SeqA protein aggregation is necessary for SeqA function

The binding of SeqA protein to hemimethylated GATC sequences is important i n the negative modulation of chromosomal initiation at oriC, and in the for mation of SeqA foci necessary for Escherichia coli chromosome segregation. Using gel-filtration chromotography and glycerol gradient sedimentation, we demonstrate that SeqA exists as a homotetramer. SeqA tetramers are able to aggregate or multimerize in a reversible, concentration-dependent manner. Using a bacterial two-hybrid system, we demonstrate that the N-terminal reg ion of SeqA, especifically the 9th amino acid residue, glutamic acid, is re quired for functional SeqA-SeqA interaction. Although the SeqA(E9K) mutant protein, containing lysine rather than glutamic acid at the 9th amino acid residue, exists as a tetramer, the mutant protein binds to hemimethylated D NA with altered binding patterns as compared with wild-type SeqA. Aggregate s of SeqA(E9K) are defective in hemimethylated DNA binding. Here we demonst rate that proper interaction between SeqA tetramers is required for both he mimethylated DNA binding and formation of active aggregates. SeqA tetramers and aggregates might be involved in the formation of SeqA foci required fo r the segregation of chromosomal DNA as well as the regulation of chromosom al initiation.