(A) Abnormal branches at the aerial hyphae of the mutant observed by contrast microscopy. The ΔcmdB and ΔcmdA-F mutants frequently produced multiple branches in aerial hyphae, both low in the hyphae (indicated by white arrows), and near the tips (black arrows). These are not common in the wide-type M145. Size bars correspond to 5 μm. (B) Observation check details of spores in M145 and null mutants of cmdB or cmdA-F under scanning
electron microscopy. Strains were inoculated on MS medium covered with cellophane at 30°C for 7 days. Samples were treated (Materials and methods) and subjected to SEM observation. The collapsed aerial hyphae and short spore chains are indicated by white arrows. (C) Chromosomes in the aerial hyphae were stained by DAPI, and observed by laser-scanning confocal microscopy. The chromosomes were not normally segregated in some of the pre-spores of the mutants, some compartments receiving none and some containing more than one chromosome (indicated by white arrows). CmdB,
an ATP/GTP-binding protein with an ABC-transporter ATPase domain, is located on the cell membrane cmdB encoded an ATP/GTP-binding protein and cmdA, C, D, E and F encoded membrane proteins. To see if CmdB protein was also located on the cell membrane, both membrane and cytoplasmic fractions were prepared from cell extracts, electrophoresed on a denatured polyacrymide gel and probed by Western-blotting Selleckchem CHIR98014 with anti-CmdB antibody. As seen in Figure 4A, CmdB protein was only detected in membrane (precipitate) but not in cytosolic (supernatant) fractions. Figure 4 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, TCL were centrifuged to obtain supernatants (S) and pellets (P) for Western blotting with CmdB polyclonal antibody. Total cell lysates was a SAHA HDAC datasheet 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. CmdB contained an ABC-transporter-ATPase domain (from positions 44 to 427) according to Superfamily 1.69 analysis http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/hmm.html. This superfamily includes several families of characterized or predicted ATPases which are predominantly involved in extrusion of DNA and peptides through membrane pores [21]. To investigate whether this domain was required for the function of CmdB, lysines at conserved positions 90 or 404 were mutated to arginines by site-directed mutagenesis (K90A or K404A).