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(ΔOD600) = 0.016x − 4.43 [general form log(ΔOD600) = 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 DH5α, (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.