FIG 3.
cis-Encoded Lpr17 sRNA is expressed in the E phase, while tsp is expressed in both the E and PE phases. (A) tsp is encoded downstream of lpg0500, and the Lpr17 sRNA is encoded on the complementary strand of tsp overlapping with its TSS and the promoter region. The coordinates of tsp and lpr17 in the Philadelphia-1 genome are indicated. The dotted line indicates the portion of the genome that was replaced with a kanamycin resistance cassette in the Δtsp mutant. (B) Northern blotting was used to investigate the expression of Lpr17 in the WT, tsp mutant (Δtsp), and complemented strain (Δtsp + ptsp) grown to exponential phase (E) and postexponential phase (PE) in AYE broth. 5S rRNA was used as a loading control. (C) An RT-PCR was performed on cDNA from the WT strain grown to the E and PE phases in AYE broth to determine if tsp is encoded on a polycistronic operon with the upstream gene lpg0500. RNA from the E and PE phases that was not reverse transcribed (No RT) as well as water were used as a negative control; genomic DNA (gDNA) of the WT strain served as a positive control. (D) RT-qPCR was used to investigate the expression of tsp in the WT grown in AYE broth to the PE phase compared to the E phase. One microgram of RNA was used for reverse transcription. The Ct values of tsp were normalized against 16S rRNA. An unpaired t test was used to access statistical significance (*, P < 0.05).