enterocolitica BT 1A strains. Chromosomal DNA was used as a template; the conditions for PCR amplification were as described earlier
. DOC-PAGE analysis of LPS LPS samples of 298 Y. enterocolitica BT 1A strains were prepared by the small scale proteinase K method as described earlier . Briefly, the bacteria were grown for 14–16 h with shaking in 2 ml of LB at 22°C (RT); the OD600 was determined, the bacteria were then pelleted by centrifugation, and the pellet was re-suspended in DOC lysis buffer (2% DOC, 4% 2-mercaptoethanol, 10% glycerol and 0.002% bromophenol blue in 1 M Tris–HCl buffer, pH 6.8) in a volume AZD2171 research buy adjusted according to the density of the culture (i.e., 100 μl / OD600 =1). The suspension was heated to 100°C for 10 min and then 2–4 μl of proteinase K (20 mg/ml) was added and the suspension was incubated overnight at 60°C. An aliquot of 10 μl was loaded on the gel and analysed in 12% DOC-PAGE and the LPS bands were visualized by silver staining as described earlier . The DOC-PAGE-based LPS classification of Y. enterocolitica and Y.
enterocolitica –like bacteria has been described elsewhere . Briefly, based on the O-polysaccharide (O-PS) the strains are classified into four main LPS types: (i) type A, LPS with homopolymeric O-PS, (ii) type B, LPS with ladder-forming heteropolymeric O-PS, (iii) type C, LPS with single-length O-PS, and (iv) type D, rough or semi-rough LPS without O-PS or with a lipid A core substituted with
a single O-repeat unit, respectively. Phage LY3023414 sensitivity assay The following bacteriophages were used in the typing scheme: фR1–37 [57, 58] that infects Yersinia expressing the outer core hexasaccharide in LPS; PY100 that infects a broad range of Yersinia strains ; фYeO3–12 that uses the Y. enterocolitica serotype O:3 O-PS as receptor [60, 61]; ϕR1-RT that is a bacteriophage originating from the sewage of Turku, Finland and infects Y. enterocolitica serotype O:3 grown at RT (Skurnik, unpublished); and ф80–18 that is a serotype O:8 O-PS specific phage . For altogether 273 Y. enterocolitica BT 1A strains, a 40 μl aliquot from a bacterial culture grown for 14–16 h at RT or 37°C with shaking in LB was mixed with 3.5 ml of molten 0.4% soft agar adjusted to 50°C, mixed briefly and poured on an LA plate. After the soft O-methylated flavonoid agar had solidified, 10 μl drops of different phage suspensions (~105 plaque forming units ml-1) were pipetted onto the surface and the plates were incubated at RT or 37°C 14–16 h. Phage sensitivity was scored as a clear lysis zone in the soft agar. Complement killing assay Blood was obtained from healthy human donors who were devoid of anti-Yersinia antibodies. Sera were pooled and stored in aliquots at −70°C. The killing assay for 298 Y. enterocolitica BT 1A strains was performed essentially as described previously . Briefly, for bactericidal assay, bacteria were grown to stationary phase overnight in 5 ml of MedECa (MedE: 0.