Fig. 3

Cell death-induced biofilm formation by the different test strains. a Microtitre-well showing biofilm formation in the wildtype along with both the mutants, complement strains and the lrgB overexpressing mazE mutant after crystal violet staining: with untreated and DNaseI treatment. b The mazE antitoxin mutant showed increased biofilm formation in case of untreated while, DNaseI treatment drastically reduced the biofilm formation in case of wildtype and mazE mutant strain. The graph depicts the correlation of the measured crystal violet absorbance of the attached cell (Absorbance 570 nm) to the planktonic cell growth (Absorbance 600 nm). Each point and the standard deviation are the measures of three independent samples per condition. (t-test: ‘*’ denotes p-value between 0.01–0.05: significant; ‘**’ denotes p-value between 0.001–0.01: highly significant; ‘n.s’ denotes not significant). c Confocal laser scanning microscopy of the three test strains obtained from the biofilm, after staining with 4 µM propidium iodide (PI). Dead cells appear as discrete red puncta when excited with a 522-nm argon-krypton laser and emission collected with a 580-nm to 630-nm bandpass filter. d Percentage of dead/total biomass were calculated for the MRSA cells from the biofilm sample. Each point and the standard deviation is the measure of three independent samples per condition. Flow cytometry analysis for e Control (unstained), f wildtype, g mazE mutant, h mazF mutant, were done with the following settings: threshold set to side scatter (SSC) and flow rate set to the lowest possible, 6 µl/min. All measurements were run for 10,000 events. Data were averaged over three identical experiments. The percentage of dead cells was analyzed using BD FACS Diva™ (Becton Dickinson)