Cloning and characterization of the Salmonella typhimurium metE gene

The metE gene of Salmonella typhimurium was cloned into plasmid pACYC184 by using a lambda gt7 -metE transducing phage as a source of metE DNA. The recombinant plasmid, designated pGS41 , carries a 12.8-kilobase-pair EcoRI insert fragment. The metE gene was subcloned from pGS41 into plasmid pBR322 on a 4.2-kilobase-pair EcoRI-HindIII fragment (plasmid pGS47 ) and a 4.5-kilobase-pair PstI fragment (plasmid pGS69 ). The location of metE in these plasmids was determined by transposon Tn5 insertional inactivation experiments. A metE-encoded polypeptide of 92,500 Mr was detected in a minicell system by using metE+ plasmids as templates. Truncated polypeptides replaced the 92,500-Mr polypeptide when plasmid derivatives containing Tn5 insertions that inactivate metE were used in the system. A comparison of the site of each Tn5 insertion and the size of the polypeptide made in the minicell system allowed us to determine the direction of transcription and translation. The location of the metE promoter was determined by using an in vitro transcription system and by RNA polymerase protection of restriction endonuclease sites.