Figure 4
Cell-wall hydrolase activity of Rv3717. (a) The cell-wall hydrolase activity of rRv3717-A was monitored by incubating live M. smegmatis cells with no (filled squares), 1 µg ml-1 (crosses), 25 µg ml-1 (filled triangles) or 50 µg ml-1 (filled circles) rRv3717-A or with BSA (filled diamonds). Error bars represent one standard deviation from the mean value of OD600 in triplicate experiments. The inset shows the mathematical fit for rRv3717-A (50 µg ml-1 reaction): log([Delta]OD600) = 0.016x - 4.43 [general form log([Delta]OD600) = ax - b, where the values of a and b are average values for each point]. (b) Zymograph analysis of rRv3717-A with cell suspensions of different heat-killed bacteria as substrates: (i) Paenibacillus sp., (ii) B. avium, (iii) E. coli DH5[alpha], (iv) E. aerogenes, (v) L. acidophilus, (vi) B. thuringiensis, (vii) B. pumilus, (viii) B. subtilis and (ix) E. coli W311. Zymograph analysis was carried out in a dose-dependent manner; in (i) lane 1 contains 4 µg, lane 2 contains 2 µg and lane 3 contains 1 µg rRv3717-A. L, Lysozyme (4 µg); B, BSA (4 µg); and R, rRv3717-A (4 µg). Lower doses are not indicated in the other panels for clarity. The zone of clearance indicates cell-wall hydrolysis. A molecular-weight marker (lane M) is included in each panel and is labelled in kDa.  [article HTML]