Hemolytic activity was determined by mixing an equal volume of bacterial cells with 1% erythrocytes in PBS. This mixture was then incubated at 37 °C for 4 h. Samples (500 μL) were withdrawn and further spun

(1300 g for 5 min) in an Eppendorf 5403 centrifuge at room temperature. The OD405 nm of supernatant was determined by spectrophotometry. As a negative control, erythrocytes were used alone. Hemagglutinin activity was determined as reported previously (Vanterpool et al., 2005a). Twenty-four-hour cultures of P. gingivalis W83 and mutants were harvested by centrifugation (10 000 g for 10 min). Cells were washed twice in PBS buffer and resuspended to a final OD600 nm Selleckchem Afatinib of 1.5. Sheep erythrocytes were washed twice with 1 × PBS and resuspended in 1 × PBS to a final concentration of 1%. An aliquot (100 μL volume) of the bacterial suspension was serially diluted twofold with PBS in wells of a round-bottom 96-well microtiter plate. An equal volume (100 μL) of 1% sheep erythrocytes was mixed

with each dilution and incubated at 4 °C for 3 h. Hemagglutination was assessed visually and the hemagglutination titer was determined as the last dilution that showed complete hemagglutination. KU-57788 mw The presence of Arg-X- and Lys-X-specific cysteine protease (Rgp and Kgp) activity was determined using a microplate reader (Bio-Rad, Hercules, CA) as reported previously (Vanterpool et al., 2005b). In brief, the activity of arginine gingipains

was measured with 1 mM BAPNA (Nα-benzoyl-dl-arginine-p-nitroanilide) in an activated protease buffer (0.2 M Tris-HCl, 0.1 M NaCl, 5 mM CaCl2, 10 mM Cediranib (AZD2171) l-cysteine, pH 7.6). Lysine gingipain activity was measured with ALNA (Ac-Lys-p-nitroanilide HCl). After incubating the substrate and culture, the reaction was stopped by the addition of 50 μL of glacial acetic acid. The OD405 nmwas then measured against a blank sample containing no bacteria. Total RNA from P. gingivalis strains was extracted using the SV Total RNA Isolation System (Promega Corp., Madison, WI) according to the manufacturer’s instructions. cDNA was synthesized using a Transcriptor High Fidelity cDNA Synthesis Kit (Roche Corp., Indianapolis, IN). The primers used are listed in Table 2. The PCR program consisted of 1 cycle of 5 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 30 s at 54 °C, and 1 min at 68 °C, with a final extension of 5 min at 68 °C. To construct ECF sigma factor isogenic mutants, PCR was used to fuse the upstream and downstream fragments of the target gene to ermF, generating a 3-kb-length fragment, which was then electrotransformed into P. gingivalis W83. To the promoter region upstream of the ATG start codon of ermF, we added a 20-base oligonucleotide 5′-TGACTAACTAGGAGGAATAA-3′ containing three stop codons separated by one nucleotide.