Hyper Accumulation of Arsenic in Mutants of Ochrobactrum tritici Silenced for Arsenite Efflux Pumps.
Bottom Line: Therefore, arsB is the main gene responsible for arsenite resistance in O. tritici.However, both genes arsB and Acr3_1 play a crucial role in the resistance mechanism, depending on the arsenite concentration in the medium.In conclusion, at moderate arsenite concentrations, the double arsB- and Acr3_1-mutant exhibited a great ability to accumulate arsenite and can be seen as a promising bioremediation tool for environmental arsenic detoxification.
Affiliation: IMAR-CMA, Coimbra, Portugal.
Ochrobactrum tritici SCII24T is a highly As-resistant bacterium, with two previously described arsenic resistance operons, ars1 and ars2. Among a large number of genes, these operons contain the arsB and Acr3 genes that encode the arsenite efflux pumps responsible for arsenic resistance. Exploring the genome of O. tritici SCII24T, an additional putative operon (ars3) was identified and revealed the presence of the Acr3_2 gene that encodes for an arsenite efflux protein but which came to prove to not be required for full As resistance. The genes encoding for arsenite efflux pumps, identified in this strain, were inactivated to develop microbial accumulators of arsenic as new tools for bioremediation. Six different mutants were produced, studied and three were more useful as biotools. O. tritici wild type and the Acr3-mutants showed the highest resistance to As(III), being able to grow up to 50 mM of arsenite. On the other hand, arsB-mutants were not able to grow at concentrations higher than 1 mM As(III), and were the most As(III) sensitive mutants. In the presence of 1 mM As(III), the strain with arsB and Acr3_1 mutated showed the highest intracellular arsenic concentration (up to 17 ng(As)/mg protein), while in assays with 5 mM As(III), the single arsB-mutant was able to accumulate the highest concentration of arsenic (up to 10 ng(As)/mg protein). Therefore, arsB is the main gene responsible for arsenite resistance in O. tritici. However, both genes arsB and Acr3_1 play a crucial role in the resistance mechanism, depending on the arsenite concentration in the medium. In conclusion, at moderate arsenite concentrations, the double arsB- and Acr3_1-mutant exhibited a great ability to accumulate arsenite and can be seen as a promising bioremediation tool for environmental arsenic detoxification.
No MeSH data available.
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Mentions: The capacity of mutated and non-mutated strains to accumulate arsenic was evaluated by exposing them to 1 mM or 5 mM of As(III) for 3 hours (Fig 4). These assays were only performed with the wild-type O. tritici SCII24T, the single arsB and Acr3_1 mutants and the double arsB/Acr3_1 mutant, because the results obtained from the arsenite resistance assays indicated no function for Acr3_2 gene. In the presence of low arsenite concentrations (1 mM) the double arsB/Acr3_1 mutant was able to accumulate up to 17 ng(As)/ng protein, while the wild-type O. tritici and the single arsB or Acr3_1 mutants accumulated only about 2 ng(As)/mg protein. After the accumulation tests, all cells showed viability. However, in assays with higher concentrations of arsenite (5 mM), arsB mutant accumulated up to 10 ng(As)/mg protein. Double arsB/Acr3_1 mutant was not tested in this condition since this mutant could not resist concentrations above 1 mM As(III). Both wild-type O. tritici and the double arsB/Acr3_1 mutant did not change their cell morphology at low arsenite concentrations (Fig 5). Moreover, the higher capacity of this double mutant to accumulate arsenic was visible in the elemental map distributions (small inserts in Fig 5) where the density of the As-specific signal was higher.
No MeSH data available.