cellular immune responses in mammals. We identified and characterized the <it>hsp60-hsp10 </it>bicistronic operon of the animal pathogen <it>Corynebacterium pseudotuberculosis</it>, a Gram-positive bacterium of the class <it>Actinobacteria</it>, which causes caseous lymphadenitis (CLA) in small ruminants.</p> <p>Findings</p> <p>To construct the DNA vaccine, the <it>hsp60 </it>gene of <it>C. pseudotuberculosis </it>was cloned in a mammalian expression vector. BALB/c mice were immunized by intramuscular injection with the recombinant plasmid (pVAX1/<it>hsp60</it>).</p> <p>Conclusion</p> <p>This vaccination induced significant anti-hsp60 IgG, IgG1 and IgG2a isotype production. However, immunization with this DNA vaccine did not confer protective immunity.</p

Reversal of the ΔdegP Phenotypes by a Novel rpoE Allele of Escherichia coli

RseA sequesters RpoE (σE) to the inner membrane of Escherichia coli when envelope stress is low. Elevated envelope stress triggers RseA cleavage by the sequential action of two membrane proteases, DegS and RseP, releasing σE to activate an envelope stress reducing pathway. Revertants of a ΔdegP ΔbamB strain, which fails to grow at 37°C due to high envelope stress, harbored mutations in the rseA and rpoE genes. Null and missense rseA mutations constitutively hyper-activated the σE regulon and significantly reduced the major outer membrane protein (OMP) levels. In contrast, a novel rpoE allele, rpoE3, resulting from the partial duplication of the rpoE gene, increased σE levels greater than that seen in the rseA mutant background but did not reduce OMP levels. A σE-dependent RybB::LacZ construct showed only a weak activation of the σE pathway by rpoE3. Despite this, rpoE3 fully reversed the growth and envelope vesiculation phenotypes of ΔdegP. Interestingly, rpoE3 also brought down the modestly activated Cpx envelope stress pathway in the ΔdegP strain to the wild type level, showing the complementary nature of the σE and Cpx pathways. Through employing a labile mutant periplasmic protein, AcrAL222Q, it was determined that the rpoE3 mutation overcomes the ΔdegP phenotypes, in part, by activating a σE-dependent proteolytic pathway. Our data suggest that a reduction in the OMP levels is not intrinsic to the σE-mediated mechanism of lowering envelope stress. They also suggest that under extreme envelope stress, a tight homeostasis loop between RseA and σE may partly be responsible for cell death, and this loop can be broken by mutations that either lower RseA activity or increase σE levels

Prediction of Extracellular Proteases of the Human Pathogen Helicobacter pylori Reveals Proteolytic Activity of the Hp1018/19 Protein HtrA

Exported proteases of Helicobacter pylori (H. pylori) are potentially involved in pathogen-associated disorders leading to gastric inflammation and neoplasia. By comprehensive sequence screening of the H. pylori proteome for predicted secreted proteases, we retrieved several candidate genes. We detected caseinolytic activities of several such proteases, which are released independently from the H. pylori type IV secretion system encoded by the cag pathogenicity island (cagPAI). Among these, we found the predicted serine protease HtrA (Hp1019), which was previously identified in the bacterial secretome of H. pylori. Importantly, we further found that the H. pylori genes hp1018 and hp1019 represent a single gene likely coding for an exported protein. Here, we directly verified proteolytic activity of HtrA in vitro and identified the HtrA protease in zymograms by mass spectrometry. Overexpressed and purified HtrA exhibited pronounced proteolytic activity, which is inactivated after mutation of Ser205 to alanine in the predicted active center of HtrA. These data demonstrate that H. pylori secretes HtrA as an active protease, which might represent a novel candidate target for therapeutic intervention strategies

MRP3 is a sex determining gene in the diatom Pseudo-nitzschia multistriata

A broad diversity of sex-determining systems has evolved in eukaryotes. However, information on the mechanisms of sex determination for unicellular microalgae is limited, including for diatoms, key-players of ocean food webs. Here we report the identification of a mating type (MT) determining gene for the diatom Pseudo-nitzschia multistriata. By comparing the expression profile of the two MTs, we find five MT-biased genes, of which one, MRP3, is expressed exclusively in MT+ strains in a monoallelic manner. A short tandem repeat of specific length in the region upstream of MRP3 is consistently present in MT+ and absent in MT- strains. MRP3 overexpression in an MT- strain induces sex reversal: the transgenic MT- can mate with another MT- strain and displays altered regulation of the other MT-biased genes, indicating that they lie downstream. Our data show that a relatively simple genetic program is involved in defining the MT in P. multistriata