HupF contributes to HupL stability under elevated oxygen tensions The existence of hupF in hydrogenase systems from bacteria synthesizing this enzyme in the presence of oxygen prompted us to study the potential role of this protein in protection against oxygen. To this aim, we analyzed the possible effect of HupF on the status of hydrogenase large subunit in cultures maintained under different

oxygen tensions (1% and 3%). The higher oxygen tension (3%) still allowed the expression of hydrogenase in R. leguminosarum wild-type strain, although at a reduced level (40% of the level Pinometostat chemical structure induced under 1% O2, Table  2). The presence

and processing status of the hydrogenase large subunit (HupL) were analyzed in crude cell extracts from microaerobic cultures through immunoblot (Figure  2). In these experiments MLN2238 supplier https://www.selleckchem.com/products/Cyclopamine.html we found that the wild-type cells contained a clear band associated to the mature form of HupL, irrespective of whether cells were induced under 1% or 3% oxygen (Figure  2A and 2B, upper panel). This band was absent in a ΔhupL mutant used as negative control (Figure  2A). Analysis of the cell extracts from the ΔhupF strain grown at 1% oxygen revealed the presence of HupL, although in the unprocessed form (Figure  2A, upper panel). Interestingly, HupL was not detected when cultures from the same mutant

strain were incubated under 3% O2 (Figure  2B). In contrast, extracts from a R. leguminosarum mutant lacking HypC, used as a hydrogenase non-processing control, showed a clear band of unprocessed HupL after exposure to both 1% and 3% oxygen tension (Figure  2A and 2B). Similar levels of an immunoreactive band corresponding to HypB were detected in all the extracts (Figure  2, lower panels), indicating that the microaerobic induction of Hup expression was equally effective for all strains in each treatment. These data suggest that, Pazopanib in vivo in the presence of 3% oxygen, HupL is either unstable or not synthesized in the absence of HupF. In order to further evaluate these possibilities, we analyzed the in vivo stability of HupL as a function of the presence/absence of HupF. To address this question, we first induced R. leguminosarum cultures for hydrogenase expression under 1% oxygen, and then the induced cells, carrying either processed HupL (wild-type strain) or unprocessed HupL (ΔhupF and ΔhypC mutants), were exposed to atmospheres containing either 1% O2 or 21% O2 for up to 3 hours. After such treatments, the amount and processing status of HupL was determined through immunoblot assay in cell extracts (Figure  3A).