Mini-Mu Transposon Mutagenesis of Ethanologenic Zymomonas mobilis

Abstract

Zymomonas mobilis is a facultatively anaerobic α-proteobacterium with a considerable potential for industrial ethanol production. An important tool in the generation of stable mutants for this organism is described in this chapter; it entails insertional mutagenesis with the help of the transposable element mini-Mu. The latter is delivered into Z. mobilis with the use of plasmid pULB113 (RP4::mini-Mu) that self-transfers in the organism at notable frequencies and remains highly stable even under nonselective conditions. Transposition of mini-Mu and subsequent mutagenesis occur readily in Z. mobilis pULB113 transconjugants and result in the generation of large numbers of random mutants. This can be demonstrated by the isolation of various auxotrophs with single or multiple nutritional requirements, the vast majority of which bears insertions at different chromosomal locations, even when exhibiting the same requirement. Therefore, transposon mutagenesis with the use of mini-Mu serves as a simple and effective tool for indiscriminate mutant production in Z. mobilis.