Elucidation of trophic interactions in an unusual single-cell nitrogen-fixing symbiosis using metabolic modeling

Marine nitrogen-fixing microorganisms are an important source of fixed nitrogen in oceanic ecosystems. The colonial cyanobacterium Trichodesmium and diatom symbionts were thought to be the primary contributors to oceanic N₂ fixation until the discovery of the unusual uncultivated symbiotic cyanobacterium UCYN-A (Candidatus Atelocyanobacterium thalassa). UCYN-A has atypical metabolic characteristics lacking the oxygen-evolving photosystem II, the tricarboxylic acid cycle, the carbon-fixation enzyme RuBisCo and de novo biosynthetic pathways for a number of amino acids and nucleotides. Therefore, it is obligately symbiotic with its single-celled haptophyte algal host. UCYN-A receives fixed carbon from its host and returns fixed nitrogen, but further insights into this symbiosis are precluded by both UCYN-A and its host being uncultured. In order to investigate how this syntrophy is coordinated, we reconstructed bottom-up genome-scale metabolic models of UCYN-A and its algal partner to explore possible trophic scenarios, focusing on nitrogen fixation and biomass synthesis. Since both partners are uncultivated and only the genome sequence of UCYN-A is available, we used the phylogenetically related Chrysochromulina tobin as a proxy for the host. Through the use of flux balance analysis (FBA), we determined the minimal set of metabolites and biochemical functions that must be shared between the two organisms to ensure viability and growth. We quantitatively investigated the metabolic characteristics that facilitate daytime N₂ fixation in UCYN-A and possible oxygen-scavenging mechanisms needed to create an anaerobic environment to allow nitrogenase to function. This is the first application of an FBA framework to examine the tight metabolic coupling between uncultivated microbes in marine symbiotic communities and provides a roadmap for future efforts focusing on such specialized systems.     

Stimulation of gastric mucosal protein synthesis by different molecular forms of gastrin.

Protein synthesis in gastric mucosa was studied by measuring the incorporation of labeled amino acids into protein by isolated gastric mucosal ribosomes in a cell-free system. In 48-hour fasted rats, administration of the synthetic analogues pentagastrin, tetragastrin and gastrin-17 or naturally occurring molecular forms of human gastrin (G-14, G-34) markedly enhanced (23-123%) the capacity of the gastric mucosal ribosomes to synthesize endogenous mRNA-directed protein in a cell-free system. In the presence of exogenous mRNA (poly-U), the gastric mucosal ribosomes from the saline-treated controls showed a higher poly(U)-directed protein synthesis, compared to each fo the gastrin-treated groups. The protein/polyphenylalanine ratio which represents a ratio of polysomes to monosomes was found increased in ribosomes from the gastrin-treated groups.     

Phytoremediation of Parboiled Rice Mill Wastewater Using Water Lettuce (Pistia Stratiotes)

Phytoremediation is an emerging technology applied for treatment of wastewater. It is a suitable option notably in developing countries as it is simple, sustainable and cost effective. In the present lab-based batch study the free floating aquatic plant water lettuce (Pistia stratiotes) is used for treatment of parboiled rice mill wastewater having low pH, high chemical oxygen demand (COD), nitrogen, and phosphate. In raw rice mill wastewater (undiluted) growth of water lettuce is found to be inhibited. Later on, two different dilution approaches (raw and facultative pond effluent 1:1; raw and tap water 1:1) are applied in order to effectively use this technology. In all cases a control (without plant) is maintained to compare the performance with the Aquatic Plant based Treatment (APT) system. In the APT system results reveal that removal of soluble COD (SCOD), ammoniacal nitrogen (NH₄-N), nitrate nitrogen (NO₃-N), and soluble phosphorus (sol. P) are upto 65%, 98%, 70%, and 65% respectively. The study highlights the efficacy of water lettuce in removing organics and nutrients from parboiled rice mill wastewater.     

Cloning and Expression of Chlorobium vibrioforme Uroporphyrinogen Decarboxylase Gene in a Salmonella Heme Auxotroph

To study the post-uroporphyrin steps in heme and chlorophyll biosynthesis in Chlorobium, we attempted to clone the uroporphyrinogen decarboxylase (hemE) gene. A Chlorobium genomic library was used to transform a restriction-minus Salmonella typhimurium strain. The recombinant DNA molecules were transduced into an auxotrophic Salmonella double mutant (hemA⁻ hemE⁻) by phage P22. Faster-growing colonies indicated complementation of the hemE mutation. Each clone was tested by backcross transduction of the mutant. Growth rates of the confirmed clones in LB medium were comparable to wild-type Salmonella. HPLC analysis of the substrate (uroporphyrinogen) and the product (coproporphyrinogen) of the decarboxylase activity was performed in one such clone. This clone showed an active hemE gene within a 4-kb insert. Received: 21 February 2002 / Accepted: 8 May 2002