Draft Genome Sequence of a Clinical Isolate of Streptococcus mutans Strain HM

We report the draft genome sequence of Streptococcus mutans strain HM isolated from a 4-year-old girl with infective endocarditis. The genomics information will provide information on the genetic diversity and virulence potential of S. mutans strain HM

Identification of a new subtype of dipeptidyl peptidase 11 and a third group of the S46-family members specifically present in the genus Bacteroides

Peptidase family S46 consists of two types of dipeptidyl-peptidases (DPPs), DPP7 and DPP11, which liberate dipeptides from the N-termini of polypeptides along with the penultimate hydrophobic and acidic residues, respectively. Their specificities are primarily defined by a single amino acid residue, Gly673 in DPP7 and Arg673 in DPP11 (numbering for Porphyromonas gingivalis DPP11). Bacterial species in the phyla Proteobacteria and Bacteroidetes generally possess one gene for each, while Bacteroides species exceptionally possess three genes, one gene as DPP7 and two genes as DPP11, annotated based on the full-length similarities. In the present study, we aimed to characterize the above-mentioned Bacteroides S46 DPPs. A recombinant protein of the putative DPP11 gene BF9343_2924 from Bacteroides fragilis harboring Gly673 exhibited DPP7 activity by hydrolyzing Leu-Leu-4-methylcoumaryl-7-amide (MCA). Another gene, BF9343_2925, as well as the Bacteroides vulgatus gene (BVU_2252) with Arg673 was confirmed to encode DPP11. These results demonstrated that classification of S46 peptidase is enforceable by the S1 essential residues. Bacteroides DPP11 showed a decreased level of activity towards the substrates, especially with P1-position Glu. Findings of 3D structural modeling indicated three potential amino acid substitutions responsible for the reduction, one of which, Asn650Thr substitution, actually recovered the hydrolyzing activity of Leu-Glu-MCA. On the other hand, the gene currently annotated as DPP7 carrying Gly673 from B. fragilis (BF9343_0130) and Bacteroides ovatus (Bovatus_03382) did not hydrolyze any of the examined substrates. The existence of a phylogenic branch of these putative Bacteroides DPP7 genes classified by the C-terminal conserved region (Ser571-Leu700) strongly suggests that Bacteroides species expresses a DPP with an unknown property. In conclusion, the genus Bacteroides exceptionally expresses three S46-family members; authentic DPP7, a new subtype of DPP11 with substantially reduced specificity for Glu, and a third group of S46 family members

Identification of Dipeptidyl-Peptidase (DPP)5 and DPP7 in Porphyromonas endodontalis, Distinct from Those in Porphyromonas gingivalis

Dipeptidyl peptidases (DPPs) that liberate dipeptides from the N-terminal end of oligopeptides are crucial for the growth of Porphyromonas species, anaerobic asaccharolytic gram negative rods that utilize amino acids as energy sources. Porphyromonas endodontalis is a causative agent of periapical lesions with acute symptoms and Asp/Glu-specific DPP11 has been solely characterized in this organism. In this study, we identified and characterized two P. endodontalis DPPs, DPP5 and DPP7. Cell-associated DPP activity toward Lys-Ala-4-methylcoumaryl-7- amide (MCA) was prominent in P. endodontalis ATCC 35406 as compared with the Porphyromonas gingivalis strains ATCC 33277, 16-1, HW24D1, ATCC 49417, W83, W50, and HNA99. The level of hydrolysis of Leu-Asp-MCA by DPP11, Gly- Pro-MCA by DPP4, and Met-Leu-MCA was also higher than in the P. gingivalis strains. MER236725 and MER278904 are P. endodontalis proteins belong to the S9- and S46-family peptidases, respectively. Recombinant MER236725 exhibited enzymatic properties including substrate specificity, and salt- and pH-dependence similar to P. gingivalis DPP5 belonging to the S9 family. However, the kcat/Km figure (194 mM21?sec21) for the most potent substrate (Lys-Ala-MCA) was 18.4-fold higher as compared to the P. gingivalis entity (10.5 mM21?sec21). In addition, P. endodontalis DPP5 mRNA and protein contents were increased several fold as compared with those in P. gingivalis. Recombinant MER278904 preferentially hydrolyzed Met-Leu-MCA and exhibited a substrate specificity similar to P. gingivalis DPP7 belonging to the S46 family. In accord with the deduced molecular mass of 818 amino acids, a 105-kDa band was immunologically detected, indicating that P. endodontalis DPP7 is an exceptionally large molecule in the DPP7/DPP11/S46 peptidase family. The enhancement of four DPP activities was conclusively demonstrated in P. endodontalis, and remarkable Lys-Ala-MCAhydrolysis was achieved by qualitative and quantitative potentiation of the DPP5 molecule

Distribution of dipeptidyl peptidase (DPP) 4, DPP5, DPP7, and DPP11 in human oral microbiota ? potent biomarkers indicating presence of periodontopathic bacteria

Dipeptidyl peptidase (DPP) 4, DPP5, DPP7, and DPP11, expressed in the periplasmic space, are crucial for energy production for Porphyromonas gingivalis, an asaccharolytic bacterium that causes periodontal disease. Bacterial DPP4 seems to be involved in regulation of blood glucose level via degradation of incretins. The present study aimed to identify four dpp orthologs in oral microbiota by database searches, and their enzymatic activities in periodontopathic and cariogenic bacteria, as well as oral specimens were determined. Search in the databases suggested that 43 species of 772 taxa possess dpp4 and other dpp genes. Most species are in the genera Bacteroides, Capnocytophaga, Porphyromonas, Prevotella, and Tannerella, indicating a limited distribution of dpp orthologs in anaerobic periodontopathic rods. In accordance with those results, activities of all four DPPs were demonstrated in P. gingivalis, Porphyromonas endodontalis, and Tannerella forsythia, while they were negligible in Treponema denticola, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. Furthermore, DPP activities were also detected in subgingival dental plaque at different intensities among individual specimens, while DPP4 activity presumably derived from human entity was solely predominant in saliva samples. These findings demonstrated that DPP activities in dental plaque serve as potent biomarkers to indicate the presence of periodontopathic bacteri

Alignment of amino acid sequences of <i>P. endodontalis</i> MER278904 and <i>P. gingivalis</i> DPP7.

<p>The amino acid sequences of putative <i>P. endodontalis</i> DPP7 (PeDPP7, MER278904) and <i>P. gingivalis</i> DPP7 (PgDPP7, PGN_1479/MER014366) were aligned by Genetyx. Hyphens represent gaps introduced for maximal matching. Common amino acids are marked by asterisks. Three amino acids essential for serine proteases are written in red letters.</p

Biochemical analysis of putative PeDPP5 (MER236725).

<p>(A) Recombinant proteins (0.3 µg) of PgDPP5 (lane 1), putative PeDPP5 (lane 2), and aliquots (5 µL) of bacterial cell suspensions of <i>P. gingivalis</i> ATCC 33277 (lane 3), KDP136 (lane 4), and <i>P. endodontalis</i> (lanes 5 and 5C) were separated on SDS-PAGE gels, then stained with CB or subjected to immunoblotting (WB). The 100-kDa bands in lanes 5 and 5C were non-specifically observed with the alkaline phosphatase-conjugated second antibody. Lane M, molecular-weight markers. (B) NaCl-concentration dependence and (C) pH dependence of the hydrolyzing activity toward Lys-Ala-MCA of PgDPP5, putative PeDPP5 (MER236725), and <i>P. endodontalis</i> cells were determined. (D) The peptidase activities of recombinant PgDPP5 and PeDPP5 were determined using various dipeptidyl MCAs. Values are shown as the mean±S.D. (n = 3).</p

DPP5 mRNA and protein levels.

<p>(A) Relative amounts of DPP5 mRNA in <i>P. gingivalis</i> ATCC 33277, KDP136, and <i>P. endodontalis</i> were measured by qPCR. Values are shown as the mean±S.D. (n = 3). *<i>p</i><0.05; **<i>p</i><0.01. (B) Inhibition ELISA was performed with recombinant PgDPP5 (white circles) and PeDPP5 (blue circles), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114221#s2" target="_blank">Materials and Methods</a>. The amounts of DPP5 in <i>P. gingivalis</i> ATCC 33277, KDP136, and <i>P. endodontalis</i> whole cell lysates were determined to be 12.0±3.0, 20.9±3.5, and 51.8±12.8 ng/µg total proteins, respectively (mean±S.D., n = 3).</p

Alignment of amino acid sequences of <i>P. endodontalis</i> MER236725 and <i>P. gingivalis</i> DPP5.

<p>The amino acid sequences of putative <i>P. endodontalis</i> DPP5 (PeDPP5, MER236725) and <i>P. gingivalis</i> DPP5 (PgDPP5, PGN_0756/MER034615) were aligned by Genetyx. Hyphens represent gaps introduced for maximal matching. Common amino acids are marked by asterisks. Three amino acids essential for serine proteases are written in red letters. Arrows indicate the N-terminal amino acid sequences determined with recombinant proteins.</p

Biochemical analysis of putative PeDPP7 (MER278904).

<p>(A) Recombinant PgDPP7 (lane 1), putative PeDPP7 (Leu³-Lys⁷¹⁰, lane 2), PgDPP11 (lane 3), and PeDPP11 (lane 4) (0.3 µg), and aliquots (5 µL) of cell suspensions of <i>P. gingivalis</i> ATCC 33277 (lane 5), KDP136 (lane 6), and <i>P. endodontalis</i> ATCC 35406 (lane 7) were separated on SDS-PAGE gels, then stained with CBB or subjected to immunoblotting (WB) against anti-PgDPP7 antiserum (300-fold dilution). Peroxidase conjugated anti-rabbit IgG (3,000-fold dilution) was used as the second antibody. Lane M, molecular-weight markers. (B) The hydrolyzing activities (mean±S.D., n = 3) of putative PeDPP7 (MER278904) and PgDPP7 were determined using dipeptidyl MCA.</p