Localization of CmdB protein, characterization of its functional domain, and detection of cmdB transcription. (A) Localization of CmdB protein. Cell lysates of strain M145 and that were treated with 0.5 M KCl or 5 mM EDTA-Na, were centrifuged to obtain supernatants (S) and pellets (P) for Western blotting with CmdB polyclonal antibody. Total cell lysates was a positive control. (B) Mutations of conserved residues in domains of the CmdB protein blocked its function. Plasmid pFX101 derivatives containing the site-mutated cmdB genes were introduced by conjugation into the cmdB null mutant. Strains were grown on MS at 30°C for 3 days. (C) RT-PCR to detect transcription of cmdB. Total RNA was isolated from MS medium grown for 16, 26, 40, 50, 62 and 74 h, and reverse-transcribed into cDNAs for PCR amplification. Transcription of 16S rRNA gene was used as an internal control.