, Tokyo, Japan) with the significance criteria of the program (P<0.05). Real-time PCR was performed using a 7900HT Fast real-time PCR system CAL-101 datasheet (Applied Biosystems). Reactions containing cDNAs from 100 ng total RNA and gene-specific primers were prepared with SYBR Green Realtime PCR Master Mix (Toyobo) according to the manufacturer’s protocol. The primers used are listed in Table S1. The thermal cycle settings used were as follows: initial denaturation at 95 °C for 1 min followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 56 °C for 15 s, and extension at 72 °C for
1 min. The expression of target genes was normalized to the endogenous 16S rRNA gene in each strain. The relative quantification of target gene expression was performed using the comparative cycle threshold (CT) method (Livak & Schmittgen, Wnt inhibitor 2001). blast searches revealed that the TF0022 ORF encodes a HTCS protein that shares homology with GppX from P. gingivalis. We sequenced a fragment containing TF0022 and the upstream
flanking region from the ATCC 43037 genome and compared the data with the existing database sequence (http://www.oralgen.lanl.gov). Although some minor differences were found at the nucleotide level, none altered any functional domains or conserved motifs in the encoding protein (Fig. 1a, DDBJ/GenBank ID: AB587729). Alignment of the TF0022 and GppX polypeptides Phospholipase D1 revealed a notable structural difference: the TF0022 protein lacks the N-terminal portion containing a transmembrane region and part of a putative periplasmic/sensor domain with a TPR motif (Fig. 1b). However, the immediate upstream ORF, TF0023, is predicted to encode a small polypeptide containing an N-terminal transmembrane region and a C-terminal TPR motif of almost the exact length needed to complement the ‘lost’ N-terminus of the TF0022 polypeptide. Indeed, both TF0022 and TF0023
were found to share homology with GppX, yielding similar blast scores (Fig. 1b). A single TPR motif typically consists of 34 amino acids (Das et al., 1998), and GppX from P. gingivalis harbors three tandem repeats of TPR (ranging from residues 155 to 254) at the center of the putative periplasmic domain (Fig. 1b). Interestingly, the TF0023 and TF0022 genes are in the same reading frame, and translation of the nucleotide sequences across the two ORFs uncovered an additional TPR motif when an 18-bp intergenic region was included (Fig. 1a). In P. gingivalis, one of the characteristic phenotypes of the disrupted gppX locus is enhanced autoaggregation (K. Nishikawa, unpublished data). In T. forsythia, ATCC 43037 wild-type cells gradually autoaggregate in broth cultures and eventually precipitate to the bottom of the test tubes. However, we noticed that the broth cultures of TF0022-ko mutant tended to precipitate faster than those of the wild-type strain.