Acid-base regulation during ammonium assimilation in Hydrodictyon africanum

Abstract The acid-base balance during ammonium (used to mean NH ₄⁺ and/or NH₃) assimilation in Hydrodictyon africanum has been measured on cells growing with about 1 mol m^?3 ammonium at an external pH of about 6.5. Measurements made included (1) ash alkalinity (corrected for intracellular ammonium) which yields net organic negative charge, (2) the accumulation of organic N in the cells and (3) the change in extracellular H⁺ (from the pH change and the buffer capacity). These measurements showed that some 0.25 excess organic negative charge (half in the cell wall, half inside the plasmalemma) accumulates per organic N synthesized, while some 1.25H⁺ accumulate in the medium per organic N synthesized. Granted a permeability (PNH₃) of some 10^?3 cm s^?1, and a finite [NH₃] in the cytoplasm of these N-assimilating cells it is likely that most of the ammonium entering these growing cells is as NH ₄⁺. This means that most of the H ⁺ appearing in the medium must have originated from inside the cell and have been subjected to active efflux at the plasmalemma: H⁺ accumulates in the medium equivalent to any NH₃ entry by requilibration from exogenous NH ₄⁺. The cell composition (net organic negative charge, organic N content) is very similar in these ammonium-grown cells to that of NO₃⁺grown cells, suggesting that there is no action of a ‘biochemical pH stat’ during longterm assimilation of NO₃⁺in H. africanum. Short-term experiments were carried out at an external pH of 7.2 in which ammonium at various concentrations were supplied to NO₃⁺-grown cells. There was in all cases a rapid influx followed by a slower uptake; at least at the lower concentrations (less than 100 μmol dm^?3) the net influx was all attributable to NH₄⁺influx via a uniporter, probably partly short-circuited by a passive NH₃ efflux due to intrinsic membrane permeability to NH₃. The net ammonium influx was in all cases associated with H⁺ accumulation in the medium. (1.3-1.7 H ⁺ per ammonium taken up); as in the growth experiments, most of the ammonium taken up was assimilated. Determinations of cytoplasmic pH showed either no effect on, or a slight decrease in, pH during ammonium assimilation; the changes that occurred were in the direction expected for actuating a ‘pH-regulating’ change in H⁺ fluxes.     

I do it my way: regulation of ammonium uptake and ammonium assimilation in Corynebacterium glutamicum

In order to utilize different nitrogen sources and to survive situations of nitrogen limitation, microorganisms have developed several mechanisms to adapt their metabolism to changes in the nitrogen supply. In this communication, recent advances in our knowledge of ammonium uptake, its assimilation, and connected regulatory systems in Corynebacterium glutamicum are discussed with respect to the situation in the bacterial model organisms Escherichia coli and Bacillus subtilis. The regulatory network of nitrogen control in C. glutamicum differs substantially from that in these bacteria, for example, by the presence of AmtR, the unique "master regulator" of nitrogen control, the absence of a NtrB/NtrC two-component signal transduction system, and a different sensing mechanism in C. glutamicum.     

Kinetic flux profiling of nitrogen assimilation in Escherichia coli

We present a new method for probing cellular metabolic fluxes that is based on the kinetics of assimilation of isotope-labeled nutrient into a diversity of downstream metabolites. In the case of nitrogen assimilation, half-maximal labeling of most metabolites occurs in 10-300 s. Fluxes measured on the basis of the kinetics of nitrogen assimilation in exponentially growing E. coli agree well with those fluxes predicted to allow optimal biomass production.     

Control of ammonium concentration in Escherichia coli fermentations

A control system has been devised for the maintenance of stable ammonium concentrations throughout a fedbatch fermentation. The control system is based on an ammonia gas-sensing electrode that monitors a pH-adjusted effluent stream from the fermentor. To overcome the time lag between the fermentor and the electrode, feedback control included metered flows of ammonium to both the fermentor and the electrode vessel. The system was used to study the growth of Escherichia coli B (ATCC 11303) at controlled ammonium concentrations of 5 to 200mM. Apparent specific growth rates, biomass and protein production, and glucose yields were essentially constant from 5 to 170mM. Above 170mM ammonium growth was inhibited. As ammonium concentration decreased from 170 to 5mM, ammonium yields increased from 1 to 24 g cell dry wt/g ammonium utilized. The results demonstrate that control of ammonium concentrations at levels so low that ammonium would be exhausted in batch fermentations can significantly increase overall ammonium yields.     

The pathways of ammonium assimilation in Rhizobium meliloti

Two pathways of ammonium assimilation are known in bacteria, one mediated by glutamate dehydrogenase, the other by glutamine synthetase and glutamate synthase. The activities of these three enzymes were measured in crude extracts from four Rhizobium meliloti wild-type strains, 2011, M15S, 444 and 12. All the strains had active glutamine synthetase and NADP-linked glutamate synthase. Assimilatory glutamate dehydrogenase activity was present in strains 2011, M15S, 444, but not in strain 12. Three glutamate synthase deficient mutants were isolated from strain 2011. They were unable to use 1 mM ammonium as a sole nitrogen source. However, increased ammonium concentration allowed these mutants to assimilate ammonium via glutamate dehydrogenase. It was found that the sole mode of ammonium assimilation in strain 12 is the glutamine synthetase-glutamate synthase route; whereas the two pathways are functional in strain 2011.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase     

The immunological dissection of the Escherichia coli molecular chaperone DnaJ

In Escherichia coli, one of the main molecular chaperones is DnaJ (hsp40), which mediates in a variety of highly conserved cellular processes including protein-folding reactions and the assembly/disassembly of protein complexes. DnaJ is characterised by the presence of four distinct domains: the J-domain, glycine/phenylalanine-rich (G/F), cysteine-rich (Zn-finger) and C-terminal regions. Truncated DnaJ polypeptides (DnaJ 1-108, DnaJ Delta1-108, DnaJ Delta1-199) representing these domains were over-produced and used as a source of immunogens for the generation of sequence-specific polyclonal antibodies. Epitope mapping was achieved by Western blotting, which demonstrated the presence of antibodies directed against these domains. These characterised affinity-purified antibodies were then used to assess the role of DnaJ in the protection of firefly luciferase from irreversible heat-inactivation. In this study we have demonstrated the involvement of J-, G/F and Zn-finger domains in the protection of luciferase from heat-inactivation. The C-terminal region had only partial involvement in luciferase protection.     

Coordinated regulation of ammonium assimilation and carbon catabolism by glyoxylate in Saccharomyces cerevisiae

The activities of citrate synthase (EC 4.1.3.7) and NADP+-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.4) of Saccharomyces cerevisiae were inhibited in vitro by glyoxylate. In the presence of glyoxylate, pyruvate and glyoxylate pools increased, suggesting that glyoxylate was efficiently transported and catabolized. Pyruvate accumulation also indicates that citrate synthase was inhibited. A decrease in the glutamate pool was also observed under these conditions. This can be attributed to an increased transamination rate and to the inhibitory effect of glyoxylate on NADP+-dependent GDH. Furthermore, the increase in the ammonium pool in the presence of glyoxylate suggests that NADP+-dependent GDH was being inhibited in vivo, since the activity of glutamine synthetase did not decrease under these conditions. We propose that the inhibition of both citrate synthase and NADP+-dependent GDH could form part of a mechanism that regulates the internal 2-oxoglutarate concentration.     

The regulation of the Escherichia coli mazEF promoter involves an unusual alternating palindrome

The Escherichia coli mazEF system is a chromosomal "addiction module" that, under starvation conditions in which guanosine-3',5'-bispyrophosphate (ppGpp) is produced, is responsible for programmed cell death. This module specifies for the toxic stable protein MazF and the labile antitoxic protein MazE. Upstream from the mazEF module are two promoters, P(2) and P(3) that are strongly negatively autoregulated by MazE and MazF. We show that the expression of this module is positively regulated by the factor for inversion stimulation. What seems to be responsible for the negative autoregulation of mazEF is an unusual DNA structure, which we have called an "alternating palindrome." The middle part, "a," of this structure may complement either the downstream fragment, "b," or the upstream fragment, "c". When the MazE.MazF complex binds either of these arms of the alternating palindrome, strong negative autoregulation results. We suggest that the combined presence of the two promoters, the alternating palindrome structure and the factor for inversion stimulation-binding site, all permit the expression of the mazEF module to be sensitively regulated under various growth conditions.     

Expression and regulation of protein K, an Escherichia coli K1 porin, in Escherichia coli K-12

Using a modified lambda phage as a vector and a procedure developed in Dr. C. Schnaitman's laboratory, we have cloned the structural gene for protein K from an Escherichia coli K1 strain to an E coli K-12 strain. The cloned inserts consist of two HindIII fragments, 4 kb and 6.5 kb in size. The protein produced by the insert is nearly identical to "authentic" protein K when chymotryptic peptides of 125I-labeled proteins are compared. Protein K was found to respond to changes in the osmolarity of the medium, being favored in trypticase soy broth (high osmolarity). This fluctuation was not dependent on a functional ompR gene. However, protein K was not expressed in strains carrying the envZ-473 mutation. Thus, protein K appears to be within a class of exported proteins whose expression is regulated by the envZ gene independent of the ompR gene.     

The regulation of porin expression in Escherichia coli: effect of turgor stress

The OmpF and OmpC porins are major outer membrane proteins of Escherichia coli. Their expression is affected by medium osmolarity such that OmpF is normally produced at low osmolarity and OmpC at high osmolarity. Potassium ion accumulation is a major means by which cells maintain their internal osmolarity in high osmolarity medium in the absence of organic osmolytes such as glycine-betaine. Starvation for potassium causes cells to become turgor stressed. The effect of turgor stress and potassium ion concentration on OmpF and OmpC expression was examined. It was found that ompF gene expression was switched off by turgor stress but there was no concomitant increase in OmpC. Instead, ompC expression responded to the accumulation of potassium ions by the cell in high osmolarity medium.     

Development of a software tool for in silico simulation of Escherichia coli using a visual programming environment

This study describes the development of a software tool, EcoSim, to assist users in implementing quantitative in silico simulation easily. It consists of four parts: extracellular environment and constraints setting mode, table for optimal metabolic flux distribution and chart for changes of substrate concentration, dynamic flux distribution viewer and dynamic hierarchical regulatory network viewer. Representation of a hierarchical regulatory network was constructed with defined modeling symbols and weight in the central Escherichia coli metabolism. All programming procedures for EcoSim were accomplished in a visual programming environment (LabVIEW). To illustrate quantitative in silico simulation with EcoSim, this program was performed on E. coli using glucose and acetate as carbon sources. The simulation results were in agreement with the experimental data obtained from the literature. EcoSim can be used to assist biologists and engineers in predicting and interpreting dynamic behaviors of E. coli under a variety of environmental conditions.     

In silico design of anaerobic growth-coupled product formation in Escherichia coli: experimental validation using a simple polyol,glycerol

Bioprocess and Biosystems Engineering - Integrated approaches using in silico model-based design and advanced genetic tools have enabled efficient production of fuels, chemicals and functional...