Growth tests starting from both nonpretreated and pretreated cells were arranged. In tests with nonpretreated cells, a preinoculum of the DBT1 was obtained in YMB medium (0.5 g L−1 K2HPO4; 0.1 g L−1 MgSO4·7H2O; 0.1 g L−1 NaCl; 0.4 g L−1 yeast extract; 10 g L−1 mannitol) after 48 h of incubation. Conversely, in tests with pretreated cells, a preinoculum of DBT1 was grown in DM supplied with DBT or phenanthrene (500 mg L−1) for 72 h to induce the PAH-degrading
genes. The cells were then collected by centrifugation (5000 g for 5 min at 4 °C) and washed twice with physiological solution (NaCl 0.9%). INNO-406 Tests were performed on YMA media (YMB added to 1.5% bacteriological agar). Naphthalene, phenanthrene, fluorene and DBT were supplied as a vapour by incubating Petri dishes containing PAH crystals placed in their base. Plates were then incubated at 27 °C and colonies were picked and restreaked on fresh media every week for a month. Total DNA for PCR amplification was prepared as follows: overnight bacterial cultures were pelleted
and resuspended buy CP-868596 in 567 μL TE buffer, 3 μL of 10% sodium dodecyl sulphate and 3 μL of 20 mg mL−1 proteinase K and incubated for 1 h at 37 °C. A 100-μL aliquot of 5 M NaCl and 80 μL CTAB/NaCl solution were then added and incubated again for 10 min at 65 °C. Samples were extracted with an equal volume of phenol/chloroform/isoamyl alcohol mixture. DNA SSR128129E was obtained after precipitation with 0.6 volumes of isopropanol and finally resuspended in 50 μL TE buffer. All PCR reactions were carried out in 25 μL of total volume containing
0.8 μM of each primer, 0.4 mM of dNTPs, 1 U of GoTaq™ DNA polymerase (Promega, Milan, Italy) and 5 μL of 5 × PCR buffer. The gene encoding for 16S rRNA gene (1500 bp) was amplified using FD1 and rp2 primers (Weisburg et al., 1991). PCR conditions were as follows: 95 °C for 5 min, then 30 cycles of 95 °C for 1 min, 50 °C for 1 min and 72 °C for 2 min, with a final extension step at 72 °C for 5 min. A specific B. fungorum recA PCR-amplification assay was performed using the primers FunF and FunR as described by Chan et al. (2003). PCR amplification for an 869-bp ORF recA was carried out according to Payne et al. (2005), while gyrB amplification was performed as described by Ait Tayeb et al. (2008). PCR products were transformed in Escherichia coli Xl1-blue using the Promega pGEM-T vector system according to the manufacturer’s instructions, sequenced on both strands and finally searched for homology using the blastn database (Altschul et al., 1997). The sequences were initially aligned using the multiple alignment program clustal_x 1.83 (Thompson et al., 1997). A phylogenetic tree was constructed based on the neighbour-joining method using the mega version 4.0 software package (Kumar et al., 2008). Bootstrap analysis was performed on the basis of 1000 bootstrap replications.