The cis -encoded BTH_s19 regulates BTH_I2095 gene expression by transcript degradation. (A) BTH_s19 is located in the IGR between BTH_I2094 and BTH_I2095 (green). Probe intensities for BTH_s19 expression in Bt CDC272 treated with the three conditions described in Figure 3A are depicted. The yellow arrowhead bars map the different BTH_s19 sequences obtained by RACE. The grey boxes labeled 5’ and 3’ denote the primers used in Northern blots in Figure 7B. (B) PCR products were generated by applying RACE to the same cDNA template using primers specific to BTH_s1, s19, and s39. (C) Total RNA (20μg) was isolated from Bt CDC272 grown in LB, LB+Kan or LB+Serum, host media (RPMI), or total RNA derived from the THP1-EC fraction of THP-1 infected cells. Results are from a single Northern blot consecutively processed with probes to 5’/BTH_s19 and 3’ region of the BTH_I2095 gene, and 5S rRNA, and is a representative of three independent experiments. (D) qPCR was performed on total RNA isolated from Bt CDC272 cultured in LB, host medium with 10% serum (RPMI), LB containing 200 μg/ml kanamycin (LB+Kan), and from the extracellular (THP1-EC) and the intracellular fraction (THP1-IC) of THP-1 infected cells. The relative RNA levels are presented as fold change of BTH_I2095 detected with the 3’ primers and were calculated as transcript ratio between each treatment relative to bacteria grown in LB. The BTH_s19/I2095 ratio is derived from the transcript levels detected with primer sets corresponding to the 5’/BTH_s19 and the 3’ region of BTH_I2095 within each condition. The average and standard deviation from three independent experiments perfomed in duplicate are presented. The “*” denotes statistical significance (p<0.05) for BTH_I2095 expression in B. thailandensis grown in LB + treatment compared to growth in LB or for BTH_s19 expression compared to BTH_I2095.