Fig. 1

Roles of four catalases (KatA1, KatA2, KatE, and KatMn), one alkyl hydroperoxidase (AhpC), and three glutathione peroxidases (Gpx1, Gpx2, and Gpx3) in the alleviation of endogenous hydrogen peroxide stress. Bars represent the average values from three independent experiments. Error bars represent the standard error of the mean. *, P < 0.001, significance calculated by Student’s t test. (a) Agarose gel electrophoresis of reverse transcription PCR (RT-PCR). Overnight-cultured S. maltophilia KJ was inoculated into fresh LB with an initial OD_450nm of 0.15 and grown for 5 h. The cDNAs were obtained using reverse transcription with random primers and PCR was performed using primer pairs (Table S1) targeting candidate genes. The smeX gene, which is not expressed in strain KJ, is used as a control for DNA contamination during cDNA preparation. (b) The expression of gpx3, katA2, and ahpC genes in logarithmic- and stationary-phase wild-type KJ cells. Overnight culture of KJ cells was inoculated into fresh LB with an initial OD_450nm of 0.15. Cells were grown aerobically for 5 h or 15 h before measuring gpx3, katA2, and ahpC transcripts using qRT-PCR. All values were normalized to gpx3 transcript of logarithmic-phase KJ cells. (c) Regulatory role of OxyR in the intrinsic expression levels of gpx3, katA2, and ahpC genes. Overnight cultures of KJ and KJΔOxyR cells were inoculated into fresh LB with an initial OD_450nm of 0.15. Cells were grown aerobically for 5 h or 15 h before measuring gpx3, katA2, and ahpC transcripts using qRT-PCR. All values were normalized to the transcript of logarithmic-phase KJ cells. (d) Regulatory role of OxyR in the intrinsic expression levels of gpx3, katA2, and ahpC genes. Overnight cultures of bacteria cells (KJ (Gpx3_xylE), KJ (pKatA2_xylE), KJ (pAhpC_xylE), KJΔOxyR (Gpx3_xylE), KJΔOxyR (pKatA2_xylE), and KJΔOxyR (pAhpC_xylE)) were inoculated into fresh LB with an initial OD_450nm of 0.15. Cells were grown aerobically for 5 h or 15 h before measuring the C23O activity. All values were normalized to the activity in KJ cells. (e) Functions of OxyR, Gpx, Kat, and AhpCF systems in response to endogenously aerobic metabolism-derived H₂O₂ stress. The growth curves of KJ and its derived isogenic mutants were measured by reading OD₄₅₀ at the time points as indicated. *, the growth difference of KJΔAhpCF and KJΔAhpCF (pAhpCF) at the 24-h time point was significant. (f) DHR 123 assay of wild-type KJ and mutants KJΔGpx3, KJΔKatA2, and KJΔAhpCF. The bacterial cells tested were cultured in LB medium containing DHR 123 for 5 h and 24 h, respectively, and the fluorescence at 550 nm was determined. The relative fluorescence is normalized to the fluorescence of wild-type KJ. (g) The expression levels of gpxs, kats, and ahpCF of KJΔGpx3, KJΔKatA2, and KJΔAhpCF in response to endogenously aerobic metabolism-derived H₂O₂ stress. Bacteria cultured overnight (KJ, KJΔGpx3, KJΔKatA2, and KJΔAhpC) were inoculated into fresh LB with an initial OD_450nm of 0.15 and grown for 5 h. The katA1, katA2, katMn, katE, ahpC, gpx1, gpx2, and gpx3 transcripts were measured using qRT-PCR. The relative transcription level for each gene was expressed as the ratio of mutant to wild-type