SYNTHESIS IN ESCHERICHIA-COLI OF 2 SMALLER ENZYMATICALLY ACTIVE ANALOGS OF COXIELLA-BURNETII MACROPHAGE INFECTIVITY POTENTIATOR (CBMIP) PROTEIN UTILIZING A SINGLE OPEN READING FRAME FROM THE CBMIP GENE

FK506-binding proteins (FKBPs) have been identified in a variety of eu karyotic and prokaryotic organisms. Macrophage infectivity potentiator (CbMip, 23.5 kDa) protein of the obligate intracellular bacterium, Co xiella burnetii, was shown previously to belong to the family of FKBPs based on sequence homology and peptidyl-prolyl cis/trans isomerase (P PIase) activity. Further characterization of the cbmip gene has identi fied two additional proteins with molecular masses of 15.5 and 15.0 kD a that are synthesized, in addition to the 23.5 kDa CbMip, when expres sed in Escherichia coli. Amino acid sequencing at the N-terminus combi ned with transcription and translation fusion expression revealed that the two proteins were synthesized from the same open reading frame of the cbmip gene, but starting at different internal translation start codons, probably by translational reinitiation. When the internal meth ionines serving as start sites were replaced with lysine by site-direc ted mutagenesis, the synthesis of 15.5 and 15.0 kDa proteins was aboli shed even though the synthesis of 23.5 kDa CbMip was intact. This conf irmed that the 15.5 and 15.0 kDa proteins are indeed generated by tran slational reinitiation and are not degradation products of the 23.5 kD a protein. Like other FKBPs, both 15.5 and 15.0 kDa proteins exhibit P PIase activity. Because they share significant sequence homology with FKBPs and have a similar PPIase activity, 15.5 and 15.0 kDa proteins a re designated as C. burnetii FKBP (Cb-FKBP) analogues I and II, respec tively. TnphoA mutagenesis demonstrated that whereas the large protein (CbMip) is secreted, Cb-FKBP analogues I and II are cytoplasmic, indi cating that structural variations could allow for different subcellula r compartmentalization of similar proteins. Western-blot analysis of l ysates of purified C. burnetii using a CbMip-specific monoclonal antib ody revealed the presence of a protein migrating at approximate to 15 kDa, indicating the presence of smaller Cb-FKBP analogue(s) in C. burn etii, although at much lower levels compared with 23.5 kDa CbMip, This unique gene organization seen with cbmip may provide the organism wit h a mechanism of efficient use of its limited genetic information to s ynthesize proteins that are structurally different yet functionally si milar.