Characterization of sialidase complementing strains.

<p>Panel A, PCR confirmation of the construction of isogenic sialidase null mutant and complementing strains. Using DNA isolated from BMC206, PCR assays using internal sialidase gene primers amplified the expected wild-type <i>nanJ</i>, <i>nanI</i> and <i>nanH</i> products of 306 bp, 467 bp and 285 bp, respectively. Using template DNA isolated from BMC207, which has a 900 bp intron insertion in each sialidase gene, the same PCR assays amplified larger products of 1206 bp, 1367 and 1185 for <i>nanJ</i>, <i>nanI</i> and <i>nanH</i>, respectively. With the same assay, DNA from each complementing strain yielded the same wild-type sized PCR products as amplified from BMC206. Lines 1 and 11, 100 bp or 1 kb molecular weight markers, respectively. Panel B, Western blot analyses for sialidase expression by complementing strains BMC2071 (for <i>nanJ</i> complementation), BMC2072 (for <i>nanI</i> complementation) and BMC2073 (for <i>nanH</i> complementation) using 8 h TH sonicated cultures. Size of protein in kDa is shown at right. Panel C, sialidase activity detected in supernatants of BMC206, BMC207, BMC2071, BMC2072 or BMC2073 after growth in TH medium for 2 h, 4 h, 6 h, 8 h, 10 h or overnight.