[Cloning of the regulator gene of Erwinia carotovora repressing pectate lyase ptlA gene expression].

Abstract

Pectate lyase synthesis in the cells of Erwinia carotovora ELA 49 is induced by polypectate. This suggested that the Erwinia chromosomes carried a regulator gene responsible for negative regulation of the pectate lyase gene expression. In the present study the regulator gene controlling expression of one of the pectate lyase structural genes was cloned and designated as ptlA gene. For this purpose a genetic system with the tester plasmid pPc624 as the main element was constructed. The tester plasmid contained cat gene (resistance to chloramphenicol) controlled by the promotor of the ptlA gene cloned on vector pPD620. Plasmid pPC624 was maintained in the E. coli cells in a number of 1-2 copies and transferred resistance to chloramphenicol in concentrations up to 100 micrograms/ml to the cells. The E. carotovora cells containing pPC624 were sensitive to chloramphenicol in media containing no inductor (sodium polypectate). In media with the inductor they were resistant to chloramphenicol. Therefore, plasmid pPC624 proved to be a suitable system for testing the regulator gene product. The E. coli cells containing plasmid pPC624 were transformed by the hybrid Ptl+ plasmids identified in the clonotheque of the Erwinia DNA EcoRI fragments. The E. coli cotransformants were characterized by chloramphenicol sensitivity which provided a conclusion that the regulator ptlR gene controlling the ptlA gene expression was localized on the DNA EcoRI fragment (7.3 kb) containing the pectate lyase ptlA and ptlB genes. Deletion analysis showed that the investigated genes were localized in the EcoRI fragment (7.3 kb) of the E. carotovora chromosomal DNA in the following order: ptlA--ptlB--ptlR.(ABSTRACT TRUNCATED AT 250 WORDS)