FIG 5.
Removal of the reporter cassette relieves polar effects on tmeB. (A) The presence of transcripts downstream of tmeA was determined by reverse transcriptase (RT) quantitative PCR. Total RNA was isolated from at 24 hpi from McCoy cells infected at an MOI of 1 with WT, L2 tmeA, L2 tmeB, or L2Rif tmeA-lx. Transcripts for tmeA, tmeB, and ct696 were detected by qRT-PCR, and signals are presented after normalization to ropD. ND, none detected. (B) Equal quantities of whole-culture material from 24-h cultures infected with equal IFUs of WT, L2 tmeA, L2Rif tmeA-lx, or L2Rif tmeA-lx ptmeA were probed in immunoblots for TmeA and TmeB. Hsp60 was used as a loading control, and proteins were visualized by chemiluminescence.