A real-time reporter system was developed to monitor the thermal induction of virulence factors in Yersinia pestis, the etiological agent of plague. The reporter system consists of a plasmid in Y. pestis in which the expression of green fluorescent protein (GFP) is under the control of the promoters for six virulence factors, yopE, sycE, yopT, yopT, yscN, and lcrE yopN, which are all components of the Type III secretion virulence mechanism of Y pestis. Induction of the expression of these genes in vivo was determined by the increase in fluorescence intensity of GFP in real time, in 96-well format. Different basal levels of expression at 26 degreesC were observed for the Y pestis promoters. Expressed as percentages of the level measured for the lac promoter (positive control), the basal expression levels before temperature shift were: yopE (15%), sycE(15%), yopK (13%), yopT(4%), lcrE (3.3%), and yscN(0.8%). Following the shift in temperature from 26 to 37 degreesC, the rates of expression of these genes increased with the yopE reporter showing the strongest degree of induction. The rates of induction of the other virulence factors after the temperature, expressed as percentages of yopE induction, were: yopK (57%), sycE (9%), yscN (3%), lcrE (3%), and yopT (2%). The thermal induction of each of these promoter fusions was repressed by calcium, and the ratios of the initial rates of thermal induction without calcium supplementation compared to the rate with calcium supplementation were: yopE (11-fold), yscN (7-fold), yopK (6-fold), lcrE (3-fold), yopT (2-fold), and sycE (1-fold). This work demonstrates a novel approach to quantify gene induction and provides a method to rapidly determine the effects of external stimuli on expression of Y pestis virulence factors in real time, in living cells, as a means to characterize virulence determinants. Published by Elsevier Inc.