Ni2+ Transport and Accumulation in Rhodospirillum rubrum

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FONTE

American Society for Microbiology

RESUMO

The cooCTJ gene products are coexpressed with CO-dehydrogenase (CODH) and facilitate in vivo nickel insertion into CODH. A Ni2+ transport assay was used to monitor uptake and accumulation of 63Ni2+ into R. rubrum and to observe the effect of mutations in the cooC, cooT, and cooJ genes on 63Ni2+ transport and accumulation. Cells grown either in the presence or absence of CO transported Ni2+ with a Km of 19 ± 4 μM and a Vmax 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 Ni2+ 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 Mn2+, Mg2+, Ca2+, and Zn2+ showed no inhibition of 63Ni2+ transport but Co2+, Cd2+, and Cu2+ inhibited transport 35, 58, and 66%, respectively. Nickel transport was inhibited by cold (50% at 4°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 Ni2+ transport. 63Ni accumulation after 2 h was four times greater in CO-induced cells than in cells not exposed to CO. The CO-stimulated 63Ni2+ accumulation coincided with the appearance of CODH activity in the culture, suggesting that the 63Ni2+ was accumulating in CODH. The cooC, cooT, and cooJ genes are required for the increased 63Ni2+ accumulation observed upon CO exposure because cells containing mutations disrupting any or all of these genes accumulated 63Ni2+ like cells unexposed to CO.

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