TTG as the initiation codon of Salmonella slyA, a gene required for survival within macrophages

The slyA gene, which has been implicated in the virulence of Salmonella ser ovar Typhimurium and its survival in macrophages, is widely distributed amo ng different Salmonella serovars, In this study, we cloned and sequenced th e translational initiation region of the slyA gene from nine different sero vars and found sequence differences in the previously proposed ATG initiati on codon but not in a TTG triplet, another putative initiation codon in the slyA gene. Therefore, we determined the actual translational initiation si te of the slyA gene by analyzing slyA genes with defined mutation in either the ATG or TTG sequences in an in vitro translation assay and a quantitati ve hemolytic assay in Escherichia coli. The replacement of TTG by TTC in th e slyA gene significantly reduced both the amount of protein synthesized an d the hemolytic activity of a transformed strain of E. coli, while replacem ent of ATG by ATC had no effect in these assays. In addition, the amino aci d sequence analysis of the His-tagged SlyA protein showed that it was ident ical with the amino acid sequence deduced from the 5' end of the slyA gene with a TTG initiation codon, Our results suggest that TTG serves as the tra nslational initiation codon for the slyA gene of Salmonella.