Backcross and rescue with recombinant proteins indicated that RSP16 depletion resulted in twitching flagella. (A) RSP16⁻ 12E8 was backcrossed with the wild-type strain cc620. 10 randomly picked progenies were scored for motility, antibiotic resistance, and the presence of RSP16 in flagella. Swimming cells (S) contained RSP16, whereas those with twitching flagella (T) did not. Absence of RSP16 was not correlated with PMM resistance. RSP11, loading control. (B) Coomassie protein gel showed Ni-NTA–purified recombinant RSP16-6his used for rescue of 12E8 (left). Approximately 2% of cells that were electroporated with 1 mg/ml of recombinant RSP16 became swimmers upon electroporation. The percentage of swimmers gradually increased within the first 12 h and decreased in 24 h or later. The efficiency of rescue was concentration dependent.