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United States Department of Agriculture

Agricultural Research Service

Title: Using Signature-Tagged Mutagenesis to Identify Salmonella Enterica Serovar Typhimurium Genes Involved in Survival in the Swine Stomach

Authors
item Bearson, Shawn
item Bearson, Bradley
item Hoffman, Ann
item Dolphin Rooney, Holly

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: February 27, 2004
Publication Date: May 23, 2004
Citation: Bearson, S.M., Bearson, B.L., Jensen, A.M., Dolphin Rooney, H. 2004. Using signature-tagged mutagenesis to identify Salmonella enterica serovar Typhimurium genes involved in survival in the swine stomach [abstract]. American Society for Microbiology. p. 369-370.

Technical Abstract: For ingested bacteria including foodborne pathogens, the environment of the stomach is the host's first line of defense. To identify genes of Salmonella enterica serovar Typhimurium involved in survival during exposure to this harsh environment (acidic pH, antimicrobial peptides, etc.), the functional genomics technique, Signature-Tagged Mutagenesis (STM) was utilized. STM is an insertional mutagenesis system with transposons carrying unique DNA sequence tags; therefore, in a complex pool of mutants, each S. typhimurium mutant is identifiable in a negative selection screen. An ex vivo swine stomach contents assay was developed using the stomach contents of 4, 6-week-old pigs that had been aseptically removed during necropsy, filter sterilized and combined equally. Thirty-four S. typhimurium STM banks (each containing 47 experimental mutants and 1 control mutant) were assayed by challenging log-phase, pH 4.4 adapted culture pools with the stomach contents for 10 minutes. The surviving mutants were grown overnight and PCR was used to amplify the STM variable regions of the mutant pools before and after challenge. The STM mutants demonstrating a decrease in survival following challenge based on Southern hybridization patterns of the PCR products were individually tested in the swine stomach contents assay. Of the 1,598 S. typhimurium mutants assayed, 21 mutants (1.31%) exhibited a 10-fold or greater decrease in survival compared to the wild-type strain. Only 3 of the 21 S. typhimurium mutants were sensitive in the acid tolerance response assay at pH 3.0, suggesting that some of the mutations may render Salmonella sensitive to other components of the swine stomach. To date, transposon insertions have been identified in the following genes: dnaK, pefA, asmA, dgt, poxR, barA, sopB, and three functionally unknown genes.

Last Modified: 12/28/2014
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