Variable opacity (Opa) outer membrane proteins account for the cell tropisms displayed by Neisseria gonorrhoeae for human leukocytes and epithelial cells.

Opacity proteins (Opa) of Neisseria gonorrhoeae, a family of variant outer membrane proteins implicated in pathogenesis, are subject to phase variation. In strain MS11, 11 different opa gene alleles have been identified, the expression of which can be turned on and off independently. Using a reverse genetic approach, we demonstrate that a single Opa protein variant of strain MS11, Opa50, enables gonococci to invade epithelial cells. The remaining variant Opa proteins show no, or very little, specificity for epithelial cells but instead confer interaction with human polymorphonuclear neutrophils (PMNs). Thus, depending on the opa allele expressed, gonococci are capable of invading epithelial cells or of interacting with human leukocytes. The respective properties of Opa proteins are maintained independent of the gonococcal strain; thus, the specificity for epithelial cells or leukocytes is intrinsic to Opa proteins. Significant homology exists in the surface exposed variable regions of two invasion supporting Opa proteins from independent strains. Efficient epithelial cell invasion is favoured by high level Opa production, however, a 10-fold reduction still allows significant invasion by gonococci. In contrast, recombinant Escherichia coli expressing Opa proteins adhered or invaded poorly under similar experimental conditions, thus indicating that additional factors besides Opa are required in the Opa-mediated interaction with human cells.     

Light modulation of phosphofructokinase in pea leaf chloroplasts

Chloroplastic phosphofructokinase, phosphorylase, phosphoglucomutase, and phosphoglucoisomerase in peas are light inactivated. The effect of light on phosphofructokinase is mimicked by dithiothreitol. DCMU, arsenite, and sulfite inhibit light modulation of the enzyme. No effect of light inactivation on the K_m (fructose-6-P) or pH optima of phosphofructokinase was observed.     

A novel allele of the P-starvation tolerance gene OsPSTOL1 from African rice (Oryza glaberrima Steud) and its distribution in the genus Oryza

Key message

We have developed allele-specific markers for molecular breeding to transfer the PSTOL1 gene from Kasalath to African mega-varieties, including NERICAs, to improve their tolerance to P-deficient soil.

Abstract

The deficiency of phosphorus (P) in soil is a major problem in Sub-Saharan Africa due to general nutrient depletion and the presence of P-fixing soils. Developing rice cultivars with enhanced P efficiency would, therefore, represent a sustainable strategy to improve the livelihood of resource-poor farmers. Recently the Pup1 locus, a major QTL for tolerance to P deficiency in soil, was successfully narrowed-down to a major gene, the protein kinase OsPSTOL1 (P-starvation tolerance), which was found to be generally absent from modern irrigated rice varieties. Our target is to improve the tolerance of African mega-varieties to P deficiency through marker-assisted introgression of PSTOL1. As a first step, we have determined the Pup1 haplotype and surveyed the presence or absence of PSTOL1 and other genes of the Pup1 locus in African mega-varieties, NERICAs (New Rice for Africa) and their Oryza glaberrima parents. Here, we report the presence of a novel PSTOL1 allele in upland NERICAs that was inherited from the O. glaberrima parent CG14. This allele showed a 35 base-pair substitution when aligned to the Kasalath allele, but maintained a fully conserved kinase domain, and is present in most O. glaberrima accessions evaluated. In-silico and marker analysis indicated that many other genes of the Kasalath Pup1 locus were missing in the O. glaberrima genome, including the dirigent-like gene OsPupK20-2, which was shown to be downstream of PSTOL1. We have developed several allele-specific markers for the use for molecular breeding to transfer the PSTOL1 gene from Kasalath to African mega-varieties, including NERICAs.     

Impact of temperature on Na2Sr(PO4)F:Eu3+ phosphor

In this article, we report the synthesis of Na₂Sr_1‐x(PO₄)F:Eu_x phosphor via a combustion method. The influence of different annealing temperatures on the photoluminescence properties was investigated. The phosphor was excited at both 254 and 393 nm. Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphors emit strong orange and red color at 593 and 612 nm, respectively, under both excitation wavelengths. Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphors annealed at 1050°C showed stronger emission intensity compared with 600, 900 and 1200°C. Moreover, Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphor was found to be more intense when compared with commercial Y₂O₃:Eu³⁺ phosphor. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparative sequence analyses of the major quantitative trait locus phosphorus uptake 1 (Pup1) reveal a complex genetic structure

The p hosphorus up take 1 ( Pup1 ) locus was identified as a major quantitative trait locus (QTL) for tolerance of phosphorus deficiency in rice. Near-isogenic lines with the Pup1 region from tolerant donor parent Kasalath typically show threefold higher phosphorus uptake and grain yield in phosphorus-deficient field trials than the intolerant parent Nipponbare. In this study, we report the fine mapping of the Pup1 locus to the long arm of chromosome 12 (15.31–15.47 Mb). Genes in the region were initially identified on the basis of the Nipponbare reference genome, but did not reveal any obvious candidate genes related to phosphorus uptake. Kasalath BAC clones were therefore sequenced and revealed a 278-kbp sequence significantly different from the syntenic regions in Nipponbare (145 kb) and in the indica reference genome of 93-11 (742 kbp). Size differences are caused by large insertions or deletions (INDELs), and an exceptionally large number of retrotransposon and transposon-related elements (TEs) present in all three sequences (45%–54%). About 46 kb of the Kasalath sequence did not align with the entire Nipponbare genome, and only three Nipponbare genes (fatty acid α-dioxygenase, dirigent protein and aspartic proteinase) are highly conserved in Kasalath. Two Nipponbare genes (expressed proteins) might have evolved by at least three TE integrations in an ancestor gene that is still present in Kasalath. Several predicted Kasalath genes are novel or unknown genes that are mainly located within INDEL regions. Our results highlight the importance of sequencing QTL regions in the respective donor parent, as important genes might not be present in the current reference genomes.     

Soil Type-Dependent Responses to Phenanthrene as Revealed by Determining the Diversity and Abundance of Polycyclic Aromatic Hydrocarbon Ring-Hydroxylating Dioxygenase Genes by Using a Novel PCR Detection System

A novel PCR primer system that targets a wide range of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD_α) genes of both Gram-positive and Gram-negative bacteria was developed and used to study their abundance and diversity in two different soils in response to phenanthrene spiking. The specificities and target ranges of the primers predicted in silico were confirmed experimentally by cloning and sequencing of PAH-RHD_α gene amplicons from soil DNA. Cloning and sequencing showed the dominance of phnAc genes in the contaminated Luvisol. In contrast, high diversity of PAH-RHD_α genes of Gram-positive and Gram-negative bacteria was observed in the phenanthrene-spiked Cambisol. Quantitative real-time PCR based on the same primers revealed that 63 days after phenanthrene spiking, PAH-RHD_α genes were 1 order of magnitude more abundant in the Luvisol than in the Cambisol, while they were not detected in both control soils. In conclusion, sequence analysis of the amplicons obtained confirmed the specificity of the novel primer system and revealed a soil type-dependent response of PAH-RHD_α gene-carrying soil bacteria to phenanthrene spiking.Polycyclic aromatic hydrocarbons (PAHs) are hydrophobic compounds composed of two or more fused aromatic rings. Although PAHs are ubiquitous in the environment (from natural oil seeps, brush fires, and plant derivatives), anthropogenic activities, such as disposal of coal-processing waste, mining accidents, petroleum wastes, and vehicle exhaust, have drastically increased their occurrence in the environment. The fate of PAHs in soil is of great interest due to their potential for bioaccumulation, persistence, transport, and toxicity. Microbe-driven aerobic degradation of PAHs is well documented (15-17). The diversity of PAH-degrading genes in soils is assumed to be huge, but the extent of diversity and how it is influenced by different soil types or their history and type of pollution are not yet fully explored. Knowledge of the genes coding for dioxygenase enzymes that catalyze the primary step of PAH degradation by incorporating molecular oxygen into the aromatic nucleus is an essential prerequisite to unraveling the contributions of microbial population networks to transformation, assimilation, and degradation of organic chemicals in soil. Recently, the complete genomes of several PAH-degrading bacteria became available and allowed new insights into degradative pathways (6, 18, 36). Organic pollutants also serve as nutrients for those microbes that have the appropriate genetic makeup to utilize them, resulting in their increased metabolic activity and abundance (4, 14). In the last decade, impressive progress was seen in techniques that allow cultivation-independent analysis of microbial communities and thus overcome the most severe limitations in studying microbial communities in natural habitats, namely, that only a rather small portion of microbes are accessible to standard cultivation conditions (1, 29). For more than a decade, cultivation-independent approaches have also been employed to unravel the responses of microbial communities in soils and sediments to PAH pollution. In all these studies, PCR amplification of PAH-degrading gene fragments from nucleic acids directly extracted from environmental samples was used to explore the abundance and diversity of PAH ring-hydroxylating dioxygenase (PAH-RHD_α) genes (4, 8, 9, 13, 14, 22, 34, 37). Despite the known biases of PCR amplification from mixed templates, these techniques allow highly sensitive and specific detection even from minute amounts of nucleic acids. In order to select suitable primer systems, previously published primer systems were analyzed for their ranges of target sequences. The existing primer systems were found to have limitations, as they often target only a rather narrow range of sequences, e.g., nahAc- or phnAc-type sequences (21, 34) or only PAH-RHD_α genes from Gram-negative bacteria (3, 13). In other studies, two-primer systems were used to target PAH-RHD_α genes of both Gram-positive and Gram-negative bacteria (4, 37). Only one primer system targeting the Rieske gene fragment was described that amplified a small fragment from PAH-RHD_α genes from both Gram-negative and Gram-positive bacteria (24). However, the amplicon size was only 78 bp and the primer might also target genes coding for dioxygenases that attack nonpolar aromatic compounds, such as benzene, toluene, and xylene. Therefore, this work aimed to design an improved primer system that targets PAH-RHD_α genes from both Gram-positive and Gram-negative bacteria and provides larger amplicon sizes. The novel primer system was tested in silico and validated by sequencing cloned PAH-RHD_α genes amplified from total-community (TC) DNA and was used in endpoint and quantitative real-time PCR (qPCR) formats. The primer system was also applied to study the responses of soil microbial communities in two different soils (a Cambisol and a Luvisol representing typical arable soils in Central Europe with different texture compositions) to artificial phenanthrene pollution.     

Lipid-binding hSH3 domains in immune cell adapter proteins

SH3 domains represent versatile scaffolds within eukaryotic cells by targeting proline-rich sequences within intracellular proteins. More recently, binding of SH3 domains to unusual peptide motifs, folded proteins or lipids has been reported. Here we show that the newly defined hSH3 domains of immune cell adapter proteins bind lipid membranes with distinct affinities. The interaction of the hSH3 domains of adhesion and degranulation promoting adapter protein (ADAP) and PRAM-1 (Promyelocytic-Retinoic acid receptor alpha target gene encoding an Adaptor Molecule-1), with phosphatidylcholine-containing liposomes is observed upon incorporation of phosphatidylserine (PS) or phosphoinositides (PIs) into the membrane bilayer. Mechanistically we show that stable association of the N-terminal, amphipathic helix with the beta-sheet scaffold favours lipid binding and that the interaction with PI(4,5)P(2)-containing liposomes is consistent with a single-site, non-cooperative binding mechanism. Functional investigations indicate that deletion of both amphipathic helices of the hSH3 domains reduces the ability of ADAP to enhance adhesion and migration in stimulated T cells.     

Erratum to: Development and application of gene-based markers for the major rice QTL Phosphorus uptake 1

Marker-assisted breeding is a very useful tool for breeders but still lags behind its potential because information on the effect of quantitative trait loci (QTLs) in different genetic backgrounds and ideal molecular markers are unavailable. Here, we report on some first steps toward the validation and application of the major rice QTL Phosphate uptake 1 (Pup1) that confers tolerance of phosphorus (P) deficiency in rice (Oryza sativa L.). Based on the Pup1 genomic sequence of the tolerant donor variety Kasalath that recently became available, markers were designed that target (1) putative genes that are partially conserved in the Nipponbare reference genome and (2) Kasalath-specific genes that are located in a large insertion-deletion (INDEL) region that is absent in Nipponbare. Testing these markers in 159 diverse rice accessions confirmed their diagnostic value across genotypes and showed that Pup1 is present in more than 50% of rice accessions adapted to stress-prone environments, whereas it was detected in only about 10% of the analyzed irrigated/lowland varieties. Furthermore, the Pup1 locus was detected in more than 80% of the analyzed drought-tolerant rice breeding lines, suggesting that breeders are unknowingly selecting for Pup1. A hydroponics experiment revealed genotypic differences in the response to P deficiency between upland and irrigated varieties but confirmed that root elongation is independent of Pup1. Contrasting Pup1 near-isogenic lines (NILs) were subsequently grown in two different P-deficient soils and environments. Under the applied aerobic growth conditions, NILs with the Pup1 locus maintained significantly higher grain weight plant⁻¹ under P deprivation in comparison with intolerant sister lines without Pup1. Overall, the data provide evidence that Pup1 has the potential to improve yield in P-deficient and/or drought-prone environments and in diverse genetic backgrounds.