Methods: We carried out transposon mutagenesis to identify the gene(s) responsible for the LPSs biosynthesis in P. gingivalis.
Results: We isolated a novel pigment-less mutant by transposon mutagenesis and identified that the transposon was inserted into the PGN_2005 gene encoding a hypothetical protein of P. gingivalis 33277. Cell surface associated gingipain activity and hemagglutination activity of the PGN_2005 mutant were lower than those of the wild-type. Interestingly, immunoblot analyses with anti-O-LPS and anti-A-LPS antibodies suggested that the molecular masses of both LPSs of the PGN_2005 mutant were lower than those of the wild-type. As the secondary structure prediction showed that PGN_2005 protein has two transmembrane regions and one periplasmic loop region, PGN_2005 protein was suggested to be a homolog of Wzz protein which plays a role in determining length of O-side chain in the inner membrane. We found i) LPSs purified form the PGN_2005 mutant were shorter than those of the wild-type, ii) PGN_2005 protein was localized in the inner membrane fraction, iii) the PGN_2005 gene conferred Wzz activity upon an Escherichia coli wzz mutant.
Conclusions: PGN_2005 protein is a functional homolog of Wzz protein. As genes homologous to the PGN_2005 gene are found only in the genus Porphyromonas, we designated the PGN_2005 gene as wzzP.