Generation of Lys-gingipain protease activity in Porphyromonas gingivalis W50 is independent of Arg-gingipain protease activities

Porphyromonas gingivalis, a black-pigmenting anaerobe implicated in the aet iology of periodontal disease, contains two loci, rgpA and rgpB, encoding t he extracellular Arg-X specific proteases (RGPs, Arg-gingipains), and kgp, which encodes a Lys-X specific protease (KGP, Lys-gingipain). The rgpA and kgp genes encode polyproteins comprising pro-peptide and catalytic domain w ith large N- and C-terminal extensions which require proteolytic processing at several Arg and Lys residues to generate mature enzymes. The product of rgpB contains only a pro-peptide and the catalytic domain which requires p rocessing at an Arg residue to generate active enzyme. An rgpA rgpB double mutant (E8) of P. gingivalis was constructed to study the role of RGPs in t he processing of KGP. A kgp mutant (K1A) was also studied to investigate th e role of KGP in the generation of RGPs. E8 was stable in the absence of th e antibiotics tetracycline and clindamycin (selection markers for rgpA and rgpB, respectively) and exhibited the same pigmentation, colony morphology and identical growth rates to the parent W50 strain in the absence of antib iotics, in both complex and chemically defined media. The KGP activity of E 8, grown in the absence of tetracycline, in whole cultures and in culture s upernatants (up to 6 d) was identical to levels in W50. However, in the pre sence of tetracycline in the growth medium, the level of KGP was reduced to 50% of levels present in whole cultures of W50. Since tetracycline had no effect on RGP or KGP activity when incorporated into assay buffer, this eff ect is most likely to be on the synthesis of Kgp polypeptide. K1A was also stable in the absence of antibiotics but was unable to pigment, and remaine d straw-coloured throughout growth. RGP activity in whole cultures of K1A w as identical to levels in W50, but RGP activity in 6 d culture supernatants was reduced to 50% of levels present in W50. Thus, although KGP is not req uired for generation of RGP activity from RgpA and RgpB polypeptides, its a bsence affects the release/transport of RGP into culture supernatant.