FIG 2.

Growth, sialidase activity, and sialidase gene expression by F4969, an isogenic nanI null mutant, and a complemented strain. (A) Cultures of F4969 were grown for 24 h at 37°C in control DMEM, DMEM with 1.6 mM sialic acid (SL), DMEM with 16 mM sialic acid (SH), DMEM with 1.6 mM glucose (GL), or DMEM with 16 mM glucose (GH), as indicated. At the designated time points, the OD₆₀₀ of cultures were determined. Results representative of three repetitions are shown. (B) Supernatant sialidase activities in supernatants from F4969 grown at 37°C for 4 or 24 h (O/N) in control DMEM versus DMEM supplemented with Neu5Ac or d-glucose, as indicated. (C) qRT-PCR analyses of nanJ, nanI, and nanH expression levels in F4969 cultures grown in DMEM with or without carbohydrate supplementation. Transcript levels were determined with 20 ng of RNA isolated from 4-h cultures grown at 37°C in control DMEM or DMEM supplemented with GL, GH, SL, or SH. Average C_T values were normalized to that of the housekeeping 16S rRNA gene, and fold differences were calculated using the comparative C_T method (2^−ΔΔCT). The value of each bar indicates the calculated fold change relative to the control. (D) Growth yields of 24-h cultures of wild-type, nanI null mutant, and complemented strains of F4969 grown at 37°C in control DMEM versus DMEM supplemented with Neu5Ac or d-glucose, as indicated. (E) Sialidase activities in supernatants from 24-h cultures of wild-type, nanI null mutant, and complemented strains of F4969 grown at 37°C in control DMEM versus DMEM supplemented with Neu5Ac or d-glucose, as indicated. For panels B to E, the data shown are mean values for three independent experiments. The error bars indicate standard deviations. *, P < 0.05 compared to the control.