Two possible 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive sites in Photosystem II of spinach chloroplasts

Two possible 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive sites were found in PS II of spinach chloroplasts, depending on the pH of the assay medium used. The low site (pH 6) can be inhibited by certain quinolines, such as 8-hydroxyquinoline at concentrations less than 50 μM. The high pH site (pH 8) can be inhibited by disodium cyanamide, folic acid, or 5,6-benzoquinoline at concentrations from 50 μM to 5 mM. With the exception of orthophenanthroline, which stimulates the high pH site but does not show much inhibition at low pH, all other inhibitors gave opposite effects at the pH values used, i.e., they stimulated at low pH or inhibited at high pH, or vice versa. Several mechanisms for the observed effects are discussed.     

The greening process in cress seedlings. I. Pigment accumulation and ultrastructure after application of 5-aminolevulinate and complexing agents

A reduced rate of greening after continuous illumination was observed in dark-grown cress seedlings ( Lepidium sativum L.) incubated with 5-aminolevulinate (ALA) or the complexing agents 2,2'-bipyridyl, 8-hydroxyquinoline or 1,10-phenanthroline. This effect cannot be explained merely by photodynamic damage caused by chlorophyll precursors which are accumulated in the dark under these conditions. Flash light experiments revealed that photoconversion of protochlorophyll(ide) to chlorophyllide was not influenced by chelator treatment. The next step in the chlorophyll pathway, the esterification of chlorophyllide, however, was inhibited. Simultaneously applicated ALA and complexing agents did not result in a synergistic reponse; on the contrary, ALA seemed to render cress plants less susceptible to the treatment with complexing agents upon subsequent irradiation. Ultrastructural studies demonstrated that grana formation in light was inhibited after pretreatment with ALA or complexing agents.     

Influence of nitrilotriacetic acid and 8-hydroxyquinoline on the production of citric acid from molasses using Aspergillus niger

The effect of nitriloacetic acid (NTA) and 8-hydroxyquinoline on the production of citric acid by Aspergillus niger was investigated. The complexing agents showed an effect only during inoculation of the microorganism. Subsequent addition after inoculation did not produce any significant increase in citric acid yield. When 200 ppm of NTA was added during inoculation, an increase of 10.9 g·dm⁻³ citric acid over that produced by the control culture was observed. 8-Hydroxyquinoline, on the other hand, produced a higher concentration of citric acid which was 34 g·dm⁻³ over that of the control culture. The use of 8-hydroxyquinoline is therefore suggested for the production of citric acid from molasses using Aspergillus niger.     

An evaluation of some complexing methods for the histochemistry of calcium.

A selection of reagents capable of complexing with calcium in various ways was compared by applying them to tissue sections known to contain calcium in a variety of physiochemical states. Their histochemical potential was evaluated according to their sensitivity, specificity, accuracy of localisation and intensity of staining. Murexide, for light microscopy, and 8-hydroxyquinoline, for fluorescence microscopy proved to be the best overall reagents. They failed to demonstrate calcium oxalate, which was well shown by naphthalhydroxamic acid.     

Determination of the dead time of a stopped-flow fluorometer

This investigation was carried out to develop a convenient alternative method for examining the performance and determining the dead time of a stopped-flow fluorometer. We examined the kinetics for the formation of the fluorescent Mg2+-8-hydroxyquinoline chelate in aqueous solutions. The reversible association of the Mg2+ ion with 8-hydroxyquinoline is a second-order process whose on and off rate constants are dependent on pH. We estimated that the Mg2+ ion chelate has a fluorescence quantum yield of 0.02 in aqueous solutions. Using this reaction we measured the dead time of a stopped-flow fluorometer at different pH values. Measurements of the dead time were found to be reproducible and accurate. The Mg2+-8-hydroxyquinoline reaction fulfills the requirements for a convenient test reaction for dead time measurement of stopped-flow fluorometers. Although the usefulness of the reaction is primarily to determine the dead times of stopped-flow instruments operating in the fluorescence mode, the reaction can also be used for testing an instrument operating in the absorbance mode.     

An evaluation of some complexing methods for the histochemistry of calcium

Summary A selection of reagents capable of complexing with calcium in various ways was compared by applying them to tissue sections known to contain calcium in a variety of physiochemical states. Their histochemical potential was evaluated according to their sensitivity, specificity, accuracy of localisation and intensity of staining. Murexide, for light microscopy, and 8-hydroxyquinoline, for fluorescence microscopy proved to be the best overall reagents. They failed to demonstrate calcium oxalate, which was well shown by naphthalhydroxamic acid.     

Ferric enterobactin binding and utilization by Neisseria gonorrhoeae

FetA, formerly designated FrpB, an iron-regulated, 76-kDa neisserial outer membrane protein, shows sequence homology to the TonB-dependent family of receptors that transport iron into gram-negative bacteria. Although FetA is commonly expressed by most neisserial strains and is a potential vaccine candidate for both Neisseria gonorrhoeae and Neisseria meningitidis, its function in cell physiology was previously undefined. We now report that FetA functions as an enterobactin receptor. N. gonorrhoeae FA1090 utilized ferric enterobactin as the sole iron source when supplied with ferric enterobactin at approximately 10 microM, but growth stimulation was abolished when an omega (Omega) cassette was inserted within fetA or when tonB was insertionally interrupted. FA1090 FetA specifically bound 59Fe-enterobactin, with a Kd of approximately 5 microM. Monoclonal antibodies raised against the Escherichia coli enterobactin receptor, FepA, recognized FetA in Western blots, and amino acid sequence comparisons revealed that residues previously implicated in ferric enterobactin binding by FepA were partially conserved in FetA. An open reading frame downstream of fetA, designated fetB, predicted a protein with sequence similarity to the family of periplasmic binding proteins necessary for transporting siderophores through the periplasmic space of gram-negative bacteria. An Omega insertion within fetB abolished ferric enterobactin utilization without causing a loss of ferric enterobactin binding. These data show that FetA is a functional homolog of FepA that binds ferric enterobactin and may be part of a system responsible for transporting the siderophore into the cell.     

Effects of pH, glucose, and chelating agents on lethality of paraquat to Escherichia coli.

Retention of paraquat by Escherichia coli B was greatest after exposure at pH 9.0 and was progressively less after exposure at pH 7.0 and 5.0, respectively. This retained paraquat was capable of persistent growth inhibition. Uptake and retention of paraquat by E. coli B was dependent upon a carbon source, such as glucose. Under comparable conditions E. coli K-12 did not retain paraquat. The lethality of paraquat was seen in TSY medium but not in VB medium. The addition of Soytone, tryptone, or yeast extract, to the VB medium allowed the lethality of paraquat to be seen. A variety of chelating agents, including EDTA, 8-hydroxyquinoline, and o-phenanthroline, prevented the lethal effect of paraquat in TSY medium. Although EDTA protected against the lethality of paraquat, it did not protect against its bacteriostatic effect.     

The interaction of europium(III) ion with nucleotides

Apparent composite stability constants have been determined at pH 7.0 and 8.0 for the interaction of Eu(III) with ADP and ATP in 0.1 M N-ethylmorpholine buffer at 20°C. The values were obtained using a competitive spectrophotometric technique with 8-hydroxyquinoline as the competing ligand and experiments were performed in the presence of relatively low concentration of europium so as to avoid precipitation of hydrolysed species of the metal ion. The data have been used, together with an assumed hydrolysis constant for europium of 10^?8 M, to calculate that the stability constant for the Eu-ADP⁰ complex is about 10⁶ M^?1. The results were not sufficiently accurate to determine the stability constant of the Eu(OH)-ADP^? complex.Values are also reported for the stability constants and molar extinction coefficients of the complexes formed by the reaction of europium and magnesium with 8-hydroxyquinoline.     

Role of the entC gene in enterobactin and menaquinone biosynthesis in Escherichia coli

Tn10 mutants of Escherichia coli MC4100 were screened for their inability to grow under iron deficiency and for their inability to grow under anaerobiosis in the presence of fumarate as an electron acceptor. A strain so obtained (E. coli PBB1) lacked the ability to convert chorismic acid to isochorismic acid. This shows that the gene (entC) encoding isochorismate synthase was mutated. E. coli PBB1 did not produce any detectable amounts of menaquinones (vitamin K2) or enterobactin. When supplemented with isochorismic acid this strain produced menaquinones, indicating that isochorismic acid is involved not only in enterobactin but also in menaquinone biosynthesis. The entC gene was isolated and was shown to be part of the enterobactin gene cluster: It was located on a DNA fragment (9 kb in length) which also carried the entA gene. The DNA fragment was identified by restriction site mapping and was compared to a previously published map of the enterobactin gene cluster. The entC gene on this fragment responds not only to conditions (iron deficiency) that stimulate enterobactin biosynthesis but also to anaerobiosis which results in increased isochorismic acid formation and increased menaquinone biosynthesis. We conclude that isochorismic acid, isochorismic synthase, and the gene (entC) encoding this enzyme are involved in catalytic events at a metabolic branch point from which both enterobactin and menaquinones originate.     

Enterobactin-mediated iron transport in Pseudomonas aeruginosa.

A pyoverdine-deficient strain of Pseudomonas aeruginosa was unable to grow in an iron-deficient minimal medium in the presence of the nonmetabolizable iron chelator ethylene diamine-di(omega-hydroxyphenol acetic acid) (EDDHA), although addition of enterobactin to EDDHA-containing minimal media did restore growth of the pyoverdine-deficient P. aeruginosa. Consistent with the apparent ability of enterobactin to provide iron to P. aeruginosa, enterobactin-dependent 55Fe3+ uptake was observed in cells of P. aeruginosa previously grown in an iron-deficient medium containing enterobactin (or enterobactin-containing Escherichia coli culture supernatant). This uptake was energy dependent, was observable at low concentrations (60 nM) of FeCl3, and was absent in cells cultured without enterobactin. A novel protein with a molecular weight of approximately 80,000 was identified in the outer membranes of cells grown in iron-deficient minimal medium containing enterobactin, concomitant with the induction of enterobactin-dependent iron uptake. A Tn501 insertion mutant lacking this protein was isolated and shown to be deficient in enterobactin-mediated iron transport at 60 nM FeCl3, although it still exhibited enterobactin-dependent growth in iron-deficient medium containing EDDHA. It was subsequently observed that the mutant was, however, capable of enterobactin-mediated iron transport at much higher concentrations (600 nM) of FeCl3. Indeed, enterobactin-dependent iron uptake at this concentration of iron was observed in both the mutant and parent strains irrespective of whether they had been cultured in the presence of enterobactin. Apparently, at least two uptake systems for ferrienterobactin exist in P. aeruginosa: one of higher affinity which is specifically inducible by enterobactin under iron-limiting conditions and the second, of lower affinity, which is also inducible under iron-limiting conditions but is independent of enterobactin for induction.     

Export of the siderophore enterobactin in Escherichia coli: involvement of a 43 kDa membrane exporter

The enterobactin system for iron transport in Escherichia coli is well characterized with the exception of the mechanism of enterobactin secretion to the extracellular environment. Escherichia coli membrane protein P43, encoded by ybdA in the chromosomal region of genes involved in enterobactin synthesis, shows strong homology to the 12-transmembrane segment major facilitator superfamily of export pumps. A P43-null mutation was created and siderophore nutrition assays, performed with a panel of E. coli strains expressing one or more outer membrane receptors for enterobactin-related compounds, demonstrated that the P43 mutant was unable to secrete enterobactin efficiently. Products released from the mutant strain were identified with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), revealing that the P43 mutant secretes little, if any, enterobactin, but elevated levels of enterobactin breakdown products 2,3- dihydroxybenzoylserine (DHBS) monomer, dimer, and trimer. These data establish that P43 is a critical component of the E. coli enterobactin secretion machinery and provides a rationale for the designation of the previous genetic locus ybdA as entS to reflect its relevant biological function.     

Improvements in the method for the electron microscopic localization of arylsulphatase activity

Summary The optimal conditions for the demonstration of arylsulphatase activity in the proximal convoluted tubule cells of the rat kidney were studied at light and electron microscopic level. 8-hydroxyquinoline sulphate, p-nitrophenyl sulphate and 2-hydroxy-5-nitrophenylsulphate were used as substrates and barium and lead as capturing ions. The effect of fixation, capturing ions, substrate concentration and pH was studied biochemically. The results of these biochemical studies were then verified histochemically. Finally a recommended method for the light and electron microscopic demonstration of arylsulphatase activity was presented.     

Iron mobilisation from lactoferrin by chelators at physiological pH

Several alpha-ketohydroxypyridine, 2-hydroxypyridine N-oxide and 8-hydroxyquinoline chelators were shown to mobilise iron from diferric 59Fe-labelled human lactoferrin at physiological pH without the use of mediators or reducing agents. 1,2-Dimethyl-3-hydroxypyrid-4-one was found to be the most effective chelator, removing 90% of 59Fe from [59Fe]lactoferrin, in contrast to desferrioxamine, which was ineffective under the same conditions.     

Accumulation of periplasmic enterobactin impairs the growth and morphology of Escherichia coli tolC mutants

TolC is the outer membrane component of tripartite efflux pumps, which expel proteins, toxins and antimicrobial agents from Gram‐negative bacteria. Escherichia coli tolC mutants grow well and are slightly elongated in rich media but grow less well than wild‐type cells in minimal media. These phenotypes have no physiological explanation as yet. Here, we find that tolC mutants have highly aberrant shapes when grown in M9‐glucose medium but that adding iron restores wild‐type morphology. When starved for iron, E. coli tolC mutants synthesize but cannot secrete the siderophore enterobactin, which collects in the periplasm. tolC mutants unable to synthesize enterobactin display no growth or morphological defects, and adding exogenous enterobactin recreates these aberrations, implicating this compound as the causative agent. Cells unable to import enterobactin across the outer membrane grow normally, whereas cells that import enterobactin only to the periplasm become morphologically aberrant. Thus, tolC mutants grown in low iron conditions accumulate periplasmic enterobactin, which impairs bacterial morphology, possibly by sequestering iron and inhibiting an iron‐dependent reaction involved in cell division or peptidoglycan synthesis. The results also highlight the need to supply sufficient iron when studying TolC‐directed export or efflux, to eliminate extraneous physiological effects.     

Aerobic biodegradation characteristics and metabolic products of quinoline by a Pseudomonas strain

A bacterial strain, BW003, which utilized quinoline as its sole C, N and energy source, was isolated and identified as Pseudomonas sp. BW003 degraded 192–911 mg/l quinoline within 3–8 h with removal rates ranging from 96% to 98%. The optimum conditions for the degradation were 30 °C and pH 8. In the process of biodegradation, at least 43% of quinoline was transformed into 2-hydroxyquinoline, then 0.69% of 2-hydroxyquinoline was transformed into 2,8-dihydroxyquinoline, and then, presumably, into 8-hydroxycoumarin. Meanwhile, at least 48% of the nitrogen in quinoline was directly transformed into ammonia-N. An extra carbon source enhanced the nitrogen transformation from ammonia-N. Further experiments showed that, besides cell synthesis, BW003 transformed less than 6% of ammonia-N into nitrate through heterotrophic nitrification. In addition, BW003 contained a large plasmid, which may be involved in quinoline metabolism. The study indicates that quinoline and its metabolic products can be eliminated from wastewater by controlling the C/N ratio using BW003 as the bioaugmentation inoculum.     

Microbial transformation of quinoline by a Pseudomonas sp

A Pseudomonas sp. isolated from sewage by enrichment culture on quinoline metabolized this substrate by a novel pathway involving 8-hydroxycoumarin. During early growth of the organism on quinoline, 2-hydroxyquinoline accumulated as the intermediate; 8-hydroxycoumarin accumulated as the major metabolite on further incubation. 2,8-Dihydroxyquinoline and 2,3-dihydroxyphenylpropionic acid were identified as the other intermediates. Inhibition of quinoline metabolism by 1 mM sodium arsenite led to the accumulation of pyruvate, whereas inhibition by 5 mM arsenite resulted in the accumulation of 2-hydroxyquinoline as the major metabolite and 2,8-dihydroxyquinoline as the minor metabolite. Coumarin was not utilized as a growth substrate by this bacterium, but quinoline-grown cells converted it to 2-hydroxyphenylpropionic acid, which was not further metabolized. Quinoline, 2-hydroxyquinoline, 8-hydroxycoumarin, and 2,3-dihydroxyphenylpropionic acid were rapidly oxidized by quinoline-adapted cells, whereas 2,8-dihydroxyquinoline was oxidized very slowly. Quinoline catabolism in this Pseudomonas sp. is therefore initiated by hydroxylation(s) of the molecule followed by cleavage of the pyridine ring to yield 8-hydroxycoumarin, which is further metabolized via 2,3-dihydroxyphenylpropionic acid.     

Microbial transformation of quinoline by a Pseudomonas sp.

A Pseudomonas sp. isolated from sewage by enrichment culture on quinoline metabolized this substrate by a novel pathway involving 8-hydroxycoumarin. During early growth of the organism on quinoline, 2-hydroxyquinoline accumulated as the intermediate; 8-hydroxycoumarin accumulated as the major metabolite on further incubation. 2,8-Dihydroxyquinoline and 2,3-dihydroxyphenylpropionic acid were identified as the other intermediates. Inhibition of quinoline metabolism by 1 mM sodium arsenite led to the accumulation of pyruvate, whereas inhibition by 5 mM arsenite resulted in the accumulation of 2-hydroxyquinoline as the major metabolite and 2,8-dihydroxyquinoline as the minor metabolite. Coumarin was not utilized as a growth substrate by this bacterium, but quinoline-grown cells converted it to 2-hydroxyphenylpropionic acid, which was not further metabolized. Quinoline, 2-hydroxyquinoline, 8-hydroxycoumarin, and 2,3-dihydroxyphenylpropionic acid were rapidly oxidized by quinoline-adapted cells, whereas 2,8-dihydroxyquinoline was oxidized very slowly. Quinoline catabolism in this Pseudomonas sp. is therefore initiated by hydroxylation(s) of the molecule followed by cleavage of the pyridine ring to yield 8-hydroxycoumarin, which is further metabolized via 2,3-dihydroxyphenylpropionic acid.