Figure 4: MukB and MatP interact in vivo and in vitro.

(a) Top: schematic of MukB protein. The Hinge construct carries residues 566–863. Bottom: space-filling representation of a dimer⁶ containing most of the residues (572–854) of the Hinge construct. (b) The bacterial two-hybrid analysis is based on functional adenylate cyclase reconstitution³¹. Proteins of interest, as indicated, were fused to the N and the C terminus of the T25 and T18 domains, respectively. Blue colouring on X-gal plates reveals positive interactions. See also Supplementary Methods and Supplementary Fig. 6a for all combinations in MatP⁺ and ΔmatP cells. (c) Size exclusion chromatography of MatPΔC18 alone (right panel), or present at seven-fold molar excess over Hinge (left panel). The elution fractions indicated in black were analysed on SDS–PAGE; shown below each graph. Because of space constraints, not all fractions are shown in the upper panels. Also see Supplementary Fig. 6b,c. (d) Co-Immunoprecipitation assay using resin-bound MatP–Flag, derived from over-expressed cell extracts, to assay for binding of MukB (Lane 1), MukBEF (Lane 2), MukBF (Lane 3), MukBE (Lane 4) and MukFE (Lane 5). MukB (Lane 6) and MukBEF (Lane 7) show no binding to the resin. Purified MukBEF (Lane 8) and MukBF (Lane 9), as used in the assay. The MW of the proteins used were: MukB-6 × His 172.6 kDa; MukF-6 × His 52.9 kDa; MukF 50.6 kDa; MukE-6 × His 29.4 kDa and MatPΔC18–Flag 16.9 kDa. (e) Co-immunoprecipitation shows MatPΔC18 is retained by MukB–Flag (lane 1) and Hinge–Flag (lane 2) attached to the anti-FLAG resin but not by the resin alone (lane 3). Lane 4: purified MatPΔC18-6 × His. Hinge proteins gave 2 bands on SDS–PAGE, maybe because of proteolytic cleavage. The MW of the proteins was: MukB–Flag 171.7 kDa; Hinge–Flag 35.2 kDa; MatPΔC18-6 × His 18.3 kDa. The lanes were derived from the same gel (shown in Supplementary Fig. 6d). MW, molecular weight.