Hemi-methylated oriC DMA binding activity found in non-specific acid phosphatase

The lacZ-hobH fusion clone, containing an Escherichia coli DNA segment loca ted at 92 min on the chromosomal map, was screened as a producer of E. coli oriC hemi-methylated binding activity. We have purified the protein encode d by this locus to near homogeneity. The protein corresponds to the monomer ic form of a non-specific acid phosphatase (NAP) whose gene has been design ated aphA. oriC DNA footprinting experiments showed protection of hemi-meth ylated probe by partially purified NAP, but not by purified preparations. Y et, gel retardation experiments with an oriC oligonucleotide demonstrated D NA binding activity of purified NAP in the presence of Mg2+. This experimen t also showed an increased affinity of the protein for the hemi-methylated probe compared with the fully or unmethylated form. Indirect immunofluoresc ence microscopy revealed the existence of discrete NAP foci at mid-cell in cells with two nucleoids but at cell poles in those with one nucleoid.