E coli bl 21

The expression plasmids of glutathione-S-transferase (GST)-fused proteins were constructed by recombining the cDNA sequences into pGEX-4T vectors (GE Healthcare Life Sciences, Pittsburgh, PA), as previously described [18 –20 , 34 (link)]. The pBluescript plasmids containing cDNA inserts were digested with EcoRI and XhoI and separated via agarose gel electrophoresis. Inserted cDNA fragments were isolated using GenElute^TM Minus EtBr Spin Columns (Merck, Darmstadt, Germany) and were ligated in frame to pGEX-4T using a Ligation-Convenience Kit (Nippon Gene, Toyama, Japan). Ligation mixtures were used to transform ECOS^TM-competent E. coli BL-21 (Nippon Gene).

P. gulae strains ATCC 51700 (fimA type A) [7 (link)], D040 (fimA type B) [7 (link)], and D049 (fimA type C) [3 (link)] were used in this study. Each strain was isolated from an oral swab specimen from a dog and confirmed to be P. gulae using a molecular biological method described previously [3 (link)]. P. gulae strains were cultured in Trypticase soy broth (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) supplemented with yeast extract (1 mg/ml), hemin (5 µg/ml), and menadione (1 µg/ml), as described previously [5 (link)].
Escherichia coli DH5α (Nippon Gene, Tokyo, Japan) and E. coli BL21 (Nippon Gene) were used as host strains for transformation of plasmid DNA. E. coli strains were grown in Luria-Bertani (LB; 1% tryptone, 0.5% yeast extract, 0.5% NaCl) medium; LB agar was prepared by the addition of 1.5% agar. When necessary, kanamycin sodium (100 µg/ml) was added to the medium.