P-064. Expression of the Phosphate Specific Transport (Pst) System of Escherichia coli O157:H7 Treated with 0.5% Sodium Benzoate

F. J. Critzer, D. H. D'Souza, D. A. Golden;
Univ. of Tennessee, Knoxville, TN.

Background: Sodium benzoate is a widely used food antimicrobial in drinks and fruit juices. A microarray study was conducted to determine the transcriptional response of Escherichia coli O157:H7 to 0.5% (w/v) sodium benzoate, and the phosphate specific transport (Pst) system was found to be highly up-regulated. This system transports inorganic phosphate into bacterial cells under phosphate limited conditions. The Pst system also mediates ciprofloxacin resistance in Mycobacterium sp. by acting in a dual role as an efflux pump and Pst system. Methods: Stationary phase E. coli O157:H7 grown in 150 ml Luria-Bertani broth (LB) was centrifuged at 4°C and the pellet was rehydrated in equal concentrations of LB containing 0 (control) and 0.5% (w/v) sodium benzoate. Each treatment was duplicated, sampled at 0 (immediately after exposure), 5, 15, 30, and 60 min. Total RNA was extracted using the Qiagen RNeasy extraction kit and analyzed with E. coli 2.0 Gene Chips (Affymetrix). Intensity signals for each probe set were normalized with a GCRMA algorithm. Significantly regulated genes (positive false discovery rate <0.05) were determined using the R-MAANOVA (microarray analysis of variance) package. Significant ontology categories affected by sodium benzoate exposure were determined with JProGO software. Results: The Pst system is composed of four subunits; PstS, a periplasmic phosphate binding protein; PstA and PstC, involved in formation of the transmembrane portion of the Pst system; and PstB, an ATPase. Increased expression of the Pst system was observed after 5 min of exposure to sodium benzoate; pstS, pstA, pstB, and pstC genes were up-regulated >2-fold (linear scale) at 5, 15, 30, and 60 min. Increased expression of several other efflux systems, such as AcrAB-TolC, was also observed. Validation of the microarry results by RT-PCR is on-going. Conclusion: The Pst system may act as an efflux pump under these stress-adapted conditions, as well as increase transport of phosphorus to aid in DNA, RNA, ATP, and phospholipid production. Understanding adaptations of E. coli O157:H7 under antimicrobial exposure is essential to better understand and implement methods to inhibit or control its survival in foods.