Role of Sodium in the RprY-Dependent Stress Response in Porphyromonas gingivalis

Abstract

Porphyromonas gingivalis is a Gram-negative oral anaerobe which is strongly associated with periodontal disease. Environmental changes in the gingival sulcus trigger the growth of P. gingivalis and a concurrent shift from periodontal health to disease. Bacteria adjust their physiology in response to environmental changes and gene regulation by two-component phospho-relay systems is one mechanism by which such adjustments are effected. In P. gingivalis RprY is an orphan response regulator and previously we showed that the RprY regulon included genes associated with oxidative stress and sodium metabolism. The goals of the present study were to identify environmental signals that induce rprY and clarify the role of the regulator in the stress response. In Escherichia coli an RprY-LacZ fusion protein was induced in sodium- depleted medium and a P. gingivalis rprY mutant was unable to grow in similar medium. By several approaches we established that sodium depletion induced up-regulation of genes involved in oxidative stress. In addition, we demonstrated that RprY interacted directly with the promoters of several molecular chaperones. Further, both genetic and transcription data suggest that the regulator acts as a repressor. We conclude that RprY is one of the regulators that controls stress responses in P. gingivalis, possibly by acting as a repressor since an rprY mutant showed a superstress reponse in sodium-depleted medium which we propose inhibited growth.

Post-translational regulation of a Porphyromonas gingivalis regulator

“…Strains of Escherichia coli were derivatives of K12 that were grown in Luria Bertani broth or plates at 37°C, and when appropriate with the addition of antibiotics (ampicillin 100 μg/ml or kanamycin 50 μg/ml). Cultures of P. gingivalis strain ATCC 33277 were grown in tryptic soy broth (TSB) as described previously [3] with erythromycin (5 μg/ml) or tetracycline (2 μg/ml) when appropriate. Cultures were grown in an anaerobic chamber (Coy) in an atmosphere of 85% nitrogen, 5% hydrogen, and 10% carbon dioxide.…”

“…DNA primers were synthesized by integrated DNA Technologies. The rprY (PGN_1185) deletion strain of ATCC 33277 was constructed as described earlier [3]. Briefly, using three-way SOEing (splicing by overlap extension) [7], PCR amplicons containing sequences 1054 bp upstream and 1047 bp downstream of the PGN_1185 ORF were ligated to an ErmF-ErmAM cassette.…”

“…In a previous report, we presented data indicating that the response regulator RprY of P. gingivalis strain ATCC 33277 played a role as a repressor in the oxidative stress response, with genes encoding AhpC, GroES, ClpB, and DnaK included in its regulon [3]. The open reading frame of RprY (PGN_1186 in ATCC 33277 and PG1089 in strain W83) encodes a 28 kDa protein with 61% identity and 74% similarity to a previously identified RprY response regulator from Bacteroides fragilis [4].…”

Two-component signal transduction systems in oral bacteria

“…The role of sodium (Na + ) depletion as a potential environmental signal to activate RprY function was further investigated [46]. In Escherichia coli , an RprY–LacZ fusion protein was induced specifically by Na + depletion; however, P. gingivalis rprY and oxyR mutants were unable to grow in a similar medium.…”

“…Taken together, these results suggest that RprY may be a constitutively expressed repressor and that, in the absence of a cognate HK, its function is modulated by the acetylation activity of Pat. The exact role of Na + depletion on expression of RprY is still unknown, although the rprY mutant has the same growth phenotype as an oxyR mutant during depletion, suggesting a role in the oxidative stress response [46]. Interestingly, it was recently demonstrated that the NQR pump of V. cholerae is a generator of ROS, specifically associated with reduced a flavin adenine dinucleotide cofactor of subunit F ( nqrF ) of the complex [45].…”