STRESS-INDUCIBLE GENE OF SALMONELLA-TYPHIMURIUM IDENTIFIED BY ARBITRARILY PRIMED PCR OF RNA

Fingerprinting of RNA by arbitrarily primed PCR (RAP) can be used to i dentify conditionally expressed genes in prokaryotes. Differential gen e expression in Salmonella typhimurium LT2 in response to peroxide tre atment was examined as a system in which to demonstrate this strategy. This treatment models the induction of bacterial protective proteins that may occur when mammalian phagocytes use peroxide to fight S. typh imurium infection. To identify genes inducible by hydrogen peroxide st ress, total RNA from peroxide-treated and untreated bacterial cultures were RAP fingerprinted with six different arbitrarily selected primer s. A 435-base RAP product that was differentially amplified by RAP usi ng the reverse sequencing primer was cloned and sequenced. Northern bl ot analysis confirmed that the RNA corresponding to this clone, RSP435 , was induced when bacteria were treated with hydrogen peroxide. The R NA was not induced in an oxyR1 mutant that constitutively expresses a subset of hydrogen peroxide-inducible genes. Using pulsed-field gel el ectrophoresis and dot blot hybridization to an array of induced Mud-P2 2 integrations, the gene corresponding to RSP435 was mapped to two pla ces, one between 19 and 21.5 min and one between 56 and 57 min. Thus, two similar or identical stress-inducible genes were found in differen t parts of the genome. Identification, cloning, and mapping of the con ditionally expressed RSP435 cDNA were performed entirely by physical m eans, demonstrating that the strategy should complement genetic method s for many prokaryotic or archaebacterial systems and should be applic able to organisms in which genetic methods are difficult to perform or have not yet been developed.