Cation-promoted association of a regulatory and target protein is controlled by protein phosphorylation.

xiv, 302 hlm, 21 c

Salmonella enterica biofilm-mediated dispersal by nitric oxide donors in association with cellulose nanocrystal hydrogels

Protected by extracellular polymers, microbes within biofilms are significantly more resistant to disinfectants. Current research has been instrumental in identifying nitric oxide donors and hydrogels as potential disinfectant additives. Nitric oxide (NO) donors are considered a very promising molecule as biofilm dispersal agents and hydrogels have recently attracted a lot of interest due to their biocompatible properties and ability to form stable thin films. When the NO donor MAHMA NONOate was dissolved in phosphate saline buffer, it was able to reduce the biomass of well-established biofilms up to 15% for at least 24 h of contact time. Encapsulation of MAHMA NONOate and molsidomine within a hydrogel composed of cellulose nanocrystals (CNC) has shown a synergistic effect in dispersing well-established biofilms: after 2 h of exposure, moderate but significant dispersion was measured. After 6 h of exposure, the number of cells transitioning from the biofilm to the planktonic state was up to 0.6 log higher when compared with non-treated biofilms. To further explore the transport processes of NO donors within hydrogels, we measured the nitric oxide flux from gels, at 25°C for a composite of 0.1 µM MAHMA NONOate–CNC. Nitric oxide diffuses up to 500 µm from the hydrogel surface, with flux decreasing according to Fick’s law. 60% of NO was released from the hydrogel composite during the first 23 min. These data suggest that the combined treatments with nitric oxide donor and hydrogels may allow for new sustainable cleaning strategies

Two transcription factors are necessary for iron homeostasis in a salt-dwelling archaeon

Because iron toxicity and deficiency are equally life threatening, maintaining intracellular iron levels within a narrow optimal range is critical for nearly all known organisms. However, regulatory mechanisms that establish homeostasis are not well understood in organisms that dwell in environments at the extremes of pH, temperature, and salinity. Under conditions of limited iron, the extremophile Halobacterium salinarum, a salt-loving archaeon, mounts a specific response to scavenge iron for growth. We have identified and characterized the role of two transcription factors (TFs), Idr1 and Idr2, in regulating this important response. An integrated systems analysis of TF knockout gene expression profiles and genome-wide binding locations in the presence and absence of iron has revealed that these TFs operate collaboratively to maintain iron homeostasis. In the presence of iron, Idr1 and Idr2 bind near each other at 24 loci in the genome, where they are both required to repress some genes. By contrast, Idr1 and Idr2 are both necessary to activate other genes in a putative a feed forward loop. Even at loci bound independently, the two TFs target different genes with similar functions in iron homeostasis. We discuss conserved and unique features of the Idr1–Idr2 system in the context of similar systems in organisms from other domains of life

A study of NO{sub x} reduction by fuel injection recirculation. Topical report, June--December, 1995

Flue-gas recirculation (FGR) is a well-known method used to control oxides of nitrogen (NO{sub x}) in industrial burner applications. Recent small- and large-scale experiments have shown that introducing the recirculated flue gases with the fuel results in a much greater reduction in NO{sub x}, per unit mass of gas recirculated, in comparison to introducing the flue gases with the combustion air. That fuel injection recirculation (FIR) is more effective than windbox FGR is quite remarkable. At present, however, there is no definitive understanding of why FIR is more effective than conventional FGR. The objective of this research is to ascertain whether or not chemical and/or molecular transport effects alone can explain the differences in NO{sub x} reduction observed between FIR and FGR. This knowledge will aid in the rational application and optimization of FIR in a wide variety of industrial applications. A combined modeling and experimental program is in progress to achieve the research objectives. This report discusses, first, computer modeling studies of counterflow diffusion flames employing detailed chemical kinetics for methane combustion and NO{sub x} formation, and, second, experimental studies of laminar, CH{sub 4}-air, jet flames

Cation-promoted association of a regulatory and target protein is controlled by protein phosphorylation.

A central question in molecular biology concerns the means by which a regulatory protein recognizes different targets. IIIGlc, the glucose-specific phosphocarrier protein of the bacterial phosphotransferase system, is also the central regulatory element of the PTS. Binding of unphosphorylated IIIGlc inhibits several non-PTS proteins, but there is little or no sequence similarity between IIIGlc binding sites on different target proteins. The crystal structure of Escherichia coli IIIGlc bound to one of its regulatory targets, glycerol kinase, has been refined at 2.6-A resolution in the presence of products, adenosine diphosphate and glycerol 3-phosphate. Structural and kinetic analyses show that the complex of IIIGlc with glycerol kinase creates an intermolecular Zn(II) binding site with ligation identical to that of the zinc peptidase thermolysin. The zinc is coordinated by the two active-site histidines of IIIGlc, a glutamate of glycerol kinase, and a water molecule. Zn(II) at 0.01 and 0.1 mM decreases the Ki of IIIGlc for glycerol kinase by factors of about 15 and 60, respectively. The phosphorylation of one of the histidines of IIIGlc, in its alternative role as phosphocarrier, provides an elegant means of controlling the cation-enhanced protein-protein regulatory interaction. The need for the target protein to supply only one metal ligand may account for the lack of sequence similarity among the regulatory targets of IIIGlc

Glycerol kinase from Escherichia coli and an Ala65→Thr mutant: the crystal structures reveal conformational changes with implications for allosteric regulation

AbstractBackground: Glycerol kinase (GK) from Escherichia coli is a velocity-modulated (V system) enzyme that has three allosteric effectors with independent mechanisms: fructose-1,6-bisphosphate (FBP); the phosphocarrier protein IIAGlc; and adenosine nucleotides. The enzyme exists in solution as functional dimers that associate reversibly to form tetramers. GK is a member of a superfamily of ATPases that share a common ATPase domain and are thought to undergo a large conformational change as an intrinsic step in their catalytic cycle. Members of this family include actin, hexokinase and the heat shock protein hsc70.Results: We report here the crystal structures of GK and a mutant of GK (Ala65→Thr) in complex with glycerol and ADP. Crystals of both enzymes contain the same 222 symmetric tetramer. The functional dimer is identical to that described previously for the IIAGlc–GK complex structure. The tetramer interface is significantly different, however, with a relative 22.3° rotation and 6.34 å translation of one functional dimer. The overall monomer structure is unchanged except for two regions: the IIAGlc-binding site undergoes a structural rearrangement and residues 230–236 become ordered and bind orthophosphate at the tetramer interface. We also report the structure of a second mutant of GK (IIe474→Asp) in complex with IIAGlc; this complex crystallized isomorphously to the wild type IIAGlc–GK complex. Site-directed mutants of GK with substitutions at the IIAGlc-binding site show significantly altered kinetic and regulatory properties, suggesting that the conformation of the binding site is linked to the regulation of activity.Conclusions: We conclude that the new tetramer structure presented here is an inactive form of the physiologically relevant tetramer. The structure and location of the orthophosphate-binding site is consistent with it being part of the FBP-binding site. Mutational analysis and the structure of the IIAGlc–GK(IIe474→Asp) complex suggest the conformational transition of the IIAGlc-binding site to be an essential aspect of IIAGlc regulation