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Genome-enabled studies of anaerobic, nitrate-dependent iron oxidation in the chemolithoautotrophic bacterium Thiobacillus denitrificans.

Beller HR, Zhou P, Legler TC, Chakicherla A, Kane S, Letain TE, A O'Day P - Front Microbiol (2013)

Bottom Line: We previously identified two c-type cytochromes involved in nitrate-dependent U(IV) oxidation in T. denitrificans and hypothesized that c-type cytochromes would also catalyze Fe(II) oxidation, as they have been found to play this role in anaerobic phototrophic Fe(II)-oxidizing bacteria.A transposon mutant with a disrupted gene associated with NADH:ubiquinone oxidoreductase (complex I) was ~35% defective relative to the wild-type strain; this strain was similarly defective in nitrate reduction with thiosulfate as the electron donor.Overall, our results indicate that nitrate-dependent Fe(II) oxidation in T. denitrificans is not catalyzed by the same c-type cytochromes involved in U(IV) oxidation, nor have other c-type cytochromes yet been implicated in the process.

View Article: PubMed Central - PubMed

Affiliation: Earth Sciences Division, Lawrence Berkeley National Laboratory Berkeley, CA, USA.

ABSTRACT
Thiobacillus denitrificans is a chemolithoautotrophic bacterium capable of anaerobic, nitrate-dependent U(IV) and Fe(II) oxidation, both of which can strongly influence the long-term efficacy of in situ reductive immobilization of uranium in contaminated aquifers. We previously identified two c-type cytochromes involved in nitrate-dependent U(IV) oxidation in T. denitrificans and hypothesized that c-type cytochromes would also catalyze Fe(II) oxidation, as they have been found to play this role in anaerobic phototrophic Fe(II)-oxidizing bacteria. Here we report on efforts to identify genes associated with nitrate-dependent Fe(II) oxidation, namely (a) whole-genome transcriptional studies [using FeCO3, Fe(2) (+), and U(IV) oxides as electron donors under denitrifying conditions], (b) Fe(II) oxidation assays performed with knockout mutants targeting primarily highly expressed or upregulated c-type cytochromes, and (c) random transposon-mutagenesis studies with screening for Fe(II) oxidation. Assays of mutants for 26 target genes, most of which were c-type cytochromes, indicated that none of the mutants tested were significantly defective in nitrate-dependent Fe(II) oxidation. The non-defective mutants included the c 1-cytochrome subunit of the cytochrome bc 1 complex (complex III), which has relevance to a previously proposed role for this complex in nitrate-dependent Fe(II) oxidation and to current concepts of reverse electron transfer. A transposon mutant with a disrupted gene associated with NADH:ubiquinone oxidoreductase (complex I) was ~35% defective relative to the wild-type strain; this strain was similarly defective in nitrate reduction with thiosulfate as the electron donor. Overall, our results indicate that nitrate-dependent Fe(II) oxidation in T. denitrificans is not catalyzed by the same c-type cytochromes involved in U(IV) oxidation, nor have other c-type cytochromes yet been implicated in the process.

No MeSH data available.


Related in: MedlinePlus

Volcano plots of log10 odds of differential expression versus log2 fold differential expression for all T. denitrificans genes under nitrate-dependent UO2-oxidizing (A), FeCO3-oxidizing (B), and Fe2+-oxidizing (C) conditions (Groups VI, V, and II, respectively; Table 1) relative to nitrate-dependent thiosulfate-oxidizing conditions (Group VII). A highly upregulated group of 16 genes is highlighted in red (see text). Two of the most highly upregulated genes are indicated with blue arrows (Tbd_2628) and green arrows (Tbd_1948).
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Figure 4: Volcano plots of log10 odds of differential expression versus log2 fold differential expression for all T. denitrificans genes under nitrate-dependent UO2-oxidizing (A), FeCO3-oxidizing (B), and Fe2+-oxidizing (C) conditions (Groups VI, V, and II, respectively; Table 1) relative to nitrate-dependent thiosulfate-oxidizing conditions (Group VII). A highly upregulated group of 16 genes is highlighted in red (see text). Two of the most highly upregulated genes are indicated with blue arrows (Tbd_2628) and green arrows (Tbd_1948).

Mentions: The search for enzyme candidates for nitrate-dependent Fe(II) oxidation extended beyond c-type cytochromes to any genes that were highly upregulated under conditions of interest. Data analysis revealed that a common group of genes was upregulated under nitrate-dependent FeCO3-, Fe2+-, and UO2-oxidizing conditions (Groups V, II, and VI, respectively; Table 1) relative to nitrate-dependent thiosulfate-oxidizing conditions (Group VII). Of the top 25 upregulated genes under each of the three conditions of interest (i.e., FeCO3-, Fe2+-, and UO2-oxidizing), a group of 16 genes belonged to all three top-25 groups. These 16 genes are shown in red in Figure 4, a volcano plot, which graphs log10 odds of differential expression vs. log2 fold differential expression for all 2,832 ORFs identified in the draft genome at the time of microarray design (the finished genome is annotated to have 2,827 ORFs; Beller et al., 2006a). Upregulation under FeCO3-, Fe2+-, and UO2-oxidizing conditions is plotted in the gray regions of Figures 4A– C, and the ORFs most highly upregulated relative to thiosulfate-oxidizing conditions are plotted furthest to the right. The group of 16 genes clearly contains many of the most highly upregulated genes under all three conditions, ranging from 5- to 39-fold upregulation and averaging 13-fold upregulation relative to thiosulfate-oxidizing conditions.


Genome-enabled studies of anaerobic, nitrate-dependent iron oxidation in the chemolithoautotrophic bacterium Thiobacillus denitrificans.

Beller HR, Zhou P, Legler TC, Chakicherla A, Kane S, Letain TE, A O'Day P - Front Microbiol (2013)

Volcano plots of log10 odds of differential expression versus log2 fold differential expression for all T. denitrificans genes under nitrate-dependent UO2-oxidizing (A), FeCO3-oxidizing (B), and Fe2+-oxidizing (C) conditions (Groups VI, V, and II, respectively; Table 1) relative to nitrate-dependent thiosulfate-oxidizing conditions (Group VII). A highly upregulated group of 16 genes is highlighted in red (see text). Two of the most highly upregulated genes are indicated with blue arrows (Tbd_2628) and green arrows (Tbd_1948).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3753534&req=5

Figure 4: Volcano plots of log10 odds of differential expression versus log2 fold differential expression for all T. denitrificans genes under nitrate-dependent UO2-oxidizing (A), FeCO3-oxidizing (B), and Fe2+-oxidizing (C) conditions (Groups VI, V, and II, respectively; Table 1) relative to nitrate-dependent thiosulfate-oxidizing conditions (Group VII). A highly upregulated group of 16 genes is highlighted in red (see text). Two of the most highly upregulated genes are indicated with blue arrows (Tbd_2628) and green arrows (Tbd_1948).
Mentions: The search for enzyme candidates for nitrate-dependent Fe(II) oxidation extended beyond c-type cytochromes to any genes that were highly upregulated under conditions of interest. Data analysis revealed that a common group of genes was upregulated under nitrate-dependent FeCO3-, Fe2+-, and UO2-oxidizing conditions (Groups V, II, and VI, respectively; Table 1) relative to nitrate-dependent thiosulfate-oxidizing conditions (Group VII). Of the top 25 upregulated genes under each of the three conditions of interest (i.e., FeCO3-, Fe2+-, and UO2-oxidizing), a group of 16 genes belonged to all three top-25 groups. These 16 genes are shown in red in Figure 4, a volcano plot, which graphs log10 odds of differential expression vs. log2 fold differential expression for all 2,832 ORFs identified in the draft genome at the time of microarray design (the finished genome is annotated to have 2,827 ORFs; Beller et al., 2006a). Upregulation under FeCO3-, Fe2+-, and UO2-oxidizing conditions is plotted in the gray regions of Figures 4A– C, and the ORFs most highly upregulated relative to thiosulfate-oxidizing conditions are plotted furthest to the right. The group of 16 genes clearly contains many of the most highly upregulated genes under all three conditions, ranging from 5- to 39-fold upregulation and averaging 13-fold upregulation relative to thiosulfate-oxidizing conditions.

Bottom Line: We previously identified two c-type cytochromes involved in nitrate-dependent U(IV) oxidation in T. denitrificans and hypothesized that c-type cytochromes would also catalyze Fe(II) oxidation, as they have been found to play this role in anaerobic phototrophic Fe(II)-oxidizing bacteria.A transposon mutant with a disrupted gene associated with NADH:ubiquinone oxidoreductase (complex I) was ~35% defective relative to the wild-type strain; this strain was similarly defective in nitrate reduction with thiosulfate as the electron donor.Overall, our results indicate that nitrate-dependent Fe(II) oxidation in T. denitrificans is not catalyzed by the same c-type cytochromes involved in U(IV) oxidation, nor have other c-type cytochromes yet been implicated in the process.

View Article: PubMed Central - PubMed

Affiliation: Earth Sciences Division, Lawrence Berkeley National Laboratory Berkeley, CA, USA.

ABSTRACT
Thiobacillus denitrificans is a chemolithoautotrophic bacterium capable of anaerobic, nitrate-dependent U(IV) and Fe(II) oxidation, both of which can strongly influence the long-term efficacy of in situ reductive immobilization of uranium in contaminated aquifers. We previously identified two c-type cytochromes involved in nitrate-dependent U(IV) oxidation in T. denitrificans and hypothesized that c-type cytochromes would also catalyze Fe(II) oxidation, as they have been found to play this role in anaerobic phototrophic Fe(II)-oxidizing bacteria. Here we report on efforts to identify genes associated with nitrate-dependent Fe(II) oxidation, namely (a) whole-genome transcriptional studies [using FeCO3, Fe(2) (+), and U(IV) oxides as electron donors under denitrifying conditions], (b) Fe(II) oxidation assays performed with knockout mutants targeting primarily highly expressed or upregulated c-type cytochromes, and (c) random transposon-mutagenesis studies with screening for Fe(II) oxidation. Assays of mutants for 26 target genes, most of which were c-type cytochromes, indicated that none of the mutants tested were significantly defective in nitrate-dependent Fe(II) oxidation. The non-defective mutants included the c 1-cytochrome subunit of the cytochrome bc 1 complex (complex III), which has relevance to a previously proposed role for this complex in nitrate-dependent Fe(II) oxidation and to current concepts of reverse electron transfer. A transposon mutant with a disrupted gene associated with NADH:ubiquinone oxidoreductase (complex I) was ~35% defective relative to the wild-type strain; this strain was similarly defective in nitrate reduction with thiosulfate as the electron donor. Overall, our results indicate that nitrate-dependent Fe(II) oxidation in T. denitrificans is not catalyzed by the same c-type cytochromes involved in U(IV) oxidation, nor have other c-type cytochromes yet been implicated in the process.

No MeSH data available.


Related in: MedlinePlus