Ammonium uptake in Rhodopseudomonas capsulata

Investigations of the uptake of ammonium (NH ₄ ⁺ ) by Rhodopseudomonas capsulata B100 supported the presence of an NH ₄ ⁺ transport system. Experimentally NH ₄ ⁺ was determined by electrode or indophenol assay and saturation kinetics were observed with two apparent K _m's of 1.7 M and 11.1 M (pH 6.8, 30°) and a V _max at saturation of 50–60 nmol/min·mg protein. The optimum pH and temperature were 7.0 and 33° C, respectively. The Q₁₀ quotient was calculated to be 1.9 at 100 M NH ₄ ⁺ , indicating enzymatic involvement. In contrast to the wild type, B100, excretion of NH ₄ ⁺ , not uptake, was observed in a glutamine auxotroph, R. capsulata G29, which is derepressed for nitrogenase and lacks glutamine synthetase activity. G29R1, a revertant of G29, also took up NH ₄ ⁺ at the same rate as wild type and had fully restored glutamine synthetase activity. Partially restored derivatives, G29R5 and G29R6, grew more slowly than wild type on NH ₄ ⁺ as the nitrogen source, remained derepressed for nitrogenase in the presence of NH ₄ ⁺ , and displayed rates of NH ₄ ⁺ uptake in proportion to their glutamine synthetase activity. Ammonium uptake and glutamine synthetase activity were also restored in R. capsulata G29 exconjugants which had received the plasmid pPS25, containing the R. capsulata glutamine synthetase structural gene. These data suggest that NH ₄ ⁺ transport is tightly coupled to assimilation.Abbreviations used CHES cyclohexylaminoethanesulfonic acid - GS glutamine synthetase - SDS sodium dodecylsulfate     

Methylammonium uptake by Rhodobacter capsulatus

The ammonium uptake system of Rhodobacter capsulatus B100 was examined using the ammonium analog methylammonium. This analog was not transported when cells were grown aerobically on ammonium. When cultured on glutamate as a nitrogen source, or when nitrogen-starved, cells would take up methylammonium. Therefore, in cells grown under nitrogen-limiting conditions, a second system of ammonium uptake (or a modified form of the first) is present which is distinguished by its capacity for transporting the analog in addition to ammonium. The methylammonium uptake system exhibited saturation kinetics with a K _m of 22 M and a V _max of about 3 nmol per min · mg protein. Ammonium completely inhibited analog transport with a K _i in the range of 1 M. Once inside the cell methylammonium was rapidly converted to -N-methylglutamine; however, a small concentration gradient of methylammonium could still be observed. Kinetic parameters reflect the effects of assimilation.The methylammonium uptake system was temperature and pH dependent, and inhibition studies indicated that energy was required for the system to be operative. A glutamine auxotroph (G29) lacking the structural gene for glutanime synthetase did not accumulate the analog, even when nitrogen starved. The Nif^- mutant J61, which is unable to express nitrogenase structural genes, also did not transport methylammonium, regardless of the nitrogen source for growth. However, the mutant exhibited wild-type ammonium uptake and glutamine synthetase activity. These data suggest that transport of ammonium is required for growth on limited nitrogen and is under the control of the Ntr system in R. capsulatus.Abbreviations CCCP carbonyl cyanide-m-chlorophenyl hydrazone - CHES cyclohexylaminoethanesulfonic acid - DMSO dimethyl sulfoxide - GMAD -N-methylglutamine - GS glutamine synthetase - MES 2-(N-morpholino) ethanesulfonic acid - MSX methionine-Dl-sulfoximine - pCMB p-chloromercuribenzoate - Tricine N-tris(hydroxymethyl)methylglycine     

The uptake and metabolism of methylamine by N2-fixing cyanobacteria

The cyanobacteria Anabaena variabilis and Nostoc CAN showed a biphasic pattern of ¹⁴CH₃NH ₃ ⁺ uptake at external pH values of 7.0 and 9.0. The initial phase of uptake, which was independent of metabolism of ¹⁴CH₃NH ₃ ⁺ , was attributed to uptake via a CH₃NH ₃ ⁺ (NH ₄ ⁺ ) transport system at pH 7.0 and probably to passive diffusion of uncharged CH₃NH₂ and trapping by protonation at pH 9.0. The second slower phase of uptake was attributed to metabolism of CH₃NH ₃ ⁺ via glutamine synthetase to form -methylglutamine which accumulates. Anabaena cylindrica showed an initial rapid uptake at pH 7.0 and pH 9.0 but metabolism of ¹⁴CH₃NH ₃ ⁺ was undetectable at pH 7.0 and was barely detectable at pH 9.0. Pretreatment of A. variabilis with l-methionine-d,l-sulphoximine to inactivate glutamine synthetase, inhibited the second phase of ¹⁴CH₃NH ₃ ⁺ uptake at both pH 7.0 and pH 9.0 and the accumulation of -methylglutamine but had no effect on the first phase of uptake. Following transfer of A. variabilis to darkness the initial phase of ¹⁴CH₃NH ₃ ⁺ uptake at pH 7.0 and 9.0 was unaffected but the subsequent metabolism via glutamine synthetase was inhibited.Abbreviations MSX l-methionine-d,l-sulphoximine - GS glutamine synthetase     

Agrobacterium-mediated gene delivery and the biology of hostrange limitations

Insertion of foreign DNA into Ti plasmid-derived vectors in Agrobacterium tumefaciens is currently the most frequently used strategy for generating transgenic plants in a wide variety of species. Limitations of the host range of Agrobacterium restrict its usefulness in many cases, particularly when dealing with monocotyledonous plants. The objective of this presentation is to briefly discuss the efficiency of the transformation process utilized by Agrobacterium tumefaciens , potential barriers to efficient transformation by Agrobacterium that result in limitation of its useful host range, and how an understanding of the successful Agrobacterium /plant cell interaction might lead to advances in a variety of DNA delivery methodologies.     

Effect of nitrate and ammonium concentration on nitrate reductase activity in five species of mycorrhizal fungi

The nitrate reducing capacity of pure cultures of Cenococcum geophilum (Sow.) Ferd. & Winge, Paxillus involutes (Batsch: Fr.) Fr. (strains 1 and 2), Piloderma croceum Erikss. & Hjortst., Suillus variegatus (Fr.) O. Kuntze (strains 1 and 2) and an ectendomycorrhizal (E-strain) fungus was measured using an in vivo nitrate reductase (EC 1.6.6.3) assay. Differences between species and strains were established. The nitrate concentration of the culture medium influenced the nitrate reductase activities of the E-strain fungus and one strain of S. variegatus. The nitrate reductase activity of certain species and strains was a function of nitrate concentration. Addition of ammonium to the growth medium did not have any significant effect on the in vivo or in vitro nitrate reductase activity. The in vivo nitrate reductase activity in the mycelia of C. geophilum and the E-strain fungus decreased during 28 day growth in modified Melin-Norkrans medium. For mycelia of Paxillus involutus, Piloderma croceum and S. variegatus grown on agar the in vitro assays showed higher nitrate reductase activity than the in vivo assays.     

Resuscitation of Vibrio cholerae O1 strain TSI-4 from a viable but nonculturable state by heat shock

Abstract Vibrio cholerae strain TSI-4 was incubated in an M9 salt solution at 15 °C for more than 100 days. The plate counts showed no viable cells on day 30, but a broth culture from that day showed the growth of bacteria. However, after 35 days the bacteria entered the nonculturable state, based on the assessment of both the plate counts and broth culture. A portion of the culture was heated at 45 °C for 1 min in a water bath and subsequently plated onto a nutrient agar plate. More than 1000 colonies were recovered after this heat-shock treatment. The recovered cells showed the same chromosomal DNA pattern in the restriction map and the same outer membrane protein pattern in SDS-PAGE. Recovery of viable cells by heat-shock was achieved in cultures grown on M9 salt but not from cultures grown in phosphate-buffered saline. This suggests that the presence of NH₄Cl in the M9 salt solution may support the growth of the bacteria in a low nutrient medium, while also playing an important role in resuscitation.     

Effect of ammonium on the regulation of nitrate and nitrite transport systems in roots of intact barley (Hordeum vulgare L.) seedlings

The effect of NH ₄ ⁺ on the regulation of NO ₃ ^– and NO ₂ ^– transport systems in roots of intact barley (Hordeum vulgareL.) seedlings grown in NO ₃ ^– or NO ₂ ^– was studied. Ammonium partially inhibited induction of both transport systems. The inhibition was less severe in NO ₂ ^– -fed than in NO ₃ ^– -fed seedlings, presumably due to lower uptake of NH ₄ ⁺ in the presence of NO ₂ ^– . In seedlings pretreated with NH ₄ ⁺ subsequent induction was inhibited only when NH ₄ ⁺ was also present during induction, even though pretreated roots accumulated high levels of NH ₄ ⁺ . This indicates that inhibition may be regulated by NH ₄ ⁺ concentration in the cytoplasm rather than its total accumulation in roots. L-Methionine sulfoximine did not relieve the inhibition by NH ₄ ⁺ , suggesting that inhibition is caused by NH ₄ ⁺ itself rather than by its assimilation product(s). Ammonium inhibited subsequent expression of NO ₃ ^– transport activity similarly in roots grown in 0.1, 1.0, or 10 mM NO ₃ ^– for 24 h (steady-state phase) or 4 d (decline phase), indicating that it has a direct, rather than general feedback effect. Induction of the NO ₃ ^– transport system was about twice as sensitive to NH ₄ ⁺ as compared to the NO ₂ ^– transport system. This may relate to higher turnover rates of membraneassociated NO ₃ ^– -transport proteins.Abbreviations Mes 2(N-morpholino)ethanesulfonic acid - MSO L-methionine sulfoximine     

Tidal flushing of ammonium from intertidal sediments of Ria Formosa,Portugal

Ammonium concentrations were determined in near-bottom water and intertidal surface sediments collected in February and July 1993 at five stations of Ria Formosa, a shallow meso-tidal coastal lagoon in southern Portugal. At each station, samples were taken a few minutes before tidal inundation, and subsequently 2, 10, 15 and 20 minutes thereafter. Ammonium concentration in near-bottom waters increased dramatically in the first 2 minutes followed by a decrease during the 18 minutes of flooding (maximum range 10.3-2.2 M). The highest levels in the flooding water were concomitant with a decrease of extractable ammonium recorded in the upper sediment layer (2 cm). Laboratory experiments indicated that ammonium is easily extracted from the sediment solids by physical perturbation, as one would expect when tidal water flushes over the intertidal area. This perturbation results in the export of ammonium from the sediment, by pulse mechanisms of short time intervals. On a daily scale this amount is two orders of magnitude higher than transport resulting from molecular diffusion.     

Observations on the influence of glutamine,asparagine and peptone on growth and t-PA production of Chinese hamster ovary (CHO) cells

When a transfected CHO cell, that produces tissue-type Plasminogen Activator, t-PA, was transferred from a medium based on 5% Fetal Calf Serum, FCS, to a medium based on 0.8% casein peptone with variable glutamine and asparagine content, it was observed, that the growth of the cells changed from anchorage dependant to suspension culture giving more reproducible cultivations. In the FCS culture t-PA was unstable, observed as a decline in t-PA concentration after 250 h. This decline in t-PA concentration was not observed in the serum free culture, although there was a decline in productivity after 200 h. This change in production profile may be attributed to either no proteolytic attack from serum or by scavenging of proteolytic activities produced by the cells from the peptone peptides. Increasing amounts of glutamine/asparagine gave higher production of t-PA in synchrony with an increasing production of ammonia/ammonium ions. Ammonia inhibition does not seem to be a key factor for this cell line as seen with many others.     

Interactions of polyamines and nitrogen nutrition in plants

Biogenic amines occupy an important position among the many nitrogenous plant compounds. Polyamines are part of the overall metabolism of nitrogenous compounds, yet they do not seem to function in the 'normal' nitrogen nutrition. Rather, these widespread polycations (e. g. putrescine, spermidine and spermine) are involved in the regulation of growth and stress, probably by binding to negatively charged macromolecules. In addition, some diamines and polyamines are metabolized to yield 'secondary 'metabolites such as nicotine and other alkaloids. Previous studies have indicated that the ratio of nitrate to ammonium nutrition affects polyamine biosynthesis and content in intact plants. Thus, an increase in putrescine accumulation was found under conditions of excess ammonium ions, relative to nitrate. Modifications of nitrogen sources in the culture medium of tobacco cell suspensions (depletion of ammonium nitrate, or potassium nitrate, or both) resulted in marked changes in the content of cellular free polyamines. Considerable changes in the content of specific polyamines were also found with exposure to specific inhibitors of polyamine biosynthesis (difluoromethyl ornithine, difluoromethyl arginine, cyclohexylamine, methylglyoxal-bis-guanylhydrazone). However, a combination of nitrogen depletion of the medium and some inhibitors resulted in a very marked over-production of spermidine and spermine. The significance of these findings is discussed in relation to the assumption that polyamines act as a metabolic buffer, and maintain cellular pH under conditions where ammonium assimilation produces an excess of protons.