RESUMEN
The aim of the present work was to engineer bacteria for the removal of Co in contaminated effluents. Radioactive cobalt ((60)Co) is known as a major contributor for person-sievert budgetary because of its long half-life and high γ-energy values. Some bacterial Ni/Co transporter (NiCoT) genes were described to have preferential uptake for cobalt. In this study, the NiCoT genes nxiA and nvoA from Rhodopseudomonas palustris CGA009 (RP) and Novosphingobium aromaticivorans F-199 (NA), respectively, were cloned under the control of the groESL promoter. These genes were expressed in Deinococcus radiodurans in reason of its high resistance to radiation as compared to other bacterial strains. Using qualitative real time-PCR, we showed that the expression of NiCoT-RP and NiCoT-NA is induced by cobalt and nickel. The functional expression of these genes in bioengineered D. radiodurans R1 strains resulted in >60 % removal of (60)Co (≥5.1 nM) within 90 min from simulated spent decontamination solution containing 8.5 nM of Co, even in the presence of >10 mM of Fe, Cr, and Ni. D. radiodurans R1 (DR-RP and DR-NA) showed superior survival to recombinant E. coli (ARY023) expressing NiCoT-RP and NA and efficiency in Co remediation up to 6.4 kGy. Thus, the present study reports a remarkable reduction in biomass requirements (2 kg) compared to previous studies using wild-type bacteria (50 kg) or ion-exchanger resins (8000 kg) for treatment of ~10(5)-l spent decontamination solutions (SDS).
Asunto(s)
Radioisótopos de Cobalto/metabolismo , Deinococcus/metabolismo , Contaminantes Ambientales/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Ingeniería Metabólica , Plantas de Energía Nuclear , Soluciones/metabolismo , Deinococcus/genética , Perfilación de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Viabilidad Microbiana , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodopseudomonas/enzimología , Rhodopseudomonas/genética , Sphingomonadaceae/enzimología , Sphingomonadaceae/genética , Activación Transcripcional/efectos de los fármacosRESUMEN
Our previous in silico studies identified a high-affinity nickel transporter, TNC, from the metal transportome of Neurospora crassa. A knockout mutant of the tnc gene in N. crassa failed to transport nickel, showed phenotypic growth defects and diminished urease activity under physiological levels of nickel. Transport assays conducted in wild type and knockout mutant strains showed that TNC transports nickel with high affinity but exhibits selectivity for other transition metal ions like cobalt. Heterologous complementation of Schizosaccharomyces pombe nickel uptake mutant by TNC further substantiates its nickel transport function. Transcriptional analysis of the nickel transporter encoding gene, tnc in N. crassa by qRT-PCR showed its constitutive expression in various phases of its life cycle. However, levels of the corresponding protein TNC were down-regulated only by increasing the nickel, but not cobalt concentration in the media. Immunolocalisation data suggested that TNC is distributed in the plasma membrane of N. crassa. Thus, the present study establishes TNC as a functional plasma membrane nickel transporter necessary for physiological acquisition of nickel in the multicellular fungi N. crassa.
