Overexpression of etcABC enhanced type 3 fimbriae production in K. pneumoniae. (A) The representative TEM images of bacteria incubated in LB broth in three independent experiments. KpWT/vc: K. pneumoniae STU1 carrying pBSK-Gm as the vector control. KpWT/etcABC: K. pneumoniae STU1 carrying pBSK::Gm::etcABC to overexpress etcABC. The magnifications of the images are (i) 15,000×, (ii) 50,000×, and (iii) 100,000×. (B) Transcriptional analysis of fimA, fimH, mrkA, and mrkD in KpWT/etcABC and KpWT/vc by RT-qPCR. The 16S rRNA gene was used as the reference. Relative gene expressions means amount of mrkA and mrkD mRNA in KpWT/etcABC compared to those in KpWT/vc and amount of fimA and fimH mRNA in KpWT/vc compared to those in KpWT/etcABC. The presented results are the means ± standard deviations of three replicates. The p-value of each group is less than 0.05 as compared with KpWT/vc. (C) Western blot analysis of MrkA, FimA, and ManA expression in each strain. WT: K. pneumoniae STU1, WT/vc: K. pneumoniae STU1 carrying pBSK-Gm as the vector control. WT/etcABC: K. pneumoniae STU1 carrying pBSK::Gm::etcABC to overexpress etcABC. ManA was used as the loading control. The blots are representatives of at least three independent experiments. (D) Immunogold electron microscopy using anti-MrkA was performed against K. pneumoniae STU1 carrying pBSK::Gm::etcABC. Bacteria displayed type 3 fimbriae. The magnification of the images is 50,000× (left), 100,000× (middle), and 150,000× (right), respectively.