A Citrobacter sp. accumulated uranyl ion (UO 2M2 ) via precipitation with phosphate ligand liberated by phosphatase activity. The onset and rate of uranyl phosphate deposition were promoted by NH M 4 , forming NH 4 UO 2 PO 4 , which has a lower solubility product than NaUO 2 PO 4 . This acceleration decoupled the rate-limiting chemical crystallization process from the biochemical phosphate ligand generation. This provided a novel approach to monitor the cell-surface-associated changes using atomic-force microscopy in conjunction with transmission electron microscopy and electron-probe X-ray microanalysis, to visualize deposition of uranyl phosphate at the cell surface. Analysis of extracted surface materials by 31

A heavy-metal-accumulating Citrobacter sp. has been used for the treatment of metal-laden industrial wastes. Metal uptake is mediated via a cell-bound phosphatase that liberates inorganic phosphate which precipitates with heavy metals as cell-bound metal phosphate. A phosphatase-def icient mutant accumulated little UOf+, while a phosphatase-overproducing mutant accumulated correspondingly more metal, with a uranium loading equivalent to the bacterial dry weight achieved after 6 h exposure of resting cells to uranyl ion in the presence of phosphatase substrate (glycerol 2-phosphate). The phosphatase, visualized by immunogold labelling in the parent and overproducing strains, but not seen in the deficient mutant, was held within the periplasmic space with, in some cells, a higher concentration at the polar regions. Enzyme was also associated with the outer membrane and found extracellularly. Accumulated uranyl phosphate was visible as cell-surface-and polar-localized deposits, identified by energy-dispersive X-ray analysis (EDAX), proton-induced X-ray emission analysis (PIXE) and X-ray diffraction analysis (XRD) as polycrystalline HUO,PO,AH,O. Nucleation sites for initiation of biocrystallization were identified at the cytoplasmic and outer membranes, prompting consideration of an in vitro biocatalytic system for metal waste remediation. Phosphatidylcholine-based liposomes with entrapped phosphatase released phosphate comparably to whole cells, as shown by 31P NMR spectroscopy in the presence of NMR-silent ' 'l2Cd2+. Application of liposome-immobilized enzyme to the decontamination of uranyl solutions was, however, limited by rapid fouling of the biocatalyst by deposited uranyl phosphate. It is suggested that the architecture of the bacterial cell surface provides a means of access of uranyl ion to the inner and outer membranes and enzymically liberated phosphate in a way that minimizes fouling in whole cells.

Growth of Pseudomonas aeruginosa ATCC 15692 was promoted when the strain was cultured in an iron-depleted succinate medium, supplemented with transferrin at 30%, 60% and 100% and lactoferrin at 60% and 100% ironsaturation. No significant differences between cell growth and pyoverdin production were observed when transferrin iron saturation was increased from 30% t o 100%; however, cell growth and pyoverdin production were strongly dependent on lactoferrin iron saturation. Lower lactoferrin iron saturation (< 30%) resulted in more pyoverdin production and reduced cell growth. Incubation of pyoverdin (19 pM) with 100 pM transferrin (So%, 60%and 100 YO iron-saturated) or lactoferrin (60 YO and 100% iron-saturated) led t o quenching of pyoverdin fluorescence. Also, 24 h incubation of pyoverdin (200 pM) with these two proteins (209 pM, 30%, 60% and 100% iron-saturated transferrin and 60% and 100% iron-saturated lactoferrin) at 25 "C resulted in increased absorbance at 460 nm. Both the fluorescence quenching and absorbance increases were iron-saturation-dependent. Taken together, these results support the conclusion that at physiological pH, P. aeruginosa pyoverdin can acquire iron from partially iron-saturated transferrin or lactoferrin.30602, USA

A Citrobacter sp. accumulates uranyl ion (UO2(2+)) as crystalline HUO2PO4.4H2O (HUP), using enzymatically generated inorganic phosphate. Ni was not removed by this mechanism, but cells already loaded with HUP removed Ni2+ by intercalative ion-exchange, forming Ni(UO2PO4)2.7H2O, as concluded by x-ray diffraction (XRD) and proton induced x-ray emission (PIXE) analyses. The loaded biomass became saturated with Ni rapidly, with a molar ratio of Ni:U in the cellbound deposit of approx. 1:6; Ni penetration was probably surface-localized. Cochallenge of the cells with Ni2+ and UO2(2+), and glycerol 2-phosphate (phosphate donor for phosphate release and metal bioprecipitation) gave sustained removal of both metals in a flow through bioreactor, with more extensively accumulated Ni. We propose 'Microbially Enhanced Chemisorption of Heavy Metals' (MECHM) to describe this hybrid mechanism of metal bioaccumulation via intercalation into preformed, biogenic crystals, and note also that MECHM can promote the removal of the transuranic radionuclide neptunium, which is difficult to achieve by conventional methods.

A Citrobacter sp. was reported previously to accumulate heavy metals as cell-bound heavy metal phosphates. Metal uptake is mediated by the activity of a periplasmic acid-type phosphatase that liberates inorganic phosphate to provide the precipitant ligand for heavy metals presented to the cells. Amino acid sequencing of peptide fragments of the purified enzyme revealed significant homology to the phoN product (acid phosphatase) of some other enterobacteria. These organisms, together with Klebsiella pneumoniae, previously reported to produce acid phosphatase, were tested for their ability to remove uranium and lanthanum from challenge solutions supplemented with phosphatase substrate. The coupling of phosphate liberation to metal bioaccumulation was limited to the metal accumulating Citrobacter sp.; therefore the participation of species-specific additional factors in metal bioaccumulation was suggested.

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13C nuclear m agnetic resonance (nm r) spectroscopy of in tact webs from the com m on garden spider Araneus diadematus has been used to dem onstrate th a t: (i) w ater retention is an im p o rtan t role for the viscid coating o f ca p tu re th re a d ; (ii) the elasticity of cap tu re th read results from w ater-induced m obility at a m olecular level, (iii) the organization and com position of stru ctu ral and cap tu re th read are different, even in the absence o f co ating; and (iv) glycoproteins m ay have a m ore im p o rtan t presence and structural role th an previously realized. D ifferent 13C -labelling patterns of webs were achieved by feeding spiders either w ith [13C]glucose or w ith [13C ]am ino acids.