Construction of a Gene Bank of Streptococcus lactis Plasmid pLM2001 Deoxyribonucleic Acid in Escherichia coli

Abstract

Plasmid pLM2001, which is essential for the fermentation of lactose by Streptococcus lactis LM0232, was isolated and cut into eight fragments with restriction endonuclease EcoRI. The deoxyribonucleic acid fragments ranged in size from approximately .8 to 10.3 kilobases as determined by agarose gel electrophoresis. EcoRI-restricted pLM-2001 deoxyribonucleic acid was shotgun cloned into the single EcoRI site of the multifunctional Escherichia coli-Streptococcus sanguis shuttle vector, pSA4, insertionally inactivating the chloroamphenicol resistance gene. Following transformation of the deoxyribonucleic acid into Escherichia coli HB101, 242 transformants containing chimeric plasmids were detected by their resistance to tetracycline and failure to grow in the presence of chloramphenicol. Plasma deoxyribonucleic acid from 98 transformants was cleaved with EcoRI and analyzed by agarose gel electrophoresis for the presence of pLM2001 inserts. Clones were obtained, which individually contained each of the eight fragments. In addition, a series of clones containing various combinations of the fragments was obtained. The availability of a system for cloning and rapidly screening for fragments of Streptococcus lactis deoxyribonucleic acid under conditions where deletion of passenger or vector deoxyribonucleic acid does not occur will facilitate studies designed to isolate and characterize metabolically important genes carried by plasmids from the group N streptococci.