New method for gene disruption in Salmonella typhimurium: Construction and characterization of an ada-deletion derivative of Salmonella typhimurium TA1535

Abstract

A new method for gene disruption in Salmonella typhimurium was developed. The key steps of this method are to produce restriction fragments with compatible ends, preligate to produce concatemers, and then transform by electrotransformation. We developed and used this method to construct a mutant of S. typhimurium TA1535 in which the resident ada-like (ada(ST)) gene was replaced with a kanamycin resistance gene to produce an ada(ST)-deletion mutant derivative. The S. typhimurium ada(ST)-deletion strain did not exhibit a higher level of mutability upon treatment with N-methyl-N'-nitro-N- nitrosoguanidine than did its wild-type parent strain. However, it did exhibit a higher sensitivity with respect to killing by N-methyl-N'-nitro-N- nitrosoguanidine. The ability of Ada(ST) to function as a transcriptional activator is discussed.