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. and appears to play a major role in the cell cycle and other cellular processes. To further understand its function, an era gene from Streptococcus pneumoniae 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 gene to complement an Escherichia coli mutant strain defective in Era production was examined. Like the full-length Era protein, the truncated Era protein was able to bind and hydrolyse GTP, but its binding activity was increased twofold and its hydrolytic activity was reduced sevenfold when compared with those of the full-length Era protein. Unlike the full-length Era protein, the truncated Era protein lost its ability to bind to the E. coli cytoplasmic membrane. The full-length era gene was able to complement the E. coli mutant deficient in Era production when carried on pACYC184, while the truncated 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 proteins in E. coli and S. pneumoniae, respectively, were then determined by Western blot analysis. In addition, the minimal amount of the S. pneumoniae 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 protein might be responsible for the binding of the protein to the cytoplasmic membrane and be essential for function.
Zhao
Genshi
G
Meier
Timothy I
TI
Peery
Robert B
RB
Matsushima
Patti
P
Skatrud
Paul L
PL
D015153:Blotting, Western; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development
D029968:Escherichia coli Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C068027:era protein, E coli; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins