Molecular regulation of iron uptake in pseudomonas aeruginosa

The microbial demand for iron is often met by the elaboration of siderophores into the surrounding medium and expression of cognate outer membrane receptors for the ferric siderophore complexes. Conditions of iron limitation, such as those encountered in vivo, cause Pseudomonas aeruginosa to express two high-affinity iron-uptake systems based on pyoverdin and pyochelin. These systems will operate both in the organism's natural habitat, soil and water, where the solubility of iron at neutral pH is extremely low, and in the human host where the availability of free iron is too low to sustain bacterial growth due to the iron-binding glycoproteins transferrin and lactoferrin. Cross-feeding and radiolabelled iron uptake experiments demonstrated that pyoverdin biosynthesis and uptake were highly heterogeneous amongst P.aeruginosa strains, that growth either in the presence of pyoverdin or pyochelin resulted in induction of specific IROMPs, and that induction of iron uptake is siderophore-specific. The P.aeruginosa Tn5 mutant PH1 is deficient in ferripyoverdin uptake and resistant to pyocin Sa, suggesting that the site of interaction of pyocin Sa is a ferripyoverdin receptor. Additional Tn5 mutants appeared to exploit different strategies to achieve pyocin Sa-resistance, involving modifications in expression of pyoverdin-mediated iron uptake, indicating that complex regulatory systems exist to enable these organisms to compete effectively for iron. Modulation of expression of IROMPs prompted a study of the mechanism of uptake of a semi-synthetic C(7) α-formamido substituted cephalosporin BRL 41897A. Sensitivity to this agent correlated with expression of the 75 kDa ferri-pyochelin receptor and demonstrated the potential of high-affinity iron uptake systems for targeting of novel antibiotics. Studies with ferri-pyoverdin uptake-deficient mutant PH1 indicated that expression of outer membrane protein G (OprG), which is usually expressed under iron-rich conditions and repressed under iron-deficient conditions, was perturbed. Attempts were made to clone the oprG gene using a degenerate probe based on the N-terminal amino acid sequence. A strongly hybridising HindIll restriction fragment was cloned and sequenced, but failed to reveal an open reading frame correspondmg to OprG. However, there appears to be good evidence that a part of the gene codmg for the hydrophilic membrane-associated ATP-binding component of a hitherto uncharacterised periplasmic- binding-protein-dependent transport system has been isolated. The full organisation and sequence of the operon, and substrate for this putative transport system, are yet: to be elucidated,

Use of ultrasonic agitation in iron group alloy electrodepositions

The effects of ultrasonic agitation on deposition from two iron group alloy plating solutions, nickel-cobalt and bright nickel-iron, have been studied. Comparison has been made with deposits plated from the same solutions using controlled air agitation. The ultrasonic equipment employed had a fixed frequency of 13 KHz but the power output from each transducer was variable up to a maximum of 350 watts. The effects of air and ultrasonic agitation on hardness, ductility, tensile strength, composition, structure, surface topography, limiting current density, cathode current efficiency and macro-throwing power were determined. Transmission and scanning electron microscopy, electron-probe microanalysis and atomic absorption spectrophotometry have been employed to study the nickel alloy deposits produced. The results obtained show that the use of Ultrasonics increased significantly the hardness of both alloy deposits and altered their composition by decreasing the cobalt and iron contents from nickel-cobalt and nickeliron solutions respectively. The ductility of coatings improved but the tensile strength did not change very much. Ultrasonic agitation gave larger grained deposits than air and they seemed to have a lower stress. Dull cobalt-nickel deposits had a similar pyramidal surface topography regardless of the type of agitation but the bright appearance of the nickel-iron was destroyed by ultrasonic agitation; an unusual ribbed pattern was produced. The use of ultrasonic agitation permitted approximately a twofold increase in the plating current density at which sound deposits could be achieved but there was only a slight increase in cathode current efficiency. Macro-throwing power of the solutions was increased slightly by the use of ultrasonic agitation. ultrasonic agitation is an expensive means of agitating plating Solutions and would be worthwhile only if significant improvements in properties could be achieved. The simultaneous improvement in hardness and ductility is a novel feature that should have useful engineering applications.

Microsegregation in nodular cast iron and its effect on mechanical properties

A review of the literature pertaining to the mechanical properties, solidification and segregation effects in nodular cast iron has been made. A series of investigations concerning the influence of microsegregation on mechanical properties of :pearlitic, ferritic and austenitic nodular cast iron have then been reported. The influence of section size on the tensile and impact properties of cornmercial purity and refined ferritic nodular cast iron has been studied. It has been shown. that an increase in section caused a decrease in impact transition temperature of the commercial purity material without greatly affecting the impact transition temperature of the purer material. This effect has been related to increased amounts of segregation effects such as cell boundary carbides in heavier sections of the commercial purity material. Microsegregation studies on the materials used in this thesis have been carried out using an electron probe microanalyser. This technique has shown that concentrations of chromium and manganese and depletions of nickel and silicon occurred at eutectic cell boundaries in nodular cast iron and were often associated with brittle carbides in these areas. These effects have been shown to be more prevalent in heavier sections. The nature of segregation during the solidification of nodular cast iron has been studied by quenching samples of nodular iron during the solidification process. Micro-analysis of such samples has shown that segregation of manganese and chromium occurs by a gradual build-up of these elements at the solid/liquid interface. The microstructures of the quenched specimens revealed carbide filaments connecting graphite nodules and areas of quenched liquid. These filaments have been used as evidence for a revised hypothesis for the solidification of nodular cast iron by a liquid diffusion mechanism. A similar series of experiments has been carried out on two high nickel austenitic irons containing 0.5 per cent manganese and 4 per cent manganese respectively. In both these materials a decrease in elongation was experienced with increasing section. This effect was more drastic in the 4 per cent manganese material which also contained much greater amounts of cell boundary carbide in heavy sections. Micro-analysis of samples of the 4 per cent manganese material quenched during solidification revealed that manganese concentrated in the liquid and that nickel concentrated in the solid during solidification. No segregation of silicon occurred in this material. Carbide filaments appeared in the microstructures of these specimens. A discussion of all the above effects in terms of current concepts is included.

Conformational analysis of the natural iron chelator myo-inositol 1,2,3-trisphosphate using a pyrene-based fluorescent mimic

myo-Inositol phosphates possessing the 1,2,3-trisphosphate motif share the remarkable ability to completely inhibit iron-catalysed hydroxyl radical formation. The simplest derivative, myo-inositol 1,2,3-trisphosphate [Ins(1,2,3)P3], has been proposed as an intracellular iron chelator involved in iron transport. The binding conformation of Ins(1,2,3)P3 is considered to be important to complex Fe3+ in a 'safe' manner. Here, a pyrene-based fluorescent probe, 4,6-bispyrenoyl-myo-inositol 1,2,3,5-tetrakisphosphate [4,6-bispyrenoyl Ins(1,2,3,5)P4], has been synthesised and used to monitor the conformation of the 1,2,3-trisphosphate motif using excimer fluorescence emission. Ring-flip of the cyclohexane chair to the penta-axial conformation occurs upon association with Fe3+, evident from excimer fluorescence induced by π-π stacking of the pyrene reporter groups, accompanied by excimer formation by excitation at 351 nm. This effect is unique amongst biologically relevant metal cations, except for Ca 2+ cations exceeding a 1:1 molar ratio. In addition, the thermodynamic constants for the interaction of the fluorescent probe with Fe3+ have been determined. The complexes formed between Fe 3+ and 4,6-bispyrenoyl Ins(1,2,3,5)P4 display similar stability to those formed with Ins(1,2,3)P3, indicating that the fluorescent probe acts as a good model for the 1,2,3-trisphosphate motif. This is further supported by the antioxidant properties of 4,6-bispyrenoyl Ins(1,2,3,5)P4, which closely resemble those obtained for Ins(1,2,3)P3. The data presented confirms that Fe3+ binds tightly to the unstable penta-axial conformation of myo-inositol phosphates possessing the 1,2,3-trisphosphate motif. © 2010 The Royal Society of Chemistry.