Cold shock influences hag expression and binding of human IgD on the surface of M. catarrhalis. A, expression level of hag mRNA. Strain O35E grown to midlogarithmic phase, was exposed for 1 h or 3 h to 26°C or 37°C. RNA was analyzed by quantitative reverse-transcription PCR to determine the amount of hag and 16S rRNA transcripts. The graph shows one of three representative experiments done in triplicate. Data are presented as means ± 1 standard deviation. B, expression of Hag following cold shock. The corresponding OMPs profiles of M. catarrhalis strains O35E and 300 were visualized by Coomassie brilliant blue staining (left panel) and Western blot analysis (right panel) after SDS-PAGE. Proteins were probed with saliva samples. The arrow indicates the position of Hag (approximately 200 kDa). Molecular weight markers in kDa are indicated to the left. C, binding of M. catarrhalis to IgD. Representative flow cytometry profiles of M. catarrhalis strain O35E, Hag-deficient mutant (O35E.hag), LOS-deficient mutant (O35E.lpxA) and clinical isolate 300 after exposure at 26°C (gray) or 37°C (black) show Hag-dependent binding to IgD. The dotted line represents the negative control (bacteria incubated with secondary antibodies only). The mean fluorescence intensity ± 1 standard deviation for 2 experiments performed is shown (D). *, p < 0.05 for 26°C versus 37°C (one-way analysis of variance).