Tadamichi Takehara

PURIFICATION AND CHARACTERIZATION OF ALKALINE PHOSPHATASE CONTAINING PHOSPHOTYROSYL PHOSPHATASE ACTIVITY FROM THE BACTERIUM PREVOTELLA INTERMEDIA

A novel alkaline phosphatase, designated PiALP, has been purified and characterized from Prevotella intermedia ATCC 25611, an anaerobe implicated in progressive periodontal disease. The enzyme was a homodimer of apparently identical subunits of M r 54 kDa. Thiol‐reducing agents completely inhibited the purified enzyme. The enzyme was highly stable even at 80°C. It exhibited substantial activity against tyrosine‐phosphate‐containing Raytide. The phosphatase activity was sensitive to orthovanadate and Zn2+ but highly resistant to okadaic acid. The amino acid sequence of peptides derived from PiALP showed a high degree of identity (65%) with alkaline phosphatases from Zymomonas mobilis and Synechococcus. The present results imply that PiALP might represent a new family of alkaline phosphotyrosyl phosphatases which has not been described previously.

A murC gene in Porphyromonas gingivalis 381.

The gene encoding a 51 kDa polypeptide of Porphyromonas gingivalis 381 was isolated by immunoblotting using an antiserum raised against P. gingivalis alkaline phosphatase. DNA sequence analysis of a 2.5 kb DNA fragment containing a gene encoding the 51 kDa protein revealed one complete and two incomplete ORFs. Database searches using the FASTA program revealed significant homology between the P. gingivalis 51 kDa protein and the MurC protein of Escherichia coli, which functions in peptidoglycan synthesis. The cloned 51 kDa protein encoded a functional product that complemented an E. coli murC mutant. Moreover, the ORF just upstream of murC coded for a protein that was 31% homologous with the E. coli MurG protein. The ORF just downstream of murC coded for a protein that was 17% homologous with the Streptococcus pneumoniae penicillin-binding protein 2B (PBP2B), which functions in peptidoglycan synthesis and is responsible for antibiotic resistance. These results suggest that P. gingivalis contains a homologue of the E. coli peptidoglycan synthesis gene murC and indicate the possibility of a cluster of genes responsible for cell division and cell growth, as in the E. coli mra region.

Kinetic and site-directed mutagenesis studies of prevotella intermedia acid phosphatase.

Site-directed mutagenesis was used to examine the roles of the conserved histidine, arginine and cysteine residues in acid phosphatase from Prevotella intermedia (PiACP). The replacement of histidine and arginine residues resulted in the elimination of the PiACP activity while the cysteine mutants retained activity. These results suggest that the histidine and arginine residues are essential for catalysis.

Characterization of the Porphyromonas gingivalis FtsZ containing a novel GTPase activity

The FtsZ protein is a GTPase that is essential for cell division. We have cloned, sequenced, and expressed the FtsZ (PgFtsZ) gene from the Porphyromonas gingivalis, an oral, anaerobic, rod-shaped bacterium implicated in progressive periodontal disease. The PgFtsZ gene consisted of 1374 bp and coded for an acidic protein with a calculated molecular mass of 50,253 Da. The deduced amino acid sequence exhibited a significant homology with E. coli FtsZ (54% identical residues). Like other prokaryotic FtsZs, PgFtsZ possessed the clear motifs for GTP binding (GGGTGTG) and hydrolysis (NLDFADV). When PgFtsZ was overexpressed in E. coli, cell division was inhibited. Recombinant PgFtsZ was purified to homogeneity and characterized. The purified PgFtsZ exhibited GTPase activity even in the absence of Mg2+, and completely retained its activity with EDTA. Furthermore, Na+ and K+ ions inhibited its GTPase activity in a dose-dependent manner. These results suggest that PgFtsZ contains an atypical GTPase activity that has not been previously described.

A conserved Ala320 in the FtsZ of Porphyromonas gingivalis is important for cell division.

We have previously cloned the gene encoding the cell division protein FtsZ, designated PgFtsZ, from Porphyromonas gingivalis, an oral anaerobic bacterium implicated in advanced periodontal disease. In the present study, we have shown that overexpression of ZΔC02, a mutant form of PgFtsZ in which 128 amino acid residues have been removed from the C-terminus, caused an inhibition of cell division in E. coli cells. However, overexpression of ZΔC03, missing 177 residues from the C-terminus, did not inhibit cell division, suggesting that the 49 residues between 281 and 329 are required for cell division. Sequence comparison of the known prokaryotic FtsZs revealed that this region contained a highly conserved domain, designated A-domain, in which Ala320 of PgFtsZ was conserved throughout a broad variety of species. Therefore, we analyzed the role of Ala320 by site-directed mutagenesis. We found that overexpression of ZA320H and ZA320R resulted in the normal phenotype, unlike the wild type. Immunoblot analysis showed that these mutant proteins were expressed at similar levels. These results suggest that Ala320 is highly conserved and is crucial for cell division.