OxyR is involved in coordinate regulation of expression of fimA and sod genes in Porphyromonas gingivalis

Abstract

Survival of Porphyromonas gingivalis in the constantly changing oral environment depends on its ability to alter gene expression. We demonstrate here that P. gingivalis activates superoxide dismutase expression in response to oxidative stress and represses expression of FimA, a subunit of major fimbriae. Coordinated expression of fimA and sod is regulated by the redox-sensing transcription factor OxyR. Mutations in the oxyR gene result in a decreased expression of sod and in an elevated expression of fimA. In addition, we provide evidence that regulation of expression of fimA and sod by OxyR is mediated by direct interaction of OxyR and the promoters of these two genes. These results suggest that OxyR plays an important role in regulation of expression of virulence genes in P. gingivalis.

Fig. 1

Coordinate expression of fimA and sod in Porphyromonas gingivalis in response to oxidative stress. Porphyromonas gingivalis 33 277 was grown on TSB plates for 48 h anaerobically. The bacteria cells were continuously incubated in the anaerobic chamber for another 3, 6, or 24 h (lanes 1, 3, 5) or exposed to air for 3, 6, or 24 h (lanes 2, 4, 6). Expression of fimA and sod was determined using RT-PCR. Glycosidase (pg1737) expression served as a control.

Fig. 2

Expression of fimA and sod in wild-type strain 33 277 and in the oxyR mutant. Expression level of fimA and sod was determined by RT-PCR on RNA isolated from 33 277 grown anaerobically (lane 1), and aerobically (lane 2), or from the oxyR mutant grown anaerobically (lane 3), and aerobically (lane 4). Glycosidase (pg1737) expression served as a control.

Fig. 3

Effect of OxyR and FimR on formation of Porphyromonas gingivalis biofilms. The ability of biofilm formation of wild-type strain 33 277, the fimR mutant (FRE), and the OxyR mutant (OxyRE) by quantization of cells bound on saliva coated 6 well plates using qPCR. The measurements were performed in triplicate; mean values are shown. Means with different letters are significantly different (P < 0.01; two-way anova and Student–Newman–Keuls Test).

Fig. 4

Interaction of rOxyR with the promoter region of fimA and sod. Electrophoretic mobility shift assays were performed in the presence or absence of rOxyR and 20 fmol biotin-labeled DNA. Increasing amounts of rOxyR were used in the assays. Asterisks indicate that 100-fold excess amounts of each specific competitor unlabeled probe were added to the reaction mixture with labeled probe.

Fig. 5

qPCR analysis of ChIP complex DNA. Immunoprecipitations were performed in the presence of anti-OxyR antibodies or preserum. The precipitated DNAs were amplified using promoter sequences of specific primers for fimA, sod, and pg1737 (as a negative control). Means with different letters are significantly different (P < 0.01; two-way anova and Student–Newman–Keuls Test).