SEQUENCE-ANALYSIS AND CHARACTERIZATION OF A 40-KILODALTON BORRELIA-HERMSII GLYCEROPHOSPHODIESTER PHOSPHODIESTERASE HOMOLOG

We report the purification, molecular cloning, and characterization of a 40-kDa glycerophosphodiester phosphodiesterase homolog from Borreli a hermsii. The 40-kDa protein was solubilized from whole organisms wit h 0.1% Triton X-100, phase partitioned into the Triton X-114 detergent phase, and purified by fast-performance liquid chromatography (FPLC). The gene encoding the 40-kDa protein was cloned from a B. hermsii chr omosomal DNA lambda EXlox expression library and identified by using a ffinity antibodies generated against the purified native protein. The deduced amino acid sequence included a 20-amino-acid signal peptide en coding a putative leader peptidase II cleavage site, indicating that t he 40-kDa protein was a lipoprotein, Based on significant homology (31 to 52% identity) of the 40-kDa protein to glycerophosphodiester phosp hodiesterases of Escherichia coli (GlpQ), Bacillus subtilis (GlpQ), an d Haemophilus influenzae (Hpd; protein D), we have designated this B. hermsii 40-kDa lipoprotein glycerophosphodiester phosphodiesterase (Gp d) homolog, the first B. hermsii lipoprotein to have a putative functi onal assignment. A nonlipidated form of the Gpd homolog was overproduc ed as a fusion protein in E. coli BL21(DE3)(pLysE) and was used to imm unize rabbits to generate specific antiserum. Immunoblot analysis with anti-Gpd serum recognized recombinant H. influenzae protein D, and co nversely, antiserum to H. influenzae protein D recognized recombinant B. hermsii Gpd (rGpd), indicating antigenic conservation between these proteins. Antiserum to rGpd also identified native Gpd as a constitue nt of purified outer membrane vesicles prepared from B. hermsii. Scree ning of other pathogenic spirochetes with anti-rGpd serum revealed the presence of antigenically related proteins in Borrelia burgdorferi, T reponema pallidum, and Leptospira kirschneri. Further sequence analysi s both upstream and downstream of the Gpd homolog showed additional ho mologs of glycerol metabolism, including a glycerol-3-phosphate transp orter (GlpT), a glycerol-3-phosphate dehydrogenase (GlpD), and a thior edoxin reductase (TrxB).