Ni(2+) transport and accumulation in Rhodospirillum rubrum

Abstract

The cooCTJ gene products are coexpressed with CO-dehydrogenase (CODH) and facilitate in vivo nickel insertion into CODH. A Ni(2+) transport assay was used to monitor uptake and accumulation of (63)Ni(2+) into R. rubrum and to observe the effect of mutations in the cooC, cooT, and cooJ genes on (63)Ni(2+) transport and accumulation. Cells grown either in the presence or absence of CO transported Ni(2+) with a K(m) of 19 +/- 4 microM and a V(max) of 310 +/- 22 pmol of Ni/min/mg of total protein. Insertional mutations disrupting the reading frame of the cooCTJ genes, either individually or all three genes simultaneously, transported Ni(2+) the same as wild-type cells. The nickel specificity for transport was tested by conducting the transport assay in the presence of other divalent metal ions. At a 17-fold excess Mn(2+), Mg(2+), Ca(2+), and Zn(2+) showed no inhibition of (63)Ni(2+) transport but Co(2+), Cd(2+), and Cu(2+) inhibited transport 35, 58, and 66%, respectively. Nickel transport was inhibited by cold (50% at 4 degrees C), by protonophores (carbonyl cyanide m-chlorophenylhydrazone, 44%, and 2,4-dinitrophenol, 26%), by sodium azide (25%), and hydroxyl amine (33%). Inhibitors of ATP synthase (N, N'-dicyclohexylcarbodiimide and oligomycin) and incubation of cells in the dark stimulated Ni(2+) transport. (63)Ni accumulation after 2 h was four times greater in CO-induced cells than in cells not exposed to CO. The CO-stimulated (63)Ni(2+) accumulation coincided with the appearance of CODH activity in the culture, suggesting that the (63)Ni(2+) was accumulating in CODH. The cooC, cooT, and cooJ genes are required for the increased (63)Ni(2+) accumulation observed upon CO exposure because cells containing mutations disrupting any or all of these genes accumulated (63)Ni(2+) like cells unexposed to CO.

FIG. 1

Initial rate of ⁶³Ni²⁺ accumulation by R. rubrum. Cells were grown in malate ammonium medium as described in Materials and Methods. Cells were harvested by centrifugation and resuspended in 100 mM MOPS buffer (pH 7.5) (○) or 100 mM MOPS buffer (pH 7.5) containing 1.0 mM MgCl₂ (■). The cells were incubated for 30 min at 30°C before the assay was initiated by the addition of 1.0 μM ⁶³NiCl₂. Nickel accumulated by the cells was measured at the indicated time point by collecting the cells by vacuum filtration, followed by three 5.0-ml washes with 10 mM EDTA in 100 mM Tris buffer (pH 8.0). The filtration assay was as described in Materials and Methods. Each data point represents a single measurement.

FIG. 2

⁶³Ni²⁺ accumulation in CO-induced R. rubrum. CO (20% gas phase) and ⁶³Ni (1 μM; 615 mCi/mmol) were added at the zero time point to R. rubrum cells (OD₆₀₀ = 2.0) in malate ammonium medium (■). The control (no CO added) contains ⁶³Ni (1 μM; 615 mCi/mmol) added to R. rubrum cells (OD₆₀₀ = 2.0) in malate ammonium medium (●). CODH activity in the cells treated with CO is also indicated (□). The in vitro CODH activity assay is described in the text. Each data point represents the average of two independent measurements. The nickel bound at 15 s was subtracted from the data.

FIG. 3

Nickel accumulation in CO-induced cooC, cooT, and cooJ mutant strains of R. rubrum. The cells were grown in malate ammonium medium lacking Ni²⁺ for 12 h with a gas phase of 20% CO–80% N₂ except as indicated. Symbols: ⧫, UR2; ■, UR2 without CO (negative control); ▴, nonpolar linker mutation in cooC (strain UR495); ○, nonpolar linker mutation in cooT (strain UR479); □, mutation in cooJ (strain UR500). All cultures except the negative control, which was not CO treated, had similar CODH activities when analyzed by the in vitro Ni²⁺ activation assay. The apo-CODH activation assay was performed on an aliquot of cell culture removed from the culture just prior to the ⁶³Ni²⁺ accumulation assay. The nickel bound at time zero was subtracted from the data.

FIG. 4

Kinetics of Ni²⁺ transport by R. rubrum. Cells grown in the absence of CO were harvested and resuspended in 100 mM MOPS (pH 7.5) and divided into 12 vials. ⁶³Ni (1.5 μM; 615 mCi/mmol) was added to each vial with the appropriate amount of unlabeled nickel to reach the indicated final nickel concentration. A sample was immediately removed and analyzed for the initial binding of nickel to the cells. Additional samples were removed at 60 and 120 s and then collected by vacuum filtration followed by three 5.0-ml washes of 10 mM EDTA in 100 mM Tris (pH 8.0). The curve represents the mean of data from three independent experiments. Cultures grown in the presence of CO or strains with cooCTJ mutationally inactivated and grown with or without CO were also tested for Ni²⁺ transport and produced curves with the same kinetic profiles within the error bars shown (data not shown).