Diversity of siderophore-mediated iron uptake systems in fluorescent pseudomonads: not only pyoverdines

Fluorescent pseudomonads are gamma-proteobacteria known for their capacity to colonize various ecological niches. This adaptability is reflected by their sophisticated and diverse iron uptake systems. The majority of fluorescent pseudomonads produce complex peptidic siderophores called pyoverdines or pseudobactins, which are very efficient iron scavengers. A tremendous variety of pyoverdines has been observed, each species producing a different pyoverdine. This variety can be used as an interesting tool to study the diversity and taxonomy of fluorescent pseudomonads. Other siderophores, including newly described ones, are also produced by pseudomonads, sometimes endowed with interesting properties in addition to iron scavenging, such as formation of complexes with other metals or antimicrobial activity. Factors other than iron limitation, and different regulatory proteins also seem to influence the production of siderophores in pseudomonads and are reviewed here as well. Another peculiarity of pseudomonads is their ability to use a large number of heterologous siderophores via different TonB-dependent receptors. A first genomic analysis of receptors in four different fluorescent pseudomonads suggests that their siderophore ligand repertoire is likely to overlap, and that not all receptors recognize siderophores as ligands.     

Export of Thermus thermophilus cytoplasmic beta-glycosidase via the E. coli Tat pathway

The Tat pathway is distinct from the Sec machinery given its unusual capacity to export folded proteins, which contain a twin-arginine (RR) signal peptide, across the plasma membrane. The functionality of the Tat pathway has been demonstrated for several Gram-negative and Gram-positive mesophilic bacteria. To assess the specificity of the Tat system, and to analyze the capacity of a mesophilic bacterial Tat system to translocate cytoplasmic proteins from hyperthermophilic bacteria, we fused the Thermus thermophilus beta-glycosidase (Glc) to the twin-arginine signal peptide of the E. coli TorA protein. When expressed in E. coli, the thermophilic RR-Glc chimera was successfully synthesized and efficiently translocated into the periplasm of the wild type strain. In contrast, the beta-glycosidase accumulated within the cytoplasm of all the tat mutants analyzed. The beta-glycosidase synthesized in these strains exhibited thermophilic properties. These results demonstrated, for the first time, the capacity of the E. coli Tat system to export cytoplasmic hyperthermophilic protein, implying an important potential of the Tat system for the production of thermostable enzymes used in bioprocessing applications.     

Structure-function relationship and regulation of two Bacillus subtilis DNA-binding proteins, HBsu and AbrB

Microorganisms use a number of small basic proteins for organization and compaction of their DNA. By their interaction with the genome, these proteins do have a profound effect on gene expression, growth behavior, and viability. It has to be distinguished between indirect effects as a consequence of the state of chromosome condensation and relaxation that influence the rate of RNA polymerase action as represented by the histone-like proteins, and direct effects by specific binding of proteins to defined DNA segments predominantly located around promoter sequences. This latter class is represented by the transition-state regulators that are involved in integrating various global stimuli and orchestrating expression of the genes under their regulation for a better adaptation to changes in growth rate. In this article we will focus on two different but abundant DNA binding proteins of the gram-positive model organism Bacillus subtilis, the histone-like HBsu as a member of the unspecific and the transition state regulator AbrB as a member of specific classes of DNA binding proteins.     

Subunit composition and functional properties of G-protein heterotrimers on rat chromaffin granules

Heterotrimeric G-proteins at the plasma membrane serve as switches between heptahelical receptors and intracellular signal cascades. Likewise endomembrane associated G-proteins may transduce signals from intracellular compartments provided they consist of a functional trimer. Using quantitative immunoelectron microscopy we found heterotrimeric G-protein subunits Galpha2, Galpha(q/11), Gbeta2 and Gbeta5 to reside on secretory granules in chromaffin cells of rat adrenal glands.Thus rat chromaffin granules are equipped with functional G-proteins that consist of a specific alpha-, beta- and probably gamma-subunit combination. Serotonin uptake into a crude rat chromaffin granule preparation was inhibited by activated Galphao2 (10 nM) to nearly the same extent as by GMppNp (50 microM) whereas GDPbetaS was ineffective. The data support the idea that vesicular G-proteins directly regulate the transmitter content of secretory vesicles. In this respect Galphao2 appears to be the main regulator of vesicular momoamine transporter activity.     

Atypical beta-adrenergic effects on insulin signaling and action in beta(3)-adrenoceptor-deficient brown adipocytes

Cross talk between adrenergic and insulin signaling systems may represent a fundamental molecular basis of insulin resistance. We have characterized a newly established beta(3)-adrenoceptor-deficient (beta(3)-KO) brown adipocyte cell line and have used it to selectively investigate the potential role of novel-state and typical beta-adrenoceptors (beta-AR) on insulin signaling and action. The novel-state beta(1)-AR agonist CGP-12177 strongly induced uncoupling protein-1 in beta(3)-KO brown adipocytes as opposed to the beta(3)-selective agonist CL-316,243. Furthermore, CGP-12177 potently reduced insulin-induced glucose uptake and glycogen synthesis. Neither the selective beta(1)- and beta(2)-antagonists metoprolol and ICI-118,551 nor the nonselective antagonist propranolol blocked these effects. The classical beta(1)-AR agonist dobutamine and the beta(2)-AR agonist clenbuterol also considerably diminished insulin-induced glucose uptake. In contrast to CGP-12177 treatment, these negative effects were completely abrogated by metoprolol and ICI-118,551. Stimulation with CGP-12177 did not impair insulin receptor kinase activity but decreased insulin receptor substrate-1 binding to phosphatidylinositol (PI) 3-kinase and activation of protein kinase B. Thus the present study characterizes a novel cell system to selectively analyze molecular and functional interactions between novel and classical beta-adrenoceptor types with insulin action. Furthermore, it indicates insulin receptor-independent, but PI 3-kinase-dependent, potent negative effects of the novel beta(1)-adrenoceptor state on diverse biological end points of insulin action.     

The Ran GTPase system in fission yeast affects microtubules and cytokinesis in cells that are competent for nucleocytoplasmic protein transport

Misregulation of the evolutionarily conserved GTPase Ran in fission yeast results in defects in several cellular processes in cells that are competent for nucleocytoplasmic protein transport. These results suggest that transport is neither the only nor the primary Ran-dependent process in living cells. The ability of Ran to independently regulate multiple cellular processes in vivo is demonstrated by showing that (i) eight different transport-competent RanGEF (guanine nucleotide exchange factor) mutants have defects in mitotic spindle formation; (ii) the RanGEF temperature-sensitive mutant pim1-d1 has abnormal actin ring structures at the septum. Overexpression of Imp2p, which specifically destabilizes these structures, restores viability. (iii) Ran-dependent processes differ in their requirements for active Ran in vivo. Microtubule function, cytokinesis, and nuclear envelope structure are the Ran-dependent processes most sensitive to the amount of Ran protein in the cell, whereas nucleocytoplasmic protein transport is the most robust. Therefore, the ability of Ran from Schizosaccharomyces pombe to independently regulate multiple cellular processes may reflect differences in its interactions with the binding proteins that mediate these functions and explain the complex phenotypic consequences of its misregulation in vivo.     

14-3-3 Proteins and photoneuroendocrine transduction: role in controlling the daily rhythm in melatonin

This paper describes the role 14-3-3 proteins play in vertebrate photoneuroendocrine transduction. 14-3-3 proteins form a complex with arylalkylamine N-acetyltransferase (AANAT), the enzyme which turns melatonin production on during the day and off at night. Complex formation is triggered at night by cAMP-dependent phosphorylation of the enzyme, and results in activation and protection against proteolysis. This enhances melatonin production >10-fold. Light exposure results in dephosphorylation of the enzyme and disassociation from 14-3-3, leading to destruction and a rapid drop in melatonin production and release and circulating levels.