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.
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Mentions: To functionally characterize the interplay between the products of the resistance determinants, mutated and non-mutated strains were tested for arsenite resistance. Bacterial growth was evaluated in solid medium LB with As(III) concentrations ranging from 0 mM to 50 mM (Fig 2). O. tritici SCII24T and Acr3_1 or Acr3_2 mutants were able to grow up to 50 mM of arsenite. However, they showed a partial inhibition at very high concentrations of As(III) (i.e. > 20 mM). On the other hand, arsB and arsB/Acr3_2 mutants exhibited lower resistance capacity than the wild strain, since they were unable to grow at As(III) concentrations above 5 mM. Moreover, at low As(III) concentrations (< 5 mM) these mutants showed lower survival rates than the wild-type strain. The double arsB/Acr3_1 or triple arsB/Acr3_1/Acr3_2 mutants were not able to grow above 1 mM As(III), being the most arsenite sensitive mutants obtained. Differences in As(III) sensitivity between wild-type O. tritici SCII24T and mutants were also evaluated in Petri dishes, by growing strains in solid LB medium and by using a filter disk assay (Fig 3). The comparison of the inhibition halos revealed that arsB/Acr3_1 or arsB/Acr3_1/Acr3_2 mutants were the most As(III) susceptible strains.. Single arsB and double arsB/Acr3_2 mutants also showed As(III) sensibility, particularly in the presence of discs saturated with concentrations higher than 250 mM, although not as evident as for the arsB/Acr3_1 or arsB/Acr3_1/Acr3_2 mutants.
No MeSH data available.