FIG. 2.

PptA-linked transposon insertion mutations perturb murF expression and PptA activity. (A) Real-time RT-PCR analysis of murF mRNA levels in mutants carrying transposon insertions. Amplification of tbpA (5) served as an internal reference and control. Negative controls were performed without reverse transcriptase treatment (not shown). The values are means ± standard errors of the mean; n = 3. The strains used were wt (N400), pptA::kan (KS9), Tn22 (pptA::Tn22; KS8), Tn5 (pptA::Tn5; KS5), Tn9 (pptA::Tn9; KS6), and Tn11 (pptA::Tn11; KS7). The Tn22 mutation reduces PilE phospho-form modification. (B and C) Intact mass analysis of PilE N400 (wt) (B) and KS8 (pptA::Tn22) (C). Species labeled as bearing PE/PC modifications also carry the hexose-2,4-diacetamido-2,4,6-trideoxyhexose (HexDATDH) dissacharide in its acetylated form. Peaks in the MS spectra that are not labeled are listed in Table S3 in the supplemental material.