Nitrogen fixation by Rhizobium leguminosarum PRE : a genetical and biochemical approach

Nitrogen fix ation by Rhizobium and Bradyrhizobium bacteria in symbiosis with their leguminous host plants forms an attractive alternative for the industrial production of nitrogenous fertilizers, both from an economic as well as an enviromnental point of view, and is the topic of many scientific research programs nowadays. Ultimate goals in many of these programs are improving the efficiency of nitrogen fix ation, the extension of the host range of the bacteria to important, non-leguminous crops and the transfer of the nitrogen fix ing ability from the bacterium to the plant. A thorough knowledge of the biochemical processes and the genetic determinants involved in both plant and bacterium, however, is a prerequisite for the achievement of any of the above mentioned goals.In this thesis a genetic and biochemical analysis of R.leguminosarum PRE is described. The organization of the nif and fix genes on the sym plasmid, involved in the reduction of dinitrogen, was studied by the construction of extended physical maps of parts of this plasmid (chapter 2). Furthermore, the region upstream of the fix ABC operon was studied in detail. A novel fix gene ( fix W), located immediately upstream of fix A, was identified and the expression of this gene was studied (chapter 3). Four regions on the PRE sym plasmid were found to contain reiterations of (parts) of functional nif and fix genes.To elucidate the route by which the enzyme nitrogenase is supplied with electrons, the membrane fraction of R.leguminosarum bacteroids was analyzed for NADH dehydrogenase activity and a bacteroid specific NADH dehydrogenase complex, DH1, was isolated (chapter 4). With the aid of specific antisera directed against the different subunits in this dehydrogenase complex, it was shown that only one subunit, with a molecular weight of 35 kD, is bacteroid specific. To investigate a possible role of complex DH1 in the electron transport to nitrogenase, an attempt was made to construct R.leguminosarum mutants disturbed in the synthesis of this 35K subunit (chapter 5). Therefore, this protein was isolated and the N-terminal amino acid sequence was determined. Based on this sequence oligodeoxynucleotide probes were synthesized and used to screen a cosmid library in order to identify the gene encoding the 35K subunit. Although this gene was not found, several other DNA fragments were cloned showing a high degree of homology to the deduced 35K-gene nucleotide sequence, which indicates that the experimental procedure followed eventually can lead to the isolation of this gene. In chapter 6 an analysis of proteins of the peribacteroid space (PBS) is described. This symbiotic compartment forms the interface between the Rhizobium bacteroid and the plant host, and the proteins of this space may have an important role in transport processes and in signal transduction between the two partners in the symbiosis. The bulk of the proteins (about 90%) was found to be excreted by the bacteroid into the PBS, whereas the remaining 10% probably is of plant origin. About one third of the PBS proteins appeared to occur also in the periplasmic space of free-living bacteria. Four bacteroid encoded PBS proteins were identified, which are not present in free-living R.leguminosarum bacteria and thus might play a role in nitrogen fix ation. These proteins might lead to the identification of novel symbiotic loci on the R.leguminosarum genome

The sequence of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus genome

The nucleotide sequence of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) DNA genome was determined and analysed. The circular genome encompasses 131 403 bp, has a G C content of 39.1 molnd contains five homologous regions with a unique pattern of repeats. Computer-assisted analysis revealed 135 putative ORFs of 150 nt or larger; 100 ORFs have homologues in Autographa californica multicapsid NPV (AcMNPV) and a further 15 ORFs have homologues in other baculoviruses such as Lymantria dispar MNPV (LdMNPV), Spodoptera exigua MNPV (SeMNPV) and Xestia c-nigrum granulovirus (XcGV). Twenty ORFs are unique to HaSNPV without homologues in GenBank. Among the six previously sequenced baculoviruses, AcMNPV, Bombyx mori NPV (BmNPV), Orgyia pseudotsugata MNPV (OpMNPV), SeMNPV, LdMNPV and XcGV, 65 ORFs are conserved and hence are considered as core baculovirus genes. The mean overall amino acid identity of HaSNPV ORFs was the highest with SeMNPV and LdMNPV homologues. Other than three 'baculovirus repeat ORFs' (bro) and two 'inhibitor of apoptosis' (iap) genes, no duplicated ORFs were found. A putative ORF showing similarity to poly(ADP-ribose) glycohydrolases (parg) was newly identified. The HaSNPV genome lacks a homologue of the major budded virus (BV) glycoprotein gene, gp64, of AcMNPV, BmNPV and OpMNPV. Instead, a homologue of SeMNPV ORF8, encoding the major BV envelope protein, has been identified. GeneParityPlot analysis suggests that HaSNPV, SeMNPV and LdMNPV (group II) have structural genomic features in common and are distinct from the group I NPVs and from the granuloviruses. Cluster alignment between group I and group II baculoviruses suggests that they have a common ancestor

Vitality and the course of limitations in activities in osteoarthritis of the hip or knee

<p>Abstract</p> <p>Background</p> <p>The objective of the study was to determine whether psychological and social factors predict the course of limitations in activities in elderly patients with osteoarthritis of the hip or knee, in addition to established somatic and cognitive risk factors.</p> <p>Methods</p> <p>A longitudinal cohort study with a follow-up period of three years was conducted. Patients (N = 237) with hip or knee osteoarthritis were recruited from rehabilitation centers and hospitals. Body functions, comorbidity, cognitive functioning, limitations in activities and psychological and social factors (mental health, vitality, pain coping and perceived social support) were assessed. Statistical analyses included univariate and multivariate regression analyses. Psychological and social factors were added to a previously developed model with body functions, comorbidity and cognitive functioning.</p> <p>Results</p> <p>In knee OA, low vitality has a negative impact on the course of self-reported and performance-based limitations in activities, after controlling for somatic and cognitive factors. In hip OA, psychological and social factors had no additional contribution to the model.</p> <p>Conclusion</p> <p>Low vitality predicts deterioration of limitations in activities in elderly patients with osteoarthritis of the knee, in addition to established somatic and cognitive risk factors. However, the contribution of vitality is relatively small. Results of this study are relevant for the group of patients with knee or hip OA, attending hospitals and rehabilitation centers.</p

Approaches and determinants to sustainably improve crop production

Abstract: Plant scientists and farmers are facing major challenges in providing food and nutritional security for a growing population, while preserving natural resources and biodiversity. Moreover, this should be done while adapting agriculture to climate change and by reducing its carbon footprint. To address these challenges, there is an urgent need to breed crops that are more resilient to suboptimal environments. Huge progress has recently been made in understanding the physiological, genetic and molecular bases of plant nutrition and environmental responses, paving the way towards a more sustainable agriculture. In this review, we present an overview of these progresses and strategies that could be developed to increase plant nutrient use efficiency and tolerance to abiotic stresses. As illustrated by many examples, they already led to promising achievements and crop improvements. Here, we focus on nitrogen and phosphate uptake and use efficiency and on adaptation to drought, salinity and heat stress. These examples first show the necessity of deepening our physiological and molecular understanding of plant environmental responses. In particular, more attention should be paid to investigate stress combinations and stress recovery and acclimation that have been largely neglected to date. It will be necessary to extend these approaches from model plants to crops, to unravel the relevant molecular targets of biotechnological or genetic strategies directly in these species. Similarly, sustained efforts should be done for further exploring the genetic resources available in these species, as well as in wild species adapted to unfavourable environments. Finally, technological developments will be required to breed crops that are more resilient and efficient. This especially relates to the development of multiscale phenotyping under field conditions and a wide range of environments, and use of modelling and big data management to handle the huge amount of information provided by the new molecular, genetic and phenotyping techniques

Improving crop yield potential: Underlying biological processes and future prospects

The growing world population and global increases in the standard of living both result in an increasing demand for food, feed and other plant‐derived products. In the coming years, plant‐based research will be among the major drivers ensuring food security and the expansion of the bio‐based economy. Crop productivity is determined by several factors, including the available physical and agricultural resources, crop management, and the resource use efficiency, quality and intrinsic yield potential of the chosen crop. This review focuses on intrinsic yield potential, since understanding its determinants and their biological basis will allow to maximize the plant's potential in food and energy production. Yield potential is determined by a variety of complex traits that integrate strictly regulated processes and their underlying gene regulatory networks. Due to this inherent complexity, numerous potential targets have been identified that could be exploited to increase crop yield. These encompass diverse metabolic and physical processes at the cellular, organ and canopy level. We present an overview of some of the distinct biological processes considered to be crucial for yield determination that could further be exploited to improve future crop productivity

A genome-wide genetic map of NB-LRR disease resistance loci in potato

Like all plants, potato has evolved a surveillance system consisting of a large array of genes encoding for immune receptors that confer resistance to pathogens and pests. The majority of these so-called resistance or R proteins belong to the super-family that harbour a nucleotide binding and a leucine-rich-repeat domain (NB-LRR). Here, sequence information of the conserved NB domain was used to investigate the genome-wide genetic distribution of the NB-LRR resistance gene loci in potato. We analysed the sequences of 288 unique BAC clones selected using filter hybridisation screening of a BAC library of the diploid potato clone RH89-039-16 (S. tuberosum ssp. tuberosum) and a physical map of this BAC library. This resulted in the identification of 738 partial and full-length NB-LRR sequences. Based on homology of these sequences with known resistance genes, 280 and 448 sequences were classified as TIR-NB-LRR (TNL) and CC-NB-LRR (CNL) sequences, respectively. Genetic mapping revealed the presence of 15 TNL and 32 CNL loci. Thirty-six are novel, while three TNL loci and eight CNL loci are syntenic with previously identified functional resistance genes. The genetic map was complemented with 68 universal CAPS markers and 82 disease resistance trait loci described in literature, providing an excellent template for genetic studies and applied research in potato