Cambronne, E.D. and Schneewind, O.
Notes: To analyze whether the presence or absence of calcium in Yersinia enterocolitica growth media affected secretion of Yersinia outer proteins (Yops), these researchers used mutational analysis on four gene products that work together to regulate yopQ expression. The various Y. enterocolitica strains were grown in TSB medium supplemented with 5mM CaCl2 or EGTA (+/-Ca2+). After a 2-hour incubation followed by a 2-hour induction, the cultures were centrifuged and the supernatant was separated from the cell pellet. After resuspension in cell lysis buffer, the pellet was subjected to French press and 100µl aliquots were used with the SV Total RNA Isolation System. The eluted total RNA was digested with DNase, phenol/chloroform extracted and ethanol precipitated. Once resuspended, 0.6µg of Yersinia RNA was used in two-step RT-PCR to amplify the yopQ gene and the amplimer was analyzed on an ethidium bromide-stained 2% agarose gel. (3071)