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Protective role of bacillithiol in superoxide stress and Fe-S metabolism in Bacillus subtilis.

Fang Z, Dos Santos PC - Microbiologyopen (2015)

Bottom Line: Interestingly, Fe-S cluster containing isopropylmalate isomerase (LeuCD) and glutamate synthase (GOGAT) showed decreased activities in BSH strain.Deficiency of BSH also resulted in decreased levels of intracellular Fe accompanied by increased levels of manganese and altered expression levels of Fe-S cluster biosynthetic SUF components.Together, this study is the first to establish a link between BSH and Fe-S metabolism in B. subtilis.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina, 27016.

No MeSH data available.


Related in: MedlinePlus

Lack of BSH leads to lower activity levels of Fe–S enzymes. (A) Activities of the Fe–S proteins glutamate synthase (GOGAT), aconitase (ACN), isopropyl malate isomerase (LeuCD) and the non-Fe–S protein malate dehydrogenase (MDH) in cell lysates of Bacillus subtilis wild type (black) and ΔbshA strain (gray) cultured in MM (filled bar) or MM containing an additional 50 μmol/L of Fe2+ (diagonal striped bar). (B) Bacillus subtilis wild-type strain (black) and ΔbshA strain (gray) cultured MM and the activity of Fe–S enzymes was quantified before (filled bar) and after the addition of 50 μmol/L of Fe2+ to cell lysates (upright striped bar). The activities of GOGAT, ACN, LeuCD, and MDH for wild-type B. subtilis were measured to be 40, 131, 17, and 1051 (nmol/min per mg total protein in crude extract), respectively. The activities of the ΔbshA strain were normalized to the activities of wild type in MM. All assays were performed in triplicates. The statistical analysis was performed using unpaired t test, P values were obtained by comparing the ΔbshA without iron to wild-type w/o Fe and to ΔbshA with Fe. Comparisons of wild-type activities with and without Fe in both panels were not statistical significant. (NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001).
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fig02: Lack of BSH leads to lower activity levels of Fe–S enzymes. (A) Activities of the Fe–S proteins glutamate synthase (GOGAT), aconitase (ACN), isopropyl malate isomerase (LeuCD) and the non-Fe–S protein malate dehydrogenase (MDH) in cell lysates of Bacillus subtilis wild type (black) and ΔbshA strain (gray) cultured in MM (filled bar) or MM containing an additional 50 μmol/L of Fe2+ (diagonal striped bar). (B) Bacillus subtilis wild-type strain (black) and ΔbshA strain (gray) cultured MM and the activity of Fe–S enzymes was quantified before (filled bar) and after the addition of 50 μmol/L of Fe2+ to cell lysates (upright striped bar). The activities of GOGAT, ACN, LeuCD, and MDH for wild-type B. subtilis were measured to be 40, 131, 17, and 1051 (nmol/min per mg total protein in crude extract), respectively. The activities of the ΔbshA strain were normalized to the activities of wild type in MM. All assays were performed in triplicates. The statistical analysis was performed using unpaired t test, P values were obtained by comparing the ΔbshA without iron to wild-type w/o Fe and to ΔbshA with Fe. Comparisons of wild-type activities with and without Fe in both panels were not statistical significant. (NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001).

Mentions: Initial growth experiments suggested the involvement of BSH in the synthesis of selected amino acids (leucine, isoleucine, glutamine, and glutamate). The biosynthetic pathways of these amino acids include Fe–S enzymes. Glutamine oxoglutarate aminotransferase (GOGAT) containing two 4Fe–4S clusters and one 3Fe–4S cluster catalyzes the reversible conversion from l-glutamine and α-ketoglutarate to l-glutamate. LeuCD, a dihydroxy-acid dehydratase in the biosynthesis of l-isoleucine, requires [4Fe–4S] cluster for its function. Likewise, the 4Fe–4S enzyme ACN in TCA cycle also indirectly contributes to synthesis of amino acids. The E. coli GSH strain displayed a decrease in ACN activity (80% of WT levels) under standard growth conditions (Gardner and Fridovich 1993a). Therefore, we hypothesized that the growth defects of the ΔbshA strain were caused by decreased Fe–S enzyme activities. To test this hypothesis, the specific activities of GOGAT, LeuCD, and ACN were determined in cell lysates from cultures harvested at mid-log phase (OD600 of 0.8–1.0 when using 1 cm cuvette). The activ-ity levels of these enzymes in the wild-type cell lysates were comparable to the previously reported activities (Bohannon and Sonenshein 1989; Alen and Sonenshein 1999), and modestly reduced in the strain lacking BSH (Fig.2A). Bacillus subtilis genome encodes for one copy of ACN which shows 51% sequence identity of the E. coli AcnA, which is the oxygen-resistant isoform of ACN. As a control, the activity of MDH, a non-Fe/S enzyme, was not significantly different in either strain. Supplementation of growth cultures with 50 μmol/L Fe2+, a condition identified to suppress BSH− phenotype in MM, led to an increase in GOGAT, ACN, and LeuCD activities in ΔbshA strain by 28%, 17%, and 16%, respectively, whereas causing no effect to the levels detected for the wild-type cell lysates (Fig.2A). Similar results were also reported in Saccharomyces cerevisiae Δgsh1 strain, in which the growth defect from deletion of GSH was also repaired upon addition of Fe3+ in the medium, and the LeuCD activity was also enhanced after 1-h exposure to Fe3+ (Kumar et al. 2011). In B. subtilis cultures, however, posthumous ex vivo addition of 50 μmol/L of Fe2+ in the cell lysates did not improved the activity levels of GOGAT, ACN, and LeuCD in both the wild-type and ΔbshA strains (Fig.2B).


Protective role of bacillithiol in superoxide stress and Fe-S metabolism in Bacillus subtilis.

Fang Z, Dos Santos PC - Microbiologyopen (2015)

Lack of BSH leads to lower activity levels of Fe–S enzymes. (A) Activities of the Fe–S proteins glutamate synthase (GOGAT), aconitase (ACN), isopropyl malate isomerase (LeuCD) and the non-Fe–S protein malate dehydrogenase (MDH) in cell lysates of Bacillus subtilis wild type (black) and ΔbshA strain (gray) cultured in MM (filled bar) or MM containing an additional 50 μmol/L of Fe2+ (diagonal striped bar). (B) Bacillus subtilis wild-type strain (black) and ΔbshA strain (gray) cultured MM and the activity of Fe–S enzymes was quantified before (filled bar) and after the addition of 50 μmol/L of Fe2+ to cell lysates (upright striped bar). The activities of GOGAT, ACN, LeuCD, and MDH for wild-type B. subtilis were measured to be 40, 131, 17, and 1051 (nmol/min per mg total protein in crude extract), respectively. The activities of the ΔbshA strain were normalized to the activities of wild type in MM. All assays were performed in triplicates. The statistical analysis was performed using unpaired t test, P values were obtained by comparing the ΔbshA without iron to wild-type w/o Fe and to ΔbshA with Fe. Comparisons of wild-type activities with and without Fe in both panels were not statistical significant. (NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001).
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Related In: Results  -  Collection

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fig02: Lack of BSH leads to lower activity levels of Fe–S enzymes. (A) Activities of the Fe–S proteins glutamate synthase (GOGAT), aconitase (ACN), isopropyl malate isomerase (LeuCD) and the non-Fe–S protein malate dehydrogenase (MDH) in cell lysates of Bacillus subtilis wild type (black) and ΔbshA strain (gray) cultured in MM (filled bar) or MM containing an additional 50 μmol/L of Fe2+ (diagonal striped bar). (B) Bacillus subtilis wild-type strain (black) and ΔbshA strain (gray) cultured MM and the activity of Fe–S enzymes was quantified before (filled bar) and after the addition of 50 μmol/L of Fe2+ to cell lysates (upright striped bar). The activities of GOGAT, ACN, LeuCD, and MDH for wild-type B. subtilis were measured to be 40, 131, 17, and 1051 (nmol/min per mg total protein in crude extract), respectively. The activities of the ΔbshA strain were normalized to the activities of wild type in MM. All assays were performed in triplicates. The statistical analysis was performed using unpaired t test, P values were obtained by comparing the ΔbshA without iron to wild-type w/o Fe and to ΔbshA with Fe. Comparisons of wild-type activities with and without Fe in both panels were not statistical significant. (NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001).
Mentions: Initial growth experiments suggested the involvement of BSH in the synthesis of selected amino acids (leucine, isoleucine, glutamine, and glutamate). The biosynthetic pathways of these amino acids include Fe–S enzymes. Glutamine oxoglutarate aminotransferase (GOGAT) containing two 4Fe–4S clusters and one 3Fe–4S cluster catalyzes the reversible conversion from l-glutamine and α-ketoglutarate to l-glutamate. LeuCD, a dihydroxy-acid dehydratase in the biosynthesis of l-isoleucine, requires [4Fe–4S] cluster for its function. Likewise, the 4Fe–4S enzyme ACN in TCA cycle also indirectly contributes to synthesis of amino acids. The E. coli GSH strain displayed a decrease in ACN activity (80% of WT levels) under standard growth conditions (Gardner and Fridovich 1993a). Therefore, we hypothesized that the growth defects of the ΔbshA strain were caused by decreased Fe–S enzyme activities. To test this hypothesis, the specific activities of GOGAT, LeuCD, and ACN were determined in cell lysates from cultures harvested at mid-log phase (OD600 of 0.8–1.0 when using 1 cm cuvette). The activ-ity levels of these enzymes in the wild-type cell lysates were comparable to the previously reported activities (Bohannon and Sonenshein 1989; Alen and Sonenshein 1999), and modestly reduced in the strain lacking BSH (Fig.2A). Bacillus subtilis genome encodes for one copy of ACN which shows 51% sequence identity of the E. coli AcnA, which is the oxygen-resistant isoform of ACN. As a control, the activity of MDH, a non-Fe/S enzyme, was not significantly different in either strain. Supplementation of growth cultures with 50 μmol/L Fe2+, a condition identified to suppress BSH− phenotype in MM, led to an increase in GOGAT, ACN, and LeuCD activities in ΔbshA strain by 28%, 17%, and 16%, respectively, whereas causing no effect to the levels detected for the wild-type cell lysates (Fig.2A). Similar results were also reported in Saccharomyces cerevisiae Δgsh1 strain, in which the growth defect from deletion of GSH was also repaired upon addition of Fe3+ in the medium, and the LeuCD activity was also enhanced after 1-h exposure to Fe3+ (Kumar et al. 2011). In B. subtilis cultures, however, posthumous ex vivo addition of 50 μmol/L of Fe2+ in the cell lysates did not improved the activity levels of GOGAT, ACN, and LeuCD in both the wild-type and ΔbshA strains (Fig.2B).

Bottom Line: Interestingly, Fe-S cluster containing isopropylmalate isomerase (LeuCD) and glutamate synthase (GOGAT) showed decreased activities in BSH strain.Deficiency of BSH also resulted in decreased levels of intracellular Fe accompanied by increased levels of manganese and altered expression levels of Fe-S cluster biosynthetic SUF components.Together, this study is the first to establish a link between BSH and Fe-S metabolism in B. subtilis.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina, 27016.

No MeSH data available.


Related in: MedlinePlus