Biochemical and molecular analyses of the C-terminal domain of Era GTPase from Streptococcus pneumoniae

Era, an essential GTPase, is present in many bacteria and Mycoplasma spp, a nd appears to play a major role in the cell cycle and other cellular proces ses. To further understand its function, an era gene from Streptococcus pne umoniae was identified and cloned, and a mutant era gene with a deletion of 68 codons from its 3'-terminus was constructed. The truncated Era protein was then purified and characterized, and the ability of the truncated era g ene to complement an Escherichia coli mutant strain defective in Era produc tion was examined. Like the full-length Era protein, the truncated Era prot ein was able to bind and hydrolyse CTP, but its binding activity was increa sed twofold and its hydrolytic activity was reduced sevenfold when compared with those of the full-length Era protein. Unlike the full-length Era prot ein, the truncated Era protein lost its ability to bind to the E. coli cyto plasmic membrane. The full-length era gene was able to complement the E. co li mutant deficient in Era production when carried on pACYC184, while the t runcated era gene failed to do so when carried on pACYC184, pBR322 or pUC18 . The cellular amounts of the truncated Era and the full-length Era protein s in E. coli and S. pneumoniae, respectively, were then determined by Weste rn blot analysis. In addition, the minimal amount of the S. pneomoniae Era protein needed for complementation of the E. coli mutant was also measured. Taken together, these results suggest that the C-terminus of the Era prote in might be responsible for the binding of the protein to the cytoplasmic m embrane and be essential for function.