Cobalt(II) Oxidation by the Marine Manganese(II)-Oxidizing Bacillus sp. Strain SG-1

The geochemical cycling of cobalt (Co) has often been considered to be controlled by the scavenging and oxidation of Co(II) on the surface of manganese [Mn(III,IV)] oxides or manganates. Because Mn(II) oxidation in the environment is often catalyzed by bacteria, we have investigated the ability of Mn(II)-oxidizing bacteria to bind and oxidize Co(II) in the absence of Mn(II) to determine whether some Mn(II)-oxidizing bacteria also oxidize Co(II) independently of Mn oxidation. We used the marine Bacillus sp. strain SG-1, which produces mature spores that oxidize Mn(II), apparently due to a protein in their spore coats (R.A. Rosson and K. H. Nealson, J. Bacteriol. 151:1027-1034, 1982; J. P. M. de Vrind et al., Appl. Environ. Microbiol. 52:1096-1100, 1986). A method to measure Co(II) oxidation using radioactive ⁵⁷Co as a tracer and treatments with nonradioactive (cold) Co(II) and ascorbate to discriminate bound Co from oxidized Co was developed. SG-1 spores were found to oxidize Co(II) over a wide range of pH, temperature, and Co(II) concentration. Leucoberbelin blue, a reagent that reacts with Mn(III,IV) oxides forming a blue color, was found to also react with Co(III) oxides and was used to verify the presence of oxidized Co in the absence of added Mn(II). Co(II) oxidation occurred optimally around pH 8 and between 55 and 65°C. SG-1 spores oxidized Co(II) at all Co(II) concentrations tested from the trace levels found in seawater to 100 mM. Co(II) oxidation was found to follow Michaelis-Menten kinetics. An Eadie-Hofstee plot of the data suggests that SG-1 spores have two oxidation systems, a high-affinity-low-rate system (K_m, 3.3 × 10^-8 M; V_max, 1.7 × 10^-15 M · spore^-1 · h^-1) and a low-affinity-high-rate system (K_m, 5.2 × 10^-6 M; V_max, 8.9 × 10^-15 M · spore^-1 · h^-1). SG-1 spores did not oxidize Co(II) in the absence of oxygen, also indicating that oxidation was not due to abiological Co(II) oxidation on the surface of preformed Mn(III,IV) oxides. These results suggest that some microorganisms may directly oxidize Co(II) and such biological activities may exert some control on the behavior of Co in nature. SG-1 spores may also have useful applications in metal removal, recovery, and immobilization processes.     

Continuous, real-time monitoring of the oxygen uptake rate (OUR) in animal cell bioreactors

A new method for real-time monitoring of the oxygen uptake rate (OUR) in bioreactors, based on dissolved oxygen (DO) measurement at two points, has been developed and tested extensively. The method has several distinct advantages over known techniques.It enables the continuous and undisturbed monitoring of OUR, which is conventionally impossible without gas analyzers. The technique does not require knowledge of k(L)a. It provides smooth, robust, and reliable signal. The monitoring scheme is applicable to both microbial and mammalian cell bioprocesses of laboratory or industrial scale. The method was successfully used in the cultivation of NSO-derived murine myeloma cell line producing monoclonal antibody. It was found that while the OUR increased with the cell density, the specific OUR decreased to approximately one-half at cell concentrations of 16 x 10(6) cells/mL, indicating gradual reduction of cell respiration activity. Apart from the laboratory scale cultivation, the method was applied to industrial scale perfusion culture, as well as to processes using other cell lines. (c) 1994 John Wiley & Sons, Inc.     

Localization of a New Type of X-Linked Liver Glycogenosis to the Chromosomal Region Xp22 Containing the Liver α-Subunit of Phosphorylase Kinase (PHKA2)

We describe here a new type of X-linked liver glycogen storage disease. The main symptoms include liver enlargement and growth retardation. The clinical and biochemical abnormalities of this glycogenosis are similar to those of classical X-linked liver glycogenosis due to phosphorylase kinase deficiency (XLG). However, in contrast to patients with XLG, the patients described here have no reduced phosphorylase kinase activity in erythrocytes and leukocytes, and no enzyme deficiency could be found. Linkage analysis of four families with this X-linked type of liver glycogenosis assigned the disease gene to Xp22. Lod scores obtained with the markers DXS987, DXS207, and DXS999 were 3.97, 2.71, and 2.40, respectively, all at 0% recombination. Multipoint linkage analysis localized the disease gene between DXS143 and DXS989 with a maximum lod score of 4.70 at θ = 0, relative to DXS987. As both the classical XLG gene and the liver α-subunit of PHK (PHKA2) are also located in Xp22, this variant type of XLG may be allelic to classical XLG, and both diseases may be caused by mutations in PHKA2. Therefore, we propose to classify XLG as XLG type I (the classical type of XLG) and XLG type II (the variant type of XLG).     

Regulation of Saccharomyces cerevisiae CDC7 function during the cell cycle.

The yeast Cdc7 function is required for the G1/S transition and is dependent on passage through START, a point controlled by the Cdc28/cdc2/p34 protein kinase. CDC7 encodes a protein kinase activity, and we now show that this kinase activity varies in the cell cycle but that protein levels appear to remain constant. We present several lines of evidence that periodic activation of CDC7 kinase is at least in part through phosphorylation. First, the kinase activity of the Cdc7 protein is destroyed by dephosphorylation of the protein in vitro with phosphatase. Second, Cdc7 protein is hypophosphorylated and inactive as a kinase in extracts of cells arrested at START but becomes active and maximally phosphorylated subsequent to passage through START. The phosphorylation pattern of Cdc7 protein is complex. Phosphopeptide mapping reveals four phosphopeptides in Cdc7 prepared from asynchronous yeast cells. Both autophosphorylation and phosphorylation in trans appear to contribute to this pattern. Autophosphorylation is shown to occur by using a thermolabile Cdc7 protein. A protein in yeast extracts can phosphorylate and activate Cdc7 protein made in Escherichia coli, and phosphorylation is thermolabile in cdc28 mutant extracts. Cdc7 protein carrying a serine to alanine change in the consensus recognition site for Cdc28 kinase shows an altered phosphopeptide map, suggesting that this site is important in determining the overall Cdc7 phosphorylation pattern.     

Overexpression of the yeast MCK1 protein kinase suppresses conditional mutations in centromere-binding protein genes CBF2 and CBF5

We find that overexpression in yeast of the yeast MCK1 gene, which encodes a meiosis and centromere regulatory kinase, suppresses the temperature-sensitive phenotype of certain mutations in essential centromere binding protein genes CBF2 and CBF5. Since Mck1p is a known serine/threonine protein kinase, this suppression is postulated to be due to Mck1p-catalyzed in vivo phosphorylation of centromere binding proteins. Evidence in support of this model was provided by the finding that purified Mck1p phosphorylates in vitro the 110 kDa subunit (Cbf2p) of the multimeric centromere binding factor CBF3. This phosphorylation occurs on both serine and threonine residues in Cbf2p.     

Effect of Root Zone Temperature on the Mineral Composition of Xylem Sap and Plasma Membrane K+-Mg++-ATPase Activity of Grafted-Cucumber and -Figleaf Gourd Root Systems

Cucumber (Cucumis sativus L.) seedlings were grafted onto cucumber-(CG) or figleaf gourd- (FG, Cucurbita ficifolia Bouché)seedlings in order to determine the effect of solution temperature(12, 22, and 32°C) on the mineral composition of xylem sapand the plasma membrane K⁺-Mg⁺⁺-ATPase activities of the roots.Low solution temperature (12°C) lowered the concentrationof NO^–₃ and H₂PO^–₄ in xylem sap of CG plants butnot of FG plants. Concentrations of K⁺, Ca⁺⁺ and Mg⁺⁺ in xylemsap were less affected than anions by solution temperature.The plasma membrane of FG plants grown in 12°C solutiontemperature showed the highest K⁺- Mg⁺⁺-ATPase activity at allATP concentrations up to 3 mM and at low reaction temperatureup to 12°C, indicating resistance of figleaf gourd to lowroot temperature. (Received December 27, 1994; Accepted March 10, 1995)