FIG 6.

ccpA null mutant strain preparation and characterization. (A) PCR confirmation of the construction of an isogenic ccpA null mutant strain. Using DNA isolated from wild-type strain CN3718, a PCR using internal ccpA gene primers amplified the expected product of about 400 bp. Using DNA template isolated from the ccpAko strain, which has a 900-bp intron insertion in the ccpA gene, the same PCR assay amplified a larger product of 1,300 bp. The leftmost, unlabeled lane contains a 100-bp molecular ruler. (B) Intron-specific Southern blot hybridization with DNA from wild-type or ccpAko strain. DNA from each strain was digested with EcoRI and electrophoresed on a 1% agarose gel. The size of the DNA fragment is indicated in kilobases. (C) RT-PCR analysis for ccpA transcription of CN3718, ccpAko, or ccpArev strain. (Top) Transcription of housekeeping gene 16S RNA gene; (bottom) transcription of ccpA gene. The leftmost, unlabeled lane contains a 100-bp molecular ruler. (D) Postinoculation change in optical density (OD₆₀₀) for cultures of wild-type CN3718, the ccpA null mutant strain ccpAko, and reversed mutant strain ccpArev growing in TH medium at 30°C. (E) ETX Western blot analysis of CN3718, ccpAko, and ccpArev strains grown in TH medium culture at 30°C for 3 h or overnight (O/N). (F) Quantitative RT-PCR analyses of etx or codY transcription for a 3-h TH culture of CN3718, ccpAko, or ccpArev strain. Average C_T values were normalized to that of the housekeeping 16S RNA gene, and the fold differences were calculated using the comparative C_T method (2^−ΔΔCT). Each bar indicates the calculated fold change relative to wild-type CN3718. All experiments were repeated three times, and mean values are shown. The error bars indicate standard deviations. *, P < 0.005 compared to wild-type strain by ordinary one-way ANOVA.