Phase variation of lipopolysaccharide of Coxiella burnetii, strain Priscilla during chick embryo yolk sac passaging

Abstract Wheat germ agglutinin was found to increase glutamine synthetase and nitrogenase activities and excretion of N₂-fixation product (NH₄⁺) in Azospirillum brasilense Sp 245. Each effect had a similar pattern and correlated well with each other. They were dynamic, had maxima in the middle of the exponential phase of growth, and were due to N-acetyl- d -glucosamine specificity of wheat germ agglutinin, since its preincubation with 10% N-acetyl- d -glucosamine caused the disappearance of the effects. When wheat germ agglutinin was replaced with Ulex europaeus agglutinin II possessing the same carbohydrate binding specificity all the above effects remained, replacing wheat germ agglutinin with concanavalin A with a different sugar specificity, or with bovine serum albumin led to their disappearance.     

Glutamine uptake by a sodium-dependent secondary transport system inCorynebacterium glutamicum

Corynebacterium glutamicum took up glutamine by a sodium-dependent secondary transport system. Both the membrane potential and the sodium gradient were driving forces. Glutamine uptake showed Michaelis-Menten kinetics, with aK _m of 36 μM and aV _max of 12.5 nmol min⁻¹ (mg dry weight)⁻¹ at pH 7. Despite a pH optimum in the alkaline range around pH 9, it was shown that uncharged glutamine is the transported species. The affinity for the cotransported sodium was relatively low; an apparentK _m of 1.4 mM was determined. Among various substrates tested, only asparagine, when added in 50-fold excess, led to an inhibition of glutamine transport. It was concluded that glutamine uptake occurs via a specific transport system in symport with at least one sodium ion.     

Evidence of covalent modification of glutamine synthetase in the purple sulfur bacterium Thiocapsa roseopersicina

Abstract It was shown that glutamine synthetase of purple sulfur bacterium Thiocapsa roseopersicina is regulated by covalent modification. This conclusion is made on the basis of results showing that: (i) incubation of cells under conditions of nitrogen deprivation in the light lead to an increase of glutamine synthetase activity; (ii) addition of ammonium to nitrogen-starved cell suspensions caused a rapid decrease of glutamine synthetase activity; (iii) inhibition of glutamine synthetase by feedback modifiers was higher in ammonium-treated cells than in those starved for a nitrogen source; (iv) treatment of purified glutamine synthetase and cell-free extracts with phosphodiesterase was accompanied by an increase of glutamine synthetase activity, indicating the cleavage of modifying residues covalently bound to glutamine synthetase molecules.     

Control of Nitrate Uptake by Phloem-Translocated Glutamine in Zea mays L. Seedlings

Abstract: The putative role of glutamine, exported from leaves to roots, as a negative feedback signal for nitrate uptake was investigated in Zea mays L. seedlings. Glutamine (Gln) was supplied by immersion of the tip-cut leaves in a concentrated solution. Nitrate (NO₃⁻) uptake was measured by its depletion in amino acid-free medium. The treatment with Gln resulted in a strong inhibition of nitrate uptake rate, accompanied by a significant enrichment of amino compounds in root tissue. The effect of N-availability on NO₃⁻ uptake was determined in split-root cultures. The plants were subjected to complete or localized N supply. Inducible NO₃⁻ uptake systems were also induced in N-deprived roots when the opposite side of the root system was supplied with KNO₃. The inhibitory effect of Gln was unaffected by localized N supply on one side of the split-root. The potential role of Gln in the shoot-to-root control of NO₃⁻ uptake is discussed.     

Microvessels Isolated from Rat Brain: Localization of Astrocyte Processes by Immunohistochemical Techniques

Microvascular networks were isolated from rat telencephalon by density gradient centrifugation. The vessels prepared were characterized morphologically at the light and electron microscope level and immunohistochemically by the localization of glutamine synthetase and glial fibrillary acidic protein, two proteins found almost exclusively in astrocytes. The vast majority of the vessels prepared contained more than just endothelial cells surrounded by a basement membrane. Many arterioles were found still retaining their smooth muscle cells. Pericytes were found in association with most of the venules and many of the capillaries. Astrocyte processes remained attached to most of the microvessels. These results show that vessels prepared from rat brain still maintain most of their complex intercellular contacts and must be viewed as a heterogeneous network of cells.     

Evolutionary relationships of superoxide dismutases and glutamine synthetases from marine species of Alteromonas,Oceanospirillum, Pseudomonas and Deleya

Evolutionary relationships among marine species assigned to the genera Alteromonas, Oceanospirillum, Pseudomonas, and Alcaligenes were determined by an immunological study of their Fe-containing superoxide dismutases (FeSOD) and glutamine synthetases (GS), two enzymes with differentially conserved amino acid sequences which are useful for determining intermediate and distant relationships, respectively. Five reference antisera were prepared against the FeSODs from Alteromonas macleodii, A. haloplanktis, Oceanospirillum commune, Pseudomonas stanieri, and Deleya pacifica. For GS, a previously prepared antiserum to the enzyme from Escherichia coli was employed. Amino acid sequence similarities for both enzymes were determined by the quantitative microcomplement fixation technique and the Ouchterlony double diffusion procedure. Six evolutionary groups were detected by FeSOD sequence similarities: three subgroups within the genus Alteromonas, the genera Oceanospirillum and Pseudomonas, and a new genus, Deleya (to accommodate marine Alcaligenes). Only four groupings were delineated by the GS data: the latter three genera and one group composed of all the species of Alteromonas. Evidence that all of these subgroups are derived from the evolutionary lineage defined by the purple sulfur photosynthetic bacteria is presented.Abbreviations Alt Alteromonas - anti-Amac, anti-Ahal, anti-Ocom, anti-Psta, anti-Dpac antisera to the Fe-containing superoxide dismutases from Alteromonas macleodii 107, Alteromonas haloplanktis 121, Oceanospirillum commune 8, Pseudomonas stanieri 146, Deleya pacifica 62 - FeSOD Fe-containing superoxide dismutase - G+C guanine plus cytosine - GS glutamine synthetase - ImD immunological distance - MnSOD Mn-containing superoxide dismutase - Oce Oceanospirillum - Pse Pseudomonas - Rm relative mobility - rRNA ribosomal RNA - SOD superoxide dismutase Dedicated to the memory of Professor Roger Y. Stanier     

Chloroplastic glutamine synthetase from tobacco leaves: a glycosylated protein

The amino acid and carbohydrate content of chloroplastic glutamine synthetase from tobacco leaves has been analysed. The enzyme subunit contanins 5% carbohydrate, mainly represented by glucosamine, galactosamine, glucose, galactose and mannose residues. The enzyme subunit displayed a single band of molecular mass 44000 Da after sodium dodecyl sulphate (SDS) electrophoresis. However, when isoelectrofocussing electrophoresis was performed, four subunits were evident differing by their charge. Furthermore, the four different subunits stained positively when tested with periodic acid Shiff reagent, showing that sugars and amino sugars were present within all the subunits.     

Recent developments on the regulation and structure of glutamine synthetase enzymes from selected bacterial groups

Abstract: The structure of glutamine synthetase (GS) enzymes from diverse bacterial groups fall into three distinct classes. GSI is the typical bacterial GS, GSII is similar to the eukaryotic GS and is found together with GSI in plant symbionts and Streptomyces , while GSIII has been found in two unrelated anaerobic rumen bacteria. In most cases, the structural gene for GS enzyme is regulated in response to nitrogen. However, different regulatory mechanisms, to ensure optimal utilization of nitrogen substrates, control the GS enzyme in each class.     

Compatible solutes in representatives of the genera Brevibacterium and Corynebacterium: Occurrence of tetrahydropyrimidines and glutamine

Abstract The aim of our investigation was to study the haloadaptation of a number of species of Gram-positive bacteria belonging to the genera Brevibacterium and Corynebacterium . We used two different HPLC-techniques and ¹³C-NMR spectroscopy for the identification of osmolytes (compatible solutes). The tetrahydropyrimidines ('ectoines') are the main compatible solutes in the genus Brevibacterium , whereas accumulation of glycine-betaine and accumulation of synthesis of glutamine is mainly responsible for osmoadaptation in the genus Corynebacterium . Pipecolic acid, formerly described as a potential osmolyte synthesized de novo in C. ammoniagenes , does not contribute markedly to the solute pool, unless supplemented to the medium.     

A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes

Media and feed optimization have fueled many-fold improvements in mammalian biopharmaceutical production, but genome editing offers an emerging avenue for further enhancing cell metabolism and bioproduction. However, the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we present a comprehensive pooled CRISPR screen for CHO cell metabolism, including ~16,000 gRNAs against ~2500 metabolic enzymes and regulators. Using this screen, we identified a glutamine response network in CHO cells. Glutamine is particularly important since it is often over-fed to drive increased TCA cycle flux, but toxic ammonia may accumulate. With the screen we found one orphan glutamine-responsive gene with no clear connection to our network. Knockout of this novel and poorly characterized lipase, Abhd11, substantially increased growth in glutamine-free media by altering the regulation of the TCA cycle. Thus, the screen provides an invaluable targeted platform to comprehensively study genes involved in any metabolic trait, and elucidate novel regulators of metabolism.