45-μm filter. Ten microlitres of culture supernatant or SDS extract was added to 100 μL of the fish serum. Subsequently, the mixture was incubated at 37 °C for 24 h. Serum opacification was determined
on the basis of OD measured using a microplate reader at 405 nm. When the OD value exceeded 0.1 compared with control (TH broth with serum, 0.5% SDS with serum), opacification activity was considered to be positive. Horse, pig, cow (GIBCO/Invitrogen, USA) and human sera (TaKaRa Bio, Inc., Japan) were also used for opacification tests with culture supernatant of fish isolate 12-06 as described above. To visualize opacification activity, 5 μL of cell cultures adjusted to an OD660 of 1.0 from strain 12-06 was dropped onto TH agar containing 10% of fish, horse, pig, cow or human sera, then incubated at 37 °C for 24 h. All used sera were heat-inactivated BIBW2992 at 55 °C for 30 min. selleck compound Genomic DNA from the representative fish isolate 12-06 was used in this study (Nomoto et al., 2004, 2006; Nishiki et al., 2010). DNA techniques were performed as described previously (Nishiki et al., 2010). Table 1 lists the primers used in this study. PCR amplification of the sof-FD gene was performed using degenerate primers SOF-d1 and SOF-d2, which were designed on the basis of several sof genes and fnbA (accession number Z22150). The PCR products
amplified with SOF-d1 and SOF-d2 were then extended by 5′- and 3′-rapid amplification of cDNA ends (RACE) PCR with the primer sets RACE SOF-fd1 and RACE SOF-fd2. The RACE-PCR was performed using the SMART RACE cDNA amplification kit according to the manufacture’s protocol (TaKaRa Bio). The entire sof-FD gene was amplified,
and subsequently TA-cloning and sequencing were performed as described previously (Nomoto et al., 2008). The amino acid sequence of sof-FD was analysed using bioedit version 7.0 (Hall, 1999) with the reference sequences of other SOFs obtained from GenBank. The signal peptide and structural domains were predicted using the signalp program (http://www.cbs.dtu.dk/services/SignalP/) and the simple modular architecture research tool (smart) version 4.0 (http://www.smart.enblheidelbergde/). To construct a recombinant plasmid, primer sets SOF-OFD1 and SOF-OFD2 were designed to contain an opacification domain referring to SOF2 (AAC32596) MG-132 obtained from S. pyogenes (Courtney et al., 1999). The amplified product was then ligated into the pBAD TOPO vector system (Invitrogen Japan K. K., Japan) and transformed into Escherichia coli TOP10 following the manufacturer’s protocols. The recombinant protein, amino acid residues 115–780 of sof-FD is referred to as rSOF-OFD. Expression of His-tagged rSOF-OFD was induced following the manufacturer’s protocol. The lysates of the recombinant E. coli TOP10 were purified by His Trap affinity columns (GE Healthcare) according to the user’s manual.