Abstract:

Abstract:Objective
To investigate the effects of the C-terminal and N-terminal deletions of FtsZ in Porphyromonas gingivalis (Pg) on the GTPase activity and to clarify the domain of PgFtsZ GTPase,and to provide experimental basis for prevention and treatment of periodontal disease.Methods Plasmids pEZ1 (Wt PgFtsZ,including Wt PgFtsZ gene),pYW1(ZΔC01,missing 73 residues from C-terminus of PgFtsZ),pYW2(ZΔC02,missing 128 residues from C-terminus of PgFtsZ),pYWN1(ZΔN01,missing 43 residues from N-terminus of PgFtsZ),and pYWN2(ZΔN02,missing 205 residues from N-terminus of PgFtsZ) were respectively introduced into E.coli BL21(DE3)pLysS to express，seperate and purify WtPgFtsZ,ZΔC01,ZΔC02,ZΔN01 and ZΔN02.pET3a vector alone was introduced into E.coli BL21(DE3)pLysS as negative control.Malachite green assay was used to measure the GTPase activities of Wt PgFtsZ,ZΔC01,ZΔC02,ZΔN01 and ZΔN02.The assemblies of the proteins were detected by sedimentation in vitro.The morphologies of E.coli that overexpressed Wt PgFtsZ and mutants proteins were observed under light microscope.Results The GTPase activity of ZΔN01 was similar to the GTPase activity of Wt PgFtsZ.However,the GTPase activities of ZΔC01,ZΔC02,and ZΔN02 were lower than that of the Wt PgFtsZ (P<0.05).10 mmol•L^-1 CaCl2 reduced the GTPase activities of Wt PgFtsZ and PgFtsZ mutants (P<0.05).In addition to ZΔN02,10 mmol?L-1 CaCl2 induced the polymerizations of Wt PgFtsZ,PgFtsZ mutants.When wild-type PgFtsZ,ZΔC01 and ZΔC02 overexpressed in E.coli,respectively,and the E.coli cells became long filamentous compared with the E.coli cells containing pET3a vector.However,ZΔN01 and ZΔN02 overexpressed in E.coli,respectively,and the E.coli morphology had similar to that of E.coli cells containing pET3a vector.Conclusion Missing 162 residues from the N-terminus of PgFtsZ between ZΔN01 and ZΔN02 and missing 128 residues from the C-terminus of PgFtsZ has relationship with the  GTPase activity of PgFtsZ.Ca2+ cation can inhibit GTPase activity of PgFtsZ and promote its polymerization in vitro.
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Key words: prophyromonas qingivalis, filamentous temperature-sensitive protein Z, N-termina, C-terminal, GTPase activity, calcium cationic