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Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1.

Clark IC, Melnyk RA, Youngblut MD, Carlson HK, Iavarone AT, Coates JD - MBio (2015)

Bottom Line: One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons.To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1.To study this phenomenon, we engineered Shewanella oneidensis MR-1 into a chlorate reducer.

View Article: PubMed Central - PubMed

Affiliation: Department of Civil and Environmental Engineering, University of California, Berkeley, California, USA.

No MeSH data available.


Related in: MedlinePlus

Growth and analyte profiles for cells with different genotypes grown in chlorate alone, chlorate plus nitrate, and nitrate alone demonstrate that (A) nrfA is not required for chlorate turnover and that (B) pICC7 rescues chlorate inhibition of nitrite reduction.
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fig5: Growth and analyte profiles for cells with different genotypes grown in chlorate alone, chlorate plus nitrate, and nitrate alone demonstrate that (A) nrfA is not required for chlorate turnover and that (B) pICC7 rescues chlorate inhibition of nitrite reduction.

Mentions: To understand the role of nrfA during chlorate reduction, we tested the ability of the purified enzyme to facilitate chlorate reduction. Using reduced methyl viologen as an electron donor, NrfA was oxidized by 1 mM nitrite, but not chlorate (Fig. 4A). This was not due to inactivation of the enzyme, as nitrite could be subsequently reduced after the addition of chlorate. In addition, chlorate did not inhibit the initial rate of nitrite reduction, even when added at 100 mM concentrations (data not shown). To further confirm this result, we tested the ability of the ΔnrfA mutant carrying plasmid pICC7 to grow on chlorate. Deleting nrfA did not prevent small amounts of growth on chlorate when cld was present (Fig. 5A). We conclude that NrfA does not reduce chlorate. Using NrfA prereduced with methyl viologen and washed to remove the reductant, we monitored the enzyme’s UV-visible (UV-Vis) spectrum after a series of chlorite injections. NrfA was rapidly oxidized by chlorite (Fig. 4B). Reduction of chlorite by NrfA could explain why deletion of this enzyme provided a growth benefit during chlorate reduction: inadvertent reaction with chlorite results in even more toxic chlorine species. However, the exact mechanism of NrfA antagonism as well as the product of oxidation by chlorite remains to be determined.


Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1.

Clark IC, Melnyk RA, Youngblut MD, Carlson HK, Iavarone AT, Coates JD - MBio (2015)

Growth and analyte profiles for cells with different genotypes grown in chlorate alone, chlorate plus nitrate, and nitrate alone demonstrate that (A) nrfA is not required for chlorate turnover and that (B) pICC7 rescues chlorate inhibition of nitrite reduction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4442138&req=5

fig5: Growth and analyte profiles for cells with different genotypes grown in chlorate alone, chlorate plus nitrate, and nitrate alone demonstrate that (A) nrfA is not required for chlorate turnover and that (B) pICC7 rescues chlorate inhibition of nitrite reduction.
Mentions: To understand the role of nrfA during chlorate reduction, we tested the ability of the purified enzyme to facilitate chlorate reduction. Using reduced methyl viologen as an electron donor, NrfA was oxidized by 1 mM nitrite, but not chlorate (Fig. 4A). This was not due to inactivation of the enzyme, as nitrite could be subsequently reduced after the addition of chlorate. In addition, chlorate did not inhibit the initial rate of nitrite reduction, even when added at 100 mM concentrations (data not shown). To further confirm this result, we tested the ability of the ΔnrfA mutant carrying plasmid pICC7 to grow on chlorate. Deleting nrfA did not prevent small amounts of growth on chlorate when cld was present (Fig. 5A). We conclude that NrfA does not reduce chlorate. Using NrfA prereduced with methyl viologen and washed to remove the reductant, we monitored the enzyme’s UV-visible (UV-Vis) spectrum after a series of chlorite injections. NrfA was rapidly oxidized by chlorite (Fig. 4B). Reduction of chlorite by NrfA could explain why deletion of this enzyme provided a growth benefit during chlorate reduction: inadvertent reaction with chlorite results in even more toxic chlorine species. However, the exact mechanism of NrfA antagonism as well as the product of oxidation by chlorite remains to be determined.

Bottom Line: One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons.To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1.To study this phenomenon, we engineered Shewanella oneidensis MR-1 into a chlorate reducer.

View Article: PubMed Central - PubMed

Affiliation: Department of Civil and Environmental Engineering, University of California, Berkeley, California, USA.

No MeSH data available.


Related in: MedlinePlus