Anaerobic Ammonia Oxidation in the Presence of Nitrogen Oxides (NOx) by Two Different Lithotrophs

The anaerobic ammonia-oxidizing activity of the planctomycete Candidatus “Brocadia anammoxidans” was not inhibited by NO concentrations up to 600 ppm and NO₂ concentrations up to 100 ppm. B. anammoxidans was able to convert (detoxify) NO, which might explain the high NO tolerance of this organism. In the presence of NO₂, the specific ammonia oxidation activity of B. anammoxidans increased, and Nitrosomonas-like microorganisms recovered an NO₂-dependent anaerobic ammonia oxidation activity. Addition of NO₂ to a mixed population of B. anammoxidans and Nitrosomonas induced simultaneous specific anaerobic ammonia oxidation activities of up to 5.5 mmol of NH₄⁺ g of protein⁻¹ h⁻¹ by B. anammoxidans and up to 1.5 mmol of NH₄⁺ g of protein⁻¹ h⁻¹ by Nitrosomonas. The stoichiometry of the converted N compounds (NO₂⁻/NH₃ ratio) and the microbial community structure were strongly influenced by NO₂. The combined activity of B. anammoxidans and Nitrosomonas-like ammonia oxidizers might be of relevance in natural environments and for technical applications.     

Improved nitrogen removal by application of new nitrogen-cycle bacteria

In order to meet increasingly stringentEuropean discharge standards, new applicationsand control strategies for the sustainableremoval of ammonia from wastewater have to beimplemented. In this paper we discuss anitrogen removal system based on the processesof partial nitrification and anoxic ammoniaoxidation (anammox). The anammox process offersgreat opportunities to remove ammonia in fullyautotrophic systems with biomass retention. Noorganic carbon is needed in such nitrogenremoval system, since ammonia is used aselectron donor for nitrite reduction. Thenitrite can be produced from ammonia inoxygen-limited biofilm systems or in continuousprocesses without biomass retention. Forsuccessful implementation of the combinedprocesses, accurate biosensors for measuringammonia and nitrite concentrations, insight inthe complex microbial communities involved, andnew control strategies have to be developed andevaluated.     

The Anaerobic Fungus Neocallimastix sp. Strain L2: Growth and Production of (Hemi)Cellulolytic Enzymes on a Range of Carbohydrate Substrates

The anaerobic fungus Neocallimastix sp. strain L2, isolated from the feces of a llama, was tested for growth on a range of soluble and insoluble carbohydrate substrates. The fungus was able to ferment glucose, cellobiose, fructose, lactose, maltose, sucrose, soluble starch, inulin, filter paper cellulose, and Avicel. No growth was observed on arabinose, galactose, mannose, ribose, xylose, sorbitol, pectin, xylan, glycerol, citrate, soya, and wheat bran. The fermentation products after growth were hydrogen, formate, acetate, ethanol, and lactate. The fermentation pattern was dependent on the carbon source. In general, higher hydrogen production resulted in decreased formation of lactate and ethanol. Recovery of the fermented carbon in products at the end of growth ranged from 50% to 80%. (Hemi)cellulolytic enzyme activities were affected by the carbon source. Highest activities were found in filtrates from cultures grown on cellulose. Growing the fungus on inulin and lactose yielded the lowest cellulolytic activities. Highest specific activities for avicelase, endoglucanase, β-glucosidase, and xylanase were obtained with Avicel as the substrate for growth (0.29, 5.9, 0.57, and 13 IU · mg⁻¹ protein, respectively). Endoglucanase activity banding patterns after SDS-PAGE were very similar for all substrates. Minor differences indicated that enzyme activities may in part be the result of secretion of different sets of isoenzymes. Received: 10 July 1996 / Accepted: 22 July 1996     

Photocontrol of anthocyanin biosynthesis in tomato

Juvenile anthocyanin biosynthesis has been studied in dark-grown seedlings of tomato (Lycopersicon esculentum Mill.) wild types (WTs) and photomorphogenic mutants. During a subsequent 24-hr period of monochromatic irradiation at different fluence rates of red light (R) the fluence-rate response relationships for induction of anthocyanin in all the WTs are similar, yet complex, showing a response at low fluence rates (LFRR) followed by a fluence rate-dependent high irradiance response (HIR). In the hypocotyl this response is restricted to the sub-epidermal layer of cells. The high-pigment-1 (hp-1) mutant exhibits a strong amplification of both response components. Theatroviolacea (atv) mutant shows strongest amplification of the HIR component. In contrast, a transgenic line overexpressing an oat phytochrome A gene (PHYA3 ⁺) shows a most dramatic amplification of the LFRR component. The far-red light (FR)-insensitive (fri) mutant, deficient in phytochrome A (phyA), lacks the LFRR component whilst retaining a normal HIR. The temporarily R-insensitive (tri) mutant, deficient in phytochrome B1 (phyB1) retains the LFRR, but lacks the HIR. Thehp-1,fri andhp-1,tri double mutant, exhibit amplified, yet qualitatively similar responses to the monogenicfri andtri mutants. Thefri,tri double mutant lacks both response components in R, but a residual response to blue light (B) remains. Similarly, theaurea (au) mutant deficient in phytochrome chromophore biosynthesis and presumably all phytochromes, lacks both response components in the R and FR regions of the spectrum. Experiments at other wavelengths demonstrate that while there is only a small response in the FR spectral region (729 nm) in tomato, there is an appreciable HIR response in the near FR at 704 nm, which is retained in thetri mutant. This suggests that the labile phyA pool participates in the HIR at this wavelength. The intense pigmentation (Ip) mutant appears to be specifically deficient in the B1 induced anthocyanin biosynthesis. Adult plants, grown under fluorescent light/dark cycles, show a reduction of anthocyanin content of young developing leaves upon application of supplemtary or end-of-day FR. The involvement of different phytochrome species in anthocyanin biosynthesis based on micro-injection studies into theau mutant and studies using type specific phytochrome mutants is discussed.     

13C and 15N NMR evidence for peripheral intercalation of uniformly labeled fusogenic peptides incorporated in a biomimetic membrane

Membrane fusion requires drastic and transient changes of bilayer curvature and here we have studied the interaction of three de novo designed synthetic hydrophobic peptides with a biomimetic three-lipid mixture by solid state NMR. An experimental approach is presented for screening of peptide-lipid interactions and their aggregation, and their embedding in a biomimetic membrane system using established proton-decoupled 13C,15N and proton spin diffusion heteronuclear 1H-13C correlation NMR methods at high magnetic field. Experiments are presented for a set of de-novo designed fusion peptides in interaction with their lipid environment. The data provide additional support for the transmembrane model for the least fusogenic peptide, L16, while the peripheral intercalation model is preferred for the fusogenic peptides LV16 and LV16G8P9. This contributes to converging evidence that peripheral intercalation is both necessary and sufficient to trigger the fusion process for a lipid mixture close to a critical point for phase separation across the bilayer.     

Expression and characterization of recombinant single-chain salmon class I MHC fused with beta2-microglobulin with biological activity

Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa beta2-microglobulin (beta2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable MHC class I molecules with biological activity. We report here the use of a bacterial expression system to produce the recombinant single-chain MHC molecules based on a specific allele Sasa-UBA*0301. This particular allele was selected because previous work has shown its association with the resistance to infectious salmon anaemia virus. The single-chain salmon MHC class I molecule has been designed and generated, in which the carboxyl terminus of beta2m is joined together with a flexible 15 or 20 amino acid peptide linker to the amino terminus of the heavy chain (Sasabeta2mUBA*0301). Monoclonal antibodies were successfully produced against both the MHC class I heavy chain and beta(2)m, and showed binding to the recombinant molecule. The recombinant complex Sasabeta2mUBA*0301 was expressed and isolated; the production was scaled up by adjusting to its optimal conditions. Subsequently, the recombinant proteins were purified by affinity chromatography using mAb against beta2m and alpha3. Eluates were analyzed by Western blot and refolded by the removal of denaturant. The correct folding was confirmed by measuring its binding capacity against mAb produced to recognize the native form of MHC molecules by biosensor analysis. This production of sufficient amounts of class I MHC proteins may represent a useful tool to study the peptide-binding specificity of MHC class I molecules, in order to design a peptide vaccine against viral pathogens.     

Algorithm of OMA for large-scale orthology inference

Background  

OMA is a project that aims to identify orthologs within publicly available, complete genomes. With 657 genomes analyzed to date, OMA is one of the largest projects of its kind.     

Hepatocystin is not secreted in cyst fluid of hepatocystin mutant polycystic liver patients

Autosomal dominant polycystic liver disease (PCLD) is characterized by multiple liver cysts and is caused by mutations in PRKCSH (hepatocystin). Mechanisms of cystogenesis are unknown, but previous studies have shown that hepatocystin is secreted in vitro. The goal of this study was to determine the fate of hepatocystin in vivo. Using immunoprecipitation, we determined that mutant hepatocystin is secreted from both apical and basolateral cell surface of MDCK cells stably transfected with mutant hepatocystin. Analysis of 60 cyst fluid samples from polycystic livers using Western blot, MALDI-TOF MS or nLC-MS/MS did not detect hepatocystin in liver cyst fluid. We did identify 163 ubiquitous serum proteins. No paracrine or autocrine factors were recognized. Although cyst fluids vary greatly in protein concentration, a PCLD specific protein pattern was not established. In conclusion, hepatocystin is not secreted in PCLD liver cyst fluid, suggesting that mutant hepatocystin is either not produced or degraded intracellularly. PCLD cysts develop from intralobular bile ductules and cyst fluid mainly contains common serum proteins comparable to that of other polycystic diseases.