The cryptic ushA gene (ushA(c)) in natural isolates of Salmonella enterica(serotype Typhimurium) has been inactivated by a single missense mutation

Two mutational mechanisms, both supported by experimental studies, have bee n proposed for the evolution of new or improved enzyme specificities in bac teria. One mechanism involves point mutation(s) in a gene conferring novel substrate specificity with partial or complete loss of the original. (wild- type) activity of the encoded product. The second mechanism involves gene d uplication followed by silencing (inactivation) of one of these duplicates. Some of these 'silent genes' may still be transcribed and translated but p roduce greatly reduced levels of functional protein; gene silencing, in thi s context, is distinct from the more common associations with bacterial par titioning sequences, and with genes which are no longer transcribed or tran slated. Whereas most Salmonella enterica strains are ushA(+), encoding an a ctive 5'-nucleotidase (UDP-sugar hydrolase), same natural isolates, includi ng most genetically related strains of serotype Typhimurium, have an ushA a llele (designated ushA(c)) which produces a protein with, comparatively, ve ry low 5'-nucleotidase activity. Previous sequence analysis of cloned ushA( c) and ushA(+) genes from serotype Typhimurium strain LT2 and Escherichia c oli, respectively, did not reveal any changes which might account for the s ignificantly different 5'-nucleotidase activities. The mechanism responsibl e for this reduced activity of UshA(c) has hitherto not been known. Sequenc e analysis of Salmonella ushA(+) and ushA(c) alleles indicated that the rel ative inactivity of UshA(c) may be due to one, or more, of four amino acid substitutions. One of these changes (S139Y) is in a sequence motif that is conserved in 5'-nucleotidases across a range of diverse prokaryotic and euk aryotic species. Site-directed mutagenesis confirmed that a Tyr substitutio n of Ser-139 in Salmonella UshA(+) was solely responsible for loss of 5'-nu cleotidase activity. If is concluded that the corresponding single missense mutation is the cause of the UshA(c) phenotype. This is the first reported instance of gene inactivation in natural isolates of bacteria via a missen se mutation. These results support a model of evolution of new enzymes invo lving a 'silent gene' which produces an inactive, or relatively inactive, p roduct, and are also consistent with the evolution of a novel, but unknown, enzyme specificity by a single amino acid change.