Localization of spiralin in Escherichia coli cells transformed with the recombinant plasmid pESI.
The expression of spiralin in the transformant strain HB101 Tsp of Escherichia coli has been investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunodetection after electrotransfer, and crossed immunoelectrophoresis. The protein has been sought in whole cells, cytoplasm, and plasma and outer membranes of the bacterium. Untransformed E. coli cells and Spiroplasma citri cells were used as negative and positive references, respectively. Contrary to earlier claims, spiralin was detected not only in the cytoplasm of E. coli, but also in the inner and outer membranes. In addition, our results show that the protein was not secreted by the bacterium. Three forms of spiralin could be distinguished with respect to differences in apparent molecular mass: 27.5, 29.5 and 30 kilodalton (kDa). The presence of the high molecular mass polypeptide in the two membranes of E. coli invalidates the hypothesis according to which the 27.5-kDa (or 28-kDa) species is a mature form derived from the 30-kDa (or 30.5-kDa) species by the amputation of a signal sequence. Since spiralin is an acyl protein, the possibility of variation in the extent of acylation of the protein in E. coli, with subsequent variation of the apparent molecular mass, should be taken into account.
Blanchard
A
A
Laboratoire de Microbiologie Fondamentale et Appliquée, Université de Rennes, France.
Wroblewski
H
H
Barroso
G
G
D000215:Acylation; D000942:Antigens, Bacterial / Q000032:analysis* / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D002462:Cell Membrane / Q000032:analysis; D003593:Cytoplasm / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000032:analysis* / Q000235:genetics / Q000648:ultrastructure; D007123:Immunoelectrophoresis, Two-Dimensional; D008970:Molecular Weight; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000032:analysis*; D013149:Spiroplasma / Q000235:genetics* / Q000276:immunology
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D011994:Recombinant Proteins; C028588:spiralin