FUNCTIONAL-PROPERTIES OF PRO REGION OF ESCHERICHIA-COLI HEAT-STABLE ENTEROTOXIN

Escherichia coli heat-stable enterotoxin Ip (STp) is synthesized as th e 72-amino-acid residue precursor consisting of three regions: pre reg ion (amino acid residues 1 to 19), pro region (amino acid residues 20 to 54), and mature ST (mST) region (amino acid residues 55 to 72). We examined the role of the pro sequence of STp in enterotoxigenicity of a strain by deleting the gene fragment encoding amino acids 22 to 57. This deletion caused a remarkable reduction of its enterotoxic activit y of culture supernatant. In order to analyze the sequence responsible for the function of the pro region, two additional deletion mutants w ere made. The deletion of the sequence covering amino acids 29 to 38, which is conserved in all sequences of ST reported, brought about a si gnificant reduction of enterotoxic activity but the deletion of the no n-conserved sequence (amino acids 40 to 53) did not. This result shows that conserved sequence is mainly responsible for the function. Subse quently, to examine the mechanism of action of the pro region, plasmid s carrying DNA sequences of hybrid proteins consisting of pre-pro-nucl ease, pre-mST-nuclease, pre-pro-mST-nuclease and pre-pro-nuclease-mST were constructed. Amino acid sequence determination and SDS-polyacryla mide gel analysis revealed that these fusion proteins were cleaved bet ween pre sequence and pro sequence during secretion and the cleaved fu sion proteins were accumulated in periplasmic space. But the amount of hybrid protein accumulated in the periplasmic space varied among the strains. That is, the amount of the pre-pro-nuclease gene product that accumulated in the periplasmic space was the highest of all fusion ge ne products. These results indicate that the existence of the mST regi on strongly interferes with the translocation of the gene product into the periplasmic space and that the pro region functions to guide the mST region into the periplasmic space.