The absorbance of the supernatants at 595 nm was estimated using a Cary50 spectrophotometer. Results for the heat-treated samples were expressed as percentage of the value for the untreated samples. Dichelobacter see more nodosus strains were subcultured onto
TAS agar plates (1.5% tryptone, 0.5% protease peptone, 0.2% yeast extract, 0.5% Lab-Lemco powder, 0.5%l-arginine, 0.15%dl-serine, 0.2% MgSO4·7H2O and 1.5% agar), incubated under anaerobic conditions for 4 days and then used to stab-inoculate fresh TAS agar plates that were incubated for a further 4 days (Kennan et al., 2001). Brilliant blue-R dye (0.25% w/v brilliant blue-R, 40% v/v methanol and 7% v/v acetic acid) was then layered over the TAS agar plates, incubated for 30 min and then treated with destaining solution (10% acetic acid, 40% methanol, 50% water) until the blue background disappeared. The diameter of the colony was measured. Student’s unpaired t-test was used to determine whether differences between assays were significant. Based on similarity to other PNPases, the predicted D. nodosus PNPase has two copies of the RNAse PH domain separated by an all-α-helical core PNPase
domain, which are followed by an RNAse KH domain and an RNAse S1 domain (Fig. 1). Two suicide plasmids were constructed (Fig. 1) to interrupt the PNPase-coding learn more region after codon 297 (pCF7), which would remove the last four domains, or codon 572 (pCF5), which would remove the S1 domain. The D. nodosus strains A198 and C305 are widely used as reference virulent and benign strains, respectively. Some D. nodosus strains are naturally competent, but all previous attempts to transform strains A198 and C305 have failed (Kennan et al., 1998). The transformation efficiency is very low and varies between D. nodosus strains (Kennan et al., 1998). For these experiments, the virulent strains A198, UNE61 and UNE64 (VCS1703A; Kennan et al., 1998) and the benign strains C305, 819, 1493 and 2483 were used. All of these virulent Galactosylceramidase strains have the intA element next to pnpA, while the benign strains have either the intB or the intD element at this position. Transformation
of these strains was attempted using pCF7, designed to truncate PNPase after amino acid 297, which would disrupt four of the five conserved domains (Fig. 1). However, despite many attempts, no transformants with disrupted pnpA genes were obtained, suggesting that a complete lack of PNPase activity is lethal. This was unexpected, because the PNPase gene has been knocked out in E. coli without affecting viability (Donovan & Kushner, 1986). However, the D. nodosus genome is very small (1.3 Mb; Myers et al., 2007) compared with E. coli and so there may be less redundancy in the RNA processing machinery. Similar transformation experiments with pCF5, designed to truncate PNPase after amino acid 572, were carried out with all strains. Tetracycline-resistant colonies were obtained using virulent strains UNE61 and UNE64, and benign strains 819, 1493 and 2483.