UbK is Involved in the Resistance of Bacillus Subtilis to Oxidative Stress.

The objective of this study was to assess the role of UbK, a novel protein kinase, in the growth of Bacillus subtilis, especially under oxidative stress conditions. Growth profiles of wild-type and ΔubK mutant strains were assessed in the presence of paraquat, an in vivo inducer of oxidative stress. Wild-type B. subtilis cells were able to efficiently survive the stress conditions, whereas the growth profile of the ΔubK mutant strain was significantly affected. Complementation of the ΔubK mutant with a plasmid coding for a wild-type UbK restored wild-type growth phenotypes. Furthermore, we used recombinant plasmids containing the genes of the active kinase (UbK) and its inactive form (E106AUbK) to transform wild-type and ΔubK mutant strains. Our results showed that an active form of UbK is needed to restore a normal growth profile. Protein kinases allow a fine-tuning of cellular processes, including those related to metabolic adaptation to environmental cues. Our findings highlight the importance of an active UbK in the bacterial growth under oxidative stress in B. subtilis. This study revealed the role of a new protein kinase, UbK, allowing B. subtilis to survive oxidative stress.

Expanding the Kinome World: A New Protein Kinase Family Widely Conserved in Bacteria.

Fine tuning of signaling pathways is essential for cells to cope with sudden environmental variations. This delicate balance is maintained in particular by protein kinases that control the activity of target proteins by reversible phosphorylation. In addition to homologous eukaryotic enzymes, bacteria have evolved some specific Ser/Thr/Tyr protein kinases without any structural resemblance to their eukaryotic counterparts. Here, we show that a previously identified family of ATPases, broadly conserved among bacteria, is in fact a new family of protein kinases with a Ser/Thr/Tyr kinase activity. A prototypic member of this family, YdiB from Bacillus subtilis, is able to autophosphorylate and to phosphorylate a surrogate substrate, the myelin basic protein. Two crystal structures of YdiB were solved (1.8 and 2.0Å) that display a unique ATP-binding fold unrelated to known protein kinases, although a conserved HxD motif is reminiscent of that found in Hanks-type protein kinases. The effect of mutations of conserved residues further highlights the unique nature of this new protein kinase family that we name ubiquitous bacterial kinase. We investigated the cellular role of YdiB and showed that a ∆ydiB mutant was more sensitive to paraquat treatment than the wild type, with ~13% of cells with an aberrant morphology. In addition, YdiE, which is known to participate with both YdiC and YdiB in an essential chemical modification of some specific tRNAs, is phosphorylated in vitro by YdiB. These results expand the boundaries of the bacterial kinome and support the involvement of YdiB in protein translation and resistance to oxidative stress in B. subtilis.

Muramyl-dipeptide-induced mitochondrial proton leak in macrophages is associated with upregulation of uncoupling protein 2 and the production of reactive oxygen and reactive nitrogen species.

The synthetic immunomodulator muramyl dipeptide (MDP) has been shown to induce, in vivo, mitochondrial proton leak. In the present work, we extended these findings to the cellular level and confirmed the effects of MDP in vitro on murine macrophages. The macrophage system was then used to analyse the mechanism of the MDP-induced mitochondrial proton leak. Our results demonstrate that the cellular levels of superoxide anion and nitric oxide were significantly elevated in response to MDP. Moreover, isolated mitochondria from cells treated with MDP presented a significant decrease in respiratory control ratio, an effect that was absent following treatment with a non-toxic analogue such as murabutide. Stimulation of cells with MDP, but not with murabutide, rapidly upregulates the expression of the mitochondrial protein uncoupling protein 2 (UCP2), and pretreatment with vitamin E attenuates upregulation of UCP2. These findings suggest that the MDP-induced reactive species upregulate UCP2 expression in order to counteract the effects of MDP on mitochondrial respiratory efficiency.