Figure 4
From: Small RNA mediated repression of subtilisin production in Bacillus licheniformis
Protease activity and Apr protein expression in B. licheniformis MW3, MW3 AprAs− and MW3 AprAs− pV2::aprAs. The strains were grown in 400 ml M9 skim milk medium for 48 h. (a) The cell culture supernatants of samples from time points 12 h, 24 h, 36 h and 48 h were used for quantitative exoprotease activity determination. The absolute value of protease activity is shown as the average of three independent experiments, each with triplicate measurements. The exoprotease activity increased from 12 h to 48 h and is clearly stronger in B. licheniformis MW3 AprAs− in all experiments compared to B. licheniformis MW3 and MW3 AprAs− pV2::aprAs. The error bars display the standard deviation and the colour legend is shown in the figure. (b) Extracellular proteins were isolated from supernatants of 400 ml M9 skim milk cultures after 48 h of growth. The protein fractions were separated by 2D-gelelectrophoresis. A gel image of B. licheniformis MW3 (green) was overlaid with the respective image of MW3 AprAs−. The volume of the three identified Apr protein spots is strongly increased in B. licheniformis MW3 AprAs− (red) compared to the original strain B. licheniformis MW3 (green). In addition, the gel image of B. licheniformis MW3 (green) was overlaid with the respective image of MW3 AprAs− pV2::aprAs (red). After vector complementation, the effect of the mutation is reversed. B. licheniformis MW3 AprAs− pV2::aprAs shows only a slight increase in Apr protein expression compared to B. licheniformis MW3. (c) To quantify the Apr expression the spot volumes (in percentage of the whole protein spot volume) were calculated. The average of the normalized spot volumes of three replicates is shown and the standard deviation is given. The Apr protein expression in the extracellular proteome of B. licheniformis MW3 AprAs− is approximately three times increased compared to the original strain B. licheniformis MW3. The Apr expression of B. licheniformis MW3 AprAs− pV2::aprAs is reduced compared to the precursor strain B. licheniformis MW3 AprAs−, but still higher than in B. licheniformis MW3.
