Role of ammona in regulation of nitrogenase synthesis and activity in Anabaena cylindrica

The regulation of nitrogenase biosynthesis and activity by ammonia was studied in the heterocystous cyanobacterium Anabaena cylindrica. Nitrogenase synthesis was measured by in vivo acetylene reduction assays and in vitro by an activity-independent, immunoelectrophoretic measurement of the Fe-Mo protein (Component I). When ammonia was added to differentiating cultures after a point when heterocyst differentiation became irreversible, FeMo protein synthesis was also insensitive to ammonia. Treating log-phase batch cultures with 100% O₂ for 30 min resulted in a loss of 90% of nitrogenase activity and a 50% loss of the FeMo protein. Recovery was inhibited by chloramphenicol but not by ammonia or urea. The addition of ammonia to log-phase cultures resulted in a decrease in specific levels of nitrogenase activity and FeMo protein that occurred at the same rate as algal growth and was independent of O₂ tension of the culture media. However, in light-limited linear-phase cultures, ammonia effected a dramatic inhibition of nitrogenase activity. These results indicate that nitrogenase biosynthesis becomes insensitive to repression by ammonia as heterocysts mature and that ammonia or its metabolites act to regulate nitrogen fixation by inhibiting heterocyst differentiation and by inhibiting nitrogenase activity through competition with nitrogenase for reductant and/or ATP, but not by directly regulating nitrogenase biosynthesis in heterocysts.     

Role of uptake hydrogenase in providing reductant for nitrogenase in Rhizobium leguminosarum bacteroids

The role of uptake hydrogenase in providing reducing power to nitrogenase was investigated in Rhizobium leguminosarum bacteroids from nodules of Pisum sativum L. (cv. Homesteader). H₂ increased the rate of C₂H₂ reduction in the absence of added substrates. Malate also increased nitrogenase (C₂H₂) activity while decreasing the effect of H₂. At exogenous malate concentrations above 0.05 mM no effect of H₂ was seen. Malate appeared to be more important as a source of reductant than of ATP. When iodoacetate was used to minimize the contribution of endogenous substrates to nitrogenase activity in an isolate in which H₂ uptake was not coupled to ATP formation, H₂ increased the rate of C₂H₂ reduction by 77%. In the presence of iodoacetate, an ATP-generating system did not enhance C₂H₂ reduction, but when H₂ was also included, the rate of C₂H₂ reduction was increased by 280% over that with the ATP-generating system alone. The data suggest that, under conditions of substrate starvation, the uptake hydrogenase in R. leguminosarum could provide reductant as well as ATP in an isolate in which the H₂ uptake is coupled to ATP formation, to the nitrogenase complex.     

Effects of in vivo treatments on the activity of nitrogenase isolated from Rhodospirillum rubrum

Nitrogenase activities of partially purified extracts of Rhodospirillum rubrum grown on different nitrogen sources were examined. Most of the nitrogenase from cells grown on N₂ or glutamate was in the inactive form. This form was also predominant in extracts from cells grown on limiting N₂ or glutamate plus N₂. The enzyme from cells grown with limiting NH⁺₄ was fully active. Nitrogenase displayed varying degrees of sensitivity to in vivo inhibition by NH⁺₄, depending on the culture conditions. However, addition of NH⁺₄ to the cultures prior to harvest did not change the proportion of the active form of the enzyme in extracts from that found in control samples. Several of these observations are inconsistent with the three component model of nitrogenase regulation of Yoch and Cantu (Yoch, D.C. and Cantu, M. (1980) J. Bacteriol, 142, 899–907). A regulatory system controlled by products of NH⁺₄ assimilation is suggested.     

Modes of reduction of nitrogenase in heterocysts isolated from Anabaena species

N₂ fixation (acetylene reduction) has been studied with heterocysts isolated from Anabaena cylindrica and Anabaena 7120. In the presence of ATP and at very low concentrations of sodium dithionite, reducing equivalents for activity of nitrogenase in these cells can be derived from several compounds. In the dark, d-glucose 6-phosphate, 6-phosphogluconate and dl-isocitrate support acetylene reduction via NADPH. In the light, reductant can be generated by Photosystem I.     

Photosynthetic electron transport,ATP synthesis and nitrogenase activity in isolated heterocysts of Anabaena cylindrica

Isolated heterocysts of the N₂-fixing blue-green alga Anabaena cylindrica contain the Photosystem I components P-700, bound and soluble ferredoxins and ferredoxin-NADP reductase. They also show Photosystem I activity being able to photoreduce both methylviologen and NADP when ascorbate+dichlorophenol-indophenol acts as reductant. They photophosphorylate (64 μmol ATP produced/mg chlorophyll $\text{a}\text{h}$) and carry out oxidative phosphorylation (8.7 μmol ATP produced/mg chlorophyll $\text{a}\text{h}$). Ninety per cent of the total cell-free extract nitrogenase activity is located in the heterocyst fraction of aerobic cultures.     

13C-NMR study of the glucose synthesis pathways in the bacterium Chlorobium thiosulfatophilum

Photoassimilation of ¹³CO₂ and acetate by the photosynthetic bacterium Chlorobium thiosulfatophilum was investigated using ¹³C-NMR as the method for determination of the labelling pattern of the glucose synthesized by the bacterium. Metabolic pathways functioning in the bacterium were identified by analysis of the multiplet structure of the spectra of tightly coupled systems. The labelling pattern showed that glucose was synthesized in C. thiosulfatophilum mainly by the gluconeogenesis pathway. In agreement with previous investigations, the reserve polysaccharide of C. thiosulfatophilum was shown to be polyglucose, with the glucose units linked predominantly by (1→4)α-glucosidic linkages.     

Inhibition of DNA synthesis does not influence the H1 histone synthesis in Tetrahymena pyriformis

In the protozoan Tetrahymena pyriformis the DNA synthesis is stopped immediately and completely after addition of one of the two DNA synthesis inhibitors methotrexate + uridine and hydroxyurea to a cell suspension. However, the present experiments show, that the accumulation of labeled H1 histone in the inhibited cells is almost totally unaffected for more than two-thirds of a cell cycle after addition of either inhibitor.     

Nitrogen fixaton in Klebsiella pneumoniae during osmotic stress effect of exogenous proline or a proline overproducing plasmid

Osmotic stress, imposed by 0.5 M NaCl or other electrolytes and non-electrolytes, caused over a 100-fold reduction in the whole-cell nitrogen fixation activity in Klebsiella pneumoniae, wild-type strain M₅A₁. This reduction of nitrogen fixation activity could be reversed by the addition of proline to the culture medium at 0.5 mM concentration. With 0.5 M NaCl, in the presence of proline, nitrogenase activity was 47-fold greater than in the absence of proline. A mutation, originally isolated in Salmonella typhimurium, which resulted in proline over-production and enhanced osmotolerance, was transferred into K. pneumoniae by F′ conjugation. Intracellular proline, synthesized at high levels because of the mutation, had similar stimulatory effects on nitrogen fixation under osmotic stress as proline provided exogenously. In the overproducing strain, the cellular level of proline is elevated as much as 125-fold during stress over that seen in the control strain. To determine the mechanism of stimulation of nitrogen fixaton by proline during stress, the biosynthesis of nitrogenase polypeptides was studied. Net nitrogenase biosynthesis and the biosynthesis of other unidentified peptides, is strongly inhibited during osmotic stress; proline reverses the inhibition. The role of proline in enhancing nitrogen fixation during osmotic stress is discussed.     

The effect of temperature change on gene expression in Paramecium primaurelia

When paramecia grown at 24°C are transferred rapidly to 32°C, DNA and protein synthesis continue uninterrupted but at higher rates. Electron microscopic observations indicate that more of the macronuclear chromatin is transcribed at the elevated temperature. This interpretation is supported by hybridization experiments which show that the percentage of the macronuclear genome transcribed into poly(A)⁺ RNA is 24°C and 35% at 32°C. Kinetic analysis of cDNA-poly(A)⁺ RNA hybridizations reveals three abundance classes of poly(A)⁺ RNA and indicates that the number of genes expressing low abundance sequences is about 9000 at 24°C and 13000 at 32°C. The intermediately abundant and highly abundant classes are represented by 100–200 and 1–3 different kinds of RNA sequence, respectively. Cross hybridization shows that changes occur throughout the distribution of abundance classes of poly(A)⁺ RNA with increase in temperature.     

The de novo and salvage pathways for the synthesis of pyrimidine residues of RNA predominate in different locations within the mouse doudenal epithelium

Summary RNA synthesis was examined by radioautography in mouse doudenal epithelium using ³H-uridine as a tracer of the salvage pathway and ³H-orotic acid as a tracer of the de novo pathway. The incorporation of the two precursors was estimated by counting silver grains in light-microscopic and electron-microscopic radioautographs at successive levels of crypt and villus. With both precursors, silver grains were found over all epithelial nuclei, but in numbers varying by location. Thus, after ³H-uridine injection, the number of grains was high over nucleolus and nucleoplasm in the base of the crypt, declined gradually in the middle and top of the crypt, and was low along the villus. After ³H-orotic acid, the number of grains was fairly low throughout, but peaked over the nucleoplasm in lower villus cells. The ³H-uridine reaction over nucleolus and nucleoplasm in crypt cells was interpreted as synthesis by the salvage pathway of ribosomal RNA and heterogeneous RNA, respectively, whereas the ³H-orotic acid reaction over the nucleoplasm of some villus cells indicated that these cells synthesized heterogeneous RNA by the de novo pathway.     

Increased synthesis of human parathyroid hormone in Escherichia coli through alterations of the 5′ untranslated region

The expression of human parathyroid hormone (hPTH) in Escherichia coli was optimized by variations of the spacing sequence between the ribosome-binding site (RBS) and the beginning of the gene (ATG) and by increasing the complementarity of the RBS to the 16 S rRNA. The expression level of 3 μg/liter increased more than 100-fold to 475 μg/liter as a direct consequence of modifications in the region 5′ of the gene.     

Electrophoretic patterns of protein synthesis and turnover in apical plasma membrane and outer bilayer of Schistosoma mansoni

Polypeptide fractions labelled with [¹⁴C]leucine and associated with fractioned inner plasma membrane and outer bilayer (envelope) from the apical double bilayer complex of the surface epithelium of the human blood fluke, Schistosoma mansoni, were analyzed by two-dimensional electrophoresis and fluorography. In contrast to the distribution of alkaline phosphatase, the polypeptide profiles of the two bilayer fractions were similar due to cross contamination between one membrane containing larger amounts of protein (inner) and the second bilayer having more heavily labelled proteins (outer bilayer). Convincing evidence for only two of 35 polypeptides could be provided for localization to the outer bilayer. These results suggest that the marker enzyme used for the inner bilayer, alkaline phosphatase, may not be homogeneously distributed in this membrane. In pulse-chase studies a correction factor for cross-contamination was derived. The rate to turnover of the polypeptide fractions was twice as fast for the outer compared to the inner membrane, this difference being consistent with the view that multilamellar bodies are the precursors of the apical double bilayer complex. Comparing the rates of surface renewal in adult and juvenile schistosomes leads to the suggestion that membrane turnover can be correlated with susceptibility to host immune effector mechanisms.     

Correlation between ATP synthesis and the decay of the carotenoid band shift after single flash activation of chromatophores from Rhodopseudomonas capsulata

ATP synthesis and the acceleration of the decay of the carotenoid absorption band shift after single flash excitation of Rhodopseudomonas capsulata chromatophores were compared. The two processes behave similarly with respect to: (1) ADP and P_i concentration; (2) inhibition by efrapeptin and venturicidin, and (3) inhibition by valinomycin/K⁺ and by ionophores.Taken together with earlier evidence for the electrochromic nature of the carotenoid band shift the data support the contention that positive charge moves outwards across the chromatophore membrane during ATP synthesis and justify the method for determination of the H⁺/ATP ratio (Petty, K.M. and Jackson, J.B. (1979) FEBS Lett. 97, 367–372).The ability of nucleotide diphosphates in the presence of P_i and Mg²⁺ to give rise to the acceleration of the carotenoid shift decay closely correlates with the rate of phosphorylation of the nucleotides in steady-state light. Nucleotide triphosphates enhance the decay in parallel with their rate of hydrolysis.Adenylyl imidodiphosphate is itself without effect on the decay of the carotenoid shift and it does not prevent the ADP-induced acceleration. The analogue does prevent the ATP effect but only after repeated flashes.     

Control of hydrogen-dependent nitrogenase activity by adenylates and electron flow in heterocysts of Anabaena variabilis (ATCC 29413)

Hydrogen-dependent nitrogenase activity was studied in heterocysts, isolated from the filamentous cyanobacterium Anabaena variabilis (ATCC 29413). Hydrogen provides reductant and ATP for nitrogenase via linear electron flow through Photosystem I. This allows for regulation of nitrogenase activity by controlling the turnover of the photosystem. When nitrogenase activity was varied by changing either the light intensity or the supply of reductant (i.e., hydrogen) or by inhibition of photosynthetic electron transport by DBMIB, no rate-dependent changes in cellular ATP concentrations were observed. This homeostasis of ATP was perturbed by addition of metronidazole, acting as alternative electron sink to nitrogenase, and by uncoupling agents like FCCP, gramicidin and nigericin. Valinomycin (in presence of KCl) exerted little effect on nitrogenase activity and adenylate pool composition. Metronidazole increased and uncoupling agents decreased cellular ATP concentration, ATP/ADP ratio and energy charge. Inhibition of nitrogenase activity by metronidazole was caused by reductant limitation; inhibition by uncoupling agents was due to energy limitation. Control exerted on nitrogenase activity by ATP (energy limitation) was more pronounced at high rates of electron flow to nitrogenase than during reductant limitation. When cellular ATP synthesis was suboptimal due to partial uncoupling, the connection of phosphorylation and nitrogenase activity by electron transport allowed for homeostasis of ATP also at a lowered cellular concentration.     

Photophosphorylation in isolated heterocysts from the blue-green alga Nostoc muscorum

Isolated heterocysts of the blue-green alga Nostoc muscorum (Anabaena 7119) exhibit high rates of photophosphorylation in systems with cyclic and non-cyclic electron transport. Cyclic photophosphorylation mediated by N-methylphenazonium methosulfate is found to be sensitive to antimycin A, but not to 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinon (DBMIB). Non-cyclic electron transport (diaminodurol → methylviologen) coupled to phosphorylation is affected by DBMIB, but not by antimycin A. Studies with uncouplers indicate that ΔpH is the main component of the protonmotive force under continuous illumination. A different effect of NH₄Cl on dark- and photophosphorylation is observed and discussed with respect to localization of respiration in blue-green algae.     

Molecular expression of xylanase gene in Cryptococcus albidus

In the yeast Cryptococcus albidus, the utilization of xylan as compared to xylose requires at least an inducible endoxylanase enzyme, secreted in the culture medium. The endoxylanase induction was monitored by immunoprecipitation of in vivo and in vitro synthesized products. The mature endoxylanase is a highly glycosylated enzyme with an apparent molecular weight of 48000. Upon chemical deglycosylation with trifluoromethanesulfonic acid, the molecular weight was reduced to 40000. Addition of tunicamycin to the culture medium resulted in the synthesis of a modified polypeptide having a molecular weight of 40000. Poly(A)-containing RNA isolated from the yeast was translated in the rabbit reticulocyte protein-synthesizing system. The appearance of a translatable xylanase mRNA was observed in xylan-grown cells but not in xylose-grown cells. The polypeptide identified as xylanase had a molecular weight of 44000. This suggests that the xylanase is synthesized as a precursor, containing a peptide signal sequence of 35 residues.     

Control of membrane polar lipid composition in Acholeplasma laidlawii a by the extent of saturated fatty acid synthesis

The low level of endogenous fatty acid synthesis in Acholeplasma laidlawii A strain EF22 was found to be caused by a deficiency of pantetheine in the lipid-depleted growth medium. By supplementing the oleic acid-containing medium with increasing concentrations of pantetheine, saturated fatty acid synthesis was stimulated (having an apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 5 μM for pantetheine) and the incorporation of endogenously synthesized fatty acids in membrane lipids increased markedly. Furthermore, carotenoid biosynthesis was stimulated. Exogenous palmitic acid was found to inhibit partially the endogenous fatty acid synthesis. A gradual stimulation of fatty acid synthesis was accompanied by a linear increase in the molar proportion between the two dominating membrane glucolipids, monoglucosyldiacylglycerol and diglucosyldiacylglycerol. The total amount of charged membrane lipids decreased upon increasing the degree of fatty acid saturation. These regulations are discussed in terms of membrane stability, and influence of membrane molecular ordering and surface charge density on lipid polar head group synthesis.