The Impact of Nitrite on Aerobic Growth of Paracoccus denitrificans PD1222

The effect of nitrite stress induced in Paracoccus denitrificans PD1222 was examined using additions of sodium nitrite to an aerobic bacterial culture. Nitrite generates a strong stress response in P. denitrificans, causing growth inhibition. This is dependent on both the concentration of nitrite present and the pH. The pH dependent effect of nitrite growth inhibition is likely a result of nitrite and free nitrous acid (FNA; pKa = 3.16) and subsequent reactive nitrogen oxides, generated from the intracellular passage of FNA into P. denitrificans. A flavohemoglobin (fhb; Pd1689) and its associated NsrR family, transcriptional regulator (Pd1690), were transcribed above a ≥2 fold expression filter (p ≤ 0.05) at 95% significance in qRT-PCR and a type II microarray transcriptional analysis at 12.5 mM nitrite in batch culture. Additionally, >25 fold expression of the flavohemoglobin was confirmed by qRT-PCR in continuous culture with nitrite. A deletion mutant determined fhb to be involved in conveying nitrite resistance at high nitrite concentrations and is linked to a stimulation of biomass generated by the presence of nitrite. The cytochrome ba3 oxidase was found to be associated with nitrite in transcriptional analysis, suggesting the uncoupling of the protonmotive force caused by the transport of FNA across the membrane, and subsequent dissociation in the cytoplasm were reduced by a method of counterbalance. No nitrate accumulation was seen and nitrous oxide levels were above that observed for atmospheric background levels. The microarray analysis was used to confirm that in batch growth at 12.5 mM nitrite addition, P. denitrificans shows an overall stress response associated with protein, DNA and lipid repair, with the addition of fhb detoxification and action of the cytochrome ba3 oxidase. It is therefore suggested that nitrite presents a pHdependent stress response in P. denitrificans, likely due to the production of associated reactive nitrogen species such as NO from the internalisation of FNA and the uncoupling of the protonmotive force

The quest for celiac-safe wheat

Gluten proteins from wheat have the unique property to interact with each other and form a network in dough preparation. In this gluten network gas bubbles can be retained that are produced by yeast, which is added during dough preparation. The result is a voluminous, viscoelastic dough for bread making. Besides these network forming properties, gluten proteins contain remarkably high amounts of the amino acids proline and glutamine. Therefore, these proteins can not be degraded completely in the human gastrointestinal tract and so called epitopes are generated in the small intestine after ingestion of gluten containing foods such as bread and cookies. Intolerance to gluten proteins can cause inflammation of the small intestine, which leads to villous atrophy (flatten mucosa) and malabsorption. This intolerance to gluten proteins is also called celiac disease. The with celiac disease occurring inflammation can lead to many symptoms such as malnutrition, weight loss, stomach pain, diarrhea, dermatitis and in the worst case lymphoma. In children, celiac disease can cause growth retardation. Gluten proteins from rye and barley, because of their similarity to gluten proteins from wheat and high proline and glutamine content, can also cause celiac disease. Celiac disease patients therefore have to avoid life-long all food products containing wheat, rye, and barley (gluten-free diet). Not only gluten protein intake but also genetic susceptibility is necessary to develop celiac disease. Therefore, relatives of celiac disease patients have a highly increased risk to develop celiac disease. About 0.52% of the Western population suffers from celiac disease of which 7097% is undiagnosed, and the prevalence of celiac disease is still increasing. The reason for this may be the consumption of many bread, cookie and pasta products, but also because wheat constituents, such as gluten and starch, are increasingly used as food additive in soups, sauces, sausage, candy, ice-cream, and even in medicines. Because bread wheat originated thousands of years ago by fusion of three different grass species, many different gluten proteins exist. Pasta wheat originated from two of the three grass species and lacks the species that contains most of the celiac disease epitopes. Not all gluten proteins can be removed because baking properties might be lost. For analysis of the presence of celiac disease epitopes, it is important that all gluten proteins are extracted from the wheat kernels. Therefore, an extraction protocol was developed to extract as many gluten proteins as possible, which allows analysis of many samples at the same time. Because thousands of different wheats exist that have never been tested for celiac disease epitopes, this extraction and detection method was applied to analyze whether modern wheats, which are used by breeders, exist with lower amounts of celiac disease epitopes. These modern wheats were subsequently compared to old wheats collected from all over the world. From these analyses it seemed that there was an increase in celiac disease epitopes in modern wheats. This might be the reason for the increase of prevalence of celiac disease. For future breeding of bread wheat it is important to select and breed not only for high yield, disease/pest resistance, and baking quality, but also for the (reduced) presence of relevant celiac disease epitopes. In older bread and pasta wheats, we observed that fewer celiac disease epitopes are present. Knowing this, a more celiac-safe bread wheat could be developed by performing crossings with a selection of wheats. In the analysis of wheat lines that miss specifically genetic parts of one of the three grass species from which bread wheat evolved, it appeared that if the part is removed that encodes celiac disease epitopes to which most patients respond, the dough properties improved compared to the control dough. By addition of similar proteins from oat (avenins), the dough properties improved even more. These gluten proteins from oat are tolerated by most celiac disease patients. The results show that it is feasible to develop wheat that contains less celiac disease epitopes without decreasing dough properties. Celiac disease patients, however, respond differently to different celiac disease epitopes which complicates the development of wheat that is suitable for all celiac disease patients. The research performed in this thesis shows that selection of wheat for the presence of celiac disease epitopes is extremely relevant because it can reduce the number of celiac disease patients in the future. At the same time, we demonstrate that celiac-safe wheat can still maintain good baking properties. Diagnosed celiac disease patients will benefit as well from celiac-safe wheat because exposure to ‘hidden’ gluten proteins will decrease. <br/

The CydDC transporter of Escherichia coli: investigating the impact of reductant export upon nitrosative stress, transcriptome/metabolome interplay, and host colonisation

CydDC of Escherichia coli is an ABC transporter that exports cysteine and glutathione from the cytoplasm to the periplasm to maintain redox homeostasis; its loss elicits a pleiotropic phenotype and causes the periplasm to become ‘over-oxidising’. In addition, the CydDC transporter is required for the assembly of cytochrome bd-I, a respiratory complex that provides tolerance to nitric oxide, a toxic radical produced by the host immune system in response to bacterial infection. The contribution of CydDC to nitric oxide tolerance and the pleiotropic phenotype of cydDC mutants implicates this exporter complex as a potential target for future therapies to combat E. coli infections. Indeed, given the rising incidence of multidrug-resistant bacterial infections, it is becoming increasingly important to develop novel strategies to combat infection. This thesis reports an investigation into the contribution of CydDC to NO tolerance, the relationship between CydDC expression and cytochrome bd-I assembly, adaptations resulting from loss of CydDC, and the requirement for CydDC for survival during infection. To gain a better understanding of how CydDC expression influences cytochrome bd-I assembly, cydDC cells were grown in the presence of exogenous cysteine and/or reduced glutathione. This work demonstrates for the first time that addition of cysteine and glutathione (i.e. both CydDC substrates) is necessary for cytochrome bd-I assembly in a cydDC strain. In vitro growth curves utilising a nitric oxide donor show that CydDC-mediated reductant export contributes to the tolerance of nitric oxide (NO) both via permitting cytochrome bd-I assembly and through a mechanism independent of this respiratory complex. This work is consistent with a model whereby NO-reactive thiols (i.e. cysteine and glutathione) exported to the periplasm can diminish the levels of NO that can enter the cytoplasm. The transcriptional response of cydDC mutants to exogenously added cysteine and glutathione was explored through microarray analysis in an attempt to gain a greater insight into the role of reduced thiol export to the periplasmic space. The addition of thiols affected genes involved in cell metabolism, respiration and led to the down-regulation of motility-related genes, providing insights into how the presence of CydDC substrates contribute to diverse cellular processes. To investigate the contribution of CydDC to survival during infection, macrophage survival assays were performed along with an infection study using a mouse model of UTI (urinary tract infection). This work demonstrates that CydDC does not contribute to bacterial survival within NO-producing macrophage cells, and that loss of CydDC has no significant effect on the ability to colonise the mouse lower urinary tract. We therefore conclude that while CydDC is important for cytochrome bd-I assembly and NO tolerance in vitro, it may not be a suitable drug target to combat pathogenic strains of E. coli

Legume Genetics and Biology

Legumes have played an important part as human food and animal feed in cropping systems since the dawn of agriculture. The legume family is arguably one of the most abundantly domesticated crop plant families. Their ability to symbiotically fix nitrogen and improve soil fertility has been rewarded since antiquity and makes them a key protein source. Pea was the original model organism used in Mendel´s discovery of the laws of inheritance, making it the foundation of modern plant genetics. This book based on Special Issue provides up-to-date information on legume biology, genetic advances, and the legacy of Mendel

Integrated statistical analysis of cDNA microarray and NIR spectroscopic data applied to a hemp dataset

Both cDNA microarray and spectroscopic data provide indirect information about the chemical compounds present in the biological tissue under consideration. In this paper simple univariate and bivariate measures are used to investigate correlations between both types of high dimensional analyses. A large dataset of 42 hemp samples on which 3456 cDNA clones and 351 NIR wavelengths have been measured, was analyzed using graphical representations. For this purpose we propose clustered correlation and clustered discrimination images. Large, tissue-related differences are seen to dominate the cDNA-NIR correlation structure but smaller, more difficult to detect, variety-related differences can be found at specific cDNA clone/NIR wavelength combination

Advances in the Biology of Phototrophic Bacteria

The application of genomic, transcriptomic, and proteomic analyses brings new dimensions to our understanding of the biology of phototrophic bacteria. Comparing gene sequences of photosynthetic reaction center proteins and a key enzyme of bacteriochlorophyll biosynthesis from more than 150 genomes demonstrates the ancient roots of phototrophic bacteria. The presence and phylogeny of biosynthetic pathways of the compatible solutes ectoine and glycine betaine define groups of marine and halophilic phototrophic bacteria. The wide range of ecological niches conquered during evolution is demonstrated by the adaptation of cyanobacterial genera Scytonema, Tolypothrix, and Nostoc to different temperature ranges and the adaptation of Heliorestis species to alkaline habitats. Differences between phototrophic purple bacteria from marine and freshwater habitats are reflected in the preference for sulfidic and non-sulfidic niches. Also, a high proportion of siderophore producers was found among isolates from freshwater sources opposed to those from salty habitats . The primary colonization of carbonate rocks by a group of novel endolithic cyanobacteria and the following successions were studied over 9 months. The genomic characterization of the aerobic Dinoroseobacter strain AAP5, the strictly anaerobic and syntrophic Prosthecochloris ethylica, and the strictly anaerobic Heliorestis convoluta is reported. Significant differences in relation to oxygen are reflected in oxygen production by some species, oxygen tolerance over a wide range of concentrations, and the use of oxygen for energy generation or a strictly anaerobic lifestyle. Relations to oxygen are highlighted in papers on photooxidative stress, regulation of iron–sulfur cluster formation, and interactions of redox regulators. In situ metatranscriptomic and proteomic studies demonstrate the high metabolic flexibility of Chloroflexus aggregans in a hot spring microbial mat and show its adaptation to the changing conditions over day and night periods by a well-coordinated regulation of key metabolic processes for both phototrophic and chemotrophic growth

Tree Peony Species Are a Novel Resource for Production of α-Linolenic Acid

Tree peony is known worldwide for its excellent ornamental and medical values, but recent reports that their seeds contain over 40% α-linolenic acid (ALA), an essential fatty acid for humans drew additional interest of biochemists. To understand the key factors that contribute to this rich accumulation of ALA, we carried out a comprehensive study of oil accumulation in developing seeds of nine wild tree peony species. The fatty acid content and composition was highly variable among the nine species; however, we selected a high- (P. rockii) and low-oil (P. lutea) accumulating species for a comparative transcriptome analysis. Similar to other oilseed transcriptomic studies, upregulation of select genes involved in plastidial fatty acid synthesis, and acyl editing, desaturation and triacylglycerol assembly in the endoplasmic reticulum was noted in seeds of P. rockii relative to P. lutea. Also, in association with the ALA content, transcript levels for fatty acid desaturases (SAD, FAD2 and FAD3), which encode for enzymes necessary for polyunsaturated fatty acid synthesis were higher in P. rockii compared to P. lutea. We further showed that the overexpression of PrFAD2 and PrFAD3 in Arabidopsis increased linoleic and α-linolenic acid content, respectively and modulated their final ratio in the seed oil. In conclusion, we identified the key steps that contribute to efficient ALA synthesis and validated the necessary desaturases in P. rockii that are responsible for not only increasing oil content but also modulating 18:2/18:3 ratio in seeds. Together, these results will aid to improve essential fatty acid content in seeds of tree peonies and other crops of agronomic interest