Kinetic parameters were computed from a Lineweaver–Burk transformation of the Michaelis–Menten equation. Data were obtained from three independent experiments. The degradation

see more of S-ethyl-l-cysteine, S-methyl-l-cysteine, l-cysteine, l-alanine, and l-serine was measured by assaying pyruvate formation, as described previously (Yoshida et al., 2002). The assays were carried out in a 100 μL reaction mixture containing 200 mM potassium phosphate buffer (pH 7.6), 0.165 mM PLP, 1 μg of purified enzyme, and several concentrations of each substrate. Data were obtained from three independent experiments. The concentration of indole in cultures of Prevotella strains was measured as described previously (Sasaki-Imamura et al., 2010). Briefly, overnight bacterial cultures, which were collected

and adjusted to an OD600 nm of about 0.5, were diluted 1/20 in fresh enriched BHI broth and then incubated for 24 h at 37 °C. After the culture was centrifuged, the supernatants (1 mL) were mixed immediately with 140 μL of Kovac’s regent [5% (w/v) p-dimethylamino-benzaldehyde, 75% (w/v) methanol, 2.5 M HCl]. Samples were Olaparib price measured spectrophotometrically at 540 nm and indole concentration was calculated based on a standard curve. Southern hybridization was performed using nonradioactive DIG-labeled PCR probes, as described previously (Yoshida et al., 2009). An aliquot of bacterial genomic DNA digested with SmaI was separated by 0.8% agarose gel by electrophoresis and then transferred

to a nylon membrane. The probes for tnaA from P. intermedia ATCC 25611 and F. nucleatum ATCC 25586 were generated by PCR with primers listed in Table S1, using a PCR DIG Labeling Mix (Roche). The membranes were hybridized for 6 h under high-stringency conditions (65 °C) Phosphatidylethanolamine N-methyltransferase with probe. Prevotella intermedia ATCC 25611 and F. nucleatum ATCC 25586 were used as positive controls; A. actinomycetemcomitans ATCC 29522 was used as a negative control. The tree was constructed by the neighbor-joining method using the computer program clustalw2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and treeview x (http://darwin.zoology.gla.ac.uk/~rpage/treeviewx/). The sequence data of 16S rRNA gene were taken from the GenBank database. The sequences of the tnaA gene and flanking regions in the type strain of P. intermedia ATCC 25611 have been submitted to the EMBL and GenBank databases through the DDBJ (accession number AB618289). Not surprisingly, the tnaA gene sequences were nearly the same between the two P. intermedia strains. The deduced amino acid sequence of P. intermedia ATCC 25611 TnaA was 70%, 44%, and 32% identical to that of P. gingivalis W83, E. coli K-12, and F. nucleatum ATCC 25586, respectively. Even though tnaB, which encodes a tryptophan permease (Edwards & Yudkin, 1982), is located immediately downstream of tnaA in E. coli K-12 and F. nucleatum ATCC 25586 (Fig.