Cloning and characterization of the gene for Salmonella typhimurium serine hydroxymethyltransferase

Abstract

A plasmid containing the glyA gene of Salmonella typhimurium LT2 was constructed in vitro using plasmid pACYC184 as the cloning vector and a lambda gt7-glyA transducing phage as the source of glyA DNA. The recombinant plasmid (pGS30) contains a 10-kb EcoRI insert fragment. Genetic and biochemical experiments established that the fragment contains a functional glyA gene. From plasmid pGS30 we subcloned a 4.4-kb SalI-EcoRI fragment containing the glyA gene and its neighboring regions (plasmid pGS38). The location and orientation of the glyA gene within the 4.4-kb insert fragment was determined in four ways: (1) comparison of the physical map of the 4.4-kb SalI-EcoRI fragment with the physical map of a 2.6-kb SalI-PvuII fragment that carries the Escherichia coli glyA gene; (2) deletion analysis; (3) transposon Tn5 insertional inactivation experiments; (4) deoxyribonucleic acid sequencing and comparison of the S. typhimurium DNA sequence with the E. coli DNA sequence. A presumptive glyA-encoded polypeptide of Mr 47000 was detected using plasmid pGS38 as template in a minicell system, but not when the glyA gene was inactivated by insertion of a Tn5 element.