The biological process for phosphate (P i ) removal is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyP). We obtained Escherichia coli mutants which accumulate a large amount of polyP. The polyP accumulation in these mutants was ascribed to a mutation of the phoU gene that encodes a negative regulator of the P i regulon. Insertional inactivation of the phoU gene also elevated the intracellular level of polyP in Synechocystis sp. strain PCC6803. The mutant could remove fourfold more P i from the medium than the wild-type strain removed.Inorganic phosphate (P i ) is recognized as one of the major nutrients causing eutrophication of lakes, bays, and waterways (18). Considerable attention has been paid to effective P i removal from wastewater (6). Many bacteria are capable of accumulating excess P i in the form of inorganic polyphosphate (polyP), which is a linear polymer of hundreds of P i residues linked by high-energy phosphoanhydride bonds (7, 9, 10). Improvement of the ability to accumulate polyP contributes to increased P i removal from wastewater (18).Previously, we have demonstrated genetic improvement of polyP accumulation in Escherichia coli (8). High levels of accumulated polyP were achieved by increasing the dosage of the E. coli genes encoding polyP kinase (ppk) and the P i -specific transport system (pstSCAB). The E. coli recombinant accumulated approximately 16% of its dry weight as phosphorus (P) (49% as P i ) (8). Over 60% of cellular P was stored in the form of polyP in the genetically engineered E. coli strain. However, growth of the E. coli recombinant was severely limited in minimal medium (8). In addition, this recombinant released polyP back into the medium when it accumulated high levels of polyP. In this paper, we report that a mutation in the phoU gene, which encodes a negative regulator of the P i regulon (3, 21), led to high levels of accumulated polyP in E. coli. phoU mutants could be easily screened on agar plates containing 5-bromo-4-chloro-3-indolylphosphate (XP) after N-methyl-NЈ-nitro-N-nitrosoguanidine mutagenesis. Therefore, isolating phoU mutants seems to be a useful way to improve the ability of bacteria to accumulate polyP. To show whether this method is effective in another bacterium, we performed insertional inactivation of the phoU gene in Synechocystis sp. strain PCC6803 and showed that the intracellular level of polyP increased in the mutant.
Isolation of E. coli mutants.The levels of polyP in E. coli MG1655 were very low (less than 1 nmol of P i residues/mg of protein) when the organism was grown on a rich medium (11). PolyP was recovered with silicate glass from cells lysed with guanidine isothiocyanate, and the polyP content was determined by a two-enzyme assay (4). PolyP was first converted to ATP by polyphosphate kinase, and then the amount of ATP was measured by a bioluminescence assay. We first selected alkaline phosphatase constitutive mutants, which could form blue colonies on agar plates containing XP (50 mg/liter) under P i -su...
Reversible and equilibrium isomerization of η 5 -bis(Me 3 Si-fluorenyl)-rare earth metal complexes to η 6 -bis(Me 3 Si-fluorene-AlR 3 )-rare earth metal complexes, which is a novel type of metallotropic tautomerism, was realized by the addition of AlR 3 to the former complexes. Resulting η 6 -complexes also changed to the initial η 5 -complexes reversibly by the addition of excess THF. η 5 -Bis(Me 3 Si-fluorenyl)Sm(THF) 2 (1) was prepared by reaction of Me 3 Sifluorenylpotassium with SmI 2 (THF) 2 , and its structure was analyzed by X-ray diffraction. η 6 -Bis(Me 3 Si-fluorene-AlMe 3 )Sm (2) was synthesized by the reaction of 1 with excess AlMe 3 . The corresponding reaction of excess AlEt 3 with 1 gave η 6 -bis(Me 3 Si-fluorene-AlEt 3 )Sm (3). The structures of 2 and 3 were determined by X-ray analyses. The reaction of bis(Me 3 Sifluorenyl)Yb(THF) 2 ( 4) with an excess amount of AlMe 3 gave η 5 -(Me 3 Si-fluorenyl)-η 6 -(Me 3 -Si-fluorene-AlMe 3 )Yb ( 5), where one equimolar AlMe 3 coordinates to the Yb atom through its two Me groups via an agostic interaction. One of the Me 3 Si-fluorenyl groups assumes η 5 -coordination, while the other Me 3 Si-fluorenyl group shows η 6 -coordination. The addition of excess THF to 5 produced (fluorenyl)Yb(THF) 4 /AlMe 4 (6) in low yield. To understand the primary factor for the alternative η 5 -and η 6 -bonding modes, we have synthesized ( i Prindenyl) 2 Yb(THF) 2 (7) without a Me 3 Si group and with a rather small indenyl ring and examined the reaction with AlR 3 .
The X-ray analysis of Me 2 Si(C 5 H 3 -3-Me 3 -Si) 2 SmCH(Me 3 Si) 2 (THF) (1) revealed a monomeric structure, while the hydrogenated complex, [Me 2 Si(C 5 H 3 -3-Me 3 Si) 2 SmH(THF)] 2 (2), exhibits binuclear µ-H structure to indicate that Me 2 SiCp′ 2 M f M(µ-Me 2 SiCp′ 2 ) 2 M rearrangement of the η 5 -η 5 ligation occurs during the hydrogenation. The resulting samarocene hydride 2 exhibits high activity as an initiator for the block copolymerization of ethylene with polar monomers such as methyl methacrylate and -caprolactone.
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