Assimilation of 13NH4+ by Azospirillum brasilense grown under nitrogen limitation and excess.

The specific activities of glutamine synthetase (GS) and glutamate synthase (GOGAT) were 4.2- and 2.2-fold higher, respectively, in cells of Azospirillum brasilense grown with N2 than with 43 mM NH4+ as the source of nitrogen. Conversely, the specific activity of glutamate dehydrogenase (GDH) was 2.7-fold higher in 43 mM NH4+-grown cells than in N2-grown cells. These results indicate that NH4+ could be assimilated and that glutamate could be formed by either the GS-GOGAT or GDH pathway or both, depending on the cellular concentration of NH4+. The routes of in vivo synthesis of glutamate were identified by using 13N as a metabolic tracer. The products of assimilation of 13NH4+ were, in order of decreasing radioactivity, glutamine, glutamate, and alanine. The formation of [13N]glutamine and [13N]glutamate by NH4+-grown cells was inhibited in the additional presence of methionine sulfoximine (an inhibitor of GS) and diazooxonorleucine (an inhibitor of GOGAT). Incorporation of 13N into glutamine, glutamate, and alanine decreased in parallel in the presence of carrier NH4+. These results imply that the GS-GOGAT pathway is the primary route of NH4+ assimilation by A. brasilense grown with excess or limiting nitrogen and that GDH has, at best, a minor role in the synthesis of glutamate.     

Azolla-Anabaena Relationship : XIII. Fixation of [N]N(2)

The major radioactive products of the fixation of [¹³N]N₂ by Azolla caroliniana Willd.-Anabaena azollae Stras. were ammonium, glutamine, and glutamate, plus a small amount of alanine. Ammonium accounted for 70 and 32% of the total radioactivity recovered after fixation for 1 and 10 minutes, respectively. The presence of a substantial pool of [¹³N]N₂-derived ¹³NH₄⁺ after longer incubation periods was attributed to the spatial separation between the site of N₂-fixation (Anabaena) and a second, major site of assimilation (Azolla). Initially, glutamine was the most highly radioactive organic product formed from [¹³N]N₂, but after 10 minutes of fixation glutamate had 1.5 times more radiolabel than glutamine. These kinetics of radiolabeling, along with the effects of inhibitors of glutamine synthetase and glutamate synthase on assimilation of exogenous and [¹³N]N₂-derived ¹³NH₄⁺, indicate that ammonium assimilation occurred by the glutamate synthase cycle and that glutamate dehydrogenase played little or no role in the synthesis of glutamate by Azolla-Anabaena.     

Covalent modification of bacterial glutamine synthetase: physiological significance

Summary Stadtman, Holzer and their colleagues (reviewed in Stadtman and Ginsburg 1974) demonstrated that the enzyme glutamine synthetase (GS) [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2] is covalently modified by adenylylation in a variety of bacterial genera and that the modification is reversible. These studies further indicated that adenylylated GS is the less active form in vitro. To assess the physiological significance of adenylylation of GS we have determined the growth defects of mutant strains (glnE) of S. typhimurium that are unable to modify GS and we have determined the basis for these growth defects. The glnE strains, which lack GS adenylyl transferase activity (ATP: [L-glutamate: ammonia ligase (ADP-forming)] adenylyltransferase, EC 2.7.7.42), show a large growth defect specifically upon shift from a nitrogen-limited growth medium to medium containing excess ammonium (NH₄ ⁺). The growth defect appears to be due to very high catalytic activity of GS after shift, which lowers the intracellular glutamate pool to 10% that under preshift conditions. Consistent with this view, recovery of a rapid growth rate on NH₄ ⁺ is accompanied by an increase in the glutamate pool. The glnE strains have normal ATP pools after shift. They synthesize very large amounts of glutamine and excrete glutamine into the medium, but excess glutamine does not seem to inhibit growth. We hypothesize that a major function for adenylylation of bacterial GS is to protect the cellular glutamate pool upon shift to NH₄ ⁺-excess conditions and thereby to allow rapid growth.     

A strategy for finding regions of similarity in complete genome sequences

MOTIVATION: Complete genomic sequences will become available in the future. New methods to deal with very large sequences (sizes beyond 100 kb) efficiently are required. One of the main aims of such work is to increase our understanding of genome organization and evolution. This requires studies of the locations of regions of similarity. RESULTS: We present here a new tool, ASSIRC ('Accelerated Search for SImilarity Regions in Chromosomes'), for finding regions of similarity in genomic sequences. The method involves three steps: (i) identification of short exact chains of fixed size, called 'seeds', common to both sequences, using hashing functions; (ii) extension of these seeds into putative regions of similarity by a 'random walk' procedure; (iii) final selection of regions of similarity by assessing alignments of the putative sequences. We used simulations to estimate the proportion of regions of similarity not detected for particular region sizes, base identity proportions and seed sizes. This approach can be tailored to the user's specifications. We looked for regions of similarity between two yeast chromosomes (V and IX). The efficiency of the approach was compared to those of conventional programs BLAST and FASTA, by assessing CPU time required and the regions of similarity found for the same data set. AVAILABILITY: Source programs are freely available at the following address: ftp://ftp.biologie.ens. fr/pub/molbio/assirc.tar.gz CONTACT: vincens@biologie.ens.fr, hazout@urbb.jussieu.fr      

Physiological sources of reductant for nitrogen fixation activity in Nostoc sp. strain UCD 7801 in symbiotic association with Anthoceros punctatus.

Pure cultures of the symbiotic cyanobacterium-bryophyte association with Anthoceros punctatus were reconstituted by using Nostoc sp. strain UCD 7801 or its 3-(3,4-dichlorophenol)-1,1-dimethylurea (DCMU)-resistant mutant strain, UCD 218. The cultures were grown under high light intensity with CO2 as the sole carbon source and then incubated in the dark to deplete endogenous reductant pools before measurements of nitrogenase activities (acetylene reduction). High rates of light-dependent acetylene reduction were obtained both before starvation in the dark and after recovery from starvation, regardless of which of the two Nostoc strains was reconstituted in the association. Rates of acetylene reduction by symbiotic tissue with the wild-type Nostoc strain decreased 99 and 96% after 28 h of incubation in the dark and after reexposure to light in the presence of 5 microM DCMU, respectively. Supplementation of the medium with glucose restored nitrogenase activity in the dark to a rate that was 64% of the illuminated rate. In the light and in the presence of 5 microM DCMU, acetylene reduction could be restored to 91% of the uninhibited rate by the exogenous presence of various carbohydrates. The rate of acetylene reduction in the presence of DCMU was 34% of the uninhibited rate of tissue in association with the DCMU-resistant strain UCD 218. This result implies that photosynthates produced immediately by the cyanobacterium can supply at least one-third of the reductant required for nitrogenase activity on a short-term basis in the symbiotic association. However, high steady-state rates of nitrogenase activity by symbiotic Nostoc strains appear to depend on endogenous carbohydrate reserves, which are presumably supplied as photosynthate from both A. punctatus tissue and the Nostoc strain.     

Plasma free amino acid profiles and nutrition proteins in chronic renal failure; effect of dialysis treatment

Summary A well preserved nutritional status is beneficial in chronically uremic patients for slowing the pace of deterioration of renal function, and delaying the need for dialysis therapy. The purpose of this study was to assess the nutritional profile of 10 patients in a steady state of advanced CRF, and of 15 patients with terminal renal failure immediately prior to their first hemodialysis session (J0), and 7, 14, 45, 60, days post start of dialysis. Patients were 18 to 65 years old with total plasma proteins 60g/1. Plasma concentrations of amino acids, nutrition proteins, apolipoproteins A1, and B were evaluated. Non inflammatory reaction was evaluated by determination of alpha-1-acid glycoprotein, and C reactive protein. The data (mean ± 1 SD) were compared with mean values of 15 healthy individuals.