Expression of Streptococcus mutans Aspartate-Semialdehyde Dehydrogenase Gene Cloned into Plasmid pBR322

Abstract

S. mutans chromosomal DNA cloned into the vector plasmid pBR322 in Escherichia coli is able to complement the metabolic defect of an aspartate-semialdehyde dehydrogenase (EC 1.2.1.11) gene (asd) deletion in the host strain. Two Asd+ recombinant plasmids, pYA570 and pYA571, containing 4.7 and 4.5 kilobases, respectively, of S. mutans chromosomal DNA inserted into the HindIII restriction endonuclease site of pBR322 in the same orientation were constructed. The S. mutans UAB62 Asd+ DNA did not hybridize with E. coli DNA which contained an intact asd gene but did hybridize with S. mutans UAB62 chromosomal DNA. Derivative Asd+ plasmids were then constructed from pYA570. One, pYA574, had a 4.5 kilobase S. mutants insert DNA in the opposite direction from pYA570. In another, pYA575, the S. mutans insert DNA was reduced in size to 1.3 kilobases. The orientation of the S. mutans DNA fragment inserted into the promoter region of the pBR322 tetracycline resistance (Tcr) gene affected expression of Tcr. Orientation of the S. mutans insert also affected the stability of the plasmid in certain E. coli strains. Restriction maps for pYA570, pYA571, pYA574 and pYA575 using the endonucleases EcoRI, BamHI, HindIII, PstI and SalI were determined. Asd+ plasmid-directed protein synthesis was studied in E. coli minicells. The plasmids pYA570, pYA574 and pYA575 each produced large amounts of a protein, with a monomeric MW of .apprx. 45,000, that was distinct from both pBR322 and E. coli specified proteins; this protein is the S. mutans asd gene product. Smaller derivatives of recombinant plasmid pYA575 that were Asd- allowed the location of the S. mutans asd gene promoter and the direction of transcription to be determined.