* Strains belonging to clonal group B are shown in lanes 10, 14, 15, 19, 21, 22, 28, 29, 31, 45, 46 and 47. Clonal group A strains are in other lanes. For clonal groups refer to [22]. The HaeIII and Sau96I restriction profiles of ureAB of biovar 1B, 2 and 4 strains were distinct from that of biovar 1A strains (See Additional file 4). As with ureAB, restriction patterns of ureC for these biovars were also quite distinct from biovar

1A strains (data not shown). Biochemical characterization The crude extract of urease of Y. enterocolitica biovar 1A strain was active over a pH range of 4.0-7.0. The maximum activity was observed at pH 5.5 (Fig. 3a). The enzyme was quite heat-stable as urease activity was recorded up to 65°C but decreased progressively at higher temperature (Fig.

selleckchem 3b). The optimum temperature for urease activity was 65°C (Fig. 3b). The urease exhibited Michaelis-Menten kinetics with Km and Vmax of 1.74 ± 0.4 mM urea and 7.29 ± 0.42 μmol of ammonia released/min/mg of protein respectively (data not shown). Figure 3 Biochemical characterization CH5183284 datasheet of Y. enterocolitica biovar 1A urease. (a) optimal pH for urease activity (b) effect of temperature on 5-Fluoracil nmr urease activity and (c) effect of click here growth phase and growth temperature on urease production; growth curve of biovar 1A strain grown at 28°C is also shown. Data points represent mean of triplicate determinations. The error bars indicate standard deviation. Y. enterocolitica biovar 1A grown at 28°C (optimum

temperature for growth) exhibited higher urease activity than that grown at 37°C (Fig. 3c). Irrespective of the growth temperature, stationary phase cells showed higher activity (Fig. 3c). The supplementation of growth medium (Luria broth) with 16.7 mM urea did not show significant difference in urease activity. However, supplementation with nickel chloride resulted in ca. 10-fold increase in the activity. 1 μM NiCl2 was sufficient to induce urease activity as no significant increase in the activity was observed with further increase in concentration up to 200 μM (See Additional file 5). On native PAGE, urease was observed as two bands with the major band having molecular weight > 545 kDa and a slowly-developing band above it (Fig. 4). The electrophoretic mobility of urease of Y. enterocolitica biovar 1A strain was shown to be different from that of biovar 1B, 2 and 4 strains though similar to the Y. intermedia urease. The isoelectric point of the crude extract urease was 5.2.