Those that showed only partial restoration of a characteristic were scored as (+). Those showed restoration of motility are called class I mutants, those that did not show a full restoration of motility are class II mutants. A subset of class II mutants which include the surface mutants D52A

and T54A fail to localize correctly as identified using immunofluorescence microscopy. The Stattic solubility dmso remaining class II mutants localize correctly, but do not restore motility. The remaining nine point mutants failed to accumulate detectable amounts of MglA and are classified as class III mutants, which are mot- and dev-. Localization patterns are shown for each motility phenotype and mutant class. Mutations at one position, Thr78, yielded mutants in classes I and II. Thr78 is conserved in the MglA homologs found in bacteria, but it represents SHP099 research buy a significant departure from the consensus found in all other prokaryotic and eukaryotic GTPases,

which use an aspartate in this position. MglA could tolerate serine in this position, but alanine and asparate abolished activity. Thr78 may represent a target for modification in MglA or may be essential for the interaction between MglA and critical effector proteins. Mutations in Ras that correspond with this region of the MglA protein are known to render Ras insensitive to GAP proteins [36, 40], thereby affecting Abemaciclib datasheet the rate of GTP hydrolysis in vivo by interaction with a critical surface feature of Ras-GAP known as the “”arginine finger”" [41]. Thus, the change of Thr78 to Asp may affect the ability of MglA to interact with other proteins in vivo. Consistent with this idea, we found that T78D was dominant to WT MglA for motility and development. These results show that threonine is critical for activity and suggest that MglA and its homologs represent a novel subfamily of GTPases. Activating mutations are predicted to shift the balance to favor more of the GTP-bound (on) state of the GTPase. While it is not possible to make a global generalization, since some of the activating mutants failed to make protein, mutants with G21V and L22V made protein and were partially

motile. The phenotype of the L22V mutant was less severe than that of the G21V next mutant, a result that is consistent with the phenotypes reported for eukaryotic GTPases [42]. G21V was a mutation based on G12V of Ras, which decreases the rate of hydrolysis, a fact confirmed in a bacterial MglA from Thermus thermophilus. kcat for a G21V mutant was 7 times lower than that of WT MglA [19]. They also reported individual movement on buffered 1.0% agar slabs. In contrast, we saw predominantly social motility in our microscopic assays, with few individually moving cells (<5%). As previously discussed, the differences in nutritional conditions as well as agar content may dictate which motility system is active. However, Leonardy et al. did not investigate the effect on motility under conditions where social motility was favored. Additionally, Leonardy et al.