Genetic Transplantation: Salmonella enterica Serovar Typhimurium as a Host To Study Sigma Factor and Anti-Sigma Factor Interactions in GeneticallyIntractable Systems

Abstract

ABSTRACT In Salmonella enterica serovar Typhimurium,σ 28 and anti-sigma factor FlgM are regulatory proteins crucial for flagellar biogenesis and motility. In this study, we used S. enterica serovar Typhimurium as an in vivo heterologous system to study σ 28 and anti-σ 28 interactions in organisms where genetic manipulation poses a significant challenge due to special growth requirements. The chromosomal copy of the S. enterica serovar Typhimurium σ 28 structural gene fliA was exchanged with homologs of Aquifex aeolicus (an extreme thermophile) and Chlamydia trachomatis (an obligate intracellular pathogen) by targeted replacement of a tetRA element in the fliA gene location using λ-Red-mediated recombination. The S. enterica serovar Typhimurium hybrid strains showed σ 28 -dependent gene expression, suggesting that σ 28 activities from diverse species are preserved in the heterologous host system. A. aeolicus mutants defective for σ 28 /FlgM interactions were also isolated in S. enterica serovar Typhimurium. These studies highlight a general strategy for analysis of protein function in species that are otherwise genetically intractable and a straightforward method of chromosome restructuring usingλ -Red-mediated recombination.