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

Joyce E. Karlinsey^* and Kelly T. Hughes

Department of Microbiology, University of Washington, Seattle, Washington 98195

Received 18 July 2005/ Accepted 28 September 2005

In Salmonella enterica serovar Typhimurium, 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 ²⁸ and anti-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 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 -Red-mediated recombination. The S. enterica serovar Typhimurium hybrid strains showed ²⁸-dependent gene expression, suggesting that ²⁸ activities from diverse species are preserved in the heterologous host system. A. aeolicus mutants defective for 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 -Red-mediated recombination.

Present address: Department of Biology, University of Utah, Salt Lake City, UT 84112.

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