The activities of many such factors are regulated by the phosphorylation of

www.selleckchem.com/mTOR.html a conserved aspartate residue in their receiver domains [42, 43]. However, the receiver domain of FlbD diverges substantially from others [37]. For example, it lacks some key residues necessary for the phosphorylation process [44]. No corresponding cognate histidine kinase for FlbD has been identified so far, and FlbD is active in the absence of phosphorylation [30, 34]. In addition, purified FliX can regulate FlbD-activated transcription in vitro, probably by affecting the oligomerization state of FlbD [35]. In this study, we further demonstrated that through a remarkably high affinity, the two proteins bind to each other to perform their regulatory activity and to escape the fate of premature degradation. Mutations in conserved regions of FliX could interrupt

the recognition between the two and hence their activity. The observed low concentrations of FliXL85K, FliXΔ117-118, and FliX 1 in JG1172 cells may be caused by their intrinsically low expression levels or their short half-life, or a combination of both. DNA or mRNA sequences of the alleles may carry intrinsic defects that inhibit the transcription or translation efficiency of the mutated genes. It is also possible that the mutations unfortunately expose target sites to intracellular proteases, making the gene products prone to degradation. Lack of protection

from FlbD may also play a role in the case of FliXL85K. No matter what might be the main cause, the final result is that the cellular levels of the three are this website about the same (Figure 4). Nevertheless, their differential binding affinities to FlbD lead to dramatically different physiological outcomes. FliXL85K completely losts the ability to interact with FlbD and exerts no influence to FlbD-mediated cellular processes. The fair amount of cellular PFKL FliXL85K (Figure 4) does not benefit the ΔfliX host in any observable way (Figure 5, 6 and 7). The mutation must have altered the gross structure of FliX and thus prevented an effective binding to FlbD. FliXΔ117-118 can still interact with FlbD to a certain degree; therefore, it is largely functional in regulating FlbD activity (Figure 5 and 6). With a strong affinity to FlbD, FliX 1 becomes constitutively active; it turns on the transcription of class III/IV genes in the absence of the class II basal body [37, 38]. The other three mutations, R71A, T130L, and L136K cause no significant effect to the expression of FliX, the interaction with FlbD, and hence the regulatory activity of the two partners. Since the three dimensional structure of FliX (or a homolog) remains to be solved, it is still Selleck Tipifarnib unclear which residues or regions of FliX and FlbD are in direct contact. An alanine scanning analysis should be helpful to probe the structural basis of the interaction.