5 mM MgCl2, DTT; pH8.7), 2 μl of Qiagen OneStep RT-PCR Enzyme Mix (Qiagen GmbH) and 10 U of RNase inhibitor. The thermal cycler program was carried out at 50°C for 30 min. A 15 min denaturation at 95°C was included prior to the initiation of PCR cycles for the Qiagen One-Step RT-PCR kit, since it contains a hot-start Taq polymerase. At the end of 27 cycles, the reaction-samples (5 μl) were analyzed on 1% agarose gels after amplification. For Northern analysis total RNA extracted from early stationary phase B. pseudomallei was separated by electrophoresis and transferred to solid matrix. Membrane was probed
with a 500-bp SphI-PstI fragment spanning dpsA labeling with α-32P-dCTP by a PCR labeling method and by X-ray exposure Buparlisib clinical trial detection as previously described (14). To investigate whether expression of oxyR regulates rpoS, the B. pseudomallei strain rpoS::lacZ was conjugated with B. pseudomallei strain oxyR−. A mutant rpoS::lacZ, oxyR strain was then selected and designated rpoS::lacZ/oxyR−. The extent to which LacZ was expressed was investigated in the log, early drug discovery stationary
and late stationary growth phases in rpoS::lacZ and rpoS::lacZ/oxyR−. As can be seen in Figure 1a, both strains showed essentially the same response curve, although late stationary phase concentrations of lacZ were somewhat higher in the rpoS::lacZ/oxyR− strain. These results suggest that rpoS expression does not require oxyR. To determine the converse, whether rpoS regulates the expression of oxyR, the B. pseudomallei strain oxyR:: CAT, which contains a chromosomal oxyR::CAT transcriptional very fusion as well as an integrated mini-transposon containing oxyR (mtoxyR+) (9), was conjugated separately with B. pseudomallei strains rpoS− and the one which carries complement rpoS (rpoS−+ pBSS1[rpoS+]), represented as RpoS, resulting in production of strains oxyR::CAT/rpoS− (chromosomal oxyR::CAT/mtoxyR+/rpoS) and oxyR::CAT/rpoS−/RpoS
(chromosomal oxyR::CAT/mtoxyR+/rpoS, +pBSS1[rpoS+]), respectively. The extent of CAT expression was assessed during log phase growth (4 hr post subculture), and the early (12 hr) and late stationary phases (24, 48, 72 hr). As can be seen in Figure 1b, significant induction of CAT expression was observed during the log to early stationary phase of growth in both strains, oxyR::CAT and oxyR::CAT/rpoS−/RpoS, in both cases declining slowly during the late stationary phase. In contrast, no induction of CAT expression was observed in strain oxyR::CAT/rpoS− (which contains no RpoS), showing that RpoS is required for the induction of oxyR gene expression under normal growth conditions.