Construction of a non-polar pilA mutant. The PCR amplicon from Figure 1 is shown in Panel A. The cloned pilABCD region of pRSM2855 is shown in Panel B. The amplicon was electroporated into E. coli DY380(pRSM2855) and the lambda recombinase genes were induced by temperature shock. Spectinomycin-resistant clones were selected. The insert region of plasmid pRSM2857 is shown in Panel C. Plasmid pRSM2857 was linearized and transformed into NTHi strain 2019 rpsL; spectinomycin-resistant clones were isolated. NTHi strain 2019 rpsL ΔpilA::spec-rpsLNg was saved, then transformed with pRSM2947 at the permissive temperature; kanamycin resistant clones were isolated. Expression of the FLP recombinase resulted in the loss of the spectinomycin resistance gene-rpsLNg cassette; growth at 37°C resulted in the loss of the plasmid. The pil region of NTHi strain 2019 rpsL ΔpilA is depicted in Panel D.