CLONING, FUNCTIONAL EXPRESSION, AND COMPLEMENTATION ANALYSIS OF AN INORGANIC PYROPHOSPHATASE FROM BARTONELLA-BACILLIFORMIS

We have cloned the inorganic pyrophosphatase gene (ppa) from the facul tative intracellular pathogen Bartonella bacilliformis and characteriz ed its encoded product. The 531-bp gene is located approximately 1 kb downstream of, and in opposite orientation to, the invasion-associated locus (ialAB) of B. bacilliformis. The predicted protein encoded by p pa is 177 amino acid residues, which is in agreement with in vitro and in vivo synthesis of a protein with an apparent molecular mass of 22- 23 kDa. The predicted B. bacilliformis pyrophosphatase (PPase) sequenc e is 53% identical and 85% similar to the E. coli PPase (EC 3.6.1.1), and contains all 12 of the amino acid residues implicated in the catal ytic active site. The. isolated B. bacilliformis PPase exhibits an act ivity of 51 +/- 2 mu mol PO4 released/(mg protein.min) at 28 degrees C and pH 8, and is sensitive to inhibition by Ca2+. In keeping with oth er prokaryotic PPases, B. bacilliformis PPase activity occurs from pH 6 to 10 (optimal pH = 8) and demonstrates high thermostability in the presence of Mg2+ (highest activity at 55 degrees C, relative activity = 80 +/- 3% at pH 8). The cloned B. bacilliformis ppa is able to genet ically complement a ppa(-) mutant strain of E. coli.