DOMAIN ANALYSIS OF THE FLIM PROTEIN OF ESCHERICHIA-COLI

The FLiM protein of Escherichia coli is required for the assembly and function of flagella, Genetic analyses and binding studies have shown that FliM interacts with several other flagellar proteins, including F liN, FliG, phosphorylated CheY, other copies of FliM, and possibly Mot A and FliF. Here, we examine the effects of a set of linker insertions and partial deletions in FliM on its binding to FliN, FliG, CheY, and phospho-CheY and on its functions in flagellar assembly and rotation. The results suggest that FliM is organized into multiple domains. A C -terminal domain of about 90 residues binds to FliN in coprecipitation experiments, is most stable when coexpressed with FliN, and has some sequence similarity to FliN. This C-terminal domain is joined to the r est of FliM by a segment (residues 237 to 247) that is poorly conserve d, tolerates linker insertion, and may be an interdomain linker. Bindi ng to FliG occurs through multiple segments of FliM, some in the C-ter minal domain and others in an N-terminal domain of 144 residues. Bindi ng of FliM to CheY and phospho-CheY was complex. In coprecipitation ex periments using purified FliM, the protein bound weakly to unphosphory lated CheY and more strongly to phospho-CheY, in agreement with previo us reports. By contrast, in experiments using FliM in fresh cell lysat es, the protein bound to unphosphorylated CheY about as well as to pho spho-CheY. Determinants for binding CheY occur both near the N terminu s of FliM, which appears most important for binding to the phosphoryla ted protein, and in the C-terminal domain, which binds more strongly t o unphosphorylated CheY. Several different deletions and linker insert ions in FliM enhanced its binding to phospho-CheY in coprecipitation e xperiments with protein from cell lysates. This suggests that determin ants for binding phospho-CheY may be partly masked in the FliM protein as it exists in the cytoplasm. A model is proposed for the arrangemen t and function of FliM domains in the flagellar motor.