Cloning and characterization of nanB, a second Streptococcus pneumoniae neuraminidase gene, and purification of the NanB enzyme from recombinant Escherichia coli

Abstract

Streptococcus pneumoniae is believed to produce more than one form of neuraminidase, but there has been uncertainty as to whether this is due to posttranslational modification of a single gene product or the existence 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 neuraminidase gene (designated nanB), which is located close to nanA on the pneumococcal chromosome (approximately 4.5 kb downstream). nanB was located on an operon separate from that of nanA, which includes at least five other open reading frames. NanB has a predicted size of 74.5 kDa after cleavage of a 29-amino-acid signal peptide. There was negligible amino acid homology between NanA and NanB, but NanB did exhibit limited homology with the sialidase of Clostridium septicum. NanB was purified 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-polyacrylamide gel electrophoresis analysis suggested that NanB has a molecular size of approximately 65 kDa. The discrepancy between this estimate and the size predicted from the nucleotide sequence is most likely a consequence of C-terminal processing or anomalous electrophoretic behavior.

Cloning of the Tannerella forsythensis (Bacteroides forsythus) siaHI gene and purification of the sialidase enzyme

“…These results could be explained as follows: (i) modification of the enzyme might occur in T. forsythensis and not in E. coli, (ii) other sialidases might exist in this bacterium. Streptococcus pneumoniae expresses two distinct sialidases (neuraminidases), NanA (Berry et al, 1988), which is cellassociated, and NanB (Berry et al, 1996). These results revealed that the purified sample from pHI-1 : 1 might be identical to that from the original bacterium T. forsythensis.…”

The flagellin N-methylase gene fliB and an adjacent serovar-specific IS200 element in Salmonella typhimurium

“…When the central parts of IS200 elements amplified by PCR from strains of E. coli and Salmonella were sequenced, their analysis showed that IS200 must have been present in the common ancestor of the two species, and that it had not been transferred between them since their divergence. This was in contrast to the IS1 element, which was found to be isogenic in both species, reflecting recent horizontal transfer (Bisercic & Ochman, 1993a). Random mutagenesis with TnlO followed by genetic mapping of those transposons which were linked to IS200 insertion sites showed that six IS200 copies in S .…”

“…The original sequence for IS200 was 708 bp in length but since an additional ' C' is found at nucleotide 1524, this would bring the length of the element to 711 bp. The additional 'C' found at this position has also been reported to be present in the IS200 elements of two Salmonella strains from the SARA reference collection (Bisercic & Ochman, 1993a), and therefore probably represents the ancestral sequence of the element. Given the available evidence it cannot be decided whether the occupied or unoccupied insertion site is ancestral.…”

“…The central part of the IS200 sequence constituted a single ORF (termed ORF2 above), transcribed in the same direction as the preceding gene. The predicted polypeptide encoded by ORF2 showed significant amino acid sequence similarity to elements from Yersinia pestis (Simonet et al, 1996), Streptococcus pneumoniae (Berry et al, 1994), Clostridium perfringens (Brynestad et al, 1994), Vibrio cholerae (Bik et al, 1996), and even an archaeobacterial phage (Schnabel et al, 1984). The nucleotide sequence of the corresponding ORFl (fliB)-fliA intergenic region from S. muenchen and two atypical S. typhimurium isolates lacking this copy of IS200 (see below) were determined by direct PCR sequencing.…”

“…Samples were subjected to 35 cycles of amplification with incubation for 1 min each at a denaturation temperature of 94"C, annealing temperature of 54°C and elongation temperature of 74 "C in a Perkin-Elmer Cetus Thermal Cycler. Primers for IS200, designed to anneal to nucleotides conserved between the two published sequences of IS200, EMBL L25848 (Bisercic & Ochman, 1993a) and EMBL X56834 (Gibert et al, 1991), were 5'-CCT AAC AGG CGC ATA CGA TC-3' (forward) and 5'-ACA TCT TGC GGT CTG GCA AC-3' (reverse) and yielded a 557 bp amplicon under the conditions described above for fliA. The identities of the amplicons obtained without adding digoxigenin-dUTP were verified by size and restriction products.…”