Genetic transplantation: Salmonella enterica serovar typhimurium as a host to study sigma factor and anti-sigma factor interactions in genetically intractable systems

In Salmonella enterica serovar Typhimurium, sigma(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 sigma(28) and anti-sigma(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 sigma(28) structural gene fliA was exchanged with homologs of Aquifex aeolicus (an extreme thermophile) and Chlamydia trachmatis (an obligate intracellular pathogen) by targeted replacement of a tetRA element in the fliA gene location using X-Red-mediated recombination. The S. enterica serovar Typhimurium hybrid strains showed sigma(28)-dependent gene expression, suggesting that sigma(28) activities from diverse species are preserved in the heterologous host system. A. aeolicus mutants defective for sigma(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 using lambda-Red-mediated recombination.