Association mapping of Stagonospora nodorum blotch resistance in modern European winter wheat varieties

Association mapping in populations relevant for wheat breeding has a large potential for validating and fine-mapping QTLs identified in F2- or DH (double haploid)-derived populations. In this study, associations between markers in the region of QSng.sfr-3BS, a major QTL for resistance to Stagonospora nodorum glume blotch (SNG), and SNG resistance were investigated by linkage and association analyses. After increasing marker density in 240 F5:7 recombinant inbred lines (RILs), QSng.sfr-3BS explained 43% of the genetic variance and peaked 0.6cM proximal from the marker SUN2-3B. Association between SNG resistance and markers mapped in the region of QSng.sfr-3BS was investigated in a population of 44 modern European winter wheat varieties. Two genetically distinct subpopulations were identified within these lines. In agreement with linkage analyses, association mapping by a least squares general linear model (GLM) at marker loci in the region of QSng.sfr-3BS revealed the highest association with SNG resistance for SUN2-3B (p<0.05). Association mapping can provide an effective mean of relating genotypes to complex quantitative phenotypes in hexaploid wheat. Linkage disequilibrium (r 2) in chromosome 3B extended less than 0.5cM in 44 varieties, while it extended about 30cM in 240 RILs, based on 91 SSR and STS marker-pair comparisons. This indicated that the association mapping population had a marker resolution potential at least 390-fold higher compared to the RIL populatio

Pembuatan Peta Zona Nilai Ekonomi Kawasan (Znek) Menggunakan Tcm (Travel Cost Method) Dan Cvm (Contingent Valuation Method) Berbasis Sistem Informasi Geografis (Studi Kasus : Candi Prambanan)

Prambanan Temple has potential as a tourist attraction. The strategic location which have historical value, makes this Place became one tourist destination areas Klaten. Based on this, we need a Zone Map Economic Value Areas (ZNEK) to the Prambanan area of the palace to estimate the economic value and benefits based on willingness to pay (WTP) tourists and the people who benefit from the region. Sampling method (respondents) were used in this research is non probability sampling with accidental sampling technique, where respondents are those who by chance / accidental encountered in the study area and can be used as a sample, if it is considered that the person who happened to be found suitable as a data source. Data processing method used is multiple linear regression analysis and calculation software WTP using Maple 17.Results obtained from the study of this final project is the Economic Value Area Zone maps with the total value of economic attraction Prambanan Temple Rp.32.851.020.029.000,- Maps generated from the integration of economic and spatial aspects can be used as an objective consideration of the decision-making process in the spatial field and economic field for the government to optimize and simplify the process of the asset\u27s management and monitoring the natural resources potential. Besides being able to provide a solution for the management of resource constraints of the economy in various regions in Indonesia, the map can be used too as a learning tool for the public society to bring awareness of the importance of potential belonging

Regulatory Characteristics of Bacillus pumilus Protease Promoters

© 2017, Springer Science+Business Media New York.Expression of extracellular protease genes of Bacilli is subject to regulation by many positive and negative regulators. Here we analyzed 5′ regulatory regions of genes encoding proteolytic proteases AprBp, GseBp, and MprBp from Bacillus pumilus strain 3–19. Gfp fusion constructs with upstream genomic regions of different lengths were created for all three genes to identify their natural promoters (regulatory regions). Our results suggest that the aprBp gene, encoding the major subtilisin-like protease, has the most extensive promoter region of approximately 445 bp, while the minor protease genes encoding glutamyl endopeptidase (gseBp) and metalloproteinase (mprBp) are preceded by promoters of 150 and 250 bp in length, respectively. Promoter analysis of PaprBp-gfpmu3 and PgseBp-gfpmu3 reporter fusion constructs in degU and spo0A mutants indicates a positive regulatory effect of DegU and Spo0A on protease expression, while the disruption of abrB, sinR, and scoC repressor genes did not significantly affect promoter activities of all protease genes. On the other hand, the expression of PaprBp-gfpmu3 and PgseBp-gfpmu3 reporters increased 1.6- and 3.0-fold, respectively, in sigD-deficient cells, indicating that the prevention of motility gene expression promotes protease expression. Our results indicate that all examined regulators regulated serine proteases production in B. subtilis

Electrical conduction of silicon oxide containing silicon quantum dots

Current-voltage measurements have been made at room temperature on a Si-rich silicon oxide film deposited via Electron-Cyclotron Resonance Plasma Enhanced Chemical Vapor Deposition (ECR-PECVD) and annealed at 750 - 1000$^\circ$C. The thickness of oxide between Si quantum dots embedded in the film increases with the increase of annealing temperature. This leads to the decrease of current density as the annealing temperature is increased. Assuming the Fowler-Nordheim tunneling mechanism in large electric fields, we obtain an effective barrier height $\phi_{eff}$ of $\sim$ 0.7 $\pm$ 0.1 eV for an electron tunnelling through an oxide layer between Si quantum dots. The Frenkel-Poole effect can also be used to adequately explain the electrical conduction of the film under the influence of large electric fields. We suggest that at room temperature Si quantum dots can be regarded as traps that capture and emit electrons by means of tunneling.Comment: 14 pages, 5 figures, submitted to J. Phys. Conden. Mat

The LIKE system, a novel protein expression toolbox for Bacillus subtilis based on the liaI promoter

Background: Bacillus subtilis is a very important Gram-positive model organism of high biotechnological relevance, which is widely used as a host for the production of both secreted and cytoplasmic proteins. We developed a novel and efficient expression system, based on the liaI promoter (PliaI) from B. subtilis, which is under control of the LiaRS antibiotic-inducible two-component system. In the absence of a stimulus, this promoter is kept tightly inactive. Upon induction by cell wall antibiotics, it shows an over 100-fold increase in activity within 10 min.Results: Based on these traits of PliaI, we developed a novel LiaRS-controlled gene expression system for B. subtilis (the " LIKE" system). Two expression vectors, the integrative pLIKE-int and the replicative pLIKE-rep, were constructed. To enhance the performance of the PliaI-derived system, site-directed mutagenesis was employed to optimize the ribosome binding site and alter its spacing to the initiation codon used for the translational fusion. The impact of these genetic modifications on protein production yield was measured using GFP as a model protein. Moreover, a number of tailored B. subtilis expression strains containing different markerless chromosomal deletions of the liaIH region were constructed to circumvent undesired protein production, enhance the positive autoregulation of the LiaRS system and thereby increase target gene expression strength from the PliaI promoter.Conclusions: The LIKE protein expression system is a novel protein expression system, which offers a number of advantages over existing systems. Its major advantages are (i) a tightly switched-off promoter during exponential growth in the absence of a stimulus, (ii) a concentration-dependent activation of PliaI in the presence of suitable inducers, (iii) a very fast but transient response with a very high dynamic range of over 100-fold (up to 1,000-fold) induction, (iv) a choice from a range of well-defined, commercially available, and affordable inducers and (v) the convenient conversion of LIKE-derived inducible expression strains into strong constitutive protein production factories. © 2012 Toymentseva et al.; licensee BioMed Central Ltd

T-cell derived acetylcholine aids host defenses during enteric bacterial infection with Citrobacter rodentium.

The regulation of mucosal immune function is critical to host protection from enteric pathogens but is incompletely understood. The nervous system and the neurotransmitter acetylcholine play an integral part in host defense against enteric bacterial pathogens. Here we report that acetylcholine producing-T-cells, as a non-neuronal source of ACh, were recruited to the colon during infection with the mouse pathogen Citrobacter rodentium. These ChAT+ T-cells did not exclusively belong to one Th subset and were able to produce IFNγ, IL-17A and IL-22. To interrogate the possible protective effect of acetylcholine released from these cells during enteric infection, T-cells were rendered deficient in their ability to produce acetylcholine through a conditional gene knockout approach. Significantly increased C. rodentium burden was observed in the colon from conditional KO (cKO) compared to WT mice at 10 days post-infection. This increased bacterial burden in cKO mice was associated with increased expression of the cytokines IL-1β, IL-6, and TNFα, but without significant changes in T-cell and ILC associated IL-17A, IL-22, and IFNγ, or epithelial expression of antimicrobial peptides, compared to WT mice. Despite the increased expression of pro-inflammatory cytokines during C. rodentium infection, inducible nitric oxide synthase (Nos2) expression was significantly reduced in intestinal epithelial cells of ChAT T-cell cKO mice 10 days post-infection. Additionally, a cholinergic agonist enhanced IFNγ-induced Nos2 expression in intestinal epithelial cell in vitro. These findings demonstrated that acetylcholine, produced by specialized T-cells that are recruited during C. rodentium infection, are a key mediator in host-microbe interactions and mucosal defenses

Identification of direct residue contacts in protein-protein interaction by message passing

Understanding the molecular determinants of specificity in protein-protein interaction is an outstanding challenge of postgenome biology. The availability of large protein databases generated from sequences of hundreds of bacterial genomes enables various statistical approaches to this problem. In this context covariance-based methods have been used to identify correlation between amino acid positions in interacting proteins. However, these methods have an important shortcoming, in that they cannot distinguish between directly and indirectly correlated residues. We developed a method that combines covariance analysis with global inference analysis, adopted from use in statistical physics. Applied to a set of >2,500 representatives of the bacterial two-component signal transduction system, the combination of covariance with global inference successfully and robustly identified residue pairs that are proximal in space without resorting to ad hoc tuning parameters, both for heterointeractions between sensor kinase (SK) and response regulator (RR) proteins and for homointeractions between RR proteins. The spectacular success of this approach illustrates the effectiveness of the global inference approach in identifying direct interaction based on sequence information alone. We expect this method to be applicable soon to interaction surfaces between proteins present in only 1 copy per genome as the number of sequenced genomes continues to expand. Use of this method could significantly increase the potential targets for therapeutic intervention, shed light on the mechanism of protein-protein interaction, and establish the foundation for the accurate prediction of interacting protein partners.Comment: Supplementary information available on http://www.pnas.org/content/106/1/67.abstrac

Coevolution of ABC Transporters and Two-Component Regulatory Systems as Resistance Modules against Antimicrobial Peptides in Firmicutes Bacteria

In Firmicutes bacteria, ATP-binding cassette (ABC) transporters have been recognized as important resistance determinants against antimicrobial peptides. Together with neighboring two-component systems (TCSs), which regulate their expression, they form specific detoxification modules. Both the transport permease and sensor kinase components show unusual domain architecture: the permeases contain a large extracellular domain, while the sensor kinases lack an obvious input domain. One of the best-characterized examples is the bacitracin resistance module BceRS-BceAB of Bacillus subtilis. Strikingly, in this system, the ABC transporter and TCS have an absolute mutual requirement for each other in both sensing of and resistance to bacitracin, suggesting a novel mode of signal transduction in which the transporter constitutes the actual sensor. We identified over 250 such BceAB-like ABC transporters in the current databases. They occurred almost exclusively in Firmicutes bacteria, and 80% of the transporters were associated with a BceRS-like TCS. Phylogenetic analyses of the permease and sensor kinase components revealed a tight evolutionary correlation. Our findings suggest a direct regulatory interaction between the ABC transporters and TCSs, mediating communication between both components. Based on their observed coclustering and conservation of response regulator binding sites, we could identify putative corresponding two-component systems for transporters lacking a regulatory system in their immediate neighborhood. Taken together, our results show that these types of ABC transporters and TCSs have coevolved to form self-sufficient detoxification modules against antimicrobial peptides, widely distributed among Firmicutes bacteria

Regulation of heterologous subtilin production in Bacillus subtilis W168

Background Subtilin is a peptide antibiotic (lantibiotic) natively produced by Bacillus subtilis ATCC6633. It is encoded in a gene cluster spaBTCSIFEGRK (spa-locus) consisting of four transcriptional units: spaS (subtilin pre-peptide), spaBTC (modification and export), spaIFEG (immunity) and spaRK (regulation). Despite the pioneer understanding on subtilin biosynthesis, a robust platform to facilitate subtilin research and improve subtilin production is still a poorly explored spot. Results In this work, the intact spa-locus was successfully integrated into the chromosome of Bacillus subtilis W168, which is the by far best-characterized Gram-positive model organism with powerful genetics and many advantages in industrial use. Through systematic analysis of spa-promoter activities in B. subtilis W168 wild type and mutant strains, our work demonstrates that subtilin is basally expressed in B. subtilis W168, and the transition state regulator AbrB strongly represses subtilin biosynthesis in a growth phase-dependent manner. The deletion of AbrB remarkably enhanced subtilin gene expression, resulting in comparable yield of bioactive subtilin production as for B. subtilis ATCC6633. However, while in B. subtilis ATCC6633 AbrB regulates subtilin gene expression via SigH, which in turn activates spaRK, AbrB of B. subtilis W168 controls subtilin gene expression in SigH-independent manner, except for the regulation of spaBTC. Furthermore, the work shows that subtilin biosynthesis in B. subtilis W168 is regulated by the two-component regulatory system SpaRK and strictly relies on subtilin itself as inducer to fulfill the autoregulatory circuit. In addition, by incorporating the subtilin-producing system (spa-locus) and subtilin-reporting system (PpsdA-lux) together, we developed “online” reporter strains to efficiently monitor the dynamics of subtilin biosynthesis. Conclusions Within this study, the model organism B. subtilis W168 was successfully established as a novel platform for subtilin biosynthesis and the underlying regulatory mechanism was comprehensively characterized. This work will not only facilitate genetic (engineering) studies on subtilin, but also pave the way for its industrial production. More broadly, this work will shed new light on the heterologous production of other lantibiotics