Effect of controlled expression of the hetR gene on heterocyst formation in the filamentous cyanobacterium Anabaena sp. PCC 7120

hetR is a central regulatory gene inducing and possibly maintaining irreversible heterocyst differentiation in filamentous cyanobacteria. A plasmid was constructed which enabled IPTG-mediated, controlled expression of hetR from a p _tac promoter in Anabaena . When introduced into a heterocyst-deficient hetR mutant, induction led to massive formation of heterocysts in a medium free of combined nitrogen. In nitrate-containing cultures, induction elicited formation of only a few heterocysts, but led to nitrogen chlorosis in vegetative cells as evidenced from degradation of phycobiliproteins. Removal of the inducer IPTG caused chlorosis and death of the organisms in nitrate-free medium, but no reversal of heterocyst formation. This indicates that constant synthesis of HetR is not the (sole) reason for irreversibility of heterocyst formation.     

The mystique of irreversibility in cyanobacterial heterocyst formation: parallels to differentiation and senescence in eukaryotic cells

The formation of cyanobacterial heterocysts is unique in the prokaryotic world: it is the only irreversible collective process. This terminal differentiation resembles senescence and differentiation in the eukaryotic urkingdom. During their cell cycle eukaryotic cells at the restriction point may reversibly proceed from a vegetative phase (G1) into a quiescent state (G0), and then may irreversibly enter the way towards differentiated or senescent cells. In parallel, at commitment point 1 vegetative cells from filamentous cyanobacteria may reversibly form proheterocysts, and then may proceed irreversibly towards mature heterocysts at commitment point 2. While the signals paving the path for differentiation or senescence in eukaryotes are largely unknown, heterocyst development is clearly triggered by nitrogen starvation. The reasons for the irreversibility in both systems are poorly understood. We discuss these questions, especially in the light of recent advances in the molecular biology of cyanobacteria, with emphasis on self-stabilizing autocatalytic cycles.     

Effect of Salicylhydroxamic Acid on Respiration, Photosynthesis, and Peroxidase Activity in Various Plant Tissues

Earlier reports from our laboratory described salicylhydroxamicacid (SHAM) stimulation of O₂ uptake by expanded soybean leavesor older green cotyledons. This stimulation could not be interpretedin terms of engagement or capacity of the cytochrome and alternativerespiratory pathways. In this report, we tested the possibilitythat a soluble peroxidase, which can be easily eluted from soybeanleaves and cotyledons, might be responsible for SHAM stimulationin whole tissue. The peroxidase catalyzes oxidation of NADHby O₂, is strongly stimulated by SHAM and benzhydroxamic acid(BHAM) and inhibited by KCN, propyl gallate and gentisic acid.This peroxidase, however, does not seem to be responsible forSHAM-stimulated O₂ uptake in whole, green tissue. In our earlier work reporting SHAM-stimulated respiration ingreen tissue, the samples had not been shielded from room light(10–20 µmol photons m^–2.s^–1). In thisreport, we show that O²-uptake rates of controls measured indarkness were always greater than those measured in room light.SHAM stimulation was not observed in the dark or in tissue withoutchlorophyll. We also found that CO₂ uptake of whole leafletsin saturating light was completely inhibited by SHAM fed throughthe transpiration stream. SHAM, therefore, is a potent inhibitorof photosynthesis. We conclude that the SHAM-stimulated respirationof green tissues we reported earlier likely was due to verylow rates of photosynthesis occurring under room light. ³Present address: SANDOZ Ltd., Agrobiological Research Station,4108 Witterswil, Switzerland ⁴Present address: WTC 1A3, Weyerhaeuser Co., Tacoma, WA 98477,U.S.A. (Received June 23, 1989; Accepted October 20, 1989)     

The 5-hydroxytryptamine(4a) receptor is palmitoylated at two different sites, and acylation is critically involved in regulation of receptor constitutive activity.

We have reported recently that the mouse 5-hydroxytryptamine(4a) (5-HT(4(a))) receptor undergoes dynamic palmitoylation (Ponimaskin, E. G., Schmidt, M. F., Heine, M., Bickmeyer, U., and Richter, D. W. (2001) Biochem. J. 353, 627-663). In the present study, conserved cysteine residues 328/329 in the carboxyl terminus of the 5-HT(4(a)) receptor were identified as potential acylation sites. In contrast to other palmitoylated G-protein-coupled receptors, the additional cysteine residue 386 positioned close to the COOH-terminal end of the receptor was also found to be palmitoylated. Using pulse and pulse-chase labeling techniques, we demonstrated that palmitoylation of individual cysteines is a reversible process and that agonist stimulation of the 5-HT(4(a)) receptor independently increases the rate of palmitate turnover for both acylation sites. Analysis of acylation-deficient mutants revealed that non-palmitoylated 5-HT(4(a)) receptors were indistinguishable from the wild type in their ability to interact with G(s), to stimulate the adenylyl cyclase activity and to activate cyclic nucleotide-sensitive cation channels after agonist stimulation. The most distinctive finding of the present study was the ability of palmitoylation to modulate the agonist-independent constitutive 5-HT(4(a)) receptor activity. We demonstrated that mutation of the proximal palmitoylation site (Cys(328) --> Ser/Cys(329) --> Ser) significantly increases the capacity of receptors to convert from the inactive (R) to the active (R*) form in the absence of agonist. In contrast, the rate of isomerization from R to R* for the Cys(386) --> Ser as well as for the triple, non-palmitoylated mutant (Cys(328) --> Ser/Cys(329) --> Ser/Cys(386) -->Ser) was similar to that obtained for the wild type.     

N2 fixation and NH 4 + assimilation in the thermophilic anaerobes Clostridium thermosaccharolyticum and Clostridium thermoautotrophicum

Inorganic nitrogen metabolism in the obligate anaerobic thermophiles Chlostridium thermosaccharolyticum and Clostridium thermoautotrophicum differs in several respects. C. thermosaccharolyticum contains a nitrogenase as inferred from NH ₄ ⁺ repressible C₂H₂ reduction, a glutamine synthetase which is partially repressed by ammonium, very labile glutamate synthase activities with both NADH and NADPH, NADPH-dependent glutamate dehydrogenase, and NH ₄ ⁺ -dependent asparagine synthetase. C. thermoautotrophicum contains no nitrogenase, but glutamine synthetase, no glutamate synthase, no glutamate dehydrogenase, but a NADH-dependent alanine dehydrogenase and a NH ₄ ⁺ -dependent asparagine synthetase.Abbreviation GOGAT glutamine-oxoglutarate amidotransferase amidotransferase (glutamate synthase)     

Ciglitazone reverses cAMP-induced post-insulin receptor resistance in rat adipocytes in vitro

Ciglitazone (cig), a thiazolidine-dione, lowers glucose and insulin levels in animal models of diabetes type II but not in controls. Since catecholamines given to rat adipocytes in vitro induce insulin resistance similar to that seen in type II diabetes in vivo, we measured the effect of cig on mono-A14-[125I]insulin binding and 3-O-methyl-D-glucose transport (GT) in isolated rat adipocytes treated with isoprenaline (iso, 10 microM). Cig (less than or equal to 5 microM) reversed (ED50 10 nM) the inhibitory effect of iso on insulin stimulation of GT. It had no effect on either basal or insulin stimulated GT. Furthermore, cig did not influence insulin binding either in the presence or absence of iso, which indicates that cig acts only on a post-insulin receptor level. Cig also reversed the inhibition of GT by both forskolin, a cyclase activator and RO20-1724, an imidazolidine phosphodiesterase inhibitor but not that of db-cAMP. It thus seems that cig does not act within the cAMP system but only neutralizes its inhibitory effect on the insulin stimulation of GT.     

Some properties of a Klebsiella pneumoniae ammonium transport negative mutant (Amt−)

The main property of an Amt^- (ammonium transport negative) mutant of Klebsiella pneumoniae is its inability to accumulate NH ₄ ⁺ intracellularly. When growing on nitrogen sources other than NH ₄ ⁺ , the mutant constantly looses NH₃ by diffusion. This loss results in poor growth. The NH₃ excretion suggests the existence of a futile cycle (NH₃ loss/NH ₄ ⁺ reabsorption) in the wild type and possibly other bacterial strains, which do not constantly excrete NH₃.Dedicated to Prof. R. H. Burris     

Alpha-bungarotoxin and the competing antibody WF6 interact with different amino acids within the same cholinergic subsite

In the nicotinic acetylcholine receptors (AChRs), the sequence segment surrounding two invariant vicinal cysteinyl residues at positions 192 and 193 of the alpha subunit contains important structural component(s) of the binding site for acetylcholine and high molecular weight cholinergic antagonists, like snake alpha-neurotoxins. At least a second sequence region contributes to the formation of the cholinergic site. Studying the binding of alpha-bungarotoxin and three different monoclonal antibodies, able to compete with alpha-neurotoxins and cholinergic ligands, to a panel of synthetic peptides as representative structural elements of the AChR from Torpedo, we recently identified the sequence segments alpha 181-200 and alpha 55-74 as contributing to form the cholinergic site (Conti-Tronconi et al., 1990). As a first attempt to elucidate the structural requirements for ligand binding to the subsite formed by the sequence alpha 181-200, we have now studied the binding of alpha-bungarotoxin and of antibody WF6 to the synthetic peptide alpha 181-200, and to a panel of peptide analogues differing from the parental sequence alpha 181-200 by substitution of a single amino acid residue. CD spectral analysis of the synthetic peptide analogues indicated that they all have comparable structures in solution, and they can therefore be used to analyze the influence of single amino acid residues on ligand binding. Distinct clusters of amino acid residues, discontinuously positioned along the sequence 181-200, seem to serve as attachment points for the two ligands studied, and the residues necessary for binding of alpha-bungarotoxin are different from those crucial for binding of antibody WF6. In particular, residues at positions 188-190 (VYY) and 192-194 (CCP) were necessary for binding of alpha-bungarotoxin, while residues W187, T191, and Y198 and the three residues at positions 193-195 (CPD) were necessary for binding of WF6. Comparison of the CD spectra of the toxin/peptide complexes, and those obtained for the same peptides and alpha-bungarotoxin in solution, indicates that structural changes of the ligand(s) occur upon binding, with a net increase of the beta-structure component. The cholinergic binding site is therefore a complex surface area, formed by discontinuous clusters of amino acid residues from different sequence regions. Such complex structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody/antigen complexes [reviewed in Davies et al. (1988)]. Within this relatively large structure, cholinergic ligands bind with multiple points of attachment, and ligand-specific patterns of the attachment points exist.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ammonium (methylammonium) transport by Klebsiella pneumoniae

Klebsiella pneumoniae can accumulate methylammonium up to 80-fold by means of a transport system as indicated by the energy requirement, saturation kinetics and a narrow pH profile around pH 6.8. Methylammonium transport (apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 100 μ}\text{M}$, $\text{V = 40 μ}\text{mol/min}$ per g dry weight at 15°C) is competitively inhibited by ammonium (apparent $\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{= 7 μ}\text{M}$). The low $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ value and the finding that methylammonium cannot serve as a nitrogen source indicate that ammonium rather than methylammonium is the natural substrate. Uphill transport is driven by a component of the protonmotive force, probably the membrane potential. The transport system is under genetic control; it is partially repressed by amino acids and completely by ammonium. Analysis of mutants suggest that the synthesis of the ammonium transport system is subject to the same ‘nitrogen control’ as nitrogenase and glutamine synthetase.