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

Sousa T, Branco R, Piedade AP, Morais PV - PLoS ONE (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: IMAR-CMA, Coimbra, Portugal.

ABSTRACT
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.


Related in: MedlinePlus

Genetic organizations of the three ars operons present in strain Ochrobactrum tritici SCII24T.Gene orientations are shown by arrows. Pattern field arrows are representative of putative genes with unknown functionality. Mutation sites are indicated by inverted triangles. Gmr and Hygr mean the insertion of gentamycin and hygromycin resistance cassettes, respectively and X means deletion of partial gene sequence.
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pone.0131317.g001: Genetic organizations of the three ars operons present in strain Ochrobactrum tritici SCII24T.Gene orientations are shown by arrows. Pattern field arrows are representative of putative genes with unknown functionality. Mutation sites are indicated by inverted triangles. Gmr and Hygr mean the insertion of gentamycin and hygromycin resistance cassettes, respectively and X means deletion of partial gene sequence.

Mentions: The genome of O. tritici SCII24T was sequenced using Illumina. Annotation of the genes confirmed the presence of the arsenic resistance genes arsB and Acr3_1 already identified in this strain, included in two different chromosomally located arsenic resistance operons [11]. Besides these two arsenic efflux systems, it was also possible to identify a third possible operon probably involved in arsenic resistance that was named putative operon ars3 (Fig 1). This one, detected in a contig carrying ParA, IncI, IncF and repC genes, comprised nine open reading frames (ORFs) and five of them showed high homology with genes already found in arsenic resistance determinants and is probably plasmid located. The first gene of this genetic region, arsC3, encoded a predicted protein of 176 aa with 98% identity and 96% similarity to an ArsR from Agrobacterium tumefaciens (WP_020810063), and also with 84% identity and 94% similarity to an ArsC from A. tumefaciens (CDN92172). The second gene, asrC4, encoded a protein of 145 aa with 96% identity and 96% similarity to an ArsC from A. tumefaciens (WP_020810062). The next gene, Acr3_2, encoded a protein of 346 aa with 95% identity and 96% similarity to an arsenic resistance protein ArsB from A. tumefaciens (WP_020810061). Downstream, the gene arsH2 was found, encoding a protein of 236 aa with 94% identity and 97% similarity to a NADPH-dependent FMN reductase from A. tumefaciens (WP_020810058). The last gene of the putative operon ars3 was arsR4, which encoded a protein of 120 aa with 91% identity and 94% similarity to an ArsR from A. tumefaciens (WP_020810055). Alignment of these new genes with those from operons ars1 and ars2 revealed that the currently identified arsR4 showed higher homology with arsR3 (70% similarity) than with the other previously identified regulators of the strain. Also, arsC3 was more similar to arsC1 (76% similarity) while arsC4 was more similar to arsC2. On the other hand, arsH2 was very similar to arsH1, exhibiting 72% of homology. Therefore, the putative operon ars3 showed an arrangement very similar to the operon ars2.


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

Sousa T, Branco R, Piedade AP, Morais PV - PLoS ONE (2015)

Genetic organizations of the three ars operons present in strain Ochrobactrum tritici SCII24T.Gene orientations are shown by arrows. Pattern field arrows are representative of putative genes with unknown functionality. Mutation sites are indicated by inverted triangles. Gmr and Hygr mean the insertion of gentamycin and hygromycin resistance cassettes, respectively and X means deletion of partial gene sequence.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4489015&req=5

pone.0131317.g001: Genetic organizations of the three ars operons present in strain Ochrobactrum tritici SCII24T.Gene orientations are shown by arrows. Pattern field arrows are representative of putative genes with unknown functionality. Mutation sites are indicated by inverted triangles. Gmr and Hygr mean the insertion of gentamycin and hygromycin resistance cassettes, respectively and X means deletion of partial gene sequence.
Mentions: The genome of O. tritici SCII24T was sequenced using Illumina. Annotation of the genes confirmed the presence of the arsenic resistance genes arsB and Acr3_1 already identified in this strain, included in two different chromosomally located arsenic resistance operons [11]. Besides these two arsenic efflux systems, it was also possible to identify a third possible operon probably involved in arsenic resistance that was named putative operon ars3 (Fig 1). This one, detected in a contig carrying ParA, IncI, IncF and repC genes, comprised nine open reading frames (ORFs) and five of them showed high homology with genes already found in arsenic resistance determinants and is probably plasmid located. The first gene of this genetic region, arsC3, encoded a predicted protein of 176 aa with 98% identity and 96% similarity to an ArsR from Agrobacterium tumefaciens (WP_020810063), and also with 84% identity and 94% similarity to an ArsC from A. tumefaciens (CDN92172). The second gene, asrC4, encoded a protein of 145 aa with 96% identity and 96% similarity to an ArsC from A. tumefaciens (WP_020810062). The next gene, Acr3_2, encoded a protein of 346 aa with 95% identity and 96% similarity to an arsenic resistance protein ArsB from A. tumefaciens (WP_020810061). Downstream, the gene arsH2 was found, encoding a protein of 236 aa with 94% identity and 97% similarity to a NADPH-dependent FMN reductase from A. tumefaciens (WP_020810058). The last gene of the putative operon ars3 was arsR4, which encoded a protein of 120 aa with 91% identity and 94% similarity to an ArsR from A. tumefaciens (WP_020810055). Alignment of these new genes with those from operons ars1 and ars2 revealed that the currently identified arsR4 showed higher homology with arsR3 (70% similarity) than with the other previously identified regulators of the strain. Also, arsC3 was more similar to arsC1 (76% similarity) while arsC4 was more similar to arsC2. On the other hand, arsH2 was very similar to arsH1, exhibiting 72% of homology. Therefore, the putative operon ars3 showed an arrangement very similar to the operon ars2.

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.

View Article: PubMed Central - PubMed

Affiliation: IMAR-CMA, Coimbra, Portugal.

ABSTRACT
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.


Related in: MedlinePlus