Limits...
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

Western blot analysis of Fe–S biosynthetic enzyme SufC after challenge with dipyridyl (DP) and paraquat (PQ). Cultures were grown to OD600 of 0.8–1.0 and challenged with stressors for 30 min. Cells were harvested, lysed. Clear extracts (50 μg of protein) were subjected to SDS-PAGE and further analyzed by western blot. All western blot assays were performed in triplicate of three independent growth experiments. A representative image from western blot containing two separate gels of wild type and ΔbshA exposed in the same film is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4554457&req=5

fig07: Western blot analysis of Fe–S biosynthetic enzyme SufC after challenge with dipyridyl (DP) and paraquat (PQ). Cultures were grown to OD600 of 0.8–1.0 and challenged with stressors for 30 min. Cells were harvested, lysed. Clear extracts (50 μg of protein) were subjected to SDS-PAGE and further analyzed by western blot. All western blot assays were performed in triplicate of three independent growth experiments. A representative image from western blot containing two separate gels of wild type and ΔbshA exposed in the same film is shown.

Mentions: In B. subtilis, the SUF system encoded by the sufCDSUB operon, is proposed to be the only Fe–S cluster biosynthesis machinery (Albrecht et al. 2010; Selbach et al. 2010; Boyd et al. 2014). The lower levels of Fe–S clusters, judged by the lower activity of Fe–S enzymes, could also be attributed to expression or inefficiency of biosynthetic apparatus in assembling Fe–S clusters in the absence of BSH and/or conditions detrimental to Fe–S metabolism. Therefore, the levels of two biosynthetic components, SufC and SufB, were determined by western blot analysis of lysates of cells cultured in MM. Expression levels of both SufC and SufB in wild type were higher than the mutant strain, whereas the internal control MnmA protein, proposed to participate in 2-thiouridine formation (Black and Dos Santos 2015), showed no significant change (Fig. S4). In E. coli, the SUF system is expressed only under oxidative stress or iron starvation to promote Fe–S biogenesis under these conditions (Outten et al. 2004). To verify the changes in expression levels of the B. subtilis SUF components under stress conditions, western blot analyses of SufC were conducted in cell lysates of MM cultures challenged with DP and PQ for 30 min. Overall, SufC protein levels were similar across various conditions in the wild-type strain (Fig.7). When the ΔbshA strain was challenged to various stresses, the levels of SufC increased to comparable levels to those observed in wild-type cultures (Fig.7). This result suggested that expression of sufC although constitutive can respond to iron starvation and oxidative stress, but only when BSH is absent.


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

Fang Z, Dos Santos PC - Microbiologyopen (2015)

Western blot analysis of Fe–S biosynthetic enzyme SufC after challenge with dipyridyl (DP) and paraquat (PQ). Cultures were grown to OD600 of 0.8–1.0 and challenged with stressors for 30 min. Cells were harvested, lysed. Clear extracts (50 μg of protein) were subjected to SDS-PAGE and further analyzed by western blot. All western blot assays were performed in triplicate of three independent growth experiments. A representative image from western blot containing two separate gels of wild type and ΔbshA exposed in the same film is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4554457&req=5

fig07: Western blot analysis of Fe–S biosynthetic enzyme SufC after challenge with dipyridyl (DP) and paraquat (PQ). Cultures were grown to OD600 of 0.8–1.0 and challenged with stressors for 30 min. Cells were harvested, lysed. Clear extracts (50 μg of protein) were subjected to SDS-PAGE and further analyzed by western blot. All western blot assays were performed in triplicate of three independent growth experiments. A representative image from western blot containing two separate gels of wild type and ΔbshA exposed in the same film is shown.
Mentions: In B. subtilis, the SUF system encoded by the sufCDSUB operon, is proposed to be the only Fe–S cluster biosynthesis machinery (Albrecht et al. 2010; Selbach et al. 2010; Boyd et al. 2014). The lower levels of Fe–S clusters, judged by the lower activity of Fe–S enzymes, could also be attributed to expression or inefficiency of biosynthetic apparatus in assembling Fe–S clusters in the absence of BSH and/or conditions detrimental to Fe–S metabolism. Therefore, the levels of two biosynthetic components, SufC and SufB, were determined by western blot analysis of lysates of cells cultured in MM. Expression levels of both SufC and SufB in wild type were higher than the mutant strain, whereas the internal control MnmA protein, proposed to participate in 2-thiouridine formation (Black and Dos Santos 2015), showed no significant change (Fig. S4). In E. coli, the SUF system is expressed only under oxidative stress or iron starvation to promote Fe–S biogenesis under these conditions (Outten et al. 2004). To verify the changes in expression levels of the B. subtilis SUF components under stress conditions, western blot analyses of SufC were conducted in cell lysates of MM cultures challenged with DP and PQ for 30 min. Overall, SufC protein levels were similar across various conditions in the wild-type strain (Fig.7). When the ΔbshA strain was challenged to various stresses, the levels of SufC increased to comparable levels to those observed in wild-type cultures (Fig.7). This result suggested that expression of sufC although constitutive can respond to iron starvation and oxidative stress, but only when BSH is absent.

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