FIG 1.

Confirmation of the S. agalactiae GD201008-001 hylB mutant and complemented strains. (A) Schematic of the strategy for producing the hylB mutant by allelic exchange mutagenesis. (B) Multiple-PCR analysis of the wild-type (WT), mutant (ΔhylB), and complemented (CΔhylB) strains. The primer combinations used in the PCR were as follows: lanes 1, 2, and 3, hylB-LF/hylB-RR; lanes 4, 5, and 6, hylB-F/hylB-R. Genomic DNA from the following strains was used as the template: the wild-type strain (lanes 1 and 4), the ΔhylB mutant strain (lanes 2 and 5), and the CΔhylB complemented strain (lanes 3 and 6). The 5-kb DNA ladder marker (M) is shown on the left. (C) mRNA expression levels of hylB upstream, downstream, and hylB self ORF in the S. agalactiae GD201008-001 wild-type, mutant, and complemented strains in vitro. The values of the hylB-associated genes in the wild-type strain were normalized to 1.0. The relative changes in gene expression ratios of selected genes were normalized to the expression of a single housekeeping gene (16S rRNA gene) and calculated as described by the 2^−ΔΔCT method. Error bars indicate SEM from three independent experiments.