Signaling system in Porphyromonas gingivalis based on a LuxS protein

Abstract

The luxS gene of quorum-sensing Vibrio harveyi is required for type 2 autoinducer production. We identified a Porphyromonas gingivalis open reading frame encoding a predicted peptide of 161 aa that shares 29% identity with the amino acid sequence of the LuxS protein of V. harveyi. Conditioned medium from a late-log-phase P. gingivalis culture induced the luciferase operon of V. harveyi, but that from a luxS insertional mutant did not. In P. gingivalis, the expression of luxS mRNA was environmentally controlled and varied according to the cell density and the osmolarity of the culture medium. In addition, differential display PCR showed that the inactivation of P. gingivalis luxS resulted in up-regulation of a hemin acquisition protein and an arginine-specific protease and reduced expression of a hemin-regulated protein, a TonB homologue, and an excinuclease. The data suggest that the luxS gene in P. gingivalis may function to control the expression of genes involved in the acquisition of hemin.

FIG. 1

Alignment of the deduced P. gingivalis LuxS sequence (obtained from the database of the The Institute for Genomic Research [http: //www.tigr.org]) with deduced LuxS sequences from other bacteria. Sequences from V. harveyi (GenBank accession no. AAD17292), E. coli (GenBank accession no. P45578), S. enterica serovar Typhimurium (GenBank accession no. AAF73475), and H. pylori (GenBank accession no. AAD07175) were aligned using the ClustalW algorithm. The amino acid residues of these sequences that are identical appear in boldface. Symbols: ∗, identity; :, strong similarity; ., weak similarity.

FIG. 2

Schematic arrangement of the luxS ORF and those upstream (nucleosidase) and downstream (traJ) of it. The ORFs themselves are indicated by arrows, with the direction of the arrow indicating the direction of transcription. The genes for LuxS and the nucleosidase are cotranscribed and encompass an additional ORF in a different reading frame. The primers used for RT-PCR are indicated with arrowheads.

FIG. 3

Induction of V. harveyi BB170 luminescence by cell-free supernatants of 33277 (parent strain), PLM1 (mutant), and V. harveyi BB170. Activation was measured by comparing the level of luminescence induced by the test strain to that induced by sterile medium.

FIG. 4

RT-PCR using RNA from cells grown under various conditions. (Top) RT-PCR of mRNA of luxS of P. gingivalis grown under various conditions. Lanes: 1, early log growth; 2, late log growth; 3, late log growth at 34°C; 4, late log growth at 80 mM NaCl; 5, late log growth at 160 mM NaCl; 6, late log growth at 240 mM NaCl. (Bottom) RT-PCR of mRNA of fimA of P. gingivalis grown under various conditions (as a control for total RNA levels). Lanes: 1, early log growth; 2, late log growth; 3, late log growth at 80 mM NaCl; 4, late log growth at 160 mM NaCl; 5, late log growth at 240 mM NaCl. Note that no constitutively expressed P. gingivalis gene that would be a more appropriate control has been reported and that fimA mRNA levels vary according to growth temperature (44).

FIG. 5

RT-PCR to confirm differential expression of genes of parent strain 33277 (P) and mutant PLM1 (M). Lanes: 1, excinuclease ABC homologue; 2, HemR; 3, RgpA; 4, P. fluorescens hemin acquisition protein homologue; 5, TonB homologue.