CLONING AND CHARACTERIZATION OF NANB, A 2ND STREPTOCOCCUS-PNEUMONIAE NEURAMINIDASE GENE, AND PURIFICATION OF THE NANB ENZYME FROM RECOMBINANT ESCHERICHIA-COLI

Streptococcus pneumoniae is believed to produce more than one form of neuraminidase, but there has been uncertainty as to whether this is du e to posttranslational modification of a single gene product or the ex istence of more than one neuraminidase-encoding gene. Only one stable pneumococcal neuraminidase gene (designated nanA) has been described. In the present study, we isolated and characterized a second neuramini dase gene (designated nanB), which is located close to nanA on the pne umococcal chromosome (approximately 4.5 kb downstream), nanB was locat ed on an operon separate from that of nanA, which includes at least fi ve other open reading frames. NanB has a predicted size of 74.5 kDa af ter cleavage of a 29-amino-acid signal peptide. There was negligible a mino acid homology between NanA and NanB, but NanB did exhibit limited homology with the sialidase of Clostridium septicum. NanB was purifie d from recombinant Escherichia coli and found to have a pH optimum of 4.5, compared with 6.5 to 7.0 for NanA. Sodium dodecyl sulfate-poly ac rylamide gel electrophoresis analysis suggested that NanB has a molecu lar size of approximately 65 kDa. The discrepancy between this estimat e and the size predicted from the nucleotide sequence is most likely a consequence of C-terminal processing or anomalous electrophoretic beh avior.