Mutational analysis of the cell division inhibitor MinC

The Min system in Escherichia coli , including MinC, MinD, and MinE, is a major negative regulator of cell division that antagonizes Z‐ring assembly at the cell poles. To gain mechanistic insight into how MinC disrupts FtsZ polymerization and determine sites of interaction between FtsZ, MinD, and MinC, we have constructed amino acid substitution mutations at surface‐exposed residues at the MinC N‐ and C‐terminal domains and assessed the function of MinC mutants in vivo and in vitro . To determine if MinC mutant proteins are defective for function in vivo , we have replaced the gene encoding minC at the native locus on the chromosome with copies of minC encoding several substitution mutations. Cells deleted for minC are on average longer than wildtype E. coli cells, and cultures also contain anucleate minicells arising from polar divisions. Several MinC mutant proteins were identified that are partially defective for MinC function in vivo. To determine if MinC mutant proteins are defective for protein interactions with either FtsZ or MinD, we purified MinC mutant proteins and tested them for activity in vitro . MinC mutant proteins were assayed for the ability to inhibit FtsZ polymer assembly in sedimentation assays, and tested for the ability to form stable polymers with MinD in a nucleotide‐dependent manner. These experiments provide important insight for the identification of surface exposed regions of MinC that mediate protein interactions with FtsZ and MinD.