
Figure 4
Cellwall hydrolase activity of Rv3717. (a) The cellwall hydrolase activity of rRv3717A 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) rRv3717A or with BSA (filled diamonds). Error bars represent one standard deviation from the mean value of OD_{600} in triplicate experiments. The inset shows the mathematical fit for rRv3717A (50 µg ml^{1} reaction): log(OD_{600}) = 0.016x  4.43 [general form log(OD_{600}) = ax  b, where the values of a and b are average values for each point]. (b) Zymograph analysis of rRv3717A with cell suspensions of different heatkilled bacteria as substrates: (i) Paenibacillus sp., (ii) B. avium, (iii) E. coli DH5, (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 dosedependent manner; in (i) lane 1 contains 4 µg, lane 2 contains 2 µg and lane 3 contains 1 µg rRv3717A. L, Lysozyme (4 µg); B, BSA (4 µg); and R, rRv3717A (4 µg). Lower doses are not indicated in the other panels for clarity. The zone of clearance indicates cellwall hydrolysis. A molecularweight marker (lane M) is included in each panel and is labelled in kDa. 