MobC C-terminus is required for oligomerization but not DNA binding. A. Bacterial two-hybrid system in vivo. The mobC orf was cloned into two sets of BACTH vectors to be linked with CyaA fragments by N-or C-terminus. Photographs document the ability to ferment maltose by double transformants of BTH101cyaA strain with plasmid encoding CyaA fragments linked to N-termini of MobC derivatives. The pLKB2 and pLKB4 represent modified empty BACTH vectors pKT25 and pUT18C, respectively. Similar results were obtained for plasmids encoding CyaA fragments linked to C- termini of MobC derivatives (data not shown). B. Glutaraldehyde cross-linking in vitro. His-tagged MobC derivatives (0.1 mg ml-1) were incubated with increasing concentrations of glutaraldehyde (0.001%, 0.005% and 0.01%). The complexes were separated on 20% polyacrylamide gels by SDS-PAGE, transferred onto a nitrocellulose membrane and visualized byWestern blotting with anti-His tag antibodies. Bands corresponding to monomeric (m), dimeric (d) and terameric (t) form of the MobC variants analyzed are marked. The protein migrating faster than MobC1-129 is probably a degradation product. C. Regulation of mobCp by truncated MobC1-129. Allele mobC1-129 was cloned into pGBT30 under tacp (pJSB5.2) and introduced into DH5α(pJSB7.9) strain. The double transformant strains were grown without IPTG. The XylE activity is expressed relative to the activity detected in DH5α(pJSB7.9)(pGBT30) strain. DH5α(pJSB7.9)(pJSB5.1 tacp-mobC) strain was used as the control. Mean values with standard deviation of at least three assays are shown. D. DNA binding by MobC1-129 in vitro. Two picomoles of 417-bp DNA fragment containing mobCp were incubated with increasing quantities (0 to 20 picomoles) of His-tagged MobC derivatives in 20 μl of binding buffer at 37°C for 15 minutes. The complexes were separated on 1.2% agarose gels run in 1xTBE and visualized by ethidium bromide staining.