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Ochrobactrum tritici  SCII24 T  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  SCII24 T , 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.

Hyper Accumulation of Arsenic in Mutants of Ochrobactrum tritici Silenced for Arsenite Efflux Pumps